Maternal and Fetal Outcomes of Pregnancies Complicated by ... · nephron endowment is associated with adult cardiovascular and renal disease, many models of reduced nephron endowment

Maternal and Fetal Outcomes of Pregnancies

Complicated by Obesity

Xiaochu Cai

Bachelor of Science (Honours)

A thesis submitted for the degree of Doctor of Philosophy at

Monash University in March 2016

Department of Physiology

Monash University

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Table of Contents

Abstract ....................................................................................................................... i

General Declaration ................................................................................................. iii

Acknowledgements .................................................................................................. iv

Publications and Conference Abstracts.................................................................. v

Abbreviations .......................................................................................................... vii

Chapter 1 GENERAL INTRODUCTION .................................................................. 1

1.1. THE MATERNAL ENVIRONMENT DETERMINES FETAL OUTCOMES ..... 4

1.2. HEMODYNAMIC ADAPTATIONS OF PREGNANCY ................................... 5

1.2.1. Cardiovascular Adaptations of Pregnancy .......................................................... 5

1.2.1.1. Systemic Vascular Resistance .................................................................... 5

1.2.1.2. Arterial Pressure ......................................................................................... 7

1.2.1.3. Cardiac Structure and Function .................................................................. 9

1.2.1.3.1. Stroke Volume, Heart Rate and Cardiac Output ................................... 9

1.2.1.3.2. Ventricular Remodelling ..................................................................... 10

1.2.2. Renal Adaptations of Pregnancy ...................................................................... 12

1.2.3. Hemodynamic Changes Post-partum ............................................................... 13

1.3. CARDIOVASCULAR AND RENAL ADAPTATIONS OF OBESITY ............ 15

1.3.1. Cardiovascular Adaptations of Obesity ............................................................. 15

1.3.1.1. Arterial Pressure ....................................................................................... 15

1.3.1.2. Cardiac Structure and Function ................................................................ 16

1.3.1.2.1. Stroke Volume, Heart Rate and Cardiac Output ................................. 16

1.3.1.2.2. Ventricular Remodelling ..................................................................... 16

1.3.2. Renal Adaptations of Obesity ........................................................................... 17

1.4. MATERNAL OUTCOMES OF PRE-PREGNANCY OBESITY .................... 18

1.4.1. Prevalence of Pre-pregnancy Obesity .............................................................. 18

1.4.2. Obstetric Complications of Obesity ................................................................... 18

1.4.3. Maternal Hemodynamic Adaptations In Obese Women ................................... 19

1.4.4. Long-Term Maternal Outcomes of Pre-pregnancy Obesity ............................... 20

1.5. MATERNAL OBESITY AND THE PROGRAMMING OF CARDIOVASCULAR AND RENAL DISEASE ...................................................... 22

1.5.1. Evidence from Human and Animal Studies ...................................................... 22

1.5.2. Maternal Obesity and The Intrauterine Environment......................................... 23

1.5.2.1. The Impact of Placenta ............................................................................. 23

1.5.2.2. Maternal Circulating Factors ..................................................................... 24

1.6. FETAL PROGRAMMING OF KIDNEY DEVELOPMENT AND ITS IMPACT ON CARDIOVASCULAR AND RENAL HEALTH ................................................. 25

1.6.1. Programming of Nephron Endowment ............................................................. 25

1.6.2. Impact of Low Nephron Endowment on Cardiovascular and Renal Health ....... 27

1.6.2.1. Nephron Endowment and Hypertension ................................................... 29

1.6.2.2. Nephron Endowment and Renal Dysfunction............................................ 29

1.6.3. Animal Model of Low Nephron Endowment ...................................................... 30

1.6.3.1. Programed and Congenital Models of Low Nephron Endowment ............. 30

1.6.3.2. GDNF Heterozygous Mice ........................................................................ 31

1.7. REGULATION OF RENAL FUNCTION IN AN ANIMAL MODEL OF LOW NEPHRON ENDOWMENT .................................................................................... 34

1.7.1. Role of NO in the Regulation of Renal Function ............................................... 34

1.7.1.1. Renal NO in Normal Kidneys .................................................................... 34

1.7.1.2. Role of NO in Nephron Deficient Kidneys ................................................. 35

1.8. HYPOTHESES AND AIMS .......................................................................... 36

Chapter 2 GENERAL METHODS .......................................................................... 39

2.1. ANIMALS ..................................................................................................... 39

2.1.1. Mouse Model of Pre-pregnancy Obesity: Chapter 3 & 4 ................................... 39

2.1.1.1. Mating....................................................................................................... 39

2.1.2. GDNF Heterozygous Mouse: Chapter 5 ........................................................... 40

2.1.2.1. Animal Origin ............................................................................................ 40

2.1.2.2. Housing and Diet ...................................................................................... 40

2.1.2.3. Genotyping ............................................................................................... 41

2.2. CONSCIOUS RENAL FUNCTION EXPERIMENTS .................................... 42

2.2.1. Assessment of Urinary Excretory Profile .......................................................... 42

2.2.2. Analysis of Urine and Plasma Samples ............................................................ 43

2.2.2.1. Urinary Osmolality and Electrolytes .......................................................... 43

2.2.2.2. Albumin Assay .......................................................................................... 43

2.2.2.2.1. Assay Principle .................................................................................. 43

2.2.2.2.2. Assay Procedure ............................................................................... 44

2.2.2.2.3. Data Analysis ..................................................................................... 44

2.2.2.3. Creatinine Clearance (Chapter 5) ............................................................. 44

2.2.3. Transcutaneous Measurement of GFR in Conscious Mice (Chapter 4) ............ 45

2.2.3.1. Principle of Transcutaneous Measurement of GFR................................... 45

2.2.3.2. Experimental Protocol ............................................................................... 46

2.2.3.2.1. Fur Depilation .................................................................................... 46

2.2.3.2.2. Experimental Procedure..................................................................... 46

2.2.3.3. Data Analysis ............................................................................................ 48

2.3. CONSCIOUS BLOOD PRESSURE MEASUREMENTS .............................. 49

2.3.1. Implantable Telemetry System for Mice............................................................ 49

2.3.2. Implantation Surgery ........................................................................................ 49

2.4. POST-MORTEM TISSUE COLLECTIONS .................................................. 51

2.4.1. Maternal and Fetal Tissues at GA19 (Chapter 3) ............................................. 51

2.4.2. Maternal tissues at 4WPW (Chapter 4) ............................................................ 51

2.4.3. GDNF HET Mice (Chapter 5) ........................................................................... 52

2.5. STEREOLOGY ............................................................................................ 53

2.5.1. Processing, Embedding and Sectioning of Fetal Kidneys ................................. 53

2.5.2. Sampling Sections ........................................................................................... 53

2.5.3. Histochemical Staining with A. hypogaea PNA ................................................. 54

2.5.4. Counting PNA-positive Glomeruli ..................................................................... 55

2.5.5. Estimating Kidney Volume................................................................................ 57

2.6. CARDIAC CINE-MAGNETIC RESONANCE IMAGING (Chapter 3) ........... 58

2.6.1. Animal Preparation ........................................................................................... 58

2.6.2. MRI scan .......................................................................................................... 59

2.6.3. Data Analysis ................................................................................................... 60

2.7. ASSESSMENT OF COLLAGEN CONTENT ............................................... 61

2.7.1. Hydroxyproline Colorimetric Assay (Chapter 4) ................................................ 61

2.7.1.1. Preparing Tissue Hydrolysates ................................................................. 61

2.7.1.2. Colorimetric Reaction and Absorbance Measurement .............................. 61

2.7.2. Histopathological and Microscopic Analysis of Collagen Content in Cardiac and Renal Tissue. ............................................................................................................... 62

2.7.2.1. Assessment of Collagen Content using PSR Staining .............................. 62

2.7.2.1.1. Picrosirius Red Staining ..................................................................... 62

2.7.2.1.2. Renal Collagen Content (Chapter 3) .................................................. 62

2.7.2.1.3. Cardiac Collagen Content (Chapter 3 & 4) ......................................... 62

2.7.2.2. Assessment of Glomerulosclerosis Using PAS Staining (Chapter 4) ........ 63

2.8. STATISTICAL ANALYSIS OF RESULTS ................................................... 65

Chapter 3 OBESITY LIMITS THE NORMAL CARDIOVASCULAR AND RENAL ADAPTATIONS OF PREGNANCY COMPROMISING FETAL KIDNEY DEVELOPMENT ....................................................................................................... 66

3.1. ABSTRACT ................................................................................................. 68

3.2. INTRODUCTION .......................................................................................... 69

3.3. METHODS ................................................................................................... 70

3.4. RESULTS .................................................................................................... 72

3.4.1. Diet-Induced Obesity in Female Mice ............................................................... 72

3.4.2. Arterial Pressure, Heart Rate and Activity ........................................................ 73

3.4.3. Cardiac MRI ..................................................................................................... 75

3.4.4. Renal Excretory Profile ..................................................................................... 76

3.4.5. Fetal Outcomes ................................................................................................ 78

3.4.6. Maternal Outcomes .......................................................................................... 81

3.5. DISCUSSION ............................................................................................... 83

3.6. CONCLUSION ............................................................................................. 87

Chapter 4 DOES PREGNANCY EXACERBATE THE CARDIOVASCULAR AND RENAL EFFECTS OF OBESITY? ............................................................................ 88

4.1. INTRODUCTION .......................................................................................... 89

4.2. METHODS ................................................................................................... 90

4.2.1. Animals ............................................................................................................ 90

4.2.2. Telemetry Recordings ...................................................................................... 90

4.2.3. Assessment of urinary excretory profile and renal function (GFR) .................... 90

4.2.4. Plasma and Tissue Collection .......................................................................... 90

4.2.5. Statistical Analysis ........................................................................................... 91

4.3. RESULTS .................................................................................................... 92

4.3.1. Post-partum Arterial Pressure and Heart Rate ................................................. 92

4.3.2. Post-partum Renal Function ............................................................................. 95

4.3.3. Maternal Outcomes .......................................................................................... 97

4.3.4. Renal and Cardiac Fibrosis ............................................................................ 100

4.4. DISCUSSION ............................................................................................. 103

4.5. CONCLUSION ........................................................................................... 106

Chapter 5 THE ROLE OF NITRIC OXIDE IN THE REGULATION OF RENAL FUNCTION AND ARTERIAL PRESSURE IN NEPHRON DEFICIENT MICE ........ 107

5.1. INTRODUCTION ........................................................................................ 108

5.2. METHODS ................................................................................................. 110

5.2.1. Animals .......................................................................................................... 110

5.2.2. Experimental Protocol .................................................................................... 110

5.2.3. Terminal Tissue Collection ............................................................................. 110

5.2.4. RT-qPCR ....................................................................................................... 111

5.2.5. Statistical Analysis ......................................................................................... 111

5.3. RESULTS .................................................................................................. 113

5.3.1. Basal Cardiovascular and Renal Excretory Profile ......................................... 113

5.3.2. Arterial Pressure and Renal Excretory Profile During NOS Inhibition ............. 115

5.3.3. Terminal Tissue Weights ................................................................................ 118

5.3.4. Gene Expression of Sodium and Water Channels .......................................... 120

5.4. DISCUSSION ............................................................................................. 122

5.5. CONCLUSION ........................................................................................... 126

Chapter 6 GENERAL DISCUSSION ................................................................... 127

6.1. BRIEF OVERVIEW AND KEY FINDINGS ................................................. 128

6.2. THE IMPACT OF PRE-PREGNANCY OBESITY ON THE MOTHER ....... 130

6.3. THE IMPACT OF PRE-PREGNANCY OBESITY ON FETAL HEALTH AND KIDNEY DEVELOPMENT ................................................................................... 132

6.4. THE RENAL FUNCTION IN NEPHRON DEFICIENT ANIMALS ............... 134

6.5. LIMITATIONS AND FUTURE DIRECTIONS ............................................. 135

6.6. CONCLUDING REMARKS ........................................................................ 137

i

Abstract

There is an increasing prevalence of maternal obesity in Western society. Maternal

obesity is associated with adverse maternal and fetal outcomes. Yet, our knowledge of the

impact of pre-pregnancy obesity on the hemodynamic adaptations of the mother during

pregnancy and postpartum is negligible. The fetal kidney is considered highly sensitive to an

adverse intrauterine environment. Surprisingly, the impact of maternal obesity on fetal kidney

development is poorly understood.

In this thesis, I established a mouse (C57BL6/J) model of diet-induced obesity and

used radiotelemetry, cardiac cine-MRI, and urine samples to characterize cardiovascular and

renal health. Obese mice demonstrated the major characteristics of human obesity pre-

pregnancy including 47% greater body weight, impaired glucose metabolism, hypertension,

cardiac hypertrophy, elevated cardiac output and albuminuria. Whilst mean arterial pressure

(MAP) and heart rate (HR) remained elevated over control mice throughout pregnancy, the

increases in MAP and HR of obese mice during late pregnancy were blunted. Obese dams

also failed to increase cardiac output, and left ventricular mass by late pregnancy and

albuminuria was exacerbated. These changes in obese dams were associated with greater

fetal loss, fetal growth restriction, altered renal morphology and, in male fetuses, a nephron

deficit (25%).

To determine the effect of pregnancy on the long-term cardiovascular and renal

health of obese mice, primiparous obese and control mice were examined 4-weeks post-

weaning and compared to time-matched nulliparous mice. Pregnancy led to greater visceral

obesity and exacerbated hypertension (light-phase) in obese mice postpartum. Total renal

and glomerular collagen content was greater in obese primiparous mice post-partum but this

was not related to renal dysfunction with GFR and albuminuria of obese mice unaffected by

pregnancy.

Maternal obesity in mice has been shown to lead to a nephron deficit in male fetuses

(Chapter 3). The ability to assess the contribution of a low nephron endowment to long-term

cardiovascular and renal health is often confounded by developmental programming of other

organs/systems in many models of nephron deficiency. Thus the choice of model is

important so that these confounding factors can be minimized. Interestingly, whilst a low

nephron endowment is associated with adult cardiovascular and renal disease, many models

of reduced nephron endowment demonstrate normal renal function and MAP. There is little

understanding of how renal function is maintained in states of nephron deficit, though nitric

oxide (NO) has been implicated. To investigate this, I used a genetic model of reduced

nephron endowment the GDNF heterozygous (HET) mouse. This model demonstrates two

ii

levels of nephron deficit. Renal function and MAP of GDNF HET mice were examined before

and after a 7-day systemic NOS inhibition (L-NAME). Nephron-deficient GDNF HET mice

with both moderate and marked nephron deficit were able to maintain normal GFR and

sodium balance in response to L-NAME. Further GDNF HET mice demonstrated a partial

escape from L-NAME-induced hypertension. These findings indicate that nephron deficient

GDNF mice do not rely heavily on NO to maintain renal function chronically.

In conclusion, findings of this thesis indicate that pre-pregnancy obesity not only

compromises the hemodynamic adaptations of pregnancy leading to poor fetal outcomes but

also has a long-term impact on the cardiovascular and renal health of the mother post-

partum. Early detection of the risk involved and the development of interventions that

enhance pregnancy-initiated hemodynamic adaptations may reduce the long-term impact of

pre-pregnancy obesity on the mother and offspring.

General Declaration

I hereby declare that this thesis contains no material which has been accepted for the award

of any other degree or diploma at any university or equivalent institution and that, to the best

of my knowledge and belief, this thesis contains no material previously published o

by another person, except where due reference is made in the text of the thesis.

Chapter 3 of this thesis was written in a format that was appropriate for submission to the

journal Hypertension. The inclusion of co

active collaboration between researchers and acknowledges input into team

Chapter Title

3 Obesity Limits The

Normal Cardiovascular

and Renal Adaptations

of Pregnancy

Compromising Fetal

Kidney Development

Candidate Name: Xiaochu Cai

Signature:

Date: 18/3/2016

Supervisor Name: Michelle Kett

Signature:

Date: 18/3/2016

iii

Declaration

his thesis contains no material which has been accepted for the award


of my knowledge and belief, this thesis contains no material previously published o



. The inclusion of co-authors reflects the fact that

active collaboration between researchers and acknowledges input into team

Publication

status

Nature and extent of

candidate’s contribution

Obesity Limits The

Normal Cardiovascular

and Renal Adaptations

of Pregnancy

Compromising Fetal

Kidney Development

Submitted

Performed a majority of

experiments, compiled and

analyzed majority of the data,

interpreted data and wrote the

manuscript 75%

Xiaochu Cai

Michelle Kett

his thesis contains no material which has been accepted for the award


of my knowledge and belief, this thesis contains no material previously published or written



authors reflects the fact that the work came from

active collaboration between researchers and acknowledges input into team-based research.

Nature and extent of

candidate’s contribution

Performed a majority of

experiments, compiled and

analyzed majority of the data,

interpreted data and wrote the

manuscript 75%

iv

Acknowledgements

First and foremost I would like to express my sincere gratitude to my supervisor, Dr

Michelle Kett. Michelle, I am so privileged to have this incredible opportunity to undertake my

research project with you. I really appreciate your patience, generosity, support, and

guidance throughout my candidature. Even in the darkest moment of this journey where the

freezer has broken down and I lost so many precious samples, your positivity and

encouragements have kept me going forward to complete this thesis after the horrific setback.

I would also like to extend my appreciation to Dr Luise Cullen-McEwen for her help

and expertise in the design and preparation of stereological analysis; to Dr James Pearson

for his time and input in cardiac MRI experiments and advice on histological analysis; and to

Prof Matthew Watt for his assistance in the measurement of plasma free fatty acids and

triacylglycerol. Thanks also go to Assoc. Prof David Nikolic-Paterson and Dr Frank Ma for

allowing me to perform collagen assays in your lab.

I also want to take this opportunity to acknowledge the scholarship funding agencies,

NHMRC and National Heart Foundation, for their financial support of my candidature.

To the people from the Renal Lab, Kate, Roger, Russell, Lucinda, Lisa, Katrina and

Rebecca, you are a wonderful group of people to be around with in the lab, in the office or in

the mouse room. Your support and guidance over the years are greatly appreciated. A

special thank-you to Katrina for helping me set up the gene expression analysis off-campus

and for your advice and feedback on this thesis. A big thank-you goes to Roger for your

brilliant advice on statistical analysis, and to Lisa for your helping hands and friendship over

the years. To Stacey and Heyley at the Ritchie centre, thank you for your assistance in my

gene expression experiments.

To Rachael Mason, thank you for being a wonderful friend to my family and me

during some of the toughest moments. Your time and effort in proofreading this thesis are

also greatly appreciated. To my personal mentor, Mark Lo, thank you for your

encouragements, time and prayers in recent years.

To my loving wife, Xiaoyu, thank you for your unconditional love, patience and

encouragements during this journey. I could not have completed this thesis without your

unreserved support. I would also like to thank my children, Muzhou and Muhan. You are both

inspiration to me on a daily basis so that I can be more than what I was the day before.

Finally, I would like to express my eternal gratitude to my parents. Thank you both for

the endless support, love and inspiration throughout my education. Mum, thank you for away

believing in me and being my biggest supporter in completing my PhD. Dad, your attitude

towards your career and life has always inspired me to be the best I can be.

v

Publications and Conference Abstracts

Publications

• Walker KA, Cai X, Caruana G, Thomas MC, Bertram JF, Kett MM, High nephron

endowment protests against salt-induced hypertension, AJP Renal Physiology 2012 July

15;303 (2): F:253-8

• Gurushighe S, Rrown RD, Cai X, Samuel CS, Ricardo SD, Thomas MC, Kett MM, Does a

nephron deficit exacerbate the renal and cardiovascular effects of obesity? PLos One

2013 Sep 3;8 (9):e73095

• Ellery, SJ, Cai, X, Walker DD, Dickinson,H, Kett MM, Transcutaneous measurement of

glomerular filtration rate in small rodents: Through the skin for the win? Nephrology

(Carlton) 2015 Mar 20(3) P:117-23

Conference Abstracts

International conferences

• Cai X, Brown RD, Thomas MC, Kett MM. The role of nitric oxide in the regulation of

arterial pressure and renal function in nephron deficient mice. (24th Scientific Meeting of

the International Society of Hypertension, Sydney, Oct 2012) (Poster)

• Cai X, Kett MM. Does obesity alter the cardiovascular and renal adaptation of pregnancy?

(AHA High Blood Pressure Research 2011 Scientific Sessions, New Orleans, USA, Sep

2013) (Poster)


(3rd ISH Young Investigator Symposium, New Orleans, USA, Sep 2013) (Poster)


(Annual Scientific Meeting of the Australian and New Zealand Obesity Society, Melbourne,

Oct 2013) (Oral)

vi

National conferences

• Cai X, Brown RD, Kett MM. The role of nitric oxide in the regulation of arterial pressure in

nephron deficient mice. (High Blood Pressure Research Council of Australia Annual

Scientific Meeting, Perth WA, Dec 2011) Selected student oral finalists


(High Blood Pressure Research Council of Australia Annual Scientific Meeting, Melbourne

VIC, Dec 2013)

vii

Abbreviations

4WPW 4 week post-weaning

7NI 7-nitro-indazole

AQP2 aquaporin 2

AT1aR angiotensin type 1a receptor

AT1R angiotensin type 1 receptor

AT2R angiotensin type 2 receptor

BMI body mass index

BSA bovine serum albumin

BW body weight

Ccre creatinine clearance

cGMP cyclic guanosine monophosphate

CKD chronic kidney disease

CNS central nervous system

CO cardiac output

DNA deoxyribonucleic acid

DOHaD Developmental Origins of Health and Disease

ECG electrocardiogram

EDV end-diastolic volume

EF ejection fraction

ELISA enzyme-linked immunosorbent assay

eNOS endothelial nitric oxide synthase

ESV end-systolic volume

ET-1 endothelin-1

FFA free fatty acid

FITC-sinistrin fluorescein-isothiocyanate labeled sinistrin

FSA filtration surface area

GA gestational age

GDNF glial cell-derived neurotrophic factor

viii

GFR glomerular filtration rate

GTT glucose tolerance test

HCG Human chorionic gonadotropin

HET heterozygous

HFD high fat diet

HPLC high performance liquid chromatography

HR heart rate

HRP horseradish peroxidase

IUGR intrauterine growth restriction

L-NAME N-nitro L-arginnin methyl ester

LV left ventricle

LVM left ventricle mass

MAP mean arterial pressure

MRI magnetic resonance imaging

mRNA messenger ribonucleic acid

NHE3 sodium-hydrogen antiporter 3

NHP non-human primates

NKCC2 sodium-potassium-chloride cotransporter 2

nNOS neuronal nitric oxide synthase

NO nitric oxide

NOS nitric oxide synthase

PAS periodic acid-schiff

PBS phosphate-buffered saline

PCR polymerase chain reaction

PN postnatal day

PNA peanut agglutinin

PNA peanut agglutinin

PSR picrosirius red

RAS renin angiotensin system

RPF renal plasma flow

ix

RPM revolutions per minute

RSNA renal sympathetic nervous system

RT-qPCR real-time quantitative polymerase chain reaction

RVR renal vascular resistance

SGA small for gestational age

SNGFR single nephron glomerular filtration rate

SV stroke volume

SVR systemic vascular resistance

t1/2 plasma half-life

TAG triacylglycerol

TGF tubuloglomerular feedback

TGFβ Transforming growth factor beta

WT wild-type

Chapter 1 General Introduction

1

Chapter 1 GENERAL INTRODUCTION

The global prevalence of adult hypertension is expected to reach 30% by 2025.199

The latest Global Burden of Disease Study revealed that elevated arterial pressure (systolic

BP >115 mmHg) has become one of the biggest risk factors for global burden of disease and

morbidity, leading to 9.4 million deaths each year.228 The etiology of hypertension is complex

and multifactorial. Factors including the central nervous system, the vasculature, dietary salt

intake, genetics and the environment, have all been implicated in the development of

hypertension. Notably, the work of Guyton and colleagues established that the kidney plays a

dominant role in the pathogenesis of hypertension.147 Further, renal transplantation studies

have demonstrated that blood pressure follows the kidney,140 suggesting that structural and

functional changes in the kidney can initiate the development of hypertension. Whilst

historically the role of the environment on the risk for hypertension has focused on childhood

and adulthood, more recently the in utero and neonatal environments have been identified as

key factors in the etiology of hypertension.

The Developmental Origins of Health and Disease (DOHaD) hypothesis evolved from

early epidemiological studies conducted by Barker and colleagues demonstrating a strong

correlation between adult mortality due to coronary heart disease and low birth weight related

infant mortality.20,23 The DOHaD hypothesis states that “adverse environments during fetal

and early postnatal development alter the structure, function and metabolism of one or more

organ systems, predisposing an individual to a greater risk of developing diseases in adult

life”.363 This phenomenon has also been described as the “Fetal Programming” of adult

diseases. It has become increasingly clear that the fetal kidney development is very sensitive

to an adverse intrauterine environment leading to permanent structural and functional

abnormalities of the kidney (renal programming), and an increased risk in developing

cardiovascular and renal disease later in life.201 The consequences of maternal undernutrition

on renal programming have been extensively studied, particularly with respect to reduced

nephron endowment. Whilst maternal undernutrition remains a significant global issue, a

more pressing concern however is the increasing burden of maternal overnutrition. As a

result of the obesity epidemic in western society, there has been an alarming increase in the

rate of obesity among women of childbearing age. It has been shown that maternal obesity is

not only associated with significant complications during pregnancy, but it also impacts on

fetal outcomes leading to greater risk of cardiovascular and metabolic disorders in offspring.


2

The impact of maternal obesity on fetal kidney development, specifically nephron endowment,

however has not been thoroughly investigated.

Pregnancy initiates a cascade of cardiovascular and renal adaptations that ultimately

lead to a marked increase in cardiac output.60 This significant elevation of cardiac output is

vital for maintaining adequate delivery of oxygen and nutrients to the fetoplacental unit.

Inadequate physiological adaptations during pregnancy lead to suboptimal fetal development

due to insufficient delivery of blood across the placenta and into the fetal circulation.360

Obesity, on the other hand, represents a state of hyperdynamic circulation, characterized by

increased cardiac output, tachycardia, hypertension, plasma volume expansion and

increased glomerular filtration rate.151 However, as this literature review will highlight, the

understanding of how this altered hemodynamic profile in obese women prior to conception

influences the hemodynamic adaptations of normal pregnancy is negligible. Such knowledge

is important when considering the implication of maternal obesity for fetal programming, and

will thus be examined in Chapter 3.

It is well recognized that normal pregnancy does not impact on the long-term

cardiovascular and renal health of the mother despite the marked adaptations of the

cardiovascular and renal systems during pregnancy.29 However pregnancy complications

such as hypertensive disorders of pregnancy, including preeclampsia, are associated with

increased risk of cardiovascular and renal disease later in life.240 Studies have also shown

that a compromised renal function prior to pregnancy not only limits the renal adaptation of

pregnancy but can also lead to the progression of chronic renal disease in the mother later in

life.32 Chronic obesity is associated with significant cardiovascular and renal abnormalities,136

yet whether pregnancy exacerbates these adverse outcomes of obesity post-birth is

unknown and will be investigated in Chapter 4.

A reduction in nephron endowment is a common outcome of adverse intrauterine

environment. Studies in Chapter 3 examined whether maternal obesity leads to a reduction

in nephron endowment in offspring. However, whilst a reduced nephron endowment is

associated with the development of hypertension and chronic renal disease, understanding

the impact of reduced nephron endowment on the cardiovascular and renal health in the

offspring is often confounded by the global effect of the adverse intrauterine environment to

other organ systems such as the heart and vasculature. There is little understanding of the

factors that control renal function in states of nephron deficit. Nitric oxide (NO) plays a

significant role in the regulation of arterial pressure and renal function in individuals with

normal nephron number. However, the role of NO in maintaining cardiovascular and renal

health in nephron deficient animal is not well understood and is the focus of studies in

Chapter 5. To eliminate the confounding global effect of intrauterine insult on cardiovascular


3

system, a unique genetic mouse model of reduced nephron endowment with 2 levels of

nephron deficit will be used.

The following review will evaluate the current literature on the cardiovascular and

renal adaptations of normal pregnancy, the hyperdynamic circulation of obesity, and

gestational and long-term outcomes of pre-pregnancy obesity. It will also highlight the gaps

in our understanding of how pre-pregnancy obesity programmes cardiovascular and renal

disease in offspring. The impact of renal programming, in particular a low nephron

endowment on the development of hypertension and renal dysfunction will be discussed.

Lastly, the regulation of renal function in normal kidneys and nephron deficient kidneys will

also be discussed.


4

A healthy maternal environment is the key for a successful pregnancy and optimal

fetal outcomes. The early epidemiological studies by Barker and colleagues facilitated the

establishment of the DOHaD Hypothesis.20-22 Initially low birth weight, as a clinical marker of

suboptimal intrauterine environment, was found to be associated with increased risk of

mortality from coronary heart disease.278 As the DOHaD hypothesis continued to evolve, it

has been recognized that low birth weight is not the only predictor of adult cardiovascular

and renal outcomes.181,182 In fact adverse intrauterine environments secondary to poor

maternal health, maternal malnutrition, maternal stress and placental dysfunction also have

significant impact on fetal outcomes.5,168,341 Maternal undernutrition has long been

recognized as an insult that leads to intrauterine growth restricted (IUGR) and small for

gestational age (SGA) babies,315,379 increasing the risks of developing cardiovascular and

metabolic disease later in life.39,315,377

Whilst it is clear that maternal nutrition contributes to adverse fetal outcomes, the

importance of adequate maternal hemodynamic adaptations for optimal fetal growth has

been largely overlooked in the literature. Maternal hemodynamic adaptations from early

pregnancy through to birth are critical for placental maturation and maximizing cardiac output

to facilitate rapid fatal growth during second half of the pregnancy.60,235,309 Further, studies

have demonstrated that poor fetal outcomes such as fetal loss, IUGR and SGA birth are

associated with inadequate maternal hemodynamic adaptations.19,103 Pre-pregnancy

cardiovascular and renal health directly influences the extent of maternal hemodynamic

adaptations, and thus impacts on maternal and fetal outcomes.7,42,128,163,360 It has been shown

that pre-existing cardiac dysfunction in the mother is associated with increased incidence of

pre-term birth, SGA babies and increased admittances to neonatal intensive care

unit.128,163,360 Women with advanced chronic kidney disease (very low glomerular filtration

rate) prior to conception also have significantly increased risk of pre-term birth, IUGR babies

and even fetal death.7 These findings highlight the importance of cardiovascular and renal

health prior to conception in the ability of cardiovascular and renal systems to adapt during

pregnancy and subsequent influence on fetal outcomes.

1.1. THE MATERNAL ENVIRONMENT DETERMINES FETAL

OUTCOMES


5

1.2. HEMODYNAMIC ADAPTATIONS OF PREGNANCY

Pregnancy is associated with profound and reversible changes to the structure and

function of the cardiovascular and renal systems, ultimately leading to an elevated cardiac

output (CO) and thus adequate delivery of oxygen and nutrients to the fetus (Figure 1.1). As

highlighted above, studies indicate that inadequate increase in CO during pregnancy is

associated with poor maternal and fetal outcomes. In this part of the literature review, both

the cardiovascular and renal adaptations of normal pregnancy will be discussed.

Figure 1.1. Hemodynamic adaptations of pregnancy. Pregnancy initiates a cascade of cardiovascular and renal adaptations that leads to a marked increase in cardiac output and therefore delivery of oxygen and nutrients to fetoplacental unit. Figure modified from Conrad et al.75

1.2.1. Cardiovascular Adaptations of Pregnancy

Cardiovascular adaptations during pregnancy include a marked fall in systemic

vascular resistance, a mid-gestational dip in arterial pressure, an increase in plasma volume,

heart rate (HR), stroke volume (SV), and cardiac output (CO) in addition to marked cardiac

hypertrophy, and each of these will be discussed (Figure 1.1).

1.2.1.1. Systemic Vascular Resistance

The cascade of hemodynamic adaptations of pregnancy is initiated by a marked fall in

systemic vascular resistance (SVR), which is caused by a significant peripheral vasodilation

�Stroke Volume


6

(Figure 1.1). Studies have shown that SVR starts to fall as early as 5 weeks gestation

reaching a nadir by 20 weeks gestation (-34%).309 SVR rises slightly during the second half

of pregnancy, however remains significantly lower than pre-pregnancy level at term (-

27%).60,236,309 This marked fall in SVR occurs prior to the completion of placentation, which

occurs between 6 to 12 weeks of gestation, suggesting that placental influence on

vasodilation during early pregnancy is negligible.50 As with humans, a persistent decrease in

SVR has been demonstrated in rats from early through to late pregnancy.53,129

The mechanisms underpinning the pregnancy-induced fall in SVR are not fully

elucidated, but studies have largely focused on the actions of relaxin and nitric oxide (NO).

Relaxin is an ovarian hormone secreted by the corpus luteum. Relaxin circulates during the

late luteal phase of the menstrual cycle, and increases markedly after conception.334 Relaxin

has been identified as major hormone that mediates pregnancy-induced reduction in

SVR.75,94 Chronic administration of relaxin to conscious non-pregnant female and male rats

mimics the hemodynamic changes observed during pregnancy, including the marked fall in

SVR and the increase in arterial compliance.74,92,93 Relaxin has been shown to promote

vasodilation via increasing downstream NO production both acutely and chronically.76 NO, a

potent vasodilator is important in relaxin-mediated systemic and renal vasodilation during

pregnancy.75 Relaxin can not only activate endothelial nitric oxide synthase (eNOS) directly

via the activation of PI3K pathway, but also increase the expression and activity of eNOS.76

Systemic NOS inhibition in pregnant rats abolishes the fall in SVR and the rise in CO during

pregnancy (Figure 1.2).53 These studies suggest that the cardiovascular adaptations of

pregnancy that are vital for successful pregnancy are NO dependent.


7

Figure 1.2. Cardiovascular and renal response to chronic NOS inhibition (L-NAME) during pregnancy. Effects of L-NAME on cardiac output (A), systemic vascular resistance (B), glomerular filtration rate (C) and renal plasma flow (D) in pregnant rats on gestational day 14. Figure from Schrier & Ohara.331 Data were originally published by Cadnapaphornchai et al 53

1.2.1.2. Arterial Pressure

Despite a marked increase in CO and plasma volume, mean arterial pressure (MAP)

falls from early gestation. This fall in MAP in early pregnancy is largely driven by the marked

fall in SVR such that the timing of the fall in these two parameters correlates with each other

during pregnancy in humans.60 Longitudinal studies in humans has found that MAP reaches

a nadir around mid-pregnancy (Figure 1.3A).152,236,309 Unlike the persistent reduction in SVR,

MAP rises post-nadir to pre-pregnancy levels by approximately 28 weeks of pregnancy.309

MAP continues to rise exceeding pre-pregnancy levels, such that at 38 weeks SBP and DBP

are 5.6% and 7.5% respectively above pre-pregnancy levels.309 Using radiotelemetry to

continuously measure MAP, studies have shown that mice also demonstrated the mid-

gestational dip in MAP (Figure 1.3B), indicating that the mouse is a suitable model to

investigate hypertensive disorders of pregnancy in the laboratory setting.48,52

A B

C D


8

Figure 1.3 The fall in MAP during pregnancy in humans and mice. (A) MAP adaptation in human pregnancy, Graph from Hall et al 152; (B) MAP adaptation in mouse pregnancy measured using radiotelemetry. Graph from Butz et al 52.

Whilst the fall in MAP in early pregnancy may be largely driven by the relaxin/NO-

mediated fall in SVR,60 additional factors are also implicated in the control of MAP during

pregnancy, including the Renin Angiotensin System (RAS). Studies in humans have found

that although RAS is upregulated during pregnancy, the presser effect of Angiotensin II (Ang

II) via Angiotensin type 1 receptor (AT1R) is attenuated in pregnant women.126 Further the

depressor effect of Ang II via angiotensin type 2 receptor (AT2R) has been shown to play

important role in the regulation of arterial pressure during pregnancy.55,256 This was

demonstrated recently by Mirabito et al 256 who found that the fall in MAP during pregnancy

was completely abolished in AT2R knockout mice.

A

B


9

1.2.1.3. Cardiac Structure and Function

1.2.1.3.1. Stroke Volume, Heart Rate and Cardiac Output

In humans, increases in both SV and HR contribute to the increase in CO during

pregnancy (Figure 1.4).279 However, SV is the primary determinant of CO during

pregnancy.179,279,309 SV increases sharply from early pregnancy resulting in a 32% rise by 20

weeks (Figure 1.4).279 Meanwhile, HR increases by 11-12% by mid-pregnancy (Figure

1.4).309 Although SV drops slightly during the third trimester, CO continues to rise up to 50%

of pre-pregnancy level by 32 weeks and remains stable towards term (Figure 1.4).179,309 The

maintenance of CO during third trimester is mainly attributed to a second increase in HR (17-

18%) in this period (Figure 1.4).179,309,322 The progressive increase in SV during early

pregnancy is mainly attributed to the increase in preload due to plasma volume expansion

(See Section 1.2.2).75,179

Figure 1.4. Change in cardiac function during human pregnancy. Percentage changes in cardiac output (CO), stroke volume (SV) and heart rate (HR) from pre-pregnancy values across pregnancy in humans. Figure modified from Ouzounian & Elkayam (2012) 279

This dramatic increase in SV and CO in humans has also been demonstrated in the

gravid rats. Slangen et al 342 reported a 28% and 20% increase in SV and CO, respectively

by gestational age (GA) 12, reaching 29% and 35% respectively by GA18, however, no

change in HR was detected in this model. In contrast, using radiotelemetry Butz and

Davisson52 showed that HR of pregnant C57BL6/J mice significant increased from early

gestation (14% GA6-8), through to mid- (18% GA11-13) and late-gestation (17% GA18-20),

an increase that was similar in timing and magnitude to that observed in humans. Using

Doppler ultrasonography, pregnant C57BL6/J mice also showed significant increase in CO

Inc

rea

se

(%

)

CO

SV

HR


10

(48%) in late pregnancy, again an increase of similar magnitude and timing as observed in

humans.213 Measurement of SV and CO in mice using Doppler ultrasonography is limited by

the need to use a fixed ventricular geometry to calculate SV, resulting a less accurate

measurement in these parameters.344 This limitation can be overcome by using a cardiac

magnetic resonance imaging (MRI) protocol specific for mice, which allows more accurate,

reproducible and repeated measurements of cardiac geometry across multiple cardiac

cycles.211 Thus in Chapter 3 MRI will be used to determine SV and CO before and during

pregnancy.

1.2.1.3.2. Ventricular Remodelling

Following plasma volume expansion, the maternal heart undergoes significant

remodelling (ventricular hypertrophy). Ventricular hypertrophy is a compensatory

enlargement of the left ventricle in response to volume or pressure overload.37 Chronic

pressure overload, such as in states of hypertension and heart failure, often leads to

concentric hypertrophy. Concentric hypertrophy is generally classified as a pathological and

is characterized by greater chamber wall thickness and small reduction or no change in

chamber volume.37 Concentric hypertrophy is associated with a parallel addition of

sarcomeres leading to an increase in myocyte cell width (Figure 1.5).37 On the other hand,

persistent volume overload, as occurs in pregnancy or endurance exercise, results in

eccentric hypertrophy, which is characterized by a proportional enlargement of chamber

volume and wall thickness (Figure 1.5).37 This type of hypertrophy is a reversible

physiological remodelling that is associated with addition of sarcomeres in series leading to

increases in myocyte cell length.139


11

Figure 1.5. Ventricular hypertrophy. Pressure overload such as hypertension and strength training causes thickening of left ventricular wall due to the addition of sarcomeres in parallel and leads to concentric hypertrophy. Volume overload such as pregnancy and endurance training leads to proportional enlargement of chamber size and wall thickness via addition of sarcomeres in series and results in a reversible eccentric hypertrophy. Figure modified from Bernardo et al 37

Ventricular hypertrophy during pregnancy is a transient and reversible process that is

not only associated with increases in left ventricular (LV) chamber size, but also involves a

significant increase in LV mass.339 Robson et al 309 showed that both LV mass and LV wall

thickness increased gradually throughout pregnancy in humans, reaching their maximum (53%

and 28% above pre-conception states, respectively) by 38 weeks of gestation. Pregnant

mice also demonstrate a marked increase in LV mass and chamber volume, however an

increase in wall thickness was not detected.105 This study in mice also confirmed that

eccentric hypertrophy at late pregnancy was associated with increase in myocyte cell

length,105 consistent with the characteristic of eccentric hypertrophy in humans.139


12

1.2.2. Renal Adaptations of Pregnancy

Pregnancy-induced systemic vasodilation is accompanied by marked vasodilation

within the renal circulation, resulting in significant increase in renal plasma flow (RPF) and

glomerular filtration rate (GFR). The landmark human study by Chapman et al 60

demonstrated that both RPF (para-aminohippurate clearance) and GFR (inulin clearance)

increased significantly by 6 weeks of gestation, consistent with the timing of the marked fall

in renal vascular resistance (RVR). The magnitude of the rise in GFR was 45-55% by the

end of first trimester and up to 67% in late gestation.60,90,305 Whilst GFR continues to rise until

late gestation (36 weeks), RPF plateaus at 12 weeks of gestation, yet remains elevated until

late pregnancy.60,305 Anatomically, the kidneys also increase in length and volume to

accommodate the physiological adaptations of the renal system during pregnancy.17,63,65

Concomitant with systemic vasodilation, a progressive expansion of plasma volume

was detected as early as 6 weeks of gestation in humans.60,184 This maternal plasma volume

expansion in early pregnancy is mainly facilitated by marked renal sodium and water

retention, which is mediated by activation of the RAS.31,231 Schrier and Briner330 proposed

that the marked systemic vasodilation during early pregnancy leads to a relative arterial

underfilling which stimulates activation of the RAS. Within the kidney, the RAS promotes the

reabsorption of sodium and water from the renal tubules.332,373 In fact, the increase in plasma

renin activity and plasma aldosterone parallels the plasma volume expansion in humans.60,373

The increase in total plasma volume in the maternal circulation from early pregnancy not only

leads to increase in preload to the heart and thus SV,60 but also facilitates ventricular

hypertrophy via volume overload.198

The reduction in RVR and elevations in RPF and GFR in human pregnancy have also

been demonstrated in the conscious gravid rats, albeit to a lesser magnitude.73 An early

study by Baylis 28 found that the rise in GFR (25%) and RPF (31%) in the gravid rat are a

result of a parallel reduction of afferent and efferent arteriolar resistance that allows single

nephron GFR to increase without a rise in glomerular hydrostatic pressure. The finding that

glomerular pressure does not rise may explain why the sustained hyperfiltration in pregnancy

is not associated with renal injury, unlike other states of hyperfiltration such as diabetic

nephropathy289 and high salt intake239. Further, similar to human pregnancy, rats also

experience progressive plasma volume expansion17,73 and significant increase in kidney

weight28,369 and kidney volume66 by mid-gestation, suggesting a similar renal adaptation of

pregnancy occur in rodents.


13

1.2.3. Hemodynamic Changes Post-partum

Functional and structural adaptations of the cardiovascular and renal systems

achieved during pregnancy return to pre-pregnancy levels during the post-partum period.

However, timing of the return of each parameter to its pre-pregnancy state is different and

often depends on the time when these parameters were assessed. In humans, MAP remains

relatively constant during the first 10 days post-partum period despite SVR increases by 30%

during this period.279 However, study by Clapp et al 70 showed that until 1 year post-partum

SVR was still 11% lower than pre-pregnancy level suggesting the adaptation of SVR during

pregnancy may take more than a year to return to pre-pregnancy state.70 The return of HR to

pre-pregnancy level has been consistently reported to occur between 12-17 weeks post-

partum.70,236 However, there is some controversy regarding the timing and magnitude of

change in SV and CO post-partum in humans. An early human study demonstrated that SV

and CO returns to pre-pregnancy level by 2 weeks post-partum.179 Mahendru et al 236 even

reported a small increase in SV 14-17 weeks post-partum compared with third trimester

measurement with CO back to pre-pregnancy levels at 17 weeks post-partum. However,

Clapp and Capeless70 found that SV falls moderately after birth and remains significantly

elevated over pre-pregnancy levels up to 1 year post-partum. Interestingly when the data for

SV was separated into first pregnancies (primigravida) and subsequent pregnancies

(multigravida), it was found that multigravida retained much higher SV at 1 year post-partum

than primigravida.70 CO followed a similar pattern in this study with CO of primigravida

dropping significantly by 12 weeks post-partum and almost completely back to pre-pregnancy

level by 24 weeks post-partum whilst CO of multigravida remained significantly higher than

pre-pregnancy levels at 24 weeks post-partum.70 Consistent with this, other studies of

predominantly primigravida women (>60%) have demonstrated that CO returns to normal

level by 17-24 weeks post-partum.236,308 These findings indicate that parity may impact the

timing and magnitude of changes in SV and CO post-partum, with multigravida more likely to

retain an elevated SV and CO for a longer period of time than primigravida post-partum. The

extensive ventricular hypertrophy that occurs during pregnancy including an increased LV

mass and LV thickness have also been shown to return to pre-pregnancy by 13 weeks post-

partum in humans.339 Further, the marked increase in GFR during human pregnancy has also

been shown to returns to pre-pregnancy level by 14-17 weeks post-partum.236

Despite the recognition that there is a vast difference in the timing of the return of

cardiovascular and renal parameters to pre-pregnancy levels, the vast majority of studies use

post-partum or even first trimester measurements as surrogate measure for pre-pregnancy

measurement.96,101,130,354 In doing so findings from these studies are confounded likely to

underestimate of the true extent of the pregnancy-induced changes in the cardiovascular and

renal systems. Thus, to properly investigate the cardiovascular and renal adaptation of


14

pregnancy and post-partum returns of these variables, a pre-pregnancy cardiovascular and

renal profile should be established. Chapter 3 & 4 will examine the impact of pre-pregnancy

obesity on cardiovascular and renal systems before and during pregnancy and post-partum.


15

1.3. CARDIOVASCULAR AND RENAL ADAPTATIONS OF OBESITY

Obesity and overweight have become a major global health burden affecting over 1.9

billion adults worldwide in 2014, with 600 million of those in the obese category (BMI over

30kg/m2).275 Findings from the Framingham Heart Study suggest more than 60% of essential

hypertension can be attributable to overweight and obesity.258 Apart from the direct link to

hypertension, obesity is known to associate with a range of cardiovascular, metabolic and

renal disorders. This section of the literature review will focus on the cardiovascular and renal

adaptations of obesity, as this is the hemodynamic background that pregnancy-induced

cardiovascular and renal adaptations must occur in pregnancies that are complicated by

obesity.

1.3.1. Cardiovascular Adaptations of Obesity

1.3.1.1. Arterial Pressure

Obesity has been recognized as one of the biggest contributors to the development of

hypertension. This phenomenon has been demonstrated in epidemiological studies208,258 and

many animal models including dogs,149 rabbits,49 rats,382 and mice.340 The mechanisms that

mediate obesity-induced hypertension are complex and multifactorial in nature. Alterations in

the central nervous system (CNS), renal sympathetic nervous activity (RSNA) and RAS have

been implicated to significantly contribute to obesity-induced hypertension. In a recent

landmark study, Simonds et al 340 demonstrated that leptin-mediated increase in sympathetic

overflow is one of the primary contributors to obesity-induced hypertension. However, the

exact mechanism linking the hypothalamic leptin signaling, sympathetic overflow and

development of hypertension remains to be elucidated. An increase in RSNA and activation

of the renal RAS also contribute to obesity-induced hypertension by promoting sodium

retention.149,150 Obese hypertensive humans demonstrated an elevation in renal

noradrenaline spillover compared to normotensive obese individuals suggesting that obesity-

induced hypertension is associated with increased RSNA.311 Diet-induced obese dogs have

increased tubular sodium reabsorption and require a higher arterial pressure to maintain

sodium balance.91,151 Renal denervation completely normalized arterial pressure in these

obese hypertensive dogs, suggesting that increased RSNA plays a significant role in obesity-

induced hypertension in this model.160,229 The contribution of RAS in obesity-induced

hypertension has also been implicated. This has been demonstrated by effective reduction of

arterial pressure by angiotensin type 1 receptor (AT1R) antagonists and angiotensin

converting enzyme inhibitors in obese animals43,307and humans,132,297 respectively.


16

Animal models are used extensively in obesity research, however these studies

predominately use male animals. Recent studies have demonstrated that sex-difference

exists such that female mice appear to be partially protected from obesity-induced

hypertension.144,368 This sex-specific protection appears to be mediated by estrogen.144,368

Nevertheless, our understanding of the impact of pre-existing obesity-induced hypertension

on pregnancy-initiated cardiovascular and renal adaptations are negligible. Further, whether

pregnancy has any impact on the long-term arterial pressure regulation in obese females is

unknown. Chapter 3 & 4 of this thesis will address some of the gaps in our knowledge.

1.3.1.2. Cardiac Structure and Function

1.3.1.2.1. Stroke Volume, Heart Rate and Cardiac Output

Human obesity is associated with elevated CO, mediated largely by an increase in

SV,72,251,252 but tachycardia also contributes.208 The increase in SV in human obesity is

mainly driven by plasma volume expansion and thus an increase in preload.364 Diet-induced

obesity in mice also leads to significant increase in SV and CO, however this has only been

demonstrated in male mice using echocardiography.36 Obesity-induced tachycardia has also

been demonstrated in animal models146,185 including diet-induced obese male340 and

female144 mice. Thus diet-induced obese female mice are excellent model to investigate the

impact of pre-existing obesity-induced cardiovascular changes on the ability of the

cardiovascular system to adapt during pregnancy. Chapter 3 will address this issue using a

robust mouse model of obesity.

1.3.1.2.2. Ventricular Remodelling

Ventricular hypertrophy is one of the major cardiac adaptations associated with

obesity.85 However, whether obesity-induced cardiac hypertrophy is a physiological or

pathological hypertrophy is controversial. Studies in 1980s by Messerli et al have that shown

both eccentric and concentric hypertrophy occur in obesity, due to synergistically increased

preload (volume expansion) and afterload (hypertension).250,252 However, using 2D

echocardiography, more recent human studies suggested that concentric LV hypertrophy is

likely to be the predominant form of LV hypertrophy in obesity.15,219,376 This was then further

confirmed using cardiac MRI among obese individuals.202

Sex differences have been identified in obesity-induced LV remodelling. Rider et al 302

found that obese men predominantly have concentric LV hypertrophy without LV chamber

dilation. Conversely, obese women are more likely to exhibit both eccentric and concentric

hypertrophy.302 Irrespective of the type of cardiac hypertrophy obese women may have, it is

clear that the LV mass is significantly elevated. What is unknown is that whether pre-existing


17

cardiac hypertrophy in obese women influences the structural and functional adaptations of

the heart during pregnancy. Chapter 3 of this thesis will address this gap in our knowledge.

1.3.2. Renal Adaptations of Obesity

During the early phase of human obesity the renal system is characterized by a state

of hyperfiltration and hyperperfusion that are associated with a reduction in RVR.58,296,301

Obesity also leads to structural changes to the kidney including renal and glomerular

hypertrophy, glomerulosclerosis and albuminuria191,196 and is an independent risk factor for

chronic kidney disease (CKD).136 In addition, animal models of obesity also demonstrated

increased renal lipid accumulation and renal collagen accumulation.95,189 It is unknown

whether pre-existing renal hyperfiltation and hyperperfusion in the kidneys of obese women

impacts the ability of the renal system to adapt during pregnancy. Further, whilst pregnancy

is known to have no long-term impact on renal function post-birth following normal pregnancy,

whether pregnancy-induced renal changes exacerbate obesity-related renal dysfunction (i.e.

albuminuria) is unknown. Studies in Chapter 3 & 4 will address these questions respectively.


18

1.4. MATERNAL OUTCOMES OF PRE-PREGNANCY OBESITY

1.4.1. Prevalence of Pre-pregnancy Obesity

As the overall prevalence of obesity has risen, so too has the prevalence of obesity

among women of reproductive age. Over 50% of the women of reproductive age in Australia,

USA, UK and other European countries are currently overweight or obese.2,226,273 Despite the

reduced fertility rate and increased risk of miscarriage associated with obesity, the

prevalence of maternal obesity has been reported as approximately 20% in UK, and US and

13% in Australia.68,162,244 However, recent data suggests that the prevalence of maternal

obesity even reached 28-33% in rural and low socio-economic groups.84,164 Maternal obesity

is not only a significant epidemic in Western world, but has also started to impact urbanized

populations of developing countries, such as in India and China.294,386

1.4.2. Obstetric Complications of Obesity

Obesity is not only recognized as a major contributor to infertility but also increases

the risk of obstetric complications after successful conception.350 Maternal obesity is now

considered the most common preventable risk factor for complicated pregnancy in the

USA.69 The complications associated with maternal obesity include, but are not restricted to,

hypertensive disorders of pregnancy, preeclampsia, gestational diabetes, renal dysfunction,

venous thromboembolism, pre-term birth, macrosomia, congenital malformations,

miscarriage, stillbirth and maternal and neonatal death.69,188,232,245,283,316 Further, maternal

obesity also increases the risk of induction of labor, post-partum hemorrhage, caesarean

delivery, and caesarean wound complications leading to lengthy hospital stays for both the

mothers and their infants.161,232 The increase in obstetric and fetal complications has resulted

in an increased burden on resources and economics in managing pregnancies that are

complicated by obesity.161,310 In order to improve maternal and fetal outcomes of obese

women, greater understanding of the implication of pre-existing obesity on the hemodynamic

adaptations of pregnancy that are crucial to fetal health is needed.

In addition to the immediate risks associated with managing maternal and fetal health

during pregnancy, an understanding of the long-term effect of maternal obesity on the mother

and offspring is also imperative in disease prevention. Unfortunately, due to limited clinical

data, our knowledge of the long-term impact of maternal obesity on the mother and offspring

comes primarily from laboratory animal research. In general, animal studies indicate that

being obese prior to, or at the beginning of, pregnancy increases the risk of offspring

developing cardiovascular and metabolic disorders in adult life, consistent with the Barker

hypothesis. Yet, the mechanisms that lead to these programming outcomes by maternal


19

obesity are unclear. Maternal environments, particularly maternal hemodynamic adaptations,

are critical in determining fetal nutritional supply and fetal growth, therefore are one of the

main focuses of this thesis.

1.4.3. Maternal Hemodynamic Adaptations In Obese Women

Adequate hemodynamic adaptations are vital for a successful pregnancy. Despite the

increasing prevalence of maternal obesity in the last two decades, only a handful of studies

have investigated the hemodynamic adaptations in obese women during pregnancy. The

mid-gestation dip in arterial pressure is a hallmark of cardiovascular adaptation of normal

pregnancy. However, whether obese women also experience this fall in arterial pressure

during pregnancy is contentious. This is largely due to the lack of pre-pregnancy data

recorded in these studies, a common limitation in studies that examine arterial pressure

during pregnancy.130,359 Further, these studies also differ in the timing and frequency of

arterial pressure measurement throughout pregnancy. Tomoda et al 354 monitored the

changes in MAP from 6 weeks of gestation through to term in normal weight and obese

women who were either primiparous or multiparous. It was found that both obese primigavida

and multigravida had significantly higher MAP over normal weight women across

pregnancy.354 Interestingly, only obese multigravida demonstrated a mid-gestational fall in

MAP whilst obese primigavida did not.354 This suggests that parity may contribute to the

adaptation of MAP in obese women. Further Abdullah et al 1 found that there was no change

in MAP between first and second trimester in morbidly obese women (BMI>45). However,

whether these women were primiparous or multiparaous was not reported.1 Importantly

studies have also demonstrated that the rise in MAP, particularly late in gestation, is

attenuated in women with greater BMI.1,123,352 This suggests that pre-existing obesity may

impact arterial pressure response in late pregnancy contributing to the complications at term.

However, no studies so far have specifically examined this matter.

The limited literature also suggests that the adaptation of CO in pregnancy

complicated by obesity might be compromised.96 Abdullah et al 1 demonstrated that morbidly

obese pregnant women had a blunted increase in CO across the trimesters compared to

non-obese controls. This appeared to be mainly due to blunted increase in SV with HR

increasing across pregnancy.1 Similarly, LV mass of obese women in this study also

remained unchanged indicating limited ventricular remodelling might have occurred during

pregnancy.1 Tomoda et al 354 also reported that the heart of obese pregnant women had a

limited capacity to increase SV in response to a mild exercise challenge. In contrast, a meta-

analysis have shown that obese women demonstrated a blunted rise in HR during pregnancy,

concomitant with the limited rise in CO.159 Further, pregnancy60 and obesity58,301 can


20

independently lead to hyperfiltration or increase in GFR and renal plasma flow, however no

study to date has specifically examined changes in renal function in obese females during

pregnancy. In order to further elucidate the impact of obesity on the hemodynamic

adaptations that occur in normal pregnancy, a well-controlled animal study that examines the

cardiovascular parameters at pre-conception state and across pregnancy is needed. Chapter

3 of this thesis will address this gap in our knowledge.

1.4.4. Long-Term Maternal Outcomes of Pre-pregnancy Obesity

Women who were obese prior to conception are likely to remain obese post-birth

exposing them to greater risks of cardiovascular and renal morbidity and mortality later in

life.112 Surprisingly, we know very little about the long-term impact of pregnancy on the

cardiovascular and renal health in obese women. A recent epidemiological study from the UK

demonstrated that maternal obesity during pregnancy is strongly associated with premature

death and greater risk of a major cardiovascular event in women later in life.223 These

increased risks were independent of common maternal complications such as preeclampsia

and low birth weight.223 In supporting of this finding in the UK population, a study examining

women from a defined region of Israel found that those who were obese before conception

had greater risk of experiencing simple cardiovascular events, early occurrence of

cardiovascular morbidity, and greater number of cardiovascular related hospitalizations

during the 10 year follow-up period.384

Whilst our knowledge of the impact of pre-pregnancy obesity on the long-term

maternal cardiovascular and renal health is limited, the common maternal complications that

obese women experience during pregnancy such as chronic hypertension, preeclampsia and

gestational diabetes have been shown to cause significant cardiovascular and renal

morbidity post-birth. As mentioned earlier (Section 1.4.2) pre-pregnancy obesity leads to a

high incidence of hypertensive disorders of pregnancy, including chronic hypertension and

preeclampsia. It is known that women who experience hypertensive disorders of pregnancy

have greater risks of developing ischemic heart disease, myocardial infarction, heart failure,

ischemic cerebrovascular disease and chronic kidney disease (CKD) later in life.157,240

Further, preeclampsia is also associated with increased risk of chronic hypertension and

CKD.61,345 Interestingly, those women who experienced preeclampsia and remained

hypertensive 2 years post-partum had greater BMI than those who became normotensive

post-birth, indicating being overweight or obese during the post-partum period might

predispose them to chronic hypertension later in life.345 Gestational diabetes is not only

associated with early onset of type II diabetes, but also a significant risk factor for the

development of chronic hypertension and renal dysfunction post-birth.35,158 Further, studies


21

have also shown that women with a compromised renal function prior to conception have

greater risks of developing CKD post-birth.32

Although obesity is associated with only mild proteinuria and glomerulosclerosis,8 it is

possible that pregnancy-related renal adaptions, particularly the glomerular hyperfiltration

could exacerbate the pre-existing renal abnormalities and lead to persistent renal injury post-

partum. Taken together, it is reasonable to predict that pre-pregnancy obesity increases the

risk of females developing chronic hypertension and renal dysfunction post-birth. To fully

elucidate the impact of pregnancy on the long-term risks of cardiovascular and renal health in

obese females, studies that incorporate both lean and obese females that are either

primiparous or virgin should be performed. Studies in Chapter 4 will address some aspects of

this issue in a mouse model of maternal obesity.


22

1.5. MATERNAL OBESITY AND THE PROGRAMMING OF

CARDIOVASCULAR AND RENAL DISEASE

1.5.1. Evidence from Human and Animal Studies

Clinical studies examining the impact of pre-pregnancy obesity on cardiovascular and

renal health of offspring in the long term are lacking. A recent epidemiological study from the

UK found that obesity in pregnancy was associated with increased premature mortality and

hospital admissions for cardiovascular events in adult offspring.299 Limited human studies

suggested a strong association between maternal obesity and hypertension in young

offspring. Studies in the UK and the Netherlands found that pre-pregnancy BMI is

independently associated with elevation of arterial blood pressure in children at 5-6 years of

age.122,124,220 Strong associations between greater pre-pregnancy maternal BMI and elevated

arterial pressure in smaller cohorts of adolescents218 and adults166 have also been found.

Fetal cardiac dysfunction104,186 and increased incidence of congenital heart defect254 has also

been linked to maternal obesity in humans.

Extensive research using animal models has confirmed this strong association

between pre-pregnancy obesity and increased risk of offspring developing cardiovascular

and metabolic disorders later in life. Although the majority of studies using high fat diet to

induce obesity do not show major increase in body weight of the dams at conception, these

studies do demonstrate programmed hypertension in young and adult offspring.40,108,109,118,318

The mechanisms underlying this programming event are unclear, however studies in rats

and rabbits have demonstrated that an increased sympathetic activation in offspring of obese

mothers is likely to be a contributor for the development of hypertension.290,319 Further,

cardiac fibrosis, contractile dysfunction and pathological cardiac hypertrophy have also been

found in offspring of obese female rodents.40,118,174,367 These findings indicate that, similar to

models of maternal undernutrition, maternal overnutrition, in particular maternal obesity is

likely to program adult diseases through an adverse intrauterine environment.

Significant renal injury can also be programmed by maternal overnutrition. Jackson et

al 187 showed that male rat offspring born to high fat/fructose-fed dams and fed a control diet

postnatally had profound renal injury including albuminuria (209%) and glomerulosclerosis

(64%) compared to the offspring of control dams. Female offspring of this model seems have

less aggressive renal injuries compared to male offspring, with exacerbated albuminuria

(~175%), TGFβ protein overexpression (~100%) and mild increases in macrophage

infiltration (~60%), but no evidence of glomerulosclerosis compared to kidneys of control

offspring.119 An abnormal kidney development is likely to be a factor contributing to the

vulnerability of this model to renal injury. Currently, no study in the literature has examined


23

the impact of pre-pregnancy obesity on fetal kidney development. Chapter 3 in this thesis will

address the gap in our knowledge using a model that has pre-existing obese phenotype.

1.5.2. Maternal Obesity and The Intrauterine Environment

Maternal obesity creates an adverse intrauterine environment with altered placental

function and abnormal maternal circulating factors such as glucose and cholesterol. This

section will discuss the evidence regarding the association of these two aspects of maternal

environment to the programming of cardiovascular and renal disease in offspring of obese

mothers.

1.5.2.1. The Impact of Placenta

The placenta plays a central role in fetal growth and development by acting as a

interface for maternal and fetal interactions. The placenta is also an important endocrine

organ crucial for a successful pregnancy. During pregnancy the placenta synthesizes

steroids such as HCG, progesterone and oestrogens, and growth hormones like placental

lactogen and placental growth hormone.78 Synthesis of these placental hormones not only

regulates reproductive function and maturation of the placenta from early pregnancy, but also

ensures an optimal intrauterine environment for adequate fetal growth during the later stage

of pregnancy.78 Impaired maturation of the placenta is associated with maternal

complications such as preeclampsia.51 In examination of term placentas of obese women

collected during caesarean section, there was increased expression of pro-inflammatory

mediators,59,304 increased oxidative stress,154,313 accumulation of macrophages,59

mitochondrial dysfunction,154 and an increased incidence of placental vascular lesions173,210

compared to the placenta of normal weight controls. Consistent with human studies, obese

female non-human primates had increased placental inflammatory cytokines, reduced blood

flow in both the uterine artery and fetal side of the placenta, and increased risk of placental

infarction contributing to the high incidence of stillbirth in this model.121,306 Despite the

structural differences between mouse and human placentas,238 similar placental pathology

has been demonstrated in mouse models of maternal obesity, confirming mouse placentas

can also be affected by maternal obesity.203,214 Placenta of obese mice demonstrated

decreased labyrinth thickness (cell proliferation), increased placental inflammation and

reduced placental efficiency.203,214 Interestingly, placentas that accompanying male fetuses

appeared to be most affected suggesting a sex-specific effect of maternal obesity in the

programming of adult disease in offspring via the placenta.203,298


24

1.5.2.2. Maternal Circulating Factors

Abnormal maternal plasma profile such as increased pro-inflammatory cytokines,

maternal hyperglycemia, hypercholesterolemia and hyperleptinemia may also program

cardiovascular and renal disease in offspring. Excess level of these maternal circulating

molecules might either be transported across the placenta into the fetal circulation such as

glucose,349 or lead to placental dysfunction secondary to excess circulating pro-inflammatory

cytokines,203 thus increasing the risk of maternal complications and developmental

abnormalities of the fetus. Offspring exposed to intrauterine hyperglycemia, irrespective of

the etiology of maternal diabetes have increased risk of congenital malformations34,148 and

greater risk of developing cardiovascular and metabolic diseases in adulthood.197,222,333,383

Renal malformations are particularly prevalent among congenital malformations associated

with gestational diabetes in humans89,269 and mice167

Further, maternal hypercholesterolemia during pregnancy in humans has also been

associated with increased susceptibility to, and faster progression, of atherosclerosis in

young children.267 Similarly, treating obese dams with cholesterol-lowering agent statins have

been shown to reduce the risks of hypertension and dyslipidemia in offspring.108,110,

indicating that maternal hypercholesterolemia associated with obesity might contribute to the

programming of cardiovascular and metabolic disease in offspring. Further, maternal

hyperleptinemia has also been linked to the greater adiposity and development of

hypertension in offspring of dams fed on a high fat diet.205,317-319 These findings indicate that

abnormal plasma profile associated with obesity during pregnancy greatly impacts the

outcomes of offspring.


25

1.6. FETAL PROGRAMMING OF KIDNEY DEVELOPMENT AND ITS

IMPACT ON CARDIOVASCULAR AND RENAL HEALTH

While the field of fetal programming was initially focused on the development of

cardiovascular diseases such as coronary heart disease, the involvement of other organ

systems and physiological processes have also been explored, including the kidney. Fetal

kidney development is extremely sensitive to adverse intrauterine environments, such as

maternal low protein diet, glucocorticoid exposure, placental insufficiency, exposure to

maternal disease (hypertension, diabetes) and toxins (pharmaceuticals,

alcohol).5,25,168,201,379209 These insults have been found to program low nephron

endowment,22,24 alteration in renal RAS88,137 and renal sympathetic nerve activity (RSNA),6,86

all of which are known to contribute to the development of hypertension and renal disease in

offspring.25,277 Nevertheless, the most sensitive aspect of renal programming is nephron

endowment, which is the focus of this thesis.

1.6.1. Programming of Nephron Endowment

Nephron endowment refers to the number of nephrons an individual is born with or

the nephron number at the termination of nephrogenesis in species such as rats and mice.

Using gold standard, unbiased stereological technique, studies have found that nephron

number in humans varies widely. The Monash Series examined 420 kidneys obtained at

autopsy from Australian aborigines, Australian Caucasian, African-American, American

Caucasian and Senegalese Africans and revealed that there is at least 13-fold range in

nephron number in these populations.100,169,170,175,247,248,291 Of importance, Hughson et al 175

demonstrated a direct correlation between nephron number and birth weight in humans,

suggesting birth weight is a strong prediction of nephron endowment. Despite this vast range

in nephron number in humans, these autopsy studies could not determine the mechanisms

or maternal insults that led to reduced nephron endowment. The type of insults that result in

a reduction in nephron endowment have been identified through research in animal models.

Adverse intrauterine environments such as placental insufficiency/intrauterine growth

restriction,24,26 maternal low protein intake,315,379 maternal glucocorticoid

exposure,263,276,277,341,374 gestational diabetes9,167 and maternal alcohol exposure133,134 have all

been shown to program low nephron endowment.

Much attention on the programming of low nephron endowment has been focused on

maternal undernutrition, yet the impact of maternal overnutrition, particularly pre-pregnancy

obesity on nephron endowment is poorly understood. In fact, no study to date has

established any correlation between pre-pregnancy obesity and nephron endowment. In a

study using rats, Armitage et al 12 found no difference in kidney weight, glomerular number


26

and glomerular volume in adult offspring of dams fed a high fat diet (HFD). However, female

rats in this study started the HFD only 10 days prior to conception and the appropriate

stereological technique (gold standard) was not used to estimate nephron number in

offspring. Although the direct evidence linking pre-pregnancy obesity and nephron deficiency

in offspring is lacking, common complications associated with pre-pregnancy obesity such as

maternal hyperglycemia and placental insufficiency, have been demonstrated to lead to a

programmed low nephron endowment.

Given pre-pregnancy obesity significantly increase the risk of maternal

hyperglycaemia and gestational diabetes,153 nephrogenesis in offspring of obese mother

could be greatly affected, resulting a reduced nephron endowment. Hokke et al 167 have

demonstrated that diabetic pregnancy in mice leads to a marked deficit in ureteric branching

morphogenesis and a reduced nephron endowment in offspring. This study also showed that

insulin therapy initiated following the onset of hyperglycemia could not reverse the deficit in

renal development, suggesting that early detection and correction of hyperglycemia might be

needed to prevent these developmental abnormalities in the kidney.167 This is particularly

relevant to those women have been screened and diagnosed with gestational diabetes late

in pregnancy, typically at 26-28 weeks, as nephrogenesis starts around 5 weeks of gestation

in humans (see Moritz & Wintour260 and Guron & Friberg145), long before the recommended

time for screening gestational diabetes Importantly, women who were obese prior to or at

time of conception are also likely to have uncontrolled hyperglycemia throughout pregnancy

and increased risk of developing gestational diabetes during pregnancy,18 implying offspring

of obese pregnant women might be at greater risk of being born with a nephron deficit and

consequently experiencing renal insufficiency later in life. Further, even without chronic

exposure to hyperglycemia from early pregnancy, Amri et al 9 have demonstrated that

glucose infusion from days 12-16 of gestation in rat pregnancy can led to a 20% reduction in

nephron number of offspring, but only among those dams whose hyperglycaemia was

transiently higher on day 13 of gestation. This suggests that even a small window of

exposure to hyperglycaemia during pregnancy could lead to nephron deficiency in offspring.

Human173 and mouse203,214 studies have shown that pre-pregnancy obesity increases

the risk of placental pathological lesions and leads to reduced placental efficiency. Placental

insufficiency in rats has been found to lead to significant growth restriction and associated

nephron deficit.24,262 It is not known whether obesity-induced placental insufficiency causes a

reduction in nephron endowment, however given the close association of pre-pregnancy

obesity and increased risk of maternal hyperglycemia and placental insufficiency, it is

reasonable to predict that pre-pregnancy obesity programmes a low nephron endowment

through an adverse intrauterine environment. Chapter 3 will examine this hypothesis in a

robust mouse model of pre-pregnancy obesity.


27

1.6.2. Impact of Low Nephron Endowment on Cardiovascular and Renal Health

Although pre-pregnancy obesity could act through multiple mechanisms to program a

low nephron endowment in offspring, these mechanisms often affect the development of

other organs/systems. In doing so, it is difficult to determine the impact of nephron deficit per

se for adult offspring in these models. A low nephron endowment is associated with

increased risk of adult cardiovascular and renal disease, however the mechanisms are not

well understood. Brenner and colleagues were the first to proposed that a reduced nephron

number leads to the development and progression of hypertension and chronic kidney

disease (Figure 1.6).46 The hypothesis proposed that a reduced nephron number would lead

to reduced glomerular filtration surface area (FSA) and thus water and sodium retention.

Consequently, there would be a rightward shift of the pressure-natriuresis relationship to

restore the adequate sodium and water excretion, but at a cost of an elevation in arterial

pressure. Arterial hypertension in turn leads to an increase in glomerular capillary pressure in

remaining glomeruli. The remaining nephrons would adapt to the increase in glomerular

capillary pressure leading to glomerular hyperfiltration and glomerular and tubular

hypertrophy.216,312 Prolonged glomerular hypertension, glomerular hyperfiltration and

glomerular hypertrophy would lead to glomerular injury such as glomerulosclerosis and thus

further loss of nephron and FSA, continuing this vicious cycle (Figure 1.6).


28

Figure 1.6. Brenner Hypothesis. A schematic illustration of a vicious cycle of progressive nephron loss, renal injury and hypertension.

The Brenner Hypothesis

Low Nephron Number

êFiltration Surface Area

Sodium and Water Retention

éArterial Pressure

éGlomerular Capillary Pressure

Renal Adaptations Glomerular Hypertrophy

Vicious Cycle

Glomerular Injury

Glomerular Hyperfiltation


29

1.6.2.1. Nephron Endowment and Hypertension

Consistent with the Brenner hypothesis, being born with a low nephron endowment is

associated with increased risk of developing hypertension later in life. In a small German

cohort, it was found that nephron number was significantly lower in those with essential

hypertension compared to normotensive controls, linking the etiology of essential

hypertension to a reduced nephron number.200 Race is also an important determinant of

nephron number. For example, a reduced nephron number has been associated with

elevated blood pressure in indigenous Australians.170,171,192 From examining the kidneys

collected at autopsy from white and African Americans, only female white Americans who

suffered from hypertension were found to have significantly lower nephron number.178

Further, in a study that investigated children who were born with a solitary kidney or with

unilateral multicystic kidneys at birth, half of them were hypertensive by 9 years of age,

indicating the significance of nephron endowment in the determination of arterial pressure in

humans.329 Animal models of programmed low nephron endowment, such as maternal low

protein diet215,375 and prenatal dexamethasone treatment263,341,374 are also associated with the

development of hypertension in adult offspring.

Despite the evidence pointing to nephron deficiency as a contributor to the

development of hypertension, it has become evident that low nephron endowment does not

always associate with hypertension, and secondary insults might be necessary for the

development of hypertension.268 As mentioned earlier, the association of low nephron

number and hypertension was only found in female white Americans, but not in male white

Americans or African Americans.178 Some renal programming models of reduced nephron

endowment even demonstrated hypotension rather than hypertension.45,271 A study from our

laboratory found that mice born with a congenital nephron deficit, the GDNF heterozygous

mice were normotensive, even up to 1-year-old.312 In support of the secondary insult

hypothesis,268 both high salt intake312 and diet-induced obesity146 caused an increase in

arterial pressure in nephron deficient GDNF heterozygous mice in a manner proportion to the

level of nephron deficit.

1.6.2.2. Nephron Endowment and Renal Dysfunction

The kidney compensates for a nephron deficit functionally by hyperfiltration to

maintain adequate GFR, and structurally through glomerular hypertrophy to normalise the

deficit in FSA.338 However, whilst these compensatory adaptations may preserve adequate

renal function in short-term, they may also predispose kidneys to greater risk of renal injury

later in life, likely due to prolonged renal hyperfiltration and elevated glomerular capillary

pressure.282,295 This has been demonstrated both in patients born with a solitary functioning

kidney and in patients that underwent unilateral nephrectomy during childhood.282,295


30

Although these patients demonstrated significantly elevated GFR in the remaining kidney,

they exhibited a gradual reduction in renal function reserve, the ability to increase GFR when

there is an increase in metabolic demand.282,295 Consistent with this abservation, Aboriginal

Australians, a population is known to have a significant nephron deficit (30%), are more likely

to suffer from chronic kidney disease and renal failure compared to non-aboriginal

Australians.170,172

Consistent with findings in human studies, some animal models of low nephron

endowment also demonstrated a gradual deterioration of renal function over time. Woods et

al 378 demonstrated that unilateral nephrectomy in neonatal rats (before completion of

nephrogenesis) led to a reduced GFR at 20 weeks of age compared to sham animals.

Similarly, in the ovine model of fetal unilateral nephrectomy, GFR of nephron deficient sheep

was lower than control sheep as early as 6 months of age.261 In contrast, it appears that

when unilateral nephrectomy is performed in adulthood, total GFR can be restored through

hyperfiltration (increased SNGFR).335,357 Further, a study from our laboratory, using a

congenital model of low nephron endowment, found that mice born with a 65% nephron

deficit exhibited normal GFR compared with controls mice when studied at 1 year of age.312

In fact, in order to maintain a normal GFR in mice with marked nephron deficit, the calculated

SNGFR (GFR divided by nephron number) in these mice was almost four fold greater than

control mice.312 Programming models of nephron deficiency also demonstrate

inconsistencies in GFR outcomes in offspring. For example, offspring of dams fed a low

protein diet were found to have reduced GFR comparing to the controls.24 However, when

nephron deficiency was induced by maternal dexamethasone treatment, offspring have a

normal GFR through adult life.203

1.6.3. Animal Model of Low Nephron Endowment

1.6.3.1. Programed and Congenital Models of Low Nephron Endowment

There is a large literature on animal models of low nephron endowment including

maternal protein restriction, maternal undernutrition, intrauterine growth restriction and

maternal glucocorticoid exposure. These models highlight how adverse maternal

environments increase the risk of offspring developing hypertension and renal disease.

However, these maternal insults are known to impact several organs and systems within the

fetus including the heart and vasculature,44,77,117,227,280 and the central nervous systems287 in

addition to the reduction in nephron number. As these extra renal systems can also influence

arterial pressure and renal function, the extent to which a reduction in nephron endowment

per se contributes to the development of hypertension and renal disease is not well

understood.201


31

In order to investigate the impact of reduced nephron endowment per se on renal

function and arterial pressure, it is important to choose a model in which other organ systems

are less likely to be affected. The glial cell line-derived neurotrophic factor (GDNF)

heterozygous mouse is an ideal genetic model of reduced nephron endowment that offers 2

distinct levels of nephron deficiency within the same genotype to compare to wild-type

littermates.312 In addition, these mice have normal arterial pressure and renal function at

least up to 1 year old, avoiding the impact of elevated arterial pressure on renal function.312

1.6.3.2. GDNF Heterozygous Mice

Glial cell line-derived neurotrophic factor (GDNF) is essential to both the initiation of

nephrogenesis and the branching of the ureteric epithelium that ultimately determines final

nephron number.259,286,320 Due to these two distinct roles of GDNF in kidney development,

GDNF null mice fail to form kidneys and die shortly after birth.286 However, GDNF

heterozygous (HET) mice are viable and born with either 2 small kidneys (HET-2K) and a

moderate (~30%) nephron deficit, or a solitary kidney (HET-1K) and a marked (~65%)

nephron deficit compared to wild-type (WT) mice (Figure 1.7A).312 This unique model thus

provides opportunity for a within genotype comparison of how renal function and arterial

pressure are regulated in mice with both moderate and marked nephron deficiency.

Glomeruli of HET-2K and HET-1K kidneys undergo compensatory glomerular

hypertrophy as they age such that average individual glomerular volumes of HET-2K and

HET-1K mice are 33% greater than WT mice (Figure 1.7B).312 The greater individual

glomerular volume of HET-2K mice offsets the 30% nephron deficit, resulting a similar total

glomerular volume compared to WT mice (Figure 1.7C). However, glomerular hypertrophy in

the solitary kidney of HET-1K mice does not fully offset the 65% deficit in nephron number

and thus the total glomerular volume (likely total FSA) of HET-1K mice is only 50% that in

WT mice (Figure 1.7C).312 This compensatory glomerular hypertrophy in GDNF HET mice

completes by 30 weeks of age,336 and thus studies in Chapter 5 will commence at this time

point.

Whilst the urinary excretory profile is similar between HET-2K and WT mice, HET-1K

mice had significantly higher water intake, urine excretion, and sodium and osmolar excretion

than HET-2K and WT mice (Figure 1.7E-H).312 It has also been shown that both GDNF HET-

2K and HET-1K mice remain normotensive (Figure 1.7D) and have normal GFR up to 1 year

of age.312 Given the significant nephron deficit and normal total GFR in HET-2K and HET-1K

mice, it was calculated that HET-2K mice had double the single nephron GFR (SNGFR) that

of WT mice and SNGFR of HET-1K mice was nearly 4-fold higher than WT mice.312 However

this significant hyperfiltration is not associated with any renal injury such as albuminuria

(Figure 1.7I), interstitial fibrosis, or glomerulosclerosis in HET-2K and HET-1K mice.312,336


32

The background strain of the GDNF colony, C57BL6/J mice, which are known to be

sclerosis-resistant, may contribute to this protection.115,224,234


33

Figure 1.7. Characteristics of GDNF WT, HET-2K and HET-1K mice on a normal-salt diet at 1-year old. A: total number of nephrons B: mean glomerular volume (Vglom). C: total glomerular volume (Total Vglom). D: 24h mean arterial pressure (MAP). E: 24h water intake. F: 24h urine excretion. G: 24h Na+ excretion. H: 24h osmolar excretion. I: 24h albumin excretion. *P<0.05 vs WT, **P<0.001 vs WT; †P<0.05 vs HET2K, ††P<0.001 vs HET2K. Figure from Ruta et al 312


34

1.7. REGULATION OF RENAL FUNCTION IN AN ANIMAL MODEL OF

LOW NEPHRON ENDOWMENT

As highlighted earlier, some models of nephron deficit demonstrate a reduction in

GFR including unilateral nephrectomized rats378 and sheep99, and in the rat model of

maternal protein restriction.379 There are also models where GFR is well maintained during

adulthood in animals with marked nephron deficiency including the GDNF HET mice.312 In

cases where GFR is normal, a significant elevation of SNGFR must occur to compensate for

the low nephron number. In fact, calculated SNGFR of nephron deficient GDNF HET-1K

mice was nearly fourfold higher than WT mice.312 To achieve this significantly elevated

SNGFR, regulation of GFR in the kidney of GDNF HET-1K mice must be altered to favor this

adaptation. Renal hemodynamic and excretory functions are tightly regulated by the

collective effort of several regulatory systems. Nitric oxide (NO), as an important signaling

molecule, plays a prominent role in the regulation of glomerular, vascular and tubular

function.207 In this section of the literature review, the functional contribution of NO in

regulation of renal function will be discussed, so will be the renal adaptations when NO

bioavailability is reduced, particularly in the setting of low nephron endowment.

1.7.1. Role of NO in the Regulation of Renal Function

1.7.1.1. Renal NO in Normal Kidneys

Nitric oxide (NO) was originally discovered as the endothelium-derived relaxing factor

that leads to vasodilation.237 NO is formed from oxygen and the amino acid L-arginine by

nitric oxide synthases (NOS) and cofactors.343 All 3 NOS isoforms, namely neuronal NOS

(nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) contribute to the synthesis of

NO in the kidney. However nNOS and eNOS are the two isoforms that are constitutively

expressed in the kidney.343 NO has been shown to regulate renal function through regulation

of renal hemodynamics and vascular resistance, mediation of pressure natriuresis, blunting

of tubuloglomerular feedback, inhibition of tubular sodium reabsorption and modulation of

renal sympathetic activity.264 In general, renal NO production promotes natriuresis and

diuresis.264 In addition, NO deficiency both in the kidney and systemically has been shown to

contribute to the pathogenesis and progression of hypertension and chronic kidney

disease.33,264 The local expression and activity of NOS in the kidney is positively linked with

the physiological action of NO.33 In rats, the highest NOS activity was found in the inner

medullary collecting duct,380 suggesting NO is strongly associated with regulation of water

and sodium reabsorption in distal tubules.


35

1.7.1.2. Role of NO in Nephron Deficient Kidneys

Renal NO plays an important role in the regulation of renal hemodynamic and

excretory function in nephron deficient animals. A reduced NO bioavailability has been

implicated in the progression of renal disease and hypertension in the remnant kidney

models,4,346,347 as well as model of programmed renal insufficiency.348 Further, aged female

sheep with congenital renal mass reduction in utero demonstrated reduced renal blood flow

and GFR, impaired renal execratory function and renal vascular dysfunction that were

associated with reduced contribution of NO.216

An upregulation of NO-facilitated vasodilation has been demonstrated to play a role in

maintaining renal function following renal mass reduction. A study by Valdivielso et al 357

demonstrated that an increase in renal NO production mediates the increase in renal blood

flow via a reduction in renal vascular resistance in the remnant kidney 2 days after unilateral

nephrectomy in rats, facilitating an increase in SNGFR in nephrectomized rats.335 Consistent

with this finding, Sigmon et al 338 found that urinary excretion of cyclic guanosine

monophosphate (cGMP), a marker of renal NO production, had a 2.5 fold increase up to 4

weeks post-uninephrectomy. The involvement of NO in renal vasculature of the remnant

kidney of rats following uninephrectomy was also demonstrated by a sharper increase in

renal vascular resistance and a greater fall in renal plasma flow compared with shame rats

when L-NAME, a non-selective NOS inhibitor, was administered acutely.357 An elevation in

renal NO production observed in rats which remained normotensive following renal ablation

(75%) has been demonstrated to be responsible for the greater sodium excretion and the

normotensive phenotype.13 These findings indicate that enhanced NO production could be

one of the adaptive mechanisms that maintain normal renal function and arterial pressure in

individuals with a reduced nephron number. The important role of NO has also been

demonstrated in renal injury-resistant rodents following 5/6 nephrectomy. For example, both

Wistar Furth rats113,114 and C57BL6/J mice234,265 have demonstrated strong dependence in

adequate NO bioavailability to maintain normal renal function and resistance to renal injuries.

A previous study from our laboratory has shown that nephron deficient GDNF HET

mice remain normotensive and have normal total GFR even through old age.312 It is not

known whether enhanced NO bioavailability contributes to the normal renal function and

arterial pressure, particularly in the HET-1K mice with marked 65% nephron deficit. Chapter

5 of this thesis will address this gap in our understanding of the regulation of renal function in

nephron deficient animals.


36

1.8. HYPOTHESES AND AIMS

Chapter 3: Obesity Limits the Normal Cardiovascular and Renal Adaptations of

Pregnancy Compromising Fetal Kidney Development

Despite the significant complications associated with pre-pregnancy obesity, the

impact of obesity on the cardiovascular and renal adaptations that occur during pregnancy is

not well understood. The altered hemodynamics in obesity has the potential to impose

significant constraint on the ability of cardiovascular and renal systems to adapt during

pregnancy. Like other adverse intrauterine environments, maternal obesity has also been

shown to increase the risk of offspring developing hypertension and renal dysfunction later in

life. Given the sensitivity of the kidney to maternal insults and the fact that common maternal

complications of maternal obesity such as hyperglycemia and placental dysfunction are

associated with reduced nephron endowment, it is reasonable to predict that nephron

endowment in offspring of obese mothers might be reduced, contributing the onset of

hypertension and renal disease in adulthood. Yet the impact of pre-pregnancy obesity on

fetal kidney development has not been investigated in a robust animal model of obesity or

using appropriate techniques and thus is poorly understood.

Hypotheses:

1) Pre-pregnancy obesity limits the normal cardiovascular and renal adaptations

of pregnancy, contributing to poor maternal and fetal outcomes

2) Pre-pregnancy obesity impacts fetal kidney development reducing nephron

endowment

Aim 1: To examine the impact of obesity on the cardiovascular and renal adaptations

during pregnancy and fetal outcomes in a robust mouse model of diet-induced obesity

Aim 2: To examine the impact of pre-pregnancy obesity on fetal kidney development

To address Aim 1, I established a mouse model of diet-induced obesity in C57BL6/J

females and used radiotelemetry (MAP and HR), cine-MRI (cardiac structure and function),

and 24 hr urine sample collected to characterize cardiovascular and renal physiology before

and during pregnancy. To address Aim 2, fetal kidneys of control and obese pregnant

C57BL6/J mice were collected at gestational age 19 and subjected to gold-standard

unbiased stereological analysis in order to estimate glomerular number, glomerular volume

and to examine general renal morphology.


37

Chapter 4: Does Pregnancy Exacerbate the Cardiovascular and Renal Outcome

of Obesity?

Despite significant structure and functional adaptions to the cardiovascular and renal

systems during normal pregnancy, pregnancy has no long-term impact on the cardiovascular

and renal health in the mothers. In contrast, pregnancies that are complicated with

hypertension, preeclampsia and gestational diabetes are often associated with adverse

cardiovascular and renal outcomes in the mothers post-birth. Importantly, these detrimental

maternal complications are commonly observed in women who were obese prior to

conception. Recent epidemiological studies indicate that maternal obesity is an independent

risk factor for major cardiovascular events and premature mortality in women post-birth.

However, the mechanisms that how maternal obesity leads to these consequences are

unclear. Given obesity is associated with compromised cardiovascular and renal health,

whether pregnancy would exacerbate cardiovascular and renal phenotypes of obesity post-

birth is currently unknown.

Hypothesis: pregnancy exacerbates the cardiovascular and renal outcomes of obesity

post-partum.

Aim 3: To examine the impact of pregnancy on the long-term cardiovascular and renal

outcomes in obese female mice

To address Aim 3, I examined obese and control female mice at 4 weeks post-

weaning (or 7 weeks post-birth) and compared them with non-pregnant, age-matched obese

and control mice. Arterial pressure (telemetry; up to 5 weeks post-birth), conscious GFR

(FITC-sinistrin Clearance), renal excretory profile, and cardiac and renal fibrosis were

examined.

Chapter 5: The Role of Nitric Oxide in the Regulation of Renal Function and

Arterial Pressure in Nephron Deficient Mice

A reduced nephron endowment is arguably the most extensively studied renal

programming outcome and has been associated with development of hypertension and renal

disease later in life. Yet, some animal models of low nephron number including the GDNF

HET mice demonstrate remarkable renal adaptations such as a significantly elevated

SNGFR and greater natriuresis, contributing to the maintenance of normal total GFR and

arterial pressure even at an old age. However, the mechanisms that mediate these renal


38

changes in nephron deficient kidneys are unclear. NO has been implicated as one of the

important regulators of renal hemodynamic and excretory function in both normal kidneys

and nephron deficient kidneys. In this Chapter, we used GDNF HET mouse, a genetic model

of reduced nephron endowment with two levels of nephron deficit, to investigate the

contribution of NO in the regulation of renal function and arterial pressure in nephron

deficient animals.

Hypothesis: Chronic NO deficiency leads to greater hypertension and renal

dysfunction in nephron deficient GDNF Heterozygous mice in a manner dependent on

the level of neohron deficit.

Aim 4: To determine the arterial pressure and renal function of GDNF WT, HET-2K and

HET-1K mice prior to and in response to systematic NOS inhibition with L-NAME.

To address Aim 4, arterial pressure (radiotelemetry) and renal excretory profile of

GDNF WT, HET-2K and HET-1K mice will be examined before and after systemic NOS

inhibition with L-NAME. Renal expression of RAS components, key water and sodium

channels will also be examined in L-NAME treated and untreated animals.

Chapter 2 General Methods

39

Chapter 2 GENERAL METHODS

2.1. ANIMALS

All experiments were conducted according to the Australian Code of Practice for the

Care and Use of Animals for Scientific Purposes and approved in advance by the Monash

University School of Biomedical Sciences Animal Ethics Committee.

2.1.1. Mouse Model of Pre-pregnancy Obesity: Chapter 3 & 4

For studies outlined in Chapter 3 & 4, a robust mouse model of diet-induced obesity

was established. Cardiovascular and renal phenotypes of these mice were characterized

before conception and the hemodynamic adaptations during pregnancy were assessed.

Cardiovascular and renal profiles of control and obese dams and time-controlled virgin

female control and obese mice were also assessed between 5-7 weeks post-birth.

Three week old female C57BL6/J mice were obtained from Monash Animal Research

Platform and housed in the experimental room maintained at 24-26 °C with 12:12 hour light-

dark cycle (light on at 6am & light off at 6pm). At 4 weeks of age, mice were separated into

individual cages and randomly allocated to receive either a control diet (CONT, Fat 7% w/w;

3.85kCal/g; AIN93G, Specialty Feeds, Australia) or high fat diet (HFD; Fat 23.5% w/w;

4.54kCal/g; SF04-001, Specialty Feeds, Australia). Mice received the diet treatment for a 10-

week period prior to baseline experiments. A subgroup of control and obese mice underwent

glucose tolerance tests (see Chapter 3 for details). Food intake and body weight of each

mouse were recorded weekly for the 10-week period. Separate cohorts of control and obese

mice were then allocated to each individual study accordingly. Mice continued to receive the

same diet until the end of the experimental protocol.

2.1.1.1. Mating

After baseline measurements female control and obese mice were allocated to be

mated or to remain virgin. For mating, female mice were paired with control-diet fed male

C57BL/6J mice from 12pm to 9 am the following day. Mice were examined for vaginal plugs

at 9 am. Gestational age (GA) 1 was defined as the 24hr period from the start of the light

cycle on the day of vaginal plug detection. Body weights were measured on the morning of


40

GA7, GA13 and GA19 (Chapter 3 only). Gestational length was recorded in mice allowed to

litter down.

2.1.2. GDNF Heterozygous Mouse: Chapter 5

For study outlined in Chapter 5, mice heterozygous for glial cell line-derived

neurotrophic factor (GDNF) were used as a congenital model of low nephron endowment.

GDNF wild-type (WT) mice were served as controls.

2.1.2.1. Animal Origin

Male GDNF heterozygous (HET) mice were initially received from Dr Heiner

Westphal (National Institutes of Health, Bethesda, MD, USA) to establish the colony at

Monash University through mating with C57BL/6J female mice.80,286 GDNF HET mice were

generated by replacing part of the third exon that encodes GDNF protein with a neo-cassette

expressing the selectable marker neomycin phosphotransferase.285,286 Tail tissue collected

from mice at 2 weeks of age were genotyped by polymerase chain reaction (PCR) analysis

(section 2.1.2.3). Genotypes of all mice were confirmed using tissue collected at post-

mortem. Whilst this study contained mice with only 2 genotypes, the GDNF WT and HET

genotypes, GDNF HET mice can be further divided into 2 groups based on their phenotype,

those born with 2 kidneys (HET-2K) and those born with a solitary kidney (HET-1K). Thus

study outlined in Chapter 5 contained 3 experimental groups, GDNF WT, GDNF HET-2K and

GDNF HET-1K groups. The separation of HET-2K mice and HET-1K mice could only be

achieved at post-mortem.

2.1.2.2. Housing and Diet

GDNF WT and HET mice were obtained from Monash Animal Research Platform at

26 weeks of age and group housed for 2 weeks in the experimental room maintained at 24-

26 °C with 12:12 hour light-dark cycle. Mice were allowed ad libitum access to tap water and

normal salt rodent chow (AIN93M, Specialty Feeds, Australia). A week later, littermates were

separated into individual cages and baseline experiments started at 30 weeks of age.


41

2.1.2.3. Genotyping

To distinguish and confirm GDNF WT and HET genotypes, tissues collected were

analysed by PCR.285,286 DNA extractions and PCR were performed using REDExtract-N-

AmpTM tissue PCR kit (Sigma-Aldrich, USA). Each PCR reaction consisted of following

components: 2 µl of H2O, 4 µl mixture of 4 primers (1 µg/µl, Geneworks, Australia), 4µl DNA

extract and 10 µl of REDExtract-N-Amp PCR Reaction Mix (supplied in REDExtract-N-AmpTM

tissue PCR kit, Sigma-Aldrich, USA). The sequences of primer 1-4 were285:

Primer 1 5’-CCAGAGAATTCCAGAGGGAAAGGTC-3’

Primer 2 5’-CAGATACATCCACACCGTTTAGCGG-3’

Primer 3 5’-GATCCCCTCAGAAGAACTCGT-3’

Primer 4 5’-CTGTGCTCGACGTTGTCACTG-3’

PCR amplifications were carried out using Mastercycler gradient (Eppendorf,

Germany) and amplification cycles were: 5 minutes at 95 °C; 40 cycles of 30 seconds at

94 °C, 55 seconds at 64.5 °C and 45 seconds at 72 °C; followed by 10 minutes at 72 °C and

held at 4°C until DNA gel electrophoresis.

PCR products were resolved into 3% agarose gel (Invitrogen, USA) stained with

SYBR SafeTM DNA gel stain (Invitrogen, USA) at 100 volts for 45 minutes and DNA gel was

viewed by GelDock viewer (Syngene, UK). Primer 1 & 2 generated a 338-bp fragment

representing WT GDNF allele and primer 3 & 4 generated a 567-bp fragment specifying the

mutant allele in HET GDNF genome (Figure 2.1).

Figure 2.1 DNA Bands of GDNF HET and WT genotypes.

338 bp

567 bp

HET HET WT WT HET HET


42

2.2. CONSCIOUS RENAL FUNCTION EXPERIMENTS

Conscious renal excretory profiles were assessed in Chapter 3, 4 & 5 from 24hr urine

samples collected in metabolic cages. In Chapter 4, glomerular filtration rate (GFR) was

assessed by transcutaneous FITC-sinistrin clearance method. In Chapter 5, GFR was

assessed by measuring renal creatinine clearance (Ccre) using urine obtained during 24hr

urine collection and plasma samples collected during radiotelemetry surgery (for baseline)

and tissue collection (for post-L-NAME).

2.2.1. Assessment of Urinary Excretory Profile

24hr urine samples were obtained by placing mice in glass metabolic cages purpose

build for mice (Figure 2.2). The metabolic cage consisted of a main chamber (5 cm in radius),

a food chamber, a water chamber, a splitter beneath the centre of the cage for separating

urine and faeces, and small collection vials for food shredding, unconsumed water, and urine

and faeces produced during the 24hr period (Figure 2.2). To acclimatize mice to the novel

environment of the metabolic cages, mice were placed in the cages for ~6 hours during the

daytime, 2 days prior to the 24 hours renal function experiments.

Figure 2.2 Image of Metabolic Cage

Main Chamber

Food Chamber Water Chamber

Urine Splitter

Urine Collection Vial Faeces Collection Vial

Food Shredding Vial Water Chamber


43

Prior to placing mice into the cage, food and water provided, and collection vials for

urine and faeces were weighed. Mice were weighed and placed in metabolic cages in the

morning (9-10 am) for 24 hours. At the end of the 24hr period, body weight of mice was

measured. The remaining food and water were measured to calculate 24hr food intake and

water consumption. Urine and faeces collection vials were weighed to calculate the volume

of urine and amount of faeces produced. Urine was then transferred to small centrifuge tubes

and clarified by centrifugation at 3000 RPM for 5 minutes at room temperature. Urine

samples were then analysed for urinary osmolality and electrolyte concentrations before

being stored at -20 °C for subsequent analysis of urinary albumin excretion and creatinine

clearance (See section 2.2.2).

2.2.2. Analysis of Urine and Plasma Samples

2.2.2.1. Urinary Osmolality and Electrolytes

Urinary osmolality was determined by freezing point depression method using the

osmometer (Advanced instrument 2020, Needham Heights, MA, USA). Urinary sodium (Na+),

potassium (K+) and chloride (Cl-) concentrations were measured by ion-selective electrodes

using EasyElectrolytes analyser (MEDICA, MA, USA). Values of urinary osmolality

(mOsmol/Kg H2O) and electrolyte concentrations (mmol/L) of each sample were then

multiplied by 24 hr urine volume to obtain 24hr excretion data, which was used to present

urinary excretory data.

2.2.2.2. Albumin Assay

Urinary albumin concentration was determined by Albuwell M kit (Exocell, PA, USA).

Albuwell M is a widely used enzyme-linked immunosorbent assay (ELISA) for quantitative

determination of albumin in mouse urine.224,312,366

2.2.2.2.1. Assay Principle

To complete the assay, albumin standard solution, urine sample and rabbit anti-

murine albumin antibody (primary antibody) are added to albumin-coated wells in a 96-well

plate. This primary antibody competitively binds to either the albumin immobilised to the

stationary phase or with that in the fluid phase (urine sample or standards). Following

washing, an anti-rabbit horseradish peroxidase (HRP) conjugate antibody (secondary

antibody) is added to the well to label the primary antibody in the stationary phase. This


44

antibody conjugate is then detected using a chromogenic reaction. The colour intensity of the

reaction is inversely proportional to the logarithm of albumin concentration in the samples.

2.2.2.2.2. Assay Procedure

Prior to the assay, urine samples were thawed to room temperature, and well mixed

before being clarified by a quick centrifugation (30sec at 3000x RPM).314 Diluted Murine

Serum Albumin (MSA) standards and urine samples were added to the 96-well plate in

triplicates using a manual micropipette. Rabbit anti-murine albumin antibody was then added

to all wells across the plate except the blank and the plate was incubated for 30 min at room

temperature. The plate was then wash with wash buffer (pH 6.8, 0.15M NaCl, 0.01M

triethanolamine, 0.05% Tween 20) for 10 cycles using a plate washer (TriContinent, CA, USA)

before each well was incubated with anti-rabbit HRP conjugate for another 30 min at room

temperature. A 10-cycle plate washing was performed again. Colour developer solution was

then added to each well and incubated for 10 min in dark at room temperature before the

colour stopper solution was added to each well. Absorbance was measured using a plate

reader at 450nm (BioRad 3350, Tokyo, Japan). Urine samples were diluted in a range of 1:3

to 1:5 to ensure values of absorbance fell within the range of the standard curve.

2.2.2.2.3. Data Analysis

The best-fit line was plotted with the log10 [MSA] on the x-axis and mean absorbance

(A450) on the y-axis in Microsoft Excel and equation of the standard curve was obtained as

shown below.

Log10 [MSA] = m A450 + b

Where m is the gradient of the standard curve generated and b is where the standard

curve intersects the y-axis. The absorbance of each sample measured was subjected to the

above standard curve to obtain log10 [MSA], which was then converted to albumin

concentration of each sample added to the well via the semi-logarithmic relationship. This

concentration of albumin was adjusted for dilution factor and then multiplied by 24hr urine

volume to obtain 24hr albumin excretion.

2.2.2.3. Creatinine Clearance (Chapter 5)

Creatinine concentrations of urine and plasma samples obtained at baseline and

post-L-NAME treatment (Chapter 5) were measured using high performance liquid


45

chromatography (HPLC) by A/Prof Merlin C Thomas’s group at the Baker IDI (Melbourne,

Australia).54,102 The creatinine clearance (Ccr) was determined using following equation:

Ccr =��Urine creatinine concentration ��µmol/L�� x Urine volume(ml/min)��

Plasma creatinine concentration (µmol/L)

2.2.3. Transcutaneous Measurement of GFR in Conscious Mice (Chapter 4)

The transcutaneous measurement of GFR using the FITC-sinistrin clearance is a new

technique that allows researchers to repeatedly measure GFR in conscious rodents including

mice, without serial sampling of urine or plasma, or laboratory assays of renal markers.326-328

Our optimised experimental protocol and methodological considerations of this newly

developed technique were published recently.111 This technique was used in study outlined in

Chapter 4. This technique, however, was not adopted in the study outlined in Chapter 5 as it

was not available in our laboratory at the time the study commenced.

2.2.3.1. Principle of Transcutaneous Measurement of GFR

Fluorescein-isothiocyanate labeled sinistrin (FITC-sinistrin) is a commercially

available exogenous marker of renal function. Sinistrin, like inulin, is freely filtered by the

glomerulus, but is not secreted or reabsorbed by the renal tubules, and not metabolized by

the body, making it an ideal renal marker to measure GFR. The NIC-Kidney Device

(Mannheim Pharma and Diagnostics, Mannheim, Germany) is a miniaturized optical device

that records the fluorescent emission of FITC-sinistrin through the depilated skin of the

mouse. Data recorded on the device over an experimental period was used to generate an

elimination kinetics curve of FITC-sinistrin (Figure 2.5). The half-life of FITC-sinistrin (t1/2) was

determined by the relative fluorescent emission signal detected by the device in the

corresponding to the single exponential excretion phase of the elimination kinetics curve

(Figure 2.5). As FITC-sinistrin is exclusively filtered by the glomerulus, the linear relationship

between the t1/2 of FITC-sinistrin and GFR can be calculated using a pre-established

conversion factor (14616. 8).328

GFR ��(μl/min)/100gBW�� =14616.8

t1/2 (min)


46

2.2.3.2. Experimental Protocol

2.2.3.2.1. Fur Depilation

One day prior to GFR measurement, mice were briefly anaesthetized by isoflurane

inhalation (~8min; Phodia, Australia), which was delivered by an anaesthesia unit (Univentor

Ltd, Zehtun, Malta) at a concentration of 4.9 % for induction and 2.1-2.4 % for maintenance

during the procedure. A small area of fur (~ 2 x 2 cm) on the flank of the mouse was shaved

using an electric shaver. Hair removal cream (Veet, Reckitt Benckiser, Australia) was then

applied and removed less than 2 minutes later with the use of a spatula. Warm water was

used to wash off the residual hair removal cream. If black pigmentation (commonly seen in

C57BL6/J mice) was present in the area where the hair was depilated, the opposite flank of

the mouse was used as this pigmentation may affect the detection of florescent signal

through the skin.

2.2.3.2.2. Experimental Procedure

The NIC-Kidney Device was adhered to one side of a double-sided adhesive patch

positioning the LEDs within the transparent window of this patch (Figure 2.3). FITC-sinistrin

was dissolved in sterile saline to make stock solution of 10-15 mg/ml. The dose of FITC-

sinistrin administered was 6-10 mg/100 g bodyweight.

Figure 2.3 Adhesive patch for device, NIC-Kidney Device and Battery. Figure from Ellery et

al 111


47

Mice were lightly anaesthetized (isoflurane). A strip of adhesive tape (10-15 cm long

& 2cm wide; Leukosilk® tape; BSM Medical, Luxembourg) was placed under the abdomen of

the mouse. The battery was then connected to the NIC-Kidney Device (Figure 2.3) and was

then mounted on top of the device using a small piece of the double-sided tape. The

powered NIC-Kidney Device was then adhered to the depilated skin area (Figure 2.4A), and

secured in place by the tape strip underneath the body to minimize movement artifact (Figure

2.4B).

Figure 2.4 Example of securing NIC-Kidney Device and battery on the back of the mouse. A: NIC-Kidney Device and battery adhered to depilated skin on the back. Battery is connected and mounted on top of the device. B: The NIC-Kidney Device was secured in place by adhesive tape while data were recording in free moving mouse in its home cage.

While the background signal was recorded (3-4 min), a latex glove filled with warm

water (50-60°C) was placed on top of the tail to warm the tail and assist dilation of the tail

vein for injection. FITC-sinistrin was then injected intravenously (tail vein) and mice were

placed back in their home cage (Figure 2.4B). Elimination kinetics of FITC-sinistrin was then

recorded in the conscious mouse for 1 hour (Figure 2.4B). At the end of the 1hr-recording

period, the tape and device were removed under brief anesthesia (isoflurane; <3 mins). Mice

were then returned to home cage for recovery.


48

2.2.3.3. Data Analysis

Device was connected to a computer and the Data analyzed by NIC-Kidney Device

software (Mannheim Pharma and Diagnostics, Mannheim, Germany) to generate the

elimination kinetics curve (Figure 2.5). A horizontal dotted line was placed along the

background signal to set the baseline signal level and a vertical dotted line was placed ~15

min post-injection to define the point where the analysis of elimination started (Figure 2.5).111

Signal was cropped at 1hr post-injection. The t1/2 of FITC-sinistrin was then determined from

the area between the vertical line (~15 min post-injection) and 1hr post-injection time by the

NIC-Kidney Device software. In cases of FITC-sinistrin solution was injected subcutaneously,

experiments were aborted and repeated the next day.

Figure 2.5 Example of FITC-sinistrin elimination kinetics curve


49

2.3. CONSCIOUS BLOOD PRESSURE MEASUREMENTS

2.3.1. Implantable Telemetry System for Mice

The implantable telemetry system and PhysioTel®PA-C10 transmitter developed by

Data Sciences International (DSI, St Paul, MN, USA) provide accurate and reliable

measurements of systolic, diastolic, and mean arterial pressures (MAP) as well as heart rate

(HR) and locomotor activity in free moving mice in their home cages.52 The receivers placed

underneath the home cages detects digitized data via radio frequency signal sent from the

transmitters and converts data into a form readily accessible by Dataquest A.R.T.TM System

(DSI, St Paul, MN, USA).

The PhysioTel®PA-C10 transmitter consists of a 5 cm-long fluid-filled catheter with a

thick-walled tip, a thermoplastic body contains electronics, sensor and battery, weighing 1.4

gram in total.255 The lumen of the catheter is filled with a low viscosity fluid. The distal 2mm of

the thin-walled tip is filled with a blood-compatible silicone gel that prevents blood from

entering the catheter lumen and clotting, allowing efficient transmission of high frequency

components of the pressure signal into the lumen of the catheter.255

2.3.2. Implantation Surgery

Mice were anaesthetised (isoflurane) and placed on a heated surgical pad to maintain

body temperature during surgery. A ventral midline incision (1-1.5cm) was made over the

neck region. Through this incision, a subcutaneous pouch was created along the right flank

using blunt dissection, for placement of the transmitter body. The left common carotid artery

was isolated and placed over curved forceps (tips 0.5 cm apart) to occlude blood flow. Three

sterile silk ligatures (Size 6-0, Dynek, Australia) were passed underneath the artery and tied

loosely around the artery. The silk tie most proximal to the head (back tie) was used to

permanently ligate the artery. A tiny incision on the artery was made using microscissors. A

small blood sample (~75 µl) was collected in a haematocrit tube through the incision and

plasma sample was used for measurement of creatinine clearance (section 2.2.2.3, for

Chapter 5 only). The catheter of the transmitter was then inserted through the incision on the

artery and advanced towards to the heart until the notch on the catheter matches the position

of the back tie. This placement ensures that the pressure-sensing tip of the catheter sits

inside of the aorta arch. The sound of pulse that generated by an AM radio was used to


50

confirm the catheter is in the artery. The catheter was secured with two silk ligatures proximal

to the heart and with the back tie at the notch to secure the catheter in place (Figure 2.5).

Figure 2.5 Implantation of Radio-telemetry Transmitters in Mice and Position of the End of Transmitter Catheter Within the Aorta Arch. Figure from Butz & Davisson52

Sterile saline (~ 1 ml) was infused into the subcutaneous pouch in the right flank and

the transmitter body inserted into the pouch (Figure 2.5). The incision in the neck region was

closed with 6-8 interrupted sutures (5-0 with needle; Dynek; Australia). Before the last suture,

10 µl of antibacterial solution (80 mg/ml of Trimethoprim, 400 mg/ml Sulfadiazine,

Trabactral®, JUROX, Australia) were administered under the skin. Mice also received

Carprofen (5 mg/kg body weight; Rimadyl® Injection, Pfizer Animal Health Group, Australia)

subcutaneously to reduce pain and inflammation. Mice were allowed to recover from surgery

in their home cage on a heated pad before they were returned to the experimental room.

Sutures were checked daily and body weights of mice were closely monitored post-surgery.

Mice were allowed to recover for 10 days before commencing baseline recording.


51

2.4. POST-MORTEM TISSUE COLLECTIONS

2.4.1. Maternal and Fetal Tissues at GA19 (Chapter 3)

At GA19, control and obese dams were weighed and anaesthetized (isoflurane). A

midline incision was made to expose the abdominal cavity. The number of viable and non-

viable fetuses and implantation sites were recorded and their location in the uterus identified.

One embryonic sac at a time, the fetus and placenta were rapidly removed and weighed.

Presence of meconium of fetal membranes and visible placental thrombosis were recorded.

Fetuses were sexed and the left fetal kidneys fixed in 4% paraformaldehyde (Sigma-Aldrich

Corp. St. Louis, MO, USA) for 2 hours then transferred to 70% ethanol until processing for

stereological analysis (see section 2.5). Fetal head width was measured between the ears

using a digital caliper (EDC-153, PI Manufacturing, Walnut, CA, USA).

Following collection of all fetal tissues, an arterial blood sample was taken from the

carotid artery of the dam for measurement of plasma free fatty acid and triacylglycerol levels

(see Chapter 3 supplement methods for details). Kidneys were rapidly excised, decapsulated,

weighed and cut into 4 equal portions transversely and immersion fixed in 10% buffered

formalin (Sigma-Aldrich, USA). One of middle portions of left kidney was processed for

histological analysis of collagen deposition (Picrosirius red staining; Section 2.7.2.1.2). The

heart was excised, and atria and ventricles were separated and weighed and all values

reported relative to tibia length.246 Ventricles (left and right) were cut in half transversely. Top

half of the ventricles were fixed in 10% buffered formalin for histological analysis of collagen

deposition (Picrosirius red staining, Section 2.7.2.1.3). Pericardial fat and liver were also

weighed. Age matched virgin control and obese mice were used as time controls.

2.4.2. Maternal tissues at 4WPW (Chapter 4)

At 4 weeks post-weaning (4WPW) or 7 weeks post-birth, control and obese dams and

age matched virgin mice (time controls) were weighed and anaesthetized (isoflurane). An

arterial blood sample was taken from the carotid artery to determine maternal plasma free

fatty acid and triacylglycerol levels (see Chapter 4 for details). Kidneys were rapidly excised,

decapsulated, weighed and cut into 4 equal portions transversely and immersion fixed in 10%

buffered formalin (Sigma-Aldrich, USA). One of middle portions of left kidney was processed

for histological assessment of glomerulosclerosis (PAS staining; Section 2.7.2.2). All 4

portions of the right kidney were snap-frozen in liquid nitrogen. One of the two middle

portions of the right kidney was used for measurement of collagen content by hydroxyproline


52

assay (section 2.7.1). The heart was excised and both atria were removed. Left ventricles

were separated and weighed and cut through the transverse plane into 3 equal portions.

Basal and apical portions of the left ventricle were then snap-frozen in liquid nitrogen. Apical

portions of the left ventricle were used for measurement of collagen content by

hydroxyproline assay (Section 2.7.1). The middle portion of the left ventricle was fixed in 10%

buffered formalin for histological analysis of collagen accumulation (Picrosirius red staining;

Section 2.7.2.1.3). Pericardial fat pad, gonadal fat pad (visceral fat pad), inguinal fat pad

(subcutaneous fat) and liver were also weighed.

2.4.3. GDNF HET Mice (Chapter 5)

Following post-L-NAME renal function experiments, mice were weighed and

anaesthetised (isoflurane). A terminal arterial blood sample was taken from the right common

carotid artery and plasma was used to analyse creatinine clearnace (Section 2.2.2.3). For

animals with two kidneys (GDNF WT and HET-2K mice), left kidney was excised, weighed,

and cut into 4 equal portions through transverse plane and immersion fixed in 4%

paraformaldehyde (Sigma-Aldrich, USA) for examination of general renal histology. The right

kidney was excised, weighed, and cut into equal 4 portions transversely. All portions of the

right kidneys were frozen in liquid nitrogen and one of the middle portions was used for RT-

PCR analysis (Section 5.2.4). In the case of the solitary kidney (GDNF HET-1K mice), the

kidney was excised, weighed, cut through transverse plane into 3 portions. The middle

portion of the kidney was frozen in liquid nitrogen for PCR analysis and the remaining

portions were immersion fixed in 4% paraformaldehyde for examination of general renal

histology. The heart was excised and atria removed, and left and right ventricles were

separated and weighed. Toes were collected to confirm the genotypes by PCR (Section

2.1.2.3).


53

2.5. STEREOLOGY

In chapter 3, fetal kidneys collected at GA19 were processed for stereological

analysis. The physical disector/fractionator stereological technique is considered the gold-

standard method for estimating nephron number in the adult kidney.83 In this project, a

recently developed variation of this technique was used for estimating number of developing

nephrons.82 This method utilizes the lectin peanut (Arachis hypogaea) agglutinin (PNA) to

histochemically identify podocytes within the glomeruli, from early S-shaped bodies through

to mature glomeruli. Once these PNA-positive structures are unambiguously identified in

systemically selected sections, the physical disector/fractionator method is then used to

accurately estimate total glomerular number (Nglom), and thus total nephron number.

2.5.1. Processing, Embedding and Sectioning of Fetal Kidneys

Kidneys were placed into microcassettes and dehydrated through a series of graded

ethanol baths and then infiltrated with paraffin wax. Before kidneys were embedded in the

mold with paraffin, a thin layer of paraffin was placed on the base of the mold to ensure that

the full block face will be obtained prior to collecting the first tissue sections. The mold was

then filled with paraffin and left to solidify.

The entire block was exhaustively sectioned at 4 µm and every section was collected.

The ribbons of sections were then transferred onto warm water bath. All sections were

collected in a similar orientation on Poly-L-Lysine-coated glass slides and allowed to dry. The

serial sectioning was performed by Dr Luise Cullen-McEwen.

2.5.2. Sampling Sections

Pairs of sections evenly spaced across the kidney were required to estimate

glomerular number. A sampling fraction was determined from the total number of sections

cut to achieve approximately 10-12 pairs of sections per kidney. Each pair of sections

consists of the n (reference section) and the n+2 (lookup section) sections. For example, if

250 sections were cut, every 25th and 27th (n=25) sections were selected, for 300 sections

every 30th and 32nd (n=30) were selected, and so on. The first section was chosen at

random (with use of a random number table) within the interval selected (i.e. 1 to n). Slides

with required sections were then selected for PNA staining.


54

2.5.3. Histochemical Staining with A. hypogaea PNA

1) Sections were deparaffinised through a series of three xylene washes and brought to

water through a series of two 100% alcohol washes and one 70% alcohol wash.

Slides were then rinsed in phosphate-buffered saline (PBS, 0.01M pH7.4, made from

PBS tablets, Medicago AB, Uppsala, Sweden).

2) Slides were incubated for 10 minutes in 2% H2O2 (Sigma-Aldrich, St Louis, MO, USA)

methanol solution and then followed with two 5-min washes in PBS.

3) Slides were then incubated for 30 min at 37°C with 0.1u/ml Neuraminidase (Sigma-

Aldrich, St Louis, MO, USA) with 1% CaCl2 in PBS, 100µl per slide. Slides were then

rinsed three times with PBS.

4) Non-specific binding was blocked by incubating sections with 2% BSA (Albumin from

Bovin serum; Sigma-Aldrich, St Louis, MO, USA) and 0.3% Triton X-100 (Sigma-

Aldrich, St Louis, MO, USA) in PBS for 30 min at room temperature (RT).

5) Without washing off BSA, slides were incubated for 1-1.5h with 20µg/ml biotinylated

PNA (Sigma-Aldrich, St Louis, MO, USA) diluted in 0.3% Triton X-100 in PBS, with

1mM CaCl2/MnCl2/MgCl2, at 37°C (also can incubate for 2h at RT or overnight at 4°C),

100µl per slide. Slides were then rinsed three times with PBS.

6) Slides were incubated with Avidin/biotin complex (ABC, from Vectastain ABC kit,

Vector Laboratories, Burlingame, CA, USA) for 1h at RT, 100µl per slide.

7) PNA staining was then developed with DAB and 0.01% H2O2 in PBS, 100µl per slide.

The color developing was confirmed under the microscope and was stopped by

placing slides in PBS washing chamber.

8) Slides were then rinsed two times with PBS before being conterstained with

haematoxylin for 20 sec.

9) Slides were washed under running tap water until the water runs clear and then

placed in Scott’s tap water for 1 min to turn sections blue.

10) Sections were then dehydrated through three washes of 100% alcohol and followed

by three washes of xylene before being coverslipped with DPX mounting media.

11) Slides were allowed to dry at room temperature before counting PNA-positive glomeruli.


55

2.5.4. Counting PNA-positive Glomeruli

Section pairs (n & n+2) were projected one at a time, at magnification of 180x onto a

piece of paper on a table in a darkened room using a microscope modified for projection

(Figure 2.6A). Developing glomeruli (PNA-positive structures in brown) from S-shaped

bodies to fully developed glomeruli were identified and marked with open circles on the paper

(Figure 2.6B). For those sections that were too large to be fully projected within the field of

view, this step was taken in stages by moving section from one side to the other using

already circled glomeruli as reference points until the entire section was analyzed. Once all

PNA-positive structures were marked on reference section, the projection was replaced with

the lookup section.

The glomeruli present in the reference section (circles on paper) were used as

reference to align the lookup section. Glomeruli that were present in the reference section

that were no longer present in the lookup section were identified as disappearing glomeruli.

The disappearing glomeruli were identified by filling the original open circles (black close

circles; Figure 2.6D). Glomeruli that were present in the reference section that were still

present in the lookup section were not counted and remained as open circles. Glomeruli that

were present in the lookup section that were not present in the reference section were

identified as appearing glomeruli. The appearing glomeruli were marked as close circles in

an alternate color (red close circles; Figure 2.6D). This process was repeated for each of the

10-12 pairs of sections selected for each kidney.


56

Figure 2.6 Estimating PNA-positive nephrons. A: micrograph of PNA-positively stained reference kidney section (n). B: All nephrons with PNA-positive podocytes are marked with open circles. C: micrograph of PNA-positively stained lookup kidney section (n+2). D: micrograph of lookup section with overlay of glomeruli identified on the reference section. Glomeruli present in the reference section not present in the lookup section are marked (disappearing glomeruli; 3 filled black circles). Glomeruli not present in the reference section but present in the lookup section are also marked (appearing glomeruli; filled red circles). Bar

= 150µm. Figure from Cullen-McEwen et al. 81

Sum of the total number of disappearing and appearing glomeruli (�� in all the

section pairs were calculated and used to calculate the total nephron number (Nglom) using

the following equation:

Nglom=1

SFF×

1

2×

1

2×Q

-,

Where Nglom is the total number of PNA-positive developing nephrons in the kidney and

1/SSF is the reciprocal of the section-sampling fraction (number of sections advanced

between section pairs). The first ½ accounts for the fact that the dissector pair of the sections

consisted of the n and the n+2 sections. The second ½ accounts for the fact the PNA-

positive structures were counted in both directions between the section pair. �� is the sum of

the total number of disappearing and appearing glomeruli in all section pairs.


57

2.5.5. Estimating Kidney Volume

Kidney volume was estimated from PNA stained kidney sections using the Cavalier

Principle.80,141 Each reference section used for estimating glomerular number was projected

at 72x magnification with full view of the each kidney on a table in the darkened room. A

stereological grid (2 x 2 cm) printed on a transparent sheet was overlaid on the projected

image. The total number of grid points landing on renal tissue was counted. Kidney volume

was then estimated using following formula:

Vkid = ∑P× a��p�� × T × 1

f

Where Vkid is the total kidney volume, ΣP is the total number of grid points counted, a(p) is

the area associated with each grid point and T is the section thickness, and 1/f is the inverse

of the section sampling fraction used.

2.6. CARDIAC CINE

In Chapter 3, female control and obese mice underwent cardiac cine

ventricle chamber dimensions and cardiac function at baseline and on GA14. This MRI

technique designed specific for

measurements of cardiac geometry across multiple cardiac cycles with minimal observer

dependency.206,211 These experiments were performed by Dr Michelle Kett and Dr James

Pearson at the Monash Biomedical Imaging Facility.

2.6.1. Animal Preparation

All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agi

Technologies, CA, USA) at Monash Biomedical Imaging facility. Mice underwent cardiac

cine-MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised

(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine

electrodes were placed subcutaneously in the right arm and left leg to detect

electrocardiogram (ECG) signals through an ECG/Temperature Module sensor (SA

Instruments Inc, NY, USA). A transducer sensor was placed on the lower back of the mouse

to monitor respiration via a Respiration/2

Once clear ECG and respiration signals were established, the cradle was then inserted into

the MR scanner for imaging. Mice were kept at an ambient temperature of 30

MRI Scanner.

Figure 2.7 Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted subcutaneously into the right fore limb and left hind limb, and alower back of the mouse for respiratory recording.


58

CARDIAC CINE-MAGNETIC RESONANCE IMAGING (Chapter 3)

In Chapter 3, female control and obese mice underwent cardiac cine


technique designed specific for mice provides accurate, reproducible and repeated


These experiments were performed by Dr Michelle Kett and Dr James

Pearson at the Monash Biomedical Imaging Facility.

Animal Preparation

All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agi


MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised

(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine




onitor respiration via a Respiration/2-CH IBP module (SA Instruments Inc, NY, USA).


the MR scanner for imaging. Mice were kept at an ambient temperature of 30

Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted subcutaneously into the right fore limb and left hind limb, and a sensor was placed on the lower back of the mouse for respiratory recording.


MAGNETIC RESONANCE IMAGING (Chapter 3)

In Chapter 3, female control and obese mice underwent cardiac cine-MRI to assess left


mice provides accurate, reproducible and repeated


These experiments were performed by Dr Michelle Kett and Dr James

All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agilent


MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised

(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine needle




CH IBP module (SA Instruments Inc, NY, USA).


the MR scanner for imaging. Mice were kept at an ambient temperature of 30-32°C within the

Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted

sensor was placed on the


59

2.6.2. MRI scan

Once the mouse was placed in the centre of the magnetic field, a series of steps was

carried out to determine the long axis of the heart before establishing a short axis for imaging

the left ventricle.

1) An initial scout scan was performed to adjust the position of the mouse within the

scanner to maximize sensitivity. A 3D Shimming was then conducted to fine-tune the

magnetic field of the gradient and transmitter coils at the center of the view, in order

to optimizes the radiofrequency intensity and contract of the signal from the heart.

2) A short axis view from the oblique angle was then obtained for the localization of the

long axis of the left ventricle in the sagittal plan.

3) The long axis of the left ventricle was located through the sagittal plane, running from

the apex of the heart thought to the aortic valve.

4) The short axis plane perpendicular to the long axis was then determined.

5) Finally, a 4-chamber view was established through the sagittal and coronal planes

and final adjustment was made to finalize the orientation of the short axis.

The images of the heart were captured in across 7 to 8 slices of 1 mm thickness

covering the entire ventricle from the aorta to the apex of the heart (Figure 2.8 A & B). An

evenly spaced 25 frames within each slice were captured across one cardiac cycle (Figure

2.8 C). These images was then used to analyze left ventricular mass, regional wall thickness

(anterior, posterior and septum), end diastolic volume, and end systolic volume.

Figure 2.8 The 4-chamber view of mouseslices of 1mm thickness covering the aorta to the apex of the heart (represents an individual slice the image taken. From each slice (across a cardiac cycle (C)

2.6.3. Data Analysis

The MRI images were

Sweden; http://segment.heiberg.se

outlined, and end-diastolic and end

calculated as the differenc

(ESV). Ejection fraction was determined by the fraction of EDV that were ejected from each

heart beat (percentage of SV/EDV). Cardiac output was calculated by multiplying SV with HR.

Left ventricular mass (LVM) was derived from chamber wall volume in assuming a density of

the cardiac muscle of 1.06g/ml. The middle short

determine wall thickness of the septum, anterior and posterior wall.


60

chamber view of mouse heart in determination of short axis plane for 7slices of 1mm thickness covering the aorta to the apex of the heart (represents an individual slice the image taken. From each slice (B), 25 frames were captured

The MRI images were analyzed using Segment software (V1.9 R2626, Medviso,

http://segment.heiberg.se).156 The epicardium and endocardium of all slices were

diastolic and end-systolic volumes determined. Stroke volume (SV) was

calculated as the difference between end-diastolic volume (EDV) and end



ular mass (LVM) was derived from chamber wall volume in assuming a density of

the cardiac muscle of 1.06g/ml. The middle short-axis slice of each heart was used to

determine wall thickness of the septum, anterior and posterior wall.


heart in determination of short axis plane for 7-8 slices of 1mm thickness covering the aorta to the apex of the heart (A). Each blue line

), 25 frames were captured

using Segment software (V1.9 R2626, Medviso,

The epicardium and endocardium of all slices were

systolic volumes determined. Stroke volume (SV) was

diastolic volume (EDV) and end-systolic volume



ular mass (LVM) was derived from chamber wall volume in assuming a density of

axis slice of each heart was used to


61

2.7. ASSESSMENT OF COLLAGEN CONTENT

2.7.1. Hydroxyproline Colorimetric Assay (Chapter 4)

Hydroxyproline is a common nonproteinogenic animo acid and it represents ~14.4%

of the amino acids in collagen.125 Therefore, hydroxyproline content in tissue hydrolysates is

a direct representation of the total collagen content of the tissue. Using the Hydroxyproline

Colorimetric Assay Kit (BioVision, CA, USA), hydroxyproline concentration was determined

by the reaction of oxidized hydroxyproline with 4-benzaldehyde (DMAB), which results in a

colorimetric (579nm) product, proportional to the hydroxyproline present in the tissue.

2.7.1.1. Preparing Tissue Hydrolysates

Kidneys (middle quadrant) and left ventricle tissue (apical portion) were dried

overnight at 62°C. Dried tissues were weighed and homogenized in a volume of dH2O

according to the dry weight of the each sample (200µl of dH2O for every 10mg of dry tissue).

Homogenates were then centrifuged for 5 min at 1250 RPM at room temperature. To a 100µl

of sample homogenate, 100µl of concentrated HCl was added. Samples were then

hydrolyzed at 120°C for 3 hours.

2.7.1.2. Colorimetric Reaction and Absorbance Measurement

Serial dilutions of hydroxyproline standard solution were preformed and each

concentration of standard was added to a 96- well plate in duplicate. Each hydrolysates was

then added to 96-well plate and subsequently dried to eliminate HCl. Chloramine T reagent

was added to each well and incubated for 5 min at room temperature. DMAB reagent was

then added to each well and incubated at 60°C for 20 minutes. A plate reader was used to

determine absorbance at 570 nm. A standard curve was generated using the absorbance

obtained and known concentration of diluted hydroxyproline standard. The slope of this

standard curve was used to convert absorbance to hydroxyproline concentration for each

sample. The dilution factors and tissue weights were then used to calculate hydroxyproline

content in the tissue.


62

2.7.2. Histopathological and Microscopic Analysis of Collagen Content in

Cardiac and Renal Tissue.

2.7.2.1. Assessment of Collagen Content using PSR Staining

2.7.2.1.1. Picrosirius Red Staining

To visualise the accumulation of collagen in the cardiac and renal tissue, picrosirius

red (PSR) was used to stain for collagen fibres. The fixed left kidneys and left ventricles were

processed and embedded into paraffin block. Tissue blocks were sectioned at 4 µm and full-

face sections were collected, flattened and dried on a glass slide. Tissue sections were

submerged in Bouin’s fixative for 1 hr at 60°C to stain the cytoplasm in yellow. Excess

Bouin’s fixative was washed off using running water and counterstained in 0.1% picrosirius

red solution for 1 hr at room temperature. The sections were washed in running water and

rehydrated through 3 changes of graded ethanol and xylene. Slides were coversliped with

DPX mounting media and left to dry overnight at room temperature.

2.7.2.1.2. Renal Collagen Content (Chapter 3)

Renal interstitial collagen content was assessed using PSR stained kidney sections

as described previously.180 PSR stained kidney sections was visualised using a polarizing

microscope (Abrio, Hinds Instrument, USA) and images acquired (CCD camera). Eight

consecutive non-overlapping fields were selected across each kidney section. In the LPS

processed image, light retarded by collagen bundles appeared white, with a maximum

retardance set at 34nm. All images were semi-quantified based on the proportional area of

collagen birefringence to tissue area. Total collagen was calculated on the polarized images

with a minimum threshold of 35 (4.67nm). Proportional area of PSR stained collagen in all

selected fields was quantified using ImageJ analysis software (Version 1.49c; National

Institutes of Health, USA). Regions of PSR staining larger than 100 pixels were defined as

renal tubular dilation and were excluded from selected fields. Results were expressed as

percentage of PSR stained area in total area chosen.

2.7.2.1.3. Cardiac Collagen Content (Chapter 3 & 4)

PSR stained left ventricle sections were scanned by Aperio AT Turbo digital Scanner

(Leica Biosystems, IL, USA) and visualized in ImageScope Viewing Software (Leica

Biosystems, IL, USA). Eight non-overlapping fields (~0.16mm2) from the subendocardium

region around the inner lumen of the left ventricle were selected (Figure 2.9). Blood vessels


63

contained in selected fields were excluded. The percentage of area with PSR-postive

staining within the selected fields in each section was quantified using ImageScope software

(Leica Biosystems, IL, USA).127

Figure 2.9 Selection of 8 non-overlapping fields in the subendocardium region of the left ventricle for analysis of cardiac collagen content. Bar = 2mm.

2.7.2.2. Assessment of Glomerulosclerosis Using PAS Staining (Chapter 4)

The transverse middle quadrant of the fixed left kidney was processed and

embedded into paraffin block. Tissue blocks were sectioned at 4 µm and full-face sections

were collected, flattened and dried on a glass slide. Slides were then stained for Periodic

acid-Schiff (PAS) and scanned using Aperio AT Turbo digital Scanner (Leica Biosystems, IL,

USA) and visualized in ImageScope Viewing Software (Leica Biosystems, IL, USA). All

glomeruli on one full-face section were selected and the tuft circled (Figure 2.10). Number of

glomeruli within each section ranged from 90-160 and did not differ between groups. The

percentage of area with PAS-positive staining within the total glomerular tuft area selected in

each section was quantified using ImageScope software.385


64

Figure 2.10 Selection of glomerular tuft area for analysis of glomerulosclerosis using PAS staining. Bar = 100μm.


65

2.8. STATISTICAL ANALYSIS OF RESULTS

Data were plotted and statistically analysed using GraphPad Prism 6 (GraphPad

software, CA, USA). The specific statistical analysis conducted for each study was detailed in

the Methods section of each chapter. All values were expressed as Mean ± SEM. P value

<0.05 were considered statistically significant.

Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy

66

Chapter 3 OBESITY LIMITS THE NORMAL

CARDIOVASCULAR AND RENAL ADAPTATIONS

OF PREGNANCY COMPROMISING FETAL KIDNEY

DEVELOPMENT


67

OBESITY LIMITS THE NORMAL CARDIOVASCULAR AND RENAL ADAPTATIONS OF

PREGNANCY COMPROMISING FETAL KIDNEY DEVELOPMENT

Xiaochu Caia, James T Pearsona,b,c, Luise Cullen-McEwend, Jeremy Colea, Kevin Yaod,

Tracey A Gasparie, Matthew J. Wattf and Michelle M Ketta

Affiliations:

aCardiovascular Disease Program, fMetabolic Disease and Obesity Program, Biomedicine

Discovery Institute and Department of Physiology, bMonash Biomedical Imaging Facility,

dDepartment of Anatomy and Developmental Biology, eDepartment of Pharmacology,

Monash University, cAustralian Synchrotron, Clayton, Victoria, Australia.

Short title: Obesity and hemodynamic adaptations of pregnancy

Publication status: Submitted

Corresponding author:

Dr. Michelle Kett

Department of Physiology,

26 Innovation Walk

Monash University,

Clayton, Victoria, Australia, 3800.

Phone: +61 3 9905 4284

Email: [email protected]


68

3.1. ABSTRACT

Maternal obesity is associated with poor maternal and fetal outcomes, yet the impact

of pre-pregnancy obesity on the normal cardiovascular and renal adaptations of pregnancy

and fetal kidney development is largely unknown and thus was the focus of this study. Four-

week old female C57BL/6J mice were fed control (7%w/w fat) or high fat (23.5%w/w fat)

chow for 10 weeks. Mean arterial pressure (MAP) and heart rate (HR; radiotelemetry),

cardiac structure and function (cine MRI) and 24hr urinary albumin excretion was assessed

before and during pregnancy. Maternal and fetal tissues were collected at GA19 and fetal

nephron number estimated (stereology). High fat feeding led to diet-induced obesity, glucose

intolerance, hypertension, tachycardia, ventricular hypertrophy and fibrosis, elevated cardiac

output and albuminuria, consistent with the obese phenotype in humans. Whilst MAP and HR

of obese mice remained elevated over control mice throughout pregnancy, the increases in

MAP and HR of obese mice during the second half of pregnancy, particularly prior to birth,

were blunted. Obese dams also failed to demonstrate an increase in cardiac output, stroke

volume or left ventricular mass by GA14, and albuminuria was exacerbated. Obesity led to

fewer viable fetuses and suboptimal fetal development at GA19 with lower body weight,

smaller kidneys, higher incidence of abnormal glomerular morphology in male and female

fetuses, and a 25% nephron deficit in male fetuses. These data indicate that obesity limits

the hemodynamic adaptations of pregnancy and leads to adverse fetal outcomes and sub-

optimal kidney development.

Key Words: obesity, hemodynamics, pregnancy, fetal outcomes, kidney development,

nephron number


69

3.2. INTRODUCTION

Over 50% of women of reproductive age in Australia, the USA, and the UK are

overweight or obese.2,226,273 Despite the reduced fertility rate and increased risk of

miscarriage associated with obesity, the prevalence of maternal obesity has been reported

as approximately 20% in the USA, and 13% in Australia.68,244 However recent data suggests

that the prevalence of maternal obesity may be higher (28-33%) in rural and low socio-

economic groups.84,164 Maternal obesity is associated with hypertensive disorders of

pregnancy, gestational diabetes, renal dysfunction and, importantly, higher rates of

congenital malformations, and maternal and fetal death.79 Apart from the immediate risks

during pregnancy and perinatally, maternal obesity is associated with the programming of

adult cardiovascular disease in humans299 and animals.40,116,318 Whilst the sensitivity of the

fetal kidney to adverse intrauterine environments is well recognized, the impact of maternal

obesity on fetal kidney development is poorly understood.

Pregnancy involves significant cardiovascular and renal adaptations to facilitate blood

flow to the fetoplacental unit. These changes include increases in blood volume, heart rate

(HR), stroke volume (SV), cardiac output (CO), and cardiac hypertrophy secondary to

volume load,60,75 whilst blood pressure falls from early pregnancy reaching a nadir mid-

pregnancy.60 Glomerular filtration rate (GFR) increases due to a fall in renal vascular

resistance, however this hyperfiltration is not associated with renal injury.60 Obesity also

induces significant cardiovascular and renal adaptations including increased blood volume,

HR, CO and GFR.151 However, in contrast to pregnancy, obesity is associated with

hypertension, pathological cardiac hypertrophy, and renal dysfunction including

albuminuria.151

Despite the significant complications associated with maternal obesity, no study to

date has examined the impact of obesity on the cardiovascular and renal adaptations that

occur during pregnancy. Further, few studies have examined the effects of maternal obesity

on kidney development in offspring. We hypothesized that obesity would limit the

cardiovascular and renal adaptations of pregnancy contributing to poor fetal outcomes. To

address this hypothesis we established a robust mouse model of diet-induced obesity in

female C57BL/6 mice and characterized the cardiovascular and renal phenotypes of these

mice and the hemodynamic adaptations during pregnancy. We also assessed fetal outcomes

with a focus on kidney development.


70

3.3. METHODS

All experiments were approved by Monash University Animal Ethics Committee and

conducted in accordance with the Australian Code of Practice for the Care and Use of

Animals for Scientific Purposes. Four-week-old female C57BL/6J mice received either a

control or a high fat diet (HFD) for 10 weeks prior to experimentation and throughout the

study (See Section 2.1.1.1 for details).

Glucose metabolism was examined via glucose tolerance tests in a sub-cohort of

mice. After 9 weeks of diet treatment, mice were fasted for 4hrs before undergoing glucose

tolerance tests (GTTs) after intraperitoneal administration of glucose. Fasting blood glucose

(tail tip blood sample) was measured (Accu-Chek Performa blood glucose meter; Roche

Diagnostics Gmbh, Germany) before all mice received 46mg of glucose ip. This dose is

equivalent to 2mg/g BW for CONT mice at this age (23g).243 Blood glucose was monitored

every 15 minute for 90 minutes.

Separate cohorts of obese (n=53) and control (n=32) mice were allocated to each

individual study. Arterial pressure, Heart rate and activity prior to conception and throughout

pregnancy were assessed using radiotelemetry (See Section 2.3) Cardiac MRI was used for

assessing left ventricle mass and cardiac function at baseline and GA14 (See Section 2.6).

Renal Excretory Profile was examined at baseline and GA13 (See Section 2.2.1). Following

baseline measurements female mice were mated overnight and gestational age (GA) 1 was

defined as the 24hr period commencing the start of the light cycle (6am) on the day of

vaginal plug detection.

Fetal and maternal outcomes were examined at GA19 (See Section 2.4.1 for greater

detail). Briefly, dams were anaesthetized (isoflurane) and the number of viable, non-viable

fetuses and implantation sites were recorded and their location in the uterus identified.

Fetuses and placentas were rapidly removed and weighed. Fetuses were sexed and the left

fetal kidney fixed in 4% paraformaldehyde for 2 hours then transferred to 70% ethanol until

processing for stereological analysis. Fetal head width was measured between ears.

Total glomerular number was estimated using unbiased stereology (See Section 2.5

for greater details).81,82 Briefly, fetal kidneys were embedded in paraffin and exhaustively

sectioned at 4 µm. 10-15 evenly spaced section pairs were systematically sampled and

histochemically stained with the lectin peanut agglutinin (PNA) to identfy glomerular

podocytes in early S-shaped bodies through to mature glomeruli. Sections were then

counterstained with hematoxylin. Section pairs were then used to estimate PNA-positive

glomeruli using the physical disector/fractionator combination as previously described.81,82 All

glomeruli in section pairs were assessed for evidence of abnormal characteristics,


71

specifically glomerular capillary dilation and enlarged Bowman’s space, and the percentage

of abnormal glomeruli calculated. Kidney volume was estimated from kidney sections using

the Cavalier Principle.80,141

Following collection of fetal tissues, a carotid arterial blood sample was taken from

the dam for determining plasma free fatty acids (FFAs) and triacylglycerol (TAG) using

enzymatic colorimetric assays (FFAs, Wako Pure Chemical Industries, Japan; TAG, GP-PAP

reagent, Roche Diagnostic, Germany respectively). Maternal kidneys were rapidly excised,

decapsulated and weighed. The left kidney was immersion fixed in 10% buffered formalin for

histological analysis of collagen deposition. The heart of the dam was excised and weighed.

The atria were removed and both atria and ventricles of the heart were weighed and all

values reported relative to tibia length.246 Top third of the ventricles were fixed in 10%

buffered formalin for histological analysis of collagen deposition. Pericardial fat and liver were

also weighed. Age-matched virgin control and obese mice were used as time controls.

Data were analyzed by unpaired t-test or two-way ANOVA, repeated measure where

appropriate. For studies that examined bodyweight and food intake, glucose metabolism,

MAP and HR, renal excretory profile and cardiac functions, two-way repeated measure

ANOVA were conducted, the factors of diet (Pd) and time (Pt), and interaction between those

factors were examined (Pd*t). For GA19 maternal tissue data, two-way ANOVA was

conducted; the factors of obesity (Pd) and pregnancy (Pp), and interaction between those

factors (Pd*p) were examined. For GA19 fetal outcome data, two-way ANOVA was

performed; the factors of diet (Pd) and sex of the fetus (Ps), and interaction between these

factors (Pd*s) were examined. Sidak post-hoc analyses were conducted where appropriate.

Values are mean ± SEM.


72

3.4. RESULTS

3.4.1. Diet-Induced Obesity in Female Mice

HFD fed mice had greater weight gain (Pd*t <0.001) and were 47% heavier than

controls prior to mating (33.3±0.6, 22.7±0.2g respectively, P<0.001; Figure 3.1A) due to

greater food and caloric intake (Pdiet<0.01-0.001; Pd*t <0.001; Figure 3.1B&C). HFD fed

obese mice had significantly higher fasting blood glucose compared to controls (8.98±0.25,

6.88±0.37mmol/L respectively; P<0.001) and impaired glucose tolerance (Pd*t <0.001,

Figure 3.1D).

Figure 3.1. Weekly body weight (A) of control (open symbols, n=32), and obese (closed symbols, n=53) mice, food intake (B) and caloric intake (C) of control (open symbols, blue line, n=19) and obese (closed symbols, red line, n=15) mice during 10-week diet treatment; Glucose tolerance tests (GTT; D) in control (n=18) and obese (n=25) mice. Data analyzed by two-way repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). Mean ± SEM.

0 1 2 3 4 5 6 7 8 9 100

5

10

15

20

25

30

35

Weeks

g

Body Weight

ControlObese

Pdiet <0.001Ptime <0.001Pd*t <0.001

0 1 2 3 4 5 6 7 8 9 100

20

40

60

80

100

120

Weeks

kC

al /

we

ek

Caloric Intake

ControlObese

Pdiet <0.001Ptime <0.001Pd*t <0.001

0 1 2 3 4 5 6 7 8 9 100

5

10

15

20

25

30

g / w

ee

k

Food Intake

ControlObese

Pdiet <0.001Ptime <0.001Pd*t <0.001

0 15 30 45 60 75 900

5

10

15

20

25

Minutes

Blo

od

Glu

co

se

(m

mo

l/L)

Glucose Tolerance

ControlObese Pdiet <0.001

Ptime <0.001Pd*t <0.001

A

D

B

C


73

3.4.2. Arterial Pressure, Heart Rate and Activity

Diet-induced obesity led to significantly higher MAP and HR during light and dark

periods (Figure 3.2A,B), but locomotor activity was not significantly different from control

mice (Figure 3.2C). Mice showed the expected changes in MAP across pregnancy

(Ptime<0.001) including the mid-gestation dip reaching a nadir on GA9 (Figure 3.2A). MAP of

obese mice remained elevated over controls for most of pregnancy (Pdiet<0.001), however

the post-dip rise of MAP in control mice was more profound, such that the groups no longer

differed from GA18. Analysis of the delta MAP across pregnancy highlighted that the timing

and the magnitude of the dip (~11mmHg) was similar between obese and control mice.

However, the rise in MAP post-dip was blunted in obese mice and, in contrast to control mice,

MAP did not rise above pregnancy levels towards term (Pd*t<0.01, Figure 3.2A).

HR increased across pregnancy as expected (Ptime<0.001, Figure 3.2B). Whilst

obese mice had elevated HR compared to control mice (Pdiet<0.01, Figure 3.2B), this effect

was lost with time and HR was no longer different between the groups from GA11 during

dark phase (GA12 light phase). The HR of obese mice changed little early in pregnancy with

the surge in HR both delayed (GA13 vs GA11) and diminished in magnitude, being

approximately half that of control mice (Figure 3.2B). Locomotor activity was lower in obese

animals (dark period only; Pdiet<0.05), decreased with pregnancy (Ptime <0.001), but the

decrease was similar in both groups (Figure 3.2C).


74

Figure 3.2. Mean arterial pressure (MAP; A), heart rate (HR; B) and locomotor activity (C)

for light period (left panel), dark period (center panel) and the change from basal levels in

dark period (∆; right panel) for control (open symbols, blue line; n=9) and obese (closed

symbols, red line; n=6) mice during pregnancy. Data analyzed by two-way repeated

measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). Mean ±

SEM.

LIGHT

80

90

100

110

120

MAP

mmHg

Pdiet <0.001Ptime <0.001

Pd*t NS

450

500

550

600

650

HRbpm

Pdiet <0.01Ptime <0.001Pd*t NS

Basal 1 7 13 190

4

8

12

16

Gestational day

ActivityCounts/min

Pdiet NS

Ptime <0.001Pd*t NS

DARK


Pd*t <0.01

Pdiet <0.001Ptime <0.001Pd*t =0.05

Basal 1 7 13 19Gestational day


Pd*t NS

DARK

-15

-10

-5

0

5

10

15

∆

Pdiet NS

Ptime <0.001Pd*t <0.001

-50

0

50

100

150

∆

Pdiet <0.01

Ptime <0.001Pd*t NS

1 7 13 19

-8

-4

0

4

Gestational day

∆

Pdiet NS

Ptime <0.001

Pd*t NS

A

B

C


75

3.4.3. Cardiac MRI

Obese mice had significantly greater left ventricle mass (LVM) and CO compared to

controls. LVM and CO increased significantly with pregnancy but the effects were blunted in

obese mice (Ptime<0.001; Pd*t<0.01; Figure 3.3A,B). Post-hoc analysis demonstrated that

while the pregnancy-induced increases in LVM and CO for control mice were profound and

highly significant (26% and 25%; P<0.001), obese mice did not show significant increases

with pregnancy (6% and 7%). Indeed at GA14, LVM and CO were no longer different

between obese and control mice (Figure 3.3A,B). The changes in CO were reflected in

changes in SV with only control mice showing a significant increase with pregnancy (21%;

P<0.01; Figure 3.3C). The increase in stroke volume with pregnancy in control mice was

due to increases in end-diastolic volume (EDV) with end-systolic volume (ESV) not changing

significantly with pregnancy (Figure 3.3D,E). ESV however tended to be higher in obese

mice. Neither obesity nor pregnancy impacted ejection fraction (Figure 3.3F). Obesity did not

impact wall thickness (data not shown).

Figure 3.3. Left ventricle mass (LVM; A), cardiac output (CO; B), stroke volume (SV; C), end-diastolic volume (EDV; D), end systolic volume (ESV; E), and ejection fraction (EF; F) in control (open symbols, blue line; n=6) and obese (closed symbols, red line; n=5) mice at baseline and GA14. Data analyzed by two-way repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). *P<0.05, ** P<0.01, ***P<0.001. Mean ± SEM.

50

60

70

80

mg

LVM

Pdiet NSPtime <0.001Pd*t <0.01

***

5

10

15

20

25

ml

/ m

in

CO

Pdiet NSPtime <0.001Pd*t <0.01

***

20

30

40

50

ul

SV

Pdiet NSPtime <0.01Pd*t =0.08

**

Baseline GA1430

40

50

60

ul

EDV

Pdiet NSPtime <0.05Pd*t NS

*

Baseline GA140

5

10

15

ul

ESV

Pdiet =0.06Ptime NSPd*t NS

Baseline GA1460

70

80

90

%

EF

Pdiet NSPtime NSPd*t NS

A CB

D FE


76

3.4.4. Renal Excretory Profile

At baseline there were no differences in 24hr water or food intake, urine or sodium

excretion, or urinary osmolality between control and obese mice, however obese mice had

significantly higher albumin excretion (P<0.001; Figure 3.4F). There was a tendency

(P=0.055; Figure 3.4A) for pregnancy to increase water intake, yet neither obesity nor

pregnancy impacted urine excretion and food intake. Sodium excretion decreased

significantly with pregnancy (Ptime<0.01, Figure 3.4E), however post-hoc analysis

demonstrated that only control mice showed a significant decrease (P<0.05). Urinary

osmolality decreased with pregnancy in both groups (Ptime<0.05, Figure 3.4C). Pregnancy

resulted in a profound increase in albuminuria in obese but not control mice (Pdiet<0.001;

Pd*t<0.05; Figure 3.4F).


77

Figure 3.4. 24hour water intake (A), food intake (B); urine osmolality (C); urinary excretion

(D); sodium excretion (E); albumin excretion (F) in control (open symbols, blue line; n=8) and

obese (closed symbols, red line; n=7) at baseline and GA13. Data analyzed by 2-way

repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t).

*P<0.05. Mean ± SEM.


78

3.4.5. Fetal Outcomes

Gestational length (control 19.6±0.1; obese 19.8±0.2 days) was similar between

groups. The number of implantation sites between groups was similar (control 7.8±0.7;

obese 7.0±1.0). However obese dams had significantly fewer viable fetuses per litter (control

7.5±0.9; obese 4.0±0.8; P<0.05).

The fetal sex ratio was similar between control and obese dams. Male and female

fetuses of obese dams had significantly lower average bodyweights per litter than fetuses of

control dams (Pdiet<0.01; Table 3.1). Whilst diet-induced obesity did not affect placental

weight, male fetuses had heavier placentas (Psex<0.01; Table 3.1). The fetal:placental

weight ratio was significantly reduced with obesity (Pdiet<0.01; Table 3.1). Post-hoc analysis

demonstrated that obesity-induced reduction in fetal:placental weight ratio was only

significant for male fetuses (P<0.05). Fetuses from obese dams had a lower average fetal

head width per litter (Pdiet<0.01), and profoundly higher incidence of placental thrombosis

(Pdiet<0.05) and meconium of fetal membranes (Pdiet<0.001; Table 3.1).

Fetal glomerular number was reduced with pre-pregnancy obesity (Pdiet<0.001;

Table 3.1). Post-hoc analysis demonstrated that this obesity-induced reduction in fetal

glomerular number was only significant for male fetuses and profound at 43% (P<0.001;

Table 3.1). The low glomerular number in male fetuses remained after correction for fetal

body weight (25% lower; P<0.01; Table 3.1). Maternal obesity also led to smaller fetal

kidney volume (Pdiet<0.05; Table 3.1), however kidney volume to bodyweight ratio was not

different between groups (Table 3.1).

Three of the 5 kidneys from male fetuses of obese dams showed either limited or

absent renal papilla formation (Figure 3.5). Further, glomeruli of male and female fetuses of

obese dams showed significant abnormalities, including glomerular capillary dilation and/or

grossly enlarged Bowman’s space. The percentage of glomeruli affected was 5 times greater

in kidneys from obese compared to control dams (Pdiet<0.001; Table 1; Figure 3.5).


79

Table 3.1. Litter Characteristics and Stereological Analysis of Fetal Kidneys at GA19. Vkid indicates kidney volume; BW, body weight and Nglom, glomerular number. Data analyzed by two-way ANOVA with factors of diet (Pdiet), sex (Psex) and interaction (Pint). *P<0.05, †P<0.01 and ‡P<0.001 vs control within the same gender. Values are mean ± SEM.

Male Female P

Control Obese Control Obese diet sex int

Litter Characteristics

Fetal Wt g 1.01±0.03 0.83±0.06 0.98±0.06 0.84±0.06 <0.001 NS NS

Placental Wt g 0.086±0.002 0.095±0.005 0.077±0.003 0.082±0.004 NS <0.01 NS

Fetal/Placental Ratio 11.8±0.6 9.0±1.0* 12.9±0.6 10.4±0.7 <0.01 NS NS

Placental Thrombosis % 0 45.8±18.7‡ 8.3±8.3 31.0±15.6‡ <0.05 NS NS

Meconium of fetal membranes % 5.6±5.6 91.7±8.3‡ 2.4±2.4 67.9±16.1‡ <0.001 NS NS

Head Width mm 6.62±0.09 6.11±0.11* 6.47±0.08 6.01±0.18* <0.01 NS NS

No. of Litters 6 6 6 7

Stereology

Vkid mm3 1.77±0.09 1.22±0.24 1.72±0.13 1.53±0.13 <0.05 NS NS

Vkid/gBW 1.62±0.04 1.44±0.20 1.70±0.11 1.79±0.07 NS NS NS

Nglom (PNA+ve) 1604±58 914±94‡ 1397±124 1100±101 <0.001 NS NS

Nglom/gBW 1474±39 1107±95† 1373±73 1280±47 <0.01 NS =0.052

Nglom/Vkid 912±30 815±102 822±62 724±45 NS NS NS

Abnormal Glomeruli % 1.6±0.4 9.2±1.6‡ 2.1±0.7 11.6±1.0‡ <0.001 NS NS

No. of Kidneys 5 5 6 7

Figure 3.5. Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower magnification (LP; top panel; scale bar 500μm) and higher magnification (HP


80

Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower magnification (LP; top panel; scale bar 500μm) and higher magnification (HP- bottom panel; scale bar100 μm).


Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower bottom panel; scale bar100 μm).


81

3.4.6. Maternal Outcomes

Weight gain was significantly lower in obese dams (Pd*t <0.01; Figure 3.6) due to

reduced weight gain between GA13-19 (11.9±0.4 vs 13.7±0.7g; P<0.05; Figure 3.6).

Pregnancy and obesity led to significantly greater kidney, heart and liver weights

(Ppreg<0.05, Pdiet <0.001; Table 3.2), however obesity did not exacerbate the pregnancy

induced increases in organ weights. Cardiac collagen content of obese primiparous

(4.08±0.36%area) and nulliparous (5.07±0.80%area) mice was significantly greater than

control primiparous and nulliparous hearts (1.96±0.44, 2.04±0.42 %area respectively;

Pdiet<0.001), however there was no effect of pregnancy. Pregnancy did not impact on

pericardial fat but obese animals had an approximately 5 fold greater pericardial fat mass

than control mice (Table 3.2). Neither obesity nor pregnancy impacted renal collagen content

(data not shown). Obese nulliparous mice had significantly greater plasma FFA compared to

control mice. Pregnancy did not affect the FFA levels in obese mice however control mice

demonstrated an increase with pregnancy (Pd*t<0.05; Table 3.2).

Figure 3.6 Body weight gain in control (open symbols, blue line) and obese (closed symbols, red line) dams at GA7 (n=12,14), GA13 (n=12,10) and GA19 (n=6,6). Data were analyzed by two-way ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t), followed by Sidak post hoc tests. *P<0.05 compared control and obese groups. Values are mean ± SEM.

7 13 190

5

10

15

Gestational day

g

Body Weight Gain

*

P diet NSP time <0.001P d*t <0.01


82

Table 3.2. Maternal body weight, total kidney weight, kidney weight/tibia ratio, heart weight, heart weight/tibia ratio, left ventricle (LV) weight, LV weight/tibia ratio, combined atria weight, atria weight/tibia ratio, pericardial fat weight, liver weight, tibia length, plasma free fatty acids and plasma triacylglycerol of control and obese dams (primiparous) at GA19 and age matched non-pregnant control and obese mice (nulliparous). Data were analyzed by two-way ANOVA with factors of Pregnancy (Ppreg), Obesity (Pdiet) and interaction (Pp*d), followed by Sidak post hoc tests comparing control and obese mice within the nulliparous groups and within the primiparous groups *P<0.05, †P<0.01 and ‡P<0.001. Values are mean ± SEM.

Nulliparous Primiparous P

Cont (10) Obese (10) Cont (6) Obese (6) Preg Diet p*d

Body Wt (g) 25.8±0.9 42.2±1.6‡ 37.2±0.7 51.8±1.6‡ <0.001 <0.001 NS

Total Kidney Wt (mg) 249±7 264±7 279±11 315±14* <0.05 <0.001 NS

Kidney Wt/Tibia ratio 14.3±0.4 15.2±0.4 16.2±0.6 18.0±0.7 <0.001 <0.05 NS

Heart Wt (mg) 105±2.5 118±3.0* 123±2.4 145±7.5† <0.001 <0.001 NS

Heart Wt/Tibia ratio 6.03±0.15 6.80±0.18* 7.18±0.15 8.28±0.38† <0.001 <0.001 NS

LV Wt (mg) 98.2±2.3 109.0±2.5* 112.3±2.5 132.4±6.9† <0.001 <0.001 NS

LV Wt/Tibia ratio 5.63±0.14 6.27±0.14* 6.54±0.16 7.57±0.35† <0.001 <0.001 NS

Atria Wt (mg) 5.44±0.33 6.69±0.35* 7.22±0.18 8.93±0.67* <0.001 <0.01 NS

Atria Wt/Tibia ratio 0.31±0.02 0.39±0.02* 0.42±0.01 0.51±0.03* <0.001 <0.01 NS

Pericardial Fat Wt (mg) 42.2±5.8 167.2±19.1‡ 28.3±6.2 171.9±23.8‡ NS <0.001 NS

Liver Wt (g) 1.11±0.06 1.53±0.13† 1.77±0.08 2.15±0.11 <0.001 <0.001 NS

Tibia Length (mm) 17.5±0.1 17.4±0.2 17.2±0.1 17.5±0.3 NS NS NS

Free Fatty Acid (mM) 0.27±0.02 0.40±0.03 0.36±0.03 0.36±0.03 NS <0.05 <0.05

Triacylglycerol (mM) 0.52±0.04 0.66±0.06* 0.57±0.06 0.61±0.08 NS NS NS


83

3.5. DISCUSSION

This study determined the impact of diet-induced obesity on the hemodynamic

adaptations of pregnancy, and fetal and maternal outcomes in late pregnancy. Our mouse

model had profound diet-induced obesity, glucose intolerance, hypertension, tachycardia,

cardiac hypertrophy and fibrosis, elevated CO and albuminuria prior to mating, consistent

with human obesity. Whilst MAP and HR of obese mice remained elevated over control mice

throughout pregnancy, the increases in MAP and HR of obese mice towards term, were

blunted. Obese dams also failed to increase CO, SV and LVM at GA14, and albuminuria was

exacerbated. Obesity led to greater fetal death and suboptimal fetal development at GA19

with lower body weight, smaller kidneys, abnormal glomerular morphology and, in males,

nephron deficiency. These results are consistent with the hypothesis that obesity limits the

normal cardiovascular adaptations of pregnancy and leads to adverse fetal outcomes.

Few studies have performed prospective longitudinal cardiovascular measurements

in women from preconception, through pregnancy and birth, and none to date in obese

women. Mice,48 like humans,236 show an early fall in arterial pressure, reaching a nadir mid-

pregnancy before rising to, or exceeding, pre-pregnancy levels near term. MAP of obese

mice remained elevated over control mice throughout pregnancy consistent with the literature

in obese women.1,354 Interestingly, both the timing and magnitude of the mid-gestation fall in

MAP were similar in obese and control mice indicating that the mechanisms driving the fall in

MAP were not affected by pre-existing obesity. The presence of the dip in arterial pressure in

obese women is contentious, most likely due to lack of pre-pregnancy values, differences in

timing and frequency of measurements during pregnancy, but parity might also contribute.

Tomodo et al 354 enrolled Japanese women at 6 weeks of gestation, measuring arterial

pressure every 3 weeks and found that multiparous obese women demonstrated the mid-

gestation dip in arterial pressure whilst primiparous obese women did not. Following the mid-

gestation dip, MAP of control mice returned to pre-pregnancy level by GA15 and continued to

rise until GA18, consistent with human studies that found that at 38 weeks of pregnancy,

systolic and diastolic blood pressure were 5.6% and 7.5% respectively above pre-conception

levels.309 In contrast, the rise of MAP in obese mice late pregnancy was significantly blunted

such that MAP did not exceed pre-pregnancy level. Human studies have also suggested that

the rise in arterial pressure, particularly late in gestation, is attenuated with increasing

BMI.1,123,352

Control mice showed a 3% rise in dark-phase HR early in pregnancy with a profound

increase (~20%) commencing GA11. These values are consistent with human data that

demonstrate an early increase in HR (4%; 6 weeks gestation)236 reaching ~20% late in


84

pregnancy.236,279 In contrast, obese mice showed negligible change in HR in early pregnancy,

with the later rise delayed in onset (GA13), and blunted in magnitude, rising by only 10%.

Indeed although HR of obese mice started off 12% greater than controls, HR was no longer

significantly different between the groups from GA11. Consistent with our mouse studies,

findings for women have shown that HR is not different between obese and normal weight

women late in gestation.1,96 However given that obesity is associated with tachycardia,362 the

similarity in HR between obese and normal weight women suggests a blunted adaptation to

pregnancy. Although no study to date has measured the change in HR in obese women

relative to preconception levels, a meta-analysis found that the increase in HR of obese

women between the first and last trimester was significantly less than non-obese women (+3

vs +14bpm).159

As in human pregnancy, control mice showed significant increases in CO (25%), SV

(21%) and LVM (26%) from pre-pregnancy to GA14, the beginning of the final trimester.

Obese mice had elevated CO (17%), SV (12%) and LVM (15%) compared to control mice

prior to conception, however the increase in these parameters (5-7%) with pregnancy was

negligible such that at GA14, CO, SV and LVM were indistinguishable between obese and

control mice. Consistent with this finding, Dennis et al 96 found that at 36 weeks gestation,

obese women had similar CO, SV and HR compared to non-obese women, though in

contrast to our study, they found LVM was greater in obese women at this time. The

changes in cardiac function from pre-pregnancy have not been measured in obese women,

however Abdullah et al 1 demonstrated that morbidly obese pregnant women had a limited

increase in CO across the trimesters compared to non-obese controls.

The mechanisms underpinning the blunted cardiovascular responses in obese mice

are unclear. Normal pregnancy is characterized by increased sympathetic drive and reduced

baroreceptor sensitivity near term, changes that are thought to contribute to the rise in

arterial pressure and heart rate at this time.135 Our data thus supports the contention of

Helmreich et al 159 that obesity results in diminished autonomic responsiveness during

pregnancy. In humans the late rise in HR is thought to maintain the elevated CO as SV

decreases towards term,279 thus the blunted HR response in obese mice may contribute to

the limited increase in CO. It is also possible that obese dams did not have the expected

plasma volume expansion, and thus increase in preload, and ultimately stroke volume with

pregnancy. Although control mice demonstrated evidence of the sodium retention expected

in pregnancy (i.e. a marked fall in sodium excretion in the absence of a change in

food/sodium intake), this was not present in obese dams where the fall in sodium excretion

was not significant. The failure to increase plasma volume would also explain the blunted


85

cardiac remodeling in obese mice, as volume overload is the primary stimulus for

hypertrophy in pregnancy.105

Obese dams exhibited exacerbated albuminuria with pregnancy compared to control

dams. This may be associated with underlying pre-existing renal injury in obese mice prior to

pregnancy. Obesity in humans and animal models is associated with increased GFR and

renal blood flow, albuminuria and renal pathology.146,151,301 Given that pregnancy also leads

to a marked increase in GFR and renal blood flow,32 this aggravated renal hyperfiltration is

likely to contribute to exacerbated albuminuria with pregnancy. Future studies that assess

GFR and renal pathology are warranted to address the gaps in our understanding of the

impact of obesity on renal health during pregnancy and post-partum.

Obesity had no impact on the number of implantation sites, however the incidence of

fetal death and resorption was increased. This is consistent with studies in human and non-

human primates (NHP) that show markedly increased risk (5-fold in the Danish Cohort)270 of

late gestation miscarriage and stillbirth with maternal obesity.121,270,316 In contrast, most

mouse models of maternal obesity do not show a reduced litter size or adverse fetal

outcomes, though for the majority of these studies, female mice were only marginally (10-

20%), albeit significantly, heavier than control mice.40,203 However, where HFD fed mice were

more than 30% heavier than control mice prior to conception, the litter size was reduced as

in our model.62,204 Maternal obesity also led to significantly reduced fetal body weight and

head width. Although pre-pregnancy obesity in humans increases the risk of large for

gestation age babies, it is also recognized as a strong independent risk factor for small for

gestation age and IUGR babies,10,293 consistent with our finding in mice. Further, placentas of

obese dams had significantly higher incidence of thrombosis, and males had lower placental

efficiency. These findings are consistent with a recent human study that found pre-pregnancy

obesity is associated with placental insufficiency and higher risk of placental pathological

lesions, including placental thrombosis.173

The greater fetal loss and low fetal weight in obese dams is consistent with our

findings of a blunted cardiovascular response in obese dams through pregnancy. Studies

have found that fetal outcomes, including birth weight, are poorer in women who fail to

demonstrate an increase in stroke volume (index of preload or plasma volume expansion),

CO or GFR with pregnancy.7,19,103 Although our study did not demonstrate a direct link

between the blunted rise in CO in obese dams and reduced blood flow to the fetoplacental

unit, Frias et al 121 found that uterine blood flow was decreased in a NHP model of maternal

obesity, contributing to the high rate of stillbirth observed in their study. Strikingly, maternal

obesity in these NHP also led to a reduction in blood flow on the fetal side of the placenta,

increased expression of placental inflammatory cytokines and higher incidence of placental


86

infarcts.121 Our finding that the reduced placental efficiency was restricted to male fetuses of

obese dams, suggests that fetal sex is a determinant of fetal outcomes with maternal obesity.

The finding that male placenta of high fat diet-fed mice had greater inflammation and

macrophage activation than female placenta,203 suggests male offspring could be more

vulnerable to maternal obesity.

Given the cardiovascular adaptations have been shown to differ between primiparous

and multiparous obese females,354 the present study focused on maternal cardiovascular

adaptations in primiparous dams. The disadvantage of investigating primiparous dams is that

it is common for them to eat their first litter, known as maternal infanticide. This was evident

in early cohorts in this study with litters eaten within the first 24 hours post-birth. Thus, to

maintain a consistent post-partum experience in these dams for the post-partum studies in

Chapter 4, all pups were removed 24 hour post-birth. Due to the loss of offspring shortly after

birth, long-term studies in the offspring of control and obese dams could not be completed in

this thesis. Future studies should investigate the programming effects of pre-pregnancy

obesity on the long-term cardiovascular and renal health of adult offspring from second or

third litters.

This is the first study to assess the impact of maternal obesity on nephron

endowment using gold-standard stereological techniques in a model representative of human

obesity, that is, where body weight is markedly elevated prior to conception. One of most

extraordinary findings of the present study was that pre-pregnancy obesity in mice led to

lower nephron number at GA19, an effect primarily seen in kidneys of male fetuses. In

addition, kidneys of both male and female fetuses from obese dams demonstrated significant

structural abnormalities including dilated glomerular capillaries and Bowman’s space, and

limited formation of renal papilla. These structural abnormalities are likely to have a long-term

impact on GFR and sodium and water homeostasis of offspring from obese dams. Together

these findings indicate that fetal kidney development is significantly impacted by pre-

pregnancy obesity. The low nephron number in offspring of obese dams is not unexpected in

light of their lower body weight. Hughson et al 175 found a linear relationship between

nephron number and birth weight in humans. The presence of reduced nephron endowment

in male fetuses after adjusting for body weight however suggests an additional sex specific

mechanism. This is consistent with the finding that nephron endowment of male offspring

was more sensitive to exposure to a low protein diet, suggesting kidney development of male

offspring might be more vulnerable to maternal insults.379 Although the mechanisms that

mediate the nephron deficit in male offspring in the present study are unclear, it is possible

that the structural and functional differences in placentas of male offspring might be


87

important, including, the greater pro-inflammatory profiles previously demonstrated in male

placentas with maternal obesity.203

3.6. CONCLUSION

In summary, findings from the present study have demonstrated, for the first time,

that pre-pregnancy obesity limits the cardiovascular and renal adaptations of pregnancy

ultimately resulting in a blunted increase in CO, and poor fetal outcomes. The present study

also revealed the detrimental impact of pre-pregnancy obesity on fetal kidney development,

particularly nephron endowment. The lower fetal weight in male offspring could not explain

the reduction in nephron number, suggesting mechanisms other than global growth

restriction may be involved, and further examination of the placenta from these fetuses may

provide deeper understanding of this sex-specific effect. In conclusion, our findings indicate

that pre-pregnancy obesity prevents normal cardiovascular and renal adaptations of

pregnancy and leads to fetal loss, growth restriction and suboptimal fetal kidney development.

The consequence of these developmental abnormalities may contribute to the development

of cardiovascular and renal diseases later in life. Interventions that enhance the

cardiovascular adaptations in pregnancies complicated by obesity may reduce the incidence

of fetal loss and growth restriction, and the long-term impact of renal programming on

cardiovascular and renal health of the offspring.

Chapter 4 Obesity & Hemodynamic Changes Post-partum

88

Chapter 4 DOES PREGNANCY EXACERBATE THE

CARDIOVASCULAR AND RENAL EFFECTS OF

OBESITY?


89

4.1. INTRODUCTION

An uncomplicated pregnancy has no long-term adverse effects on the cardiovascular

and renal health (see Section 1.2.2). However recent studies have reported that obesity

during pregnancy is strongly associated with premature death, greater risk of major and

simple cardiovascular events, and greater number of hospitalizations of these women due to

cardiovascular events 10 years post-partum.223,384 Obesity, per se, is associated with

significant cardiovascular and renal consequences (see Section 1.3). Further, pre-pregnancy

obesity is closely associated with significant maternal complications, including gestational

hypertension, preeclampsia, and gestational diabetes.79 Importantly, these complications

have been recognized as independent risk factors for the development of significant

cardiovascular and renal disorders, such as chronic hypertension, type 2 diabetes, and CKD

in women later in life.35,61,158,240 Surprisingly, no study to date has formally investigated the

cardiovascular and renal health of females following a pregnancy complicated by obesity.

In Chapter 3 we reported that the obese dams demonstrated blunted increases in

MAP and HR towards term, and limited adaptations in SV, CO and LVM in late pregnancy.

Further, pregnancy also led to an exacerbation of albuminuria in obese dams during

pregnancy. In this chapter, we will use this mouse model of pre-pregnancy obesity to

examine the impact of pregnancy on obese female mice post-partum. We hypothesized that

pregnancy exacerbates the cardiovascular and renal outcomes of obesity and leads to

greater cardiovascular and renal morbidities. To address this hypothesis, we examined

arterial pressure, heart rate, GFR, renal excretory profiles, and cardiac and renal fibrosis in

control and obese mice 4 weeks post-weaning (4WPW) and compared them with non-

pregnant (nulliparous), age-matched control and obese mice.


90

4.2. METHODS

4.2.1. Animals



Animals for Scientific Purposes. Four-week-old female C57BL/6J mice received either a

control or a high fat diet (HFD) for 10 weeks prior to experimentation and throughout the

study (See Section 2.1.1.1 for details). Following baseline (pre-pregnancy) measurement, a

subgroup of control and obese mice were then mated with male mice and became pregnant

(see Section 2.1.1.1). The rest of control and obese remained virgin and served as time-

matched non-pregnant (nulliparous) groups to be compared with primiparous control and

obese mice post-partum.

4.2.2. Telemetry Recordings

Radiotelemetry transmitters were implanted in mice used in Chapter 3 at the

completion of the initial 10-week diet treatment and pre-pregnancy data were recorded

before mating (see section 2.3.2). Mean arterial pressure (MAP), heart rate (HR) and

locomotor activity were continuously recorded throughout pregnancy and post-partum until

PN36. Recording ceased at this time point as the battery life of the telemetry transmitters

were exhausted.

4.2.3. Assessment of urinary excretory profile and renal function (GFR)

Urinary excretory profile (see section 2.2.1) was assessed at before mating (pre-

pregnancy) and then again at 4WPW in primiparous and time-matched nulliparous control

and obese mice. Transcutaneous measurements of GFR were performed only at 4WPW in

conscious primiparous and time-match nulliparous control and obese mice (see Section 2.2.3

for details).111

4.2.4. Plasma and Tissue Collection

At 4WPW, plasma and tissues were collected from primiparous and time-matched

nulliparous control and obese mice (see Section 2.4.2). Briefly mice were anaesthetised

(Isoflurane) and an arterial blood sample was taken from the carotid artery for the

measurement of plasma FFA and TAG concentrations. Plasma free fatty acids (FFAs) and

triacylglycerol (TAG) were determined by enzymatic colorimetric assays (FFAs, Wako Pure

Chemical Industries, Japan; TAG, GP-PAP reagent, Roche Diagnostic, Germany


91

respectively). The kidneys and heart were rapidly excised and weighed. One of middle

portions of the right kidney and the apical portions of the left ventricle were used for

hydroxyproline assay to assess total collagen content in the tissue (see Section 2.4.2 &

2.7.1). One of middle portions of the left kidney was subject to histological analysis of

glomerulosclerosis (PAS staining; see Section 2.7.2.2). The middle portion of the left

ventricle was subjected to histological analysis of cardiac fibrosis (Picrosirius red staining;

see Section 2.7.2.1.3). Peri-cardial fat pad, gonadal fat pad (visceral fat) and inguinal fat pad

(subcutaneous fat) were also weighed.

4.2.5. Statistical Analysis

Data were analyzed by unpaired t-tests and two-way ANOVA, repeated measure

where appropriate. Sidak post-hoc analyses were conducted where appropriate. To assess

the change in MAP, HR and locomotor activity from pre-pregnancy levels in control and

obese primiparous mice, two-way repeated measure ANOVA was conducted, factors of

obesity (Pobe) and time (Ptime), and interaction (Pint) between those factors were examined.

For MAP and HR of obese mice at pre-pregnancy and PN36, two-way repeated measure

ANOVA was conducted, factors of pregnancy (Ppreg) and time (Ptime), and interaction (Pint)

between those factors were examined. The change in MAP and HR from pre-pregnancy to

PN36 of primiparous and time-matched nulliparous obese mice was analyzed by unpaired t-

tests. For GFR, urinary excretion profile, maternal plasma lipid profile, maternal tissue data,

total collagen contents and histological analysis of collagen accumulation, two-way ANOVA

was conducted, factors of obesity (Pobe) and pregnancy (Ppreg), and interaction (Pint)

between those factors were examined. Values are mean ± SEM. P<0.05 was considered

statistically significant.


92

4.3. RESULTS

4.3.1. Post-partum Arterial Pressure and Heart Rate

Obese dams had elevated 24h MAP and HR over control dams throughout the post-

partum period up to PN36 (Figure 4.1A&G). MAP fell rapidly by approximately 15 mmHg in

both groups post-partum reaching pre-pregnancy levels by approximately day 6 (Figure

4.1A&B), whilst HR did not change significantly with time post-partum (Figure 4.1G&H). The

change in 24h MAP and HR from pre-pregnancy levels were not significantly different

between obese and control mice across this period (Figure 4.1B&H). Similar results were

found in 24h diastolic blood pressure and systolic blood pressure (Data not shown).

Locomotor activity of control and obese dams were indistinguishable over the post-partum

period (Figure 4.1I&J).

When examining the MAP data closely, control and obese mice appear to drift apart

from PN29, with 24h MAP of obese mice rising gradually, whilst 24h MAP of control mice

remained relatively stable until the end of recording (Figure 4.1A&B). Interestingly, this effect

was only significant in light-phase MAP (Pint<0.05; Figure 4.1C&D) and not present in dark-

phase MAP (Figure 4.1E&F). In comparing the light-phase MAP at PN36, the rise in light-

phase MAP from pre-pregnancy in control mice was 2.1± 1.9 mmHg (Figure 4.1D). However

in obese mice this rise was more than 6 fold higher at 14.2 ± 3.0 mmHg (P<0.05; Figure

4.1D).

To examine whether the greater rise in MAP was simply an effect of obesity, we

compared the change in MAP from pre-pregnancy to PN36 of primiparous obese mice with

nulliparous obese mice over the same period. The change in light phase MAP of primiparous

obese mice at PN36 was 78% higher than nulliparous obese mice (8.0 ± 1.1 mmHg; P<0.05;

Figure 4.2D). However, this effect of pregnancy on MAP in obese mice was not found in 24h

MAP or dark-phase MAP (Figure 4.2A,B,E&F). Interestingly, we found that whilst the rise in

HR in primiparous obese mice from pre-pregnancy levels was minimal and similar to

primiparous control mice (Figure 4.1H), nulliparous obese mice demonstrated a greater

increase in HR over this time compared to primiparous obese mice (Pint<0.05; P<0.05;

Figure 4.2G&H).


93

Figure 4.1 MAP(24hr, light & dark phases), HR and locomotor activity at pre-pregnancy state and postnatal day 1 to 36, and the change from pre-pregnancy levels for control (blue line) and obese (red line) primiparous mice. A) 24h MAP; B) Delta 24h MAP;C) MAP in light phase; D) Delta MAP in light phase; E) MAP in dark phase; F) Delta MAP in dark phase; G) 24h HR; H) Delta 24h HR; I) 24h locomotor activity;J) Delta 24h locomotor activity. Data analyzed by two-way repeated measures ANOVA with factors of obesity (Pobe), time (Ptime) and interaction (Pint), followed by Sidak post-hoc analysis. Mean±SEM

1 4 8 12 16 20 24 28 32 3680

90

100

110

120

130

140

mm

Hg

24h MAP

CONT (n=5)

Obese (n=3)

Pre-Preg

1 4 8 12 16 20 24 28 32 3670

80

90

100

110

120

130

mm

Hg

Light MAP

Pre-Preg

1 4 8 12 16 20 24 28 32 3680

90

100

110

120

130

140

mm

Hg

Dark MAP

Pre-Preg

1 4 8 12 16 20 24 28 32 36400

450

500

550

600

650

bp

m

24h HR

Pre-Preg

1 4 8 12 16 20 24 28 32 360

5

10

Post-natal Day

Co

un

ts/m

in

24h Activity

Pre-Preg

1 4 8 12 16 20 24 28 32 36-10

0

10

20

30

∆m

mH

g

Delta 24h MAP

CONT (n=5)

Obese (n=3) P int NS P time <0.001P obe NS

1 4 8 12 16 20 24 28 32 36-10

0

10

20

30

∆m

mH

g

Delta Light MAP

P int <0.05 P time <0.001P obe NS

1 4 8 12 16 20 24 28 32 36-10

0

10

20

30

∆m

mH

gDelta Dark MAP

P int NSP time <0.001P obe NS

1 4 8 12 16 20 24 28 32 36-100

-50

0

50

100

∆b

pm

Delta 24h HRP int NS P time NSP obe NS

1 4 8 12 16 20 24 28 32 36-10

-5

0

5

10

Post-natal Day

∆c

ou

nts

/min

Delta 24h ActivityP int NSP time NSP obe NS

A B

C D

FE

I J

G H


94

Figure 4.2 MAP (24h, light & dark phases) and HR at pre-pregnancy state and postnatal day 36 for nulliparous (n=5) and primiparous (n=3) obese mice. A) 24h MAP; B) Delta 24h MAP; C) Dark-phase MAP; D) Delta dark-phase MAP; E) Light-phase MAP; F) Delta light-phase MAP; G) 24h HR; H) Delta 24h HR; N: nulliparous; P: primiparous; Absolute data were analysed by two-way ANOVA with factors of pregnancy (Ppreg), time (Ptime) and interaction (Pint), followed by Sidak post-hoc analysis. Delta data was analysed by unpaired t-tests. *P<0.05; **P<0.01. Data are presented as Mean±SEM.

24h MAP

Pre-preg PN360

9090

100

110

120

130

140

mm

Hg

Nulliparous

Primiparous

P int NS

P time <0.001P preg <0.05

Dark MAP

Pre-preg PN360

9090

100

110

120

130

140

mm

Hg

Nulliparous

Primiparous

P int NS P time <0.05P preg NS

Light MAP

Pre-preg PN360

9090

100

110

120

130

140

mm

Hg

Nulliparous

Primiparous

P int <0.05 P time <0.001P preg <0.05

**

24h HR

Pre-preg PN360

400500

550

600

650

700

bp

m

Nulliparous

Primiparous

P int <0.05 P time <0.01

P preg NS

N P0

5

10

15

20

∆m

mH

g

Delta 24h MAP

N P0

5

10

15

20

∆m

mH

g

Delta Dark MAP

N P0

5

10

15

20

∆m

mH

g

Delta Light MAP

*

N P0

10

20

30

40

50

∆b

pm

Delta 24h HR

*

A

HG

FE

DC

B


95

4.3.2. Post-partum Renal Function

Neither obesity nor pregnancy had an impact on 24h urine or sodium excretion at

4WPW, with values similar across the groups of primaparous and nulliparous control and

obese mice (Figure 4.3A&C). The changes in urine and sodium excretion from pre-

pregnancy level were also not impacted by obesity or pregnancy (Figure 4.3B&D). Obese

primiparous and nulliparous mice had significantly elevated albumin excretion compared to

control mice at 4WPW (Pobe <0.001: Figure 4.3E). Pregnancy had no effect on albumin

excretion whether assessed as absolute excretion at 4WPW (Figure 4.3E) or as the change

in albumin excretion from pre-pregnancy levels (Figure 4.3F).

GFR was measured only at 4WPW using the transcutaneous FITC-sinistrin clearance

technique. There was no evidence of renal dysfunction in any of the groups with T1/2 and

calculated GFR were similar across all 4 groups of mice (Figure 4.4A&B).


96

Figure 4.3 Urinary excretory profiles at 4WPW and change from pre-pregnancy level. A) Urine volume; B) Delta Urine volume; C) Urinary sodium excretion; D) Delta sodium excretion; E) Albumin excretion; F) Delta Albumin excretion. N: nulliparous; P: primiparous; N-Control (n=8); N-Obese (n=7); P-Control (n=7); P-Obese (n=8); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. Data are presented as Mean±SEM.

Urine Excretion

N P N P0.0

0.5

1.0

1.5

2.0

2.5

ml

P Int NSP Preg NS P Obe NS

Control Obese

Na+ Excretion

N P N P0

50

100

150

µm

ol


Control Obese

Albumin Excretion

N P N P0

5

10

15

20

25

µg

/24h

P Int NSP Preg NS P Obe <0.001

Control Obese

Delta Urine Excretion

N P N P-0.4

-0.2

0.0

0.2

0.4

0.6

∆m

l


Control Obese

Delta Na+ Excretion

N P N P-40

-20

0

20

40

60

∆µ

mo

l P Int NSP Preg NS P Obe NS

Control Obese

Delta Albumin Excretion

N P N P0

5

10

15

∆µ

g/2

4h

P Int NSP Preg NS P Obe <0.05

Control Obese

A

E

DC

F

B


97

Figure 4.4 Transcutaneous measurement of GFR. A) T1/2; B) Calculated GFR. N: nulliparous; P: primiparous; N-Control (n=7); N-Obese (n=9); P-Control (n=5); P-Obese (n=7); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. Data are presented as Mean±SEM.

4.3.3. Maternal Outcomes

Plasma FFA and TAG concentrations were measured at 4WPW. Plasma FFA level

was not affected by pregnancy or obesity (Figure 4.5A). In control groups, primiparous mice

had significantly lower plasma TAG level than nulliparous mice (P<0.05; Pint<0.05; Figure

4.5B). However, this effect was not observed in obese mice (Figure 4.5B).

Figure 4.5 Plasma lipid profile at 4WPW. A) Plasma FFA, B) Plasma TAG. N: nulliparous; P: primiparous; N-Control (n=9); N-Obese (n=9); P-Control (n=7); P-Obese (n=9). Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.

N P N P0

5

10

15

20

25

min

T1/2

P Int NSP Preg NSP Obe NS

Control Obese

N P N P0

300

600

900

1200

1500

1800

ul/m

in/1

00

gB

W

Calculated GFR

P Int NSP Preg NSP Obe NS

Control Obese

A B

N P N P0.0

0.2

0.4

0.6

0.8

1.0

mM

Plasma FFA

Pint NSPpreg NSPobe NS

Control Obese

N P N P0.0

0.2

0.4

0.6

0.8

1.0

mM

Plasma TAG

Pint <0.05Ppreg NSPobe <0.01

*

Control Obese

A B


98

At 4WPW, obese mice were 75% heavier than control mice (Pobe<0.01; Figure 4.6A).

This large difference was due to a greater weight gain in obese mice, 16.8± 0.9 g from pre-

pregnancy values, compared to 6.5± 0.5 g in control mice (Pobe<0.01; Figure 4.6B).

Pregnancy also increased weight gain, but this effect was only present among obese mice

(P<0.05; Figure 4.6B). Differences in gonadal fat may have contributed to this greater weight

gain. Gonadal fat mass was significantly increased with pregnancy (Pint<0.01) such that

primiparous obese mice (5.4 ± 0.3 g) had 74% greater gonadal fat mass than nulliparous

obese mice (3.1± 0.3 g; P<0.001; Figure 4.6C). Obesity had significant effects on inguinal

and peri-cardial fat mass with values 3 fold greater than control mice (Pobe<0.001 Figure

4.6D&E). However, pregnancy had no impact on inguinal or peri-cardial fat mass (Figure

4.6D&E). Obese mice had significantly greater kidney and ventricle weights, however

pregnancy did not impact these organ weights at 4WPW (Pobe<0.001; Figure 4.6F-I).


99

Figure 4.6 Maternal body weight (BW) and tissue weight at 4WPW for primiparous (P) and time-matched nulliparous (N) control and obese mice. A) BW at 4WPW; B) Delta BW against pre-pregnancy BW; C) Gonadal fat pad; D) Inguinal fat pad; E) Peri-cardial fat pad; F) Total kidney weight; G) Total ventricle weight; H) Left ventricle weight; I) Right ventricle weight. N: nulliparous; P: primiparous; Data analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05; ***P<0.001. Data are presented as Mean±SEM.

N P N P0

10

20

30

40

50

60

70

80

g

BW Pint NSPpreg <0.05Pobe <0.001

n =10 n =11n =15n =13

Control Obese

N P N P0

1

2

3

4

5

6

7

g

Gonadal Fat Pint <0.01Ppreg <0.001Pobe <0.001

***

n = 8

n = 3n = 6n = 6

Control Obese

N P N P0

100

200

300

400m

gPeri-cardial fat

Pint NSPpreg NSPobe <0.001

n = 7n = 8n = 9n = 8

Control Obese

N P N P0

100

200

300

400

500

mg

Total KidneyPint NSPpreg NSPobe <0.001

n =10 n =11n =15n =13

Control Obese

N P N P0

20

40

60

80

100

120

140

mg

Left VentriclePint NSPpreg NSPobe <0.001

n =10 n =11n =14n =13

Control Obese

N P N P0

10

20

30

∆g

∆BW

Pint NSPpreg <0.05Pobe <0.001 *

n =10 n =11n =15n =13

Control Obese

N P N P0

1

2

3

g

Inguinal Fat

Pint NSPpreg NSPobe <0.001

n = 8 n = 3n = 6n = 6

Control Obese

N P N P0

20

40

60

80

100

120

140

160

mg

Total Ventricle Pint NSPpreg NSPobe <0.001

n =10 n =11n =14n =13

Control Obese

N P N P0

10

20

30

40

mg

Right VentriclePint NSPpreg NSPobe <0.001

n =10 n =11n =14n =13

Control Obese

A CB

D E F

IHG


100

4.3.4. Renal and Cardiac Fibrosis

Pregnancy resulted in a profound increase in renal hydroxyproline content that was

dependent on group (Pint <0.05; Ppreg<0.05; Pobe<0.05; Figure 4.7A). Post-hoc analysis

showed that this effect of pregnancy was only significant among obese mice with renal

collagen content 44% greater in primiparous obese compared with nulliparous obese mice,

(P<0.01; Figure 4.7A). Neither pregnancy nor obesity had any impact on hydroxyproline

content of the LV (Figure 4.7B). In examining the level of glomerulosclerosis using PAS

staining, there was no global effect of obesity or pregnancy (Figure 4.8A&B). However, post-

hoc analysis showed that percentage area of PAS-positive staining in glomeruli of

primiparous obese mice was greater than that of the nulliparous obese mice (P<0.05; Figure

4.8B). Percentage area of picrosirius red (PSR) staining within transverse sections of the

subendocardium region of the mid-ventricle wall was used as marker of cardiac fibrosis

(Figure 4.8A&B). Percentage area of PSR staining in the subendocardium of mid-ventricle

was significantly elevated with pregnancy at 4WPW (Ppreg<0.05; Figure 4.9B). However,

post-hoc analysis demonstrated that this effect of pregnancy was only present among obese

mice, but not in control mice (P<0.05; Figure 4.9B).

Figure 4.7 Total renal and left ventricular (LV) collagen content. A) Renal hydroxyproline

content; B) LV hydroxyproline content. N: nulliparous; P: primiparous; N-Control (n=7); N-

Obese (n=8); P-Control (n=5); P-Obese (n=5); Data were analyzed by two-way ANOVA with

factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-

hoc analysis. **P<0.01. Data are presented as Mean±SEM.


101

Figure 4.8 Assessment of glomerulosclerosis. A) Representative Brightfield images of renal PAS staining in control and obese mice that were either nulliparous or primiparous; Scale bar = 100 µm B) Quantification of percentage PAS positive staining within the glomeruli. N: nulliparous; P: primiparous; N-Control (n=6); N-Obese (n=8); P-Control (n=5); P-Obese (n=5); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.

Nulliparous Primiparous C

ontr

ol

Obe

se

A

B

N P N P0

10

20

30

%PAS positive staining

GlomerulosclerosisPint NSPpreg NSPobe NS

Control Obese

*


102

Figure 4.9 Assessment of cardiac fibrosis. A) Representative Brightfield images of PSR staining in the subendocardium region of the left ventricle in control and obese mice that were either nulliparous or primiparous. Scale bar = 300 µm B) Quantification of percentage PSR positive staining within the fields selected. N: nulliparous; P: primiparous; N-Control (n=5); N-Obese (n=8); P-Control (n=4); P-Obese (n=5). Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.

Nulliparous Primiparous

Con

trol

O

bese

A

N P N P0.0

0.5

1.0

1.5

2.0

% P

SR p

osit

ive

stai

nin

g

LV Fibrosis

*

Pint NSPpreg <0.05Pobe NS

Control Obese

B


103

4.4. DISCUSSION

This study aimed to determine the impact of pregnancy on cardiovascular and renal

outcomes post-partum in a model of obesity. There were three major findings in the present

study. Firstly, pregnancy exacerbated obesity-induced hypertension post-partum. Secondly,

pregnancy led to greater renal and glomerular fibrosis in obese mice, in the absence of an

exacerbated effect on albuminuria or renal function post-partum. Lastly, pregnancy led to

greater weight gain, greater accumulation of visceral fat, and a small but significant elevated

cardiac fibrosis in obese mice post-partum. These findings indicate that pregnancy has a

mild impact on cardiovascular and renal health in obese females post-partum. Evidence of

exacerbated hypertension and greater renal and cardiac fibrosis may however predispose

obese females to greater risks of cardiovascular and renal morbidity later in life.

In the present study, we found that MAP of obese mice remained elevated over the

MAP of control mice during the post-partum period. The Initial response in MAP post-partum

was similar, with MAP of control and obese mothers returning to pre-pregnancy levels by

PN6. Similarly, studies in humans have found that arterial pressure returned to pre-

pregnancy values when measured at 12-14 weeks post-partum70,236. Whilst MAP of control

mice remained stable post-partum, obese mice showed a rise in MAP beginning on day

PN29. Moreover, this increase in arterial pressure was only present during the light phase

when mice are at rest. This finding is consistent with what has been shown in human obesity,

where SBP, DBP and pulse pressure of obese individuals during night-time were higher than

lean subjects. 208 Further, study by Kang et al193 also demonstrated that obese women had

more profound elevation of night-time blood pressure than obese men when compared to

respective lean counterparts, indicating that obese hypertensive women might have a greater

risk of developing cardiovascular events during night-time. It has also been reported that

night-time high blood pressure in humans was related to an increased risk of chronic kidney

disease and all-cause death, suggesting that night-time blood pressure has significant

clinical relevance.194 This greater rise in light phase MAP of obese compared to control

primiparous mice at PN36 was profound (14.2 versus 2.1 mmHg respectively). Importantly

the present study also demonstrated that pregnancy exacerbated hypertension in obese

mice with light-phase MAP of primiparous obese mice was greater than nulliparous obese

mice over the same period (14.2 versus 8.0 mmHg respectively). This indicates that the

impact of pregnancy and obesity on MAP was greater than the impact by obesity alone.

The findings of this chapter are consistent with studies demonstrating that women

who experience all forms of hypertensive disorders of pregnancy not only have higher pre-

pregnancy BMI but also exhibit greater risk of developing subsequent hypertension, stroke,

CKD and diabetes compared with normotensive controls.240,372 Interestingly, Goel et al 131


104

demonstrated that women who developed hypertension post-partum, regardless of whether

they were hypertensive or normotensive during pregnancy, had significantly elevated BMI

compared with women who were normotensive post-partum. Taken together, these studies

suggest that being overweight or obese contributes significantly to the development of post-

partum hypertension. No study has prospectively examined cardiovascular parameters in

obese women prior to and during pregnancy, and chronically post-partum. Future studies

should address this gap in our knowledge.

Whilst tachycardia in obese mice was not exacerbated by pregnancy in present study,

persistent tachycardia is an independent risk factor for the development atherosclerosis and

coronary heart disease.281 In examining the impact of pregnancy and obesity in the

development of cardiac fibrosis using a hydroxyproline assay, we found neither pregnancy

nor obesity had an impact on total collagen content between the groups. However, using

PSR staining we demonstrated a greater level of cardiac fibrosis with pregnancy in obese

mice. It is important to note that the portion of the tissue used in hydroxyproline assay was

restricted to the one-third of the LV tissue at the apical end, which is generally considered to

be less prone to fibrosis. In contrast, the histological analysis of collagen deposition using

PSR staining was performed on transverse sections of the mid-ventricle of the heart. The

fields selected for this analysis was restricted to the subendocardium region of the ventricular

wall, the region that experiences the greatest force and stress during contraction and filling in

a cardiac cycle, and thus more likely to develop fibrosis. Given the myocardial shortening in

subendocardium region is the dominant force generator of myocardial contraction,57,71 the

development of fibrosis in this region would have a substantial impact on the contractile

function of the heart. Given that the increase in cardiac fibrosis was only observed in obese

mice during the post-partum period suggests that obese females might be at a high risk of

developing cardiac dysfunction post-partum due to a greater level of cardiac fibrosis.

However, the mechanisms that mediate the greater collagen deposition in the heart of obese

mice post-partum are yet to be elucidated.

To the best of my knowledge, this is the first study that has specifically and

comprehensively examined maternal renal structure and function pos-tpartum in an animal

model of pre-pregnancy obesity. In contrast to what we hypothesized, pregnancy had

minimal impact on renal function in obese mice post-partum. At 4WPW, we found that neither

obesity nor pregnancy had an impact on renal excretory function or GFR. Although albumin

excretion was greater in obese mice, pregnancy did not exacerbate albuminuria. Pregnancy

however increased renal collagen content in obese mice. Obese primiparous mice had 44%

greater total renal collagen deposition and a 28% increase in glomerular collagen content

compared with nulliparous obese mice, suggesting pregnancy leads to some degree of renal


105

and glomerular injury in obese mice post-partum. Whilst no human studies have specifically

addressed the impact of pregnancy on renal health in obesity, similar studies have been

performed post-partum in preeclamptic women who demonstrate hypertension and

albuminuria. Consistent with our study, preeclamptic women have demonstrated comparable

GFR to control subjects by 4 weeks post-partum, despite a lower GFR detected at post-

partum day 1.165 Although preeclamptic women had normal GFR, a high percentage of these

women experienced persistant hypertension (16%) and overt proteinuria (22%) 3 months

post-partum.209 Further, some women who had preeclampsia exhibited permanent renal

injury including glomerulosclerosis and focal interstitial scarring.155 These findings indicate

that whilst renal function in women who have had preeclampsia might be preserved short-

term post-partum, the presence of renal injury and persistent hypertension may increase the

rise of CKD later in life.

Another major health risk in women post-partum is weight retention and this is

exacerbated with obesity. In the present study, we demonstrated that pregnancy facilitates

excess weight gain in obese mice to a large extent by promoting accumulation of excess

visceral fat. Our result is consistent with findings in humans that high pre-pregnancy BMI is a

strong predictor of post-partum weight retention and high prevalence of obesity both short-

term (3 months) and up to 2 years post-partum.112,142,241,337 Further, although obese mice in

the present study had only relatively mild cardiovascular and renal phenotype at 4WPW, it is

recognized that visceral obesity is associated with a greater risk of comorbidities such as

type 2 diabetes, chronic hypertension, coronary heart disease, renal dysfunction and chronic

inflammation.351 Thus, having excess abdominal fat in addition to persistent obesity and

hypertension in women post-partum, is likely to predispose women to a greater risk of

cardiovascular and renal morbidity and mortality later in life, supporting the epidemiological

findings.223,384

Several limitations of the present study should be considered. Firstly, renal function

including GFR and albuminuria were examined at a single time point (4WPW) as a snapshot

of the renal health during the post-partum period. Multiple measurements of renal function

post-partum in a longitudinal setting would likely to provide further understanding into the

potential for developing CKD in this model. Secondly, we did not obtain any radiotelemetry

data in nulliparous control mice, thus we were unable to differentiate the impact of pregnancy

on arterial pressure post-partum between control and obese mice. However the literature

suggests that arterial pressure is not adversely affected by parity in uncomplicated

pregnancies.143 Thirdly, although we detected a clear difference in the level of fibrosis in the

kidney, the evidence of fibrosis in heart was inconsistent. This inconsistency is likely due to

the differences in the section of the ventricle used for each analysis. Future studies should


106

focus on the development of fibrosis in the mid-ventricular wall of the heart. In both the heart

and kidney, it would be also interesting to examine the subtypes of collagen present in these

tissues and abundance of collagen-producing myofibroblasts using biochemical and

immunohistochemical techniques.249,281 Lastly, our study only followed cardiovascular

phenotype for a short period post-partum. Unfortunately due to limitations in battery life of the

radiotelemetry transmiters we were unable to track arterial pressure beyond PN36. In the

current protocol mice were recorded continuously for 85-100 days. Future studies could use

intermittent recording to increase the longevity of the radiotelemetry transmitters to assess

the impact of pregnancy on arterial pressure of obese females in the long-term. Further, it

would be also important to investigate whether obese primiparous mice are more vulnerable

to secondary cardiovascular and renal insults such as diabetes and high salt diet during later

life.

4.5. CONCLUSION

The present study demonstrates that pregnancy exacerbates hypertension in obese

mice during light phase over a short period post-partum, but does not lead to overt renal

dysfunction during this period. However, excess weight gain and greater level of renal and

cardiac fibrosis in obese mice post-partum would likely contribute to the progression of

cardiovascular and renal morbidities if these mice were to followed for a longer period post-

partum. Our findings suggest that pregnancy is likely to be detrimental for obese women in

terms of long-term cardiovascular and renal health. Early detection and appropriate

interventions should be considered by health professionals in managing the risk factors for

the early onset of cardiovascular and renal morbidities in obese mothers post-partum.

Chapter 5 Renal Function & Nephron Deficiency

107

Chapter 5 THE ROLE OF NITRIC OXIDE IN THE

REGULATION OF RENAL FUNCTION AND

ARTERIAL PRESSURE IN NEPHRON DEFICIENT

MICE


108

5.1. INTRODUCTION

Human studies have shown that nephron number varies up to 13 fold among

populations.38,248,291 Studies in animal models would suggest this is due to both genetic

influence and the intrauterine environment which is highly susceptible to maternal

malnutrition, disease, drug exposure and, as identified in Chapter 3, overnutrition,201 Brenner

and colleagues suggested that individuals born with a low nephron number are at a greater

risk of developing hypertension and progressive renal disease.46 This hypothesis has been

supported by findings obtained in animal models of nephron deficiency87,216,277,374 and human

studies where offspring show elevated arterial pressure, low glomerular filtration rate (GFR)

and renal fibrosis.170,176,200,321,329 However not all studies in this field support the Brenner

hypothesis with studies in humans177 and animal45,212,234,312 with low nephron number

demonstrating normal arterial pressure and renal function even at an old age. One such

study from our laboratory demonstrated that both phenotypes of GDNF heterozygous (HET)

mice (HET-2K -30% deficit; HET-1K -65% deficit) had a normal total GFR up to 14months of

age.312 With a normal GFR but low nephron number, this study indicated that single nephron

GFR (SNGFR) of HET-2K mice was doubled, and HET-1K mice almost fourfold higher than

WT mice.312 However, the mechanism by which the single nephron hyperfiltration is

maintained in this state of nephron deficit is unclear.

One of the most prominent regulators of renal function is nitric oxide (NO). NO is

formed during the conversion of L-arginine to L-citrulline by NO synthases (NOS) and

cofactors.343 In the kidney, NO plays a significant role in the regulation of renal

hemodynamics, blunting tubuloglomerular feedback (TGF), inhibition of tubular sodium

reabsorption and modulation of renal sympathetic activity.264 In general, renal NO production

promotes natriuresis and diuresis.183,264 Neuronal NOS (nNOS) and endothelial NOS (eNOS)

are the main NOS isoforms abundantly expressed in the kidney. Whilst nNOS is primarily

expressed in macula densa, thick ascending limb, vasa recta and throughout collecting duct

of the nephron,16,242,380 eNOS has been found mainly in renal vasculature, thick ascending

limb, as well as collecting duct of the nephron.16,264,356 Of importance, the high abundance of

nNOS in the macula densa has been implicated as the source of NO production that is

responsible for blunting TGF mechanism, which is a potent regulator of GFR and arterial

pressure.47,230,370,371 Indeed, reduced NO bioavailability has been shown to contribute to the

pathogenesis and progression of hypertension and chronic kidney disease in humans.11,27,233

A recent study using an ovine model of congenital nephron deficiency demonstrated that

age-related renal dysfunction was associated with a reduction in NO-mediated regulation of

renal hemodynamics and sodium excretion, and significant vascular dysfunction due to

reduced contribution of NO.216 Further, Muller et al 265 demonstrated that chronic systematic


109

NOS inhibition with either the non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester

(L-NAME) or the nNOS selective inhibitor, 7-nitro-indazole (7NI) rendered the renal injury

resistant C57BL/6J mice susceptible to renal mass reduction-induced chronic kidney disease.

These findings led us to hypothesize that NO contributes significantly to the maintenance of

normal GFR and excretory function in animals with a nephron deficit, and in the absence of

NO, renal function deteriorates.

To test our hypothesis, we used GDNF HET mice for two reasons. Firstly, this genetic

mouse model of low nephron endowment allows us to examine the regulation of renal

function and arterial pressure without the confounding influence of globally programmed

cardiovascular dysfunction that often results from maternal undernutrition and overnutrition.

Secondly, this model provides two distinct levels of nephron deficit within one genotype and

both GDNF HET-2K and HET-1K mice have normal GFR and remain normotensive even

through old age. The aim of present study was to determine the arterial pressure and renal

function of GDNF WT, HET-2K, and HET-1K mice prior to and in response to systematic NO

inhibition with L-NAME. We predicted that chronic NO deficiency would lead to greater

hypertension and renal dysfunction in GDNF HET mice in a manner dependent on the level

of nephron deficit.


110

5.2. METHODS

5.2.1. Animals



Animals for Scientific Purposes. Thirty-week-old male GDNF HET mice and WT littermates

were obtained from Monash Animal Research Platform, Monash University. Genotype of the

mice was determined by PCR as described in Section 2.1.2.3. Mice had ad libitum access to

a maintenance diet (AIN93M, Specialty Feed, Australia) and tap water, and were housed in

the experimental room maintained at 24-26 °C with 12:12 hour light-dark cycle.

5.2.2. Experimental Protocol

At 30 weeks of age, mice were placed in metabolic cages to obtain 24hour urine

samples for assessment of basal renal excretory profile (electrolytes, osmolality, albumin

excretion and creatinine concentration; see Section 2.2.1). Mice were then anaesthetized

(Isoflurane, 2.2-2.6%) and radiotelemetry probes (PA-C10, DSI, MN, USA) implanted for the

measurement of conscious arterial pressure (see Section 2.3).312,365 A small blood sample

(~60μl) was collected from the left carotid artery to determine basal plasma creatinine

concentration. Mice were given a 10-day recovery period following which basal arterial

pressures, heart rate and locomotor activity were recorded continuously for 7 days. At the

end of the 7 days of basal recording, mice were administered L-NAME (0.5mg/ml L-NAME

Hydrochloride; Sigma-Aldrich, USA) in the drinking water for a further 7 days.138 Mice were

then placed in metabolic cages again to obtain 24hr urine samples for assessment of renal

excretory profile.

5.2.3. Terminal Tissue Collection

Following the final 24hour urine sample collection, mice were anaesthetised

(Isoflurane) and an arterial blood sample taken from the right carotid artery for the

measurement of plasma creatinine concentration. The kidneys were rapidly excised,

decapsulated, weighed and cut into equal 4 portions transversely. All portions of the right

kidney were frozen in liquid nitrogen and a middle portion used for RT-PCR analysis. In the

case of the solitary kidney (GDNF HET-1K mice), the kidney was excised, weighed, cut

through transverse plane into 3 portions. The middle portion of the kidney was frozen in liquid

nitrogen for PCR analysis. The heart was excised and the left ventricle isolated and weighed.


111

Tissues from non-treated, age-matched cohort (time controls) of GDNF WT, HET-2K and

HET-1K mice were collected in the same manner.

5.2.4. RT-qPCR

To assess relative renal gene expression levels, total RNA was extracted from frozen

kidney tissues of L-NAME treated and untreated GDNF WT, HET-2K and HET-1K mice using

an RNeasy Mini Kit (Giagen, Hilden, Germany). RNA was then reverse transcribed into

cDNAs using iScript Reverse Transcription Supermix for RT-qPCR (BIORAD, Hercules, CA,

USA) according to manufacturer’s instructions. Gene expression analysis for the NOS

isoforms (NOS1, NOS2 & NOS3), aquaporin 2 (AQP2), sodium-hydrogen antiporter 3

(NHE3), sodium-potassium-chloride cotransporter 2 (NKCC2), angiotensin type 1a receptor

(AT1aR), angiotensin type 2 receptor (AT2R) were performed with TaqMan gene expression

assays using the Applied Biosystems 7900HT Fast Real-Time PCR system (Applied

Biosystems by Life Technologies, Foster City, CA, USA). TaqMan gene expression assays

(Dye: FAM-MGB; Applied Biosystems) used for genes of interest are Nos1

(Mm00435175_m1) for NOS1/nNOS, Nos2 (Mm00440502_m1) for NOS2/iNOS, Nos3

(Mm00435217_m1) for NOS3/eNOS, Slc12a1 (Mm01275821_m1) for NKCC2, Slc9a3

(Mm01352473_m1) for NHE3, Aqp2 (Mm00437575_m1) for AQP2, Agtr1a

(Mm01957722_s1) for AT1aR, and Agtr2 (Mm01341373_m1) for AT2R. RT-qPCR reactions

were run in triplicate and duplexed with 18S as endogenous housekeeping gene (Dye: VIC-

Primer limited; Eukaryotic 18S rRNA endogenous control for Agtr1a and Agtr2; Rn18s,

Mm03928990_m1 for other genes; Applied Biosystems by Life Technologies, CA, USA).

Reactions were assembled on a 384-well PCR plate using an automated liquid handler

(CAS-1200 liquid handler, Giagen, Germany). RT-qPCR Data was analyzed by Applied

Biosystems Sequence Detection (SDS) version 2.4 software. The relative expression of

mRNA levels was calculated using the comparative ΔCt (threshold cycle number) method.323

The relative fold changes in gene expression for each target gene was calculated against

untreated GDNF WT group.

5.2.5. Statistical Analysis

Basal telemetry data and renal excretory profile data were analyzed using one-way

ANOVA, followed by Tukey post-hoc analysis. Change in MAP, HR and locomotor activity

during L-NAME treatement was analyzed by two-way repeated measure ANOVA considering

factors of group (Pg) and time (Pt) and interaction between group and time (Pgxt), followed

by Bonferroni post-hoc analysis. Renal excretory profile before and after L-NAME treatement


112

was analyzed by two-way repeated measure ANOVA considering factors of group (Pg) and

treatment (Pt) and interaction between group and treatment (Pgxt), followed by Bonferroni

post-hoc analysis. For terminal tissue weight and gene expression analysis, two-way ANOVA

were conducted considering factors of group (Pg) and treatment (Pt) and interaction between

group and treatment (Pgxt), followed by Bonferroni post-hoc analysis. Values are mean ±

SEM. P<0.05 was considered statistically significant.


113

5.3. RESULTS

5.3.1. Basal Cardiovascular and Renal Excretory Profile

WT, HET-2K and HET-1K mice did not differ in 24hr mean arterial pressure (MAP;

104.1±10, 103.0±2.1, 101.1±2.1 mmHg respectively; Figure 5.1A) or heart rate (HR, 486±11,

500±9, 510±17 bpm Figure 5.1B) under basal conditions. There were also no differences in

systolic or diastolic pressures between the groups, or any differences between groups during

light or dark phase of the day (data not shown). Locomotor activity of HET-2K and HET-1K

mice (3.6±0.3, 5.1±0.4 Counts/min respectively) was not different to WT mice (4.3±0.5

Counts/min) though HET-1K mice were more active than HET-2K mice (P<0.05).

Water intake and urine excretion were not significantly different between WT and

HET-2K mice. Both water intake and urine excretion were significantly greater in HET-1K

mice compared with WT and HET-2K mice under basal conditions (P<0.01-0.001; Figure

5.1C&D). HET-1K mice excreted significantly more sodium than WT and HET-2K mice

during 24 hr urine collection period (P<0.01 and P<0.05 respectively, Figure 5.1F). The high

sodium excretion was reflected in higher food intake in HET-1K mice compared with WT

mice (P<0.05; Figure 5.1E), however the comparison with HET-2K mice did not reach

statistical significance. In addition, there were no differences between WT, HET-2K and HET-

1K mice in creatinine clearance, nor in 24hr albumin excretion under basal conditions (Figure

5.1G&H).


114

Figure 5.1 Basal arterial pressure and heart rate (WT n=8, HET-2K n=8 and HET-1K n=7) and renal excretory profile (WT n=11, HET-2K n=14 and HET-1K n=8). A: Basal 24hr mean arterial pressure (MAP). B: Basal 24hr heart rate (HR). Water intake. C: Water intake. D: Urine excretion. E: Food intake. F: Sodium excretion. G: Creatinine clearance (Ccre). H: Albumin excretion. Date analyzed using one-way ANOVA with Tukey post-hoc analysis. *P<0.05; **P<0.01; ***P<0.001.

24h MAP

WT HET-2K HET-1K

0

20

40

60

80

100

120

mm

Hg

24h HR

WT HET-2K HET-1K

0

100

200

300

400

500

600

bp

m

A

E

C

B

F

DWater Intake

WT HET-2K HET-1K

0

30

60

90

120

150

180

µl/2

4h

/gB

W

*****

Urine Excretion

WT HET-2K HET-1K

0

20

40

60

80

100

µl/2

4h

/gB

W

******

Food Intake

WT HET-2K HET-1K

0.00

0.03

0.06

0.09

0.12

0.15

0.18

g/g

BW

*

Sodium Excretion

WT HET-2K HET-1K

0

2

4

6

8

µm

ol/2

4h

/gB

W

***

Ccre

WT HET-2K HET-1K

0

2

4

6

8

10

12

µl/m

in/g

BW

Albumin Excretion

WT HET-2K HET-1K

0.00

0.02

0.04

0.06

0.08

0.10

0.12

µg

/24

h/g

BW

HG


115

5.3.2. Arterial Pressure and Renal Excretory Profile During NOS Inhibition

In response to systematic NOS inhibition with L-NAME, there were similar rises in

MAP of WT, HET-2K and HET-1K mice during the first 24 hours (17.9±0.6, 16.1±0.9,

18.4±1.4 mmHg respectively; Figure 5.2A&B). However the groups differed in their response

over the following 6 days (Pgxt<0.001; Figure 5.2B). The initial rise in MAP of WT mice was

maintained over the next 6 days (Figure 5.2B). However, the MAP response to L-NAME in

HET-2K and HET-1K mice was gradually attenuated over the following 6 days. By the last

day of recording, the rise in MAP of WT mice from the basal level was 14.0±1.3 mmHg whilst

the rise in MAP of HET-2K and HET-1K mice had lessened to 12.0±1.7 and 7.6±2.0 mmHg

respectively (Figure 5.2B). However, post-hoc analysis demonstrated that only HET-1K mice

had significantly lower MAP than WT mice on day 7 of L-NAME treatment (P<0.01). HR of all

groups demonstrated an initial fall in response to L-NAME (Figure 5.2C) before gradually

returning to basal level (Pt<0.01; Figure 5.2D). There was no significant difference in HR

response to L-NAME between groups (Figure 5.2D). Locomotor activity was not altered in

any of the groups following L-NAME treatment. (Data not shown)

Figure 5.2 Mean arterial pressure (MAP) and heart rate (HR) during 7 day of L-NAME treatment in WT (n=8), HET-2K (n=8) and HET-1K (n=7) mice. A: Absolute MAP from basal condition to day 7 of NOS inhibition (L-NAME treatment). B: Change in 24hr MAP in response to NOS inhibition. C: Absolute HR. D: Change in 24hr HR in response to NOS inhibition. Data (B&D) were analyzed using two-way repeated measures ANOVA with factors of group (Pg), time (Pt) and interaction (Pgxt) and Bonferroni’s post-hoc analysis was conducted. NS, not significant.

24h MAP during NOS inhibition

Days of NOS Inhibition

mm

Hg

Basal 1 2 3 4 5 6 790

100

110

120

130

HET1K (n=7)

WT (n=8)

HET2K (n=8)

24h HR during NO inhibition

Basal 1 2 3 4 5 6 7400

450

500

550


bp

m

WT (n=8)

HET2K (n=8)

HET1K (n=7)

Change in 24h MAP

1 2 3 4 5 6 70

5

10

15

20


∆ m

mH

g

Pgxt <0.001Pg <0.05Pt <0.001

Change in 24h HR

1 2 3 4 5 6 7

-80

-60

-40

-20

0

20


∆ b

pm

Pgxt NSPg NSPt <0.001

A

C

B

D


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At the completion of arterial pressure recordings, 24h urine samples were collected

again while mice continued L-NAME treatment. There was a significant effect of L-NAME on

water intake, food intake, urine and sodium excretion (Pt<0.05-0.001; Figure 5.3A-D),

however this effect was primarily found in HET-1K mice. The higher 24h water intake and

urine excretion of HET-1K mice were abolished by L-NAME treatment (water intake Pgxt

<0.01, urine excretion Pgxt <0.001; Post-hoc analysis P<0.001; Figure 5.3A&B). Post-hoc

analysis showed that urine excretion of HET-2K was also significantly reduced with L-NAME

treatment (P<0.01), however this was not reflected in a significant fall in water intake (Figure

5.3A&B). The greater 24h sodium excretion of HET-1K mice was abolished by L-NAME

treatment such that they no longer differed from WT and HET-2K mice (Pgxt <0.01, Post-hoc

analysis P<0.001; Figure 5.3D). Food intake in HET-1K mice reflected the changes in

sodium excretion such that L-NAME treatment normalized the differences in food intake

between the WT and HET-1K mice (Pt<0.05; Post-hoc analysis P<0.05; Figure 5.3C). L-

NAME treatment did not alter creatinine clearance among groups (Figure 5.3E). Albumin

excretion was reduced with L-NAME treatment (Pt<0.01), but no difference was detected

among groups (Figure 5.3F).


117

Figure 5.3 Renal excretory profile (WT n=11, HET-2K n=14 & HET-1K n=8) and creatinine clearance (WT n=6, HET-2K n=9 & HET-1K n=7) under basal conditions and after L-NAME treatment. A: 24h water intake. B: 24h urine excretion. C: 24h food intake. D: 24h sodium excretion. E: Creatinine clearance (Ccre). F: 24h albumin excretion. Date analyzed using two way repeated measures ANOVA with factors of group (Pg), treatment (Pt) and interaction (Pgxt), followed by Bonferroni’s post-hoc analysis. NS = not significant. *P<0.05; **P<0.01; ***P<0.001.


118

5.3.3. Terminal Tissue Weights

Body weights of GDNF WT, HET-2K and HET-1K mice was not influence by L-NAME

treatment and no difference was detected among groups within each cohort (Table 5.1). As

expected, total kidney weight of untreated control HET-2K and HET-1K mice was

significantly lower than WT mice (Pg<0.001, Post-hoc analysis P<0.001; Table 5.1). Although

the solitary kidney of HET-1K was substantially heavier than a single kidney of HET-2K and

WT mice (data not shown), the solitary kidney of HET-1K mice weighed significantly less

than the total kidney weight of HET2K and WT mice (Post-hoc analysis P<0.01-0.001; Table

5.1). L-NAME treatment did not alter the differences in kidney weight observed in untreated

control cohort (Table 5.1). Similar effects of group and L-NAME treatment were found for

kidney weights when corrected for body weight (Table 5.1). Left ventricle weight was not

affected by group or L-NAME treatment (Table 5.1).


119

Table 5.1 Terminal tissue weights. Kid: B Wt, total kidney to body weight ratio; LV, left ventricle; LV:B Wt, left ventricle to body weight ratio; WT, wild-type; HET-2K, GDNF HET mice born with 2 kidneys; HET-1K, GDNF HET mice born with 1 kidney; Data were analyzed by Two-way ANOVA considering factors of treatment (t), group (g), and interaction (gxt), followed by Bonferroni’s post-hoc analysis. For all the parameters of L-NAME treated group, WT (n=16), HET-2K (n=14) and HET-1K (n=10). For untreated control group, WT (n=18), HET-2K (n=10) and HET-1K (n=11) except for LV Wt and LV:Bwt Ratio of untreated control group, WT (n=15), HET-2K (n=5) and HET-1K (n=9). § P<0.05, # P<0.01, * P<0.001, compared to WT within group. † P<0.01 ^ P<0.001 compared to HET-2K within group. NS, not significant.

Untreated control L-NAME treated P

WT HET-2K HET-1K WT HET-2K HET-1K t g g x t

Body Wt, g 36.0±1.0 34.4±1.3 34.8±1.5 34.7±1.1 34.0±1.0 33.6±0.8 NS NS NS

Kidney Wt, mg 347±8 283±11* 223±9*† 339±13 296±12§ 228±12*^ NS <0.001 NS

Kid:B Wt, mg/g 9.79±0.36 8.26±0.26# 6.46±0.27*† 9.76±0.25 8.72±0.26§ 6.81±0.35*† NS <0.001 NS

LV Wt, mg 107±3 106±8 102±3 105±2 109±4 105±5 NS NS NS

LV:B Wt, mg/g 3.06±0.13 3.09±0.15 2.96±0.13 3.05±0.08 3.21±0.10 3.14±0.17 NS NS NS


120

5.3.4. Gene Expression of Sodium and Water Channels

There were no differences in relative gene expression of renal NOS isoforms (NOS1,

NOS2, NOS3), NHE2, NKCC2 and AT2R in L-NAME treated and untreated GDNF WT, HET-

2K and HET-1K mice (Table 5.2). There was a tendency for L-NAME to increase renal AQP2

expression in GDNF HET-2K and HET-1K mice, however these differences did not reach

statistical significance (Pt=0.08; Table 5.2). There was an increase in AT1aR gene

expression in response to L-NAME, but this effect was dependent on group (Pg<0.05,

Pgxt<0.01, Table 5.2). Post-hoc analysis showed that L-NAME resulted in a 1.5 fold increase

in AT1aR expression in kidneys of WT mice (P<0.05), but L-NAME did not change AT1aR

gene expression in the kidneys of HET-2K and HET-1K mice (Table 5.2).


121

Table 5.2 Relative gene expression. NOS, nitric oxide synthase; AQP2, aquaporin 2, NHE3, sodium-hydrogen antiporter 3; NKCC2, sodium-potassium-chloride cotransporter 2; AT1aR, angiotensin type 1a receptor; AT2R, angiotensin type 2 receptor; WT, wild-type; HET-2K, GDNF HET mice born with 2 kidneys; HET-1K, GDNF HET mice born with 1 kidney; Data analyzed by Two-way ANOVA considering factors of treatment (Pt), group (Pg) and interaction (Pgxt), followed by Bonferroni’s post-hoc analysis. For untreated cohort, WT (n=6), HET-2K (n=7) and HET-1K (n=6). For L-NAME treated cohort, WT (n=5), HET-2K (n=5) and HET-1K (n=5). †P<0.05 when compared to untreated WT mice. NS, not significant.

Untreated control L-NAME treated P

Genes WT HET-2K HET-1K WT HET-2K HET-1K t g g x t

NOS1 1.00±0.18 0.96±0.09 1.03±0.11 1.10±0.24 1.22±0.13 1.20±0.15 NS NS NS

NOS2 1.00±0.16 0.83±0.17 0.76±0.11 0.91±0.09 0.71±0.03 0.84±0.10 NS NS NS

NOS3 1.00±0.10 1.11±0.11 1.05±0.14 1.09±0.08 1.34±0.11 1.08±0.12 NS NS NS

AQP2 1.00±0.15 1.00±0.21 0.88±0.15 1.11±0.29 1.66±0.70 1.68±0.45 NS NS NS

NHE3 1.00±0.07 1.01±0.09 0.93±0.08 1.01±0.06 1.07±0.08 0.95±0.04 NS NS NS

NKCC2 1.00±0.18 0.88±0.04 0.73±0.07 0.86±0.07 1.19±0.21 0.98±0.14 NS NS NS

AT1aR 1.00±0.03 1.27±0.14 0.98±0.04 1.46±0.18† 0.91±0.07 0.93±0.10 NS P<0.05 P<0.01

AT2R 1.00±0.17 0.89±0.18 0.66±0.19 0.89±0.09 0.98±0.16 0.61±0.04 NS NS NS


122

5.4. DISCUSSION

The aim of the present study was to examine the arterial pressure and renal function

responses to systematic NOS inhibition in nephron deficient GDNF HET mice. There are

three major findings of the present study. Firstly, in stark contrast to our hypothesis, L-

NAME-induced hypertension was not exacerbated in GDNF HET mice, instead GDNF HET

mice, particularly HET-1K mice demonstrated a marked escape from the initial hypertensive

response to L-NAME treatment over the 7 days. Secondly, L-NAME treatment abolished

greater diuresis and natriuresis present in the GDNF HET-1K mice, and did not reduce GFR

or induce albuminuria. Lastly, the upregulation of renal AT1aR gene expression in WT mice

in response to NOS inhibition was absent in GDNF HET-2K and HET-1K mice. Taken

together, these findings indicate that maintenance of normal arterial pressure and renal

function in nephron deficient GDNF HET mice may not be strongly dependent on NO

bioavailability.

Using radiotelemetry, the present study found that 30-week old GDNF HET-2K and

HET-1K mice had very similar basal MAP and HR as WT mice, consistent with previous

studies.312,336 Brenner et al 46 suggested that a reduced filtration surface area (FSA) is

responsible for the development of hypertension in individuals born with low nephron

endowment. We have previously shown that glomeruli of HET-2K mice undergo significant

hypertrophy such that by 30 weeks of age total glomerular volume and, therefore total FSA

are not different from WT mice.336 Thus, it is perhaps not surprising that HET-2K mice remain

normotensive. However, although glomeruli of HET-1K mice undergo the same magnitude of

hypertrophy, this does not offset the 65% nephron deficit leaving total glomerular volume

(therefore FSA) only 50% of WT and HET-2K mice.312 Yet, we have consistently

demonstrated that the markedly reduced nephron number and FSA are not associated with

hypertension in HET-1K mice under basal conditions. Again consistent with our previous

studies, we found that urine and sodium excretion were significantly higher in 30-week old

HET-1K mice compared to WT and HET-2K mice.312 GFR and albumin excretion were also

similar among the 3 groups of mice, in agreement with our previous findings.312

In response to L-NAME, urine and sodium excretion of WT mice were not altered

from basal levels. Carlstrom et al 56 also found that L-NAME given at the same dose as the

current study did not affect diuresis in rats after 7 days. However in contrast to our study,

they found L-NAME for 7 days reduced sodium excretion.56 Given that NO promotes

natriuresis, it is likely that other natriuretic mechanisms might be upregulated to maintain a

sodium balance in WT mice when NO synthesis is blocked. Sodium excretion of HET-2K

mice was also not changed in response to L-NAME treatment. However, we did observe an

anti-diuretic effect of L-NAME on HET-2K mice. The effect of L-NAME was however more


123

profound in HET-1K reducing urine and sodium excretion of GDNF HET-1K mice to the

levels seen in HET-2K and WT mice. This anti-diuretic and anti-natriuretic effect of L-NAME

indicates NO plays a dominant role in promoting diuresis and natriuresis in HET-1 mice.

However, the anti-diuretic and anti-natriuretic effects of L-NAME found in HET-1 mice could

not be explained by changes in the level of mRNA expression of water (AQP2) and sodium

channels (NHE2 & NKCC2) in the renal tissues. Although not statistically significant, AQP2

mRNA expression in L-NAME treated HET-1K kidneys was 90% higher than untreated HET-

1K kidneys, indicating that AQP2 gene expression might be upregulated during L-NAME

treatment, potentially contributing to the anti-diuretic effects. Whilst the mRNA expression of

renal AQP2 did not change, we cannot exclude the possibility that changes may have

occurred at the level of AQP2 protein abundance, particularly at the apical membrane of the

collecting duct, which is likely to be responsible for the changes in renal excretory profile

seen in HET-1K mice. To further investigate the role of AQP2 in the diuresis, abundance of

membrane-bound AQP2 protein should be assessed.64,257,381 The present study also could

not detect any effect of nephron deficiency nor L-NAME on gene expression of two sodium

channels, NHE3 and NKCC2, despite demonstrating a significant anti-natriuretic effect of L-

NAME in HET-1K mice. Further examination of the abundance and activity of other major

renal sodium channels in the renal tubule might provide insight into the possible mechanisms

that may explain the anti-natriuretic effect of L-NAME in HET-1K mice.

Consistent with our previous finding in 1-year old mice,312 a normal GFR was found in

30-week old nephron deficient HET-2K and HET-1K mice in the present study, indicating

these mice must have a significantly elevated SNGFR to compensate for the nephron deficit.

Previous reports calculated SNGFR (GFR divided by nephron number) of HET-1K mice to be

4 fold greater, and HET-2K mice doubled, that of WT mice.312 However, the mechanism(s)

that maintains this elevated SNGFR in HET-2K and HET-1K mice is unclear. An altered TGF

response has been suggested to play a role in the regulation of SNGFR in nephron deficient

rodents. It was reported that the sensitivity of TGF was reduced following uninephrectomy in

rats, which facilitated an increase in SNGFR in remaining kidney preventing a fall in total

GFR.266,335 Therefore there is a possibility that the sensitivity of TGF in nephron deficient

GDNF HET mice is significantly decreased compared with WT mice, allowing a marked

elevation in SNGFR to occur in HET mice.

Macula densa derived NO is known to blunt the sensitivity of the TGF mechanism

facilitating a higher SNGFR.47,274,353,370 Acute NOS inhibition with L-NAME or nNOS-specific

inhibition with 7-NI in control rats results in a significant fall in SNGFR and total GFR due to

an enhanced TGF sensitivity.47,274 Following nephron loss, there is evidence of greater NO

bioavailability.357 Further, acute blockade of NOS with L-NAME led to a fall (~30%) in

SNGFR in the remaining kidney of rats following uninephrectomy.41 We predicted that there


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would be a greater dependence on NO to maintain normal GFR in nephron deficient GDNF

HET mice. Thus the current study investigated the impact of NOS inhibition with L-NAME on

GFR predicting that GFR would be reduced in a manner dependent on the level of nephron

deficit. However after 7 days of L-NAME, GFR of all three groups was not different from

baseline measurements indicating that NO may play little role in the chronic regulation of

GFR in these mice. In examining the literature it is clear that the chronic responses to L-

NAME is different to the acute setting. Although Ollerstam et al 274 demonstrated a reduction

in SNGFR and enhanced TGF sensitivity in rats in response to acute NOS inhibition, they

found that after chronically treating rats with 7NI for 4 weeks, GFR was not different to

untreated rats. Further, the enhanced TGF sensitivity that was present in the acute setting of

nNOS inhibition was no longer present after chronic NOS inhibition.274 These findings

suggests that other factors are involved in the maintenance of GFR within the normal range

in chronic NOS deficiency. These factors may include formation of prostaglandins and

adenosine receptor signaling in the control of renin production in macula densa cells,120,284,358

as well as the contribution of RAS in the regulation of TGF.325

Future studies should clarify the role of NO, particularly nNOS at the macula densa in

in determining TGF sensitivity and the control of SNGFR and thus total GFR. In addition,

treating rats with L-NAME for extended periods (4-8 weeks) has been shown to cause a

reduction in GFR in addition to the significant renal injury such as glomerulosclerosis and

proteinuria.30,300 Thus future studies should also examine the impact of long-term NOS

inhibition on GFR and renal excretory function in nephron deficient GDNF mice in order to

determine whether the progression to renal injury is enhanced in GDNF HET mice

The most unexpected finding in the present study was the blood pressure response

to L-NAME in GDNF HET mice. MAP of GDNF WT mice had risen by 18mmHg during the

first day of L-NAME treatment, consistent with what has been shown in the literature.225 HET-

2K and HET-1K mice showed a similar initial rise in MAP. The elevated MAP of WT mice

was well maintained during next 6 days. Interestingly, instead of further exacerbation of

hypertension as we hypothesized, the increase in MAP of GDNF HET-2K and HET-1K mice

was gradually attenuated over the next 6 days. The escape from hypertension was more

profound in HET-1K mice than HET-2K mice, such that by the 7th-day post-L-NAME

treatment, MAP of HET-1K mice fell to be less than half of the initial rise. This fall in MAP in

HET-1K animals could not be explained by any further enhancement of diuresis and

natriuresis in these animals, instead we actually observed a reduction of sodium and water

excretion to WT levels.

One potential explanation for the escape of L-NAME-induced hypertension in HET-2K

and HET-1K mice may be related to the difference in response of the renin-angiotensin

system (RAS) following L-NAME treatment. WT mice showed an increase in AT1aR


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expression in response to L-NAME treatment, consistent with what has been demonstrated

previously.292,303 Interestingly L-NAME-induced hypertension is prevented when rats were

treated with the AT1 receptor antagonist Losartan,190 suggesting activation of the AT1

receptor directly contributes to L-NAME-induced hypertension. Thus, the upregulation of

AT1aR gene expression in WT mice may facilitate the sustained L-NAME-induced

hypertension in these mice. In contrast, the upregulation of renal AT1aR gene expression

was absent in HET-2K and HET-1K mice. This may explain the partial escape of L-NAME-

induced hypertension. It is unclear why L-NAME does not lead to upregulation of the AT1a

receptor in GDNF HET mice. Future studies may require examination of plasma renin activity

and renal angiotensin II level to further confirm the status of RAS activation in L-NAME

treated WT and HET mice. Apart from the RAS, collecting duct-derived endothelin (ET-1) has

also been shown to play a role in pressure-induced changes in diuresis and natriuresis in L-

NAME-induced hypertension.3,324 Treatment with L-NAME leads to a rather moderate

increase in blood pressure in collecting duct-specific ET-1 knockout mice compared to a

marked hypertension in controls, suggesting that NO derived from ET-1 signaling in the

collecting duct contribute greatly to the regulation of blood pressure.324 Investigation into the

status of endothelin-mediated NO pathway before and after NOS inhibition in GDNF HET

mice should be considered in future studies.

Our results indicate that NO is critical in maintaining elevated diuresis and natriuresis

in HET-1K mice. However, to our surprise, NO seems less important in the long-term

maintenance of arterial pressure and GFR in GDNF HET mice. There are several limitations

to the present study that should be considered. Firstly, renal excretory profiles were only

examined at baseline and at the end of 7 days L-NAME treatment. It would be ideal to collect

urine samples daily from the beginning of the L-NAME treatment in addition to the conscious

arterial pressure measurement, in order to monitor potential changes in pressure-natriuresis

relationship throughout L-NAME treatment. Secondly, we only examined the blood pressure

response to 7 days of L-NAME treatment.. It is possible that the partial escape of

hypertension and well-maintained real function in response to NOS inhibition observed in

nephron deficient GDNF HET mice were only transient. Studies that have treated rodents

with L-NAME for a longer period (e.g. 6-12 weeks) result in significant hypertension,265

profound renal injury including albuminuria,98,195 glomerulosclerosis,265,300 and a fall in GFR300.

Future studies should consider examining the cardiovascular and renal outcomes of GDNF

HET mice in response to long-term NOS inhibition in order to verify the longevity of this

protection against hypertension and renal dysfunction in these mice. Thirdly, in the present

study the gene expression analysis was limited to renal NOS isoforms, RAS receptors, and

key water and sodium channels. As mentioned earlier, further investigation of the protein

abundance of water and sodium channels in the apical membrane of the distal tubules, and


126

the state of renal RAS activation may help to elucidate the mechanisms behind our

physiological observations.

5.5. CONCLUSION

In the present study, we predicted that renal function would deteriorate in nephron

deficient GDNF HET mice in response to NO deficiency in a manner dependent on the level

of nephron deficit. In contrast, our findings indicate NO may have little contribution in the

long-term regulation of renal function and blood pressure in nephron deficient GDNF HET

mice. Further elucidation of the mechanisms underpinning may help us to understand why

some nephron deficient animals are able to maintain a normal GFR and be protected from

hyperfiltration-related renal injury. A better understanding of these protective mechanisms in

nephron deficient kidneys would be useful in developing therapeutic strategies for individuals

with a significant nephron deficit who are at a greater risk of developing progressive renal

disease and chronic hypertension.

Chapter 6 General Discussion

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Chapter 6 GENERAL DISCUSSION


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6.1. BRIEF OVERVIEW AND KEY FINDINGS

There is an increasing prevalence of pregnancies complicated by obesity, with more

than one in five pregnancies in Western society affected.226,273 Pregnancies that occur in

obese women are often associated with adverse maternal and fetal outcomes that are likely

to have a persistent impact. In the last decade, numerous studies have demonstrated a

strong association between maternal obesity and the development of cardiovascular and

renal dysfunction in young and adult offspring. Nevertheless, the mechanisms that are

involved in the programming of cardiovascular and renal disease in offspring by the adverse

intrauterine environment generated by obesity are not well understood. Importantly, the lack

of knowledge of this process means the strategies in managing the wellbeing of the mother

and fetus in pregnancies complicated by obesity is very limited. Thus, this thesis was aimed

to address this gap in the field of research.

One overlooked aspect of obesity-induced fetal programming in the literature is

whether obesity alters the cardiovascular and renal adaptations of pregnancy, and thereby

contributes to adverse fetal outcomes. Therefore, in Chapter 3, I investigated the impact of

pre-pregnancy obesity on pregnancy-induced cardiovascular and renal adaptations,

specifically examining the changes in MAP, HR, SV, CO and renal excretory function.

Chapter 3 also examined the impact of maternal obesity on fetal development. Given

complications that are commonly associated with maternal obesity such as placental

insufficiency and gestational diabetes are also associated with a reduced nephron

endowment in offspring, we focused our attention on examining fetal kidney development, in

particular, nephron number in this mouse model of pre-pregnancy obesity. The key findings

include:

� The diet-induced obesity model used in this thesis was appropriate to test the

hypothesis. This model of pre-pregnancy exhibited a similar phenotype to human

obesity including profound diet-induced obesity, glucose intolerance, hypertension,

tachycardia, cardiac hypertrophy and fibrosis, elevated CO and albuminuria.

� Pre-pregnancy obesity limited the cardiovascular adaptations of pregnancy including

blunting the increases in MAP, HR, SV, CO and LVM during late pregnancy, and

exacerbated obesity-induced albuminuria.

� Pre-pregnancy obesity led to increased fetal death, fetal growth restriction, small

head size and kidneys, and reduced placental efficiency.


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� Pre-pregnancy obesity led to significantly developmental abnormalities of the fetal

kidney including an abnormal renal and glomerular morphology, and in males, a

reduced nephron number.

These findings indicate that adaptations of cardiovascular and renal excretory systems were

indeed limited by pre-existing obesity, and may contribute to fetal loss, growth restriction,

significant glomerular abnormalities, and, in male fetuses, a nephron deficit.

In examining the impact of obesity during pregnancy, studies in the literature have

focused on the programming of adult disease in the offspring. Few studies have considered

the wellbeing of the mother post-birth with only handful of epidemiological studies that have

specifically investigated the cardiovascular outcomes in obese women more than 10 years

post-partum. These epidemiological studies did however indicate that there is a close

association between cardiovascular-related mortality and pre-pregnancy obesity.223,384 Thus

studies in Chapter 4 followed control and obese gravid mice post-partum and compared the

cardiovascular and renal phenotypes of these mice with non-pregnant time controls. The key

findings include:

� Pregnancy led to greater visceral obesity and exacerbated hypertension (light-phase)

in obese mice post-partum.

� Total renal and glomerular collagen content was greater in obese primiparous mice

post-partum but this was not related to renal dysfunction with GFR and albuminuria

of obese mice unaffected by pregnancy.

These findings indicate that pregnancy exacerbates obesity-induced hypertension in the

inactive phase of the day, but did not lead to overt renal dysfunction post-birth.

A variety of adverse intrauterine environments can program low nephron endowment

in the offspring, including as demonstrated in Chapter 3, maternal obesity. A low nephron

endowment is associated with the development of hypertension and renal insufficiency later

in life. However, not all models of reduced nephron endowment demonstrate hypertension or

renal dysfunction. One such model is the GDNF heterozygous (GDNF HET) that has been

well characterized by our laboratory as normotensive when examined at 14months of age

with no evidence of renal dysfunction or disease. An important question to ask is how

nephron deficient kidneys function in order to maintain normal renal function. Chapter 5

investigated the role of NO in maintaining renal function and normal arterial pressure in

nephron deficient GDNF HET mice. The key findings include:


130

� Nephron-deficient GDNF HET mice with both moderate and marked nephron deficit

were able to maintain a normal GFR and sodium balance in response to 7 days of

L-NAME treatment.

� Nephron-deficient GDNF HET mice demonstrated a partial escape from L-NAME-

induced hypertension when compared with WT mice. The absence of an

upregulated renal AT1R expression that was demonstrated in WT mice in response

to NO inhibition might underpin the partial escape of GDNF HET mice to L-NAME

induced hypertension and well-maintained renal function in nephron deficient GDNF

Het mice.

� NOS inhibition normalized the exaggerated diuresis and natriuresis in GDNF HET-

1K mice suggesting NO plays a dominant role in promoting diuresis and natriuresis

in HET-1 mice.

These findings indicate that GDNF HET mice do not rely heavily on NO to maintain normal

blood pressure and renal function.

6.2. THE IMPACT OF PRE-PREGNANCY OBESITY ON THE MOTHER

Research into the detrimental impact of pre-pregnancy obesity has to a large extent

focused on the programming of cardiovascular and metabolic morbidities in offspring.

However, the mechanisms that contribute to this programming process are poorly

understood. An important aspect missing in the literature is what happens to the obese

mother during pregnancy and post-birth. The vital role of adequate adaptations in the

cardiovascular and renal systems during pregnancy has been overlooked in the field of fetal

programming and certainly has not been specifically examined in the context of pre-

pregnancy obesity. Studies in Chapter 3 of this thesis have addressed this gap in the field,

and for the first time, demonstrated that obesity indeed limits the cardiovascular and renal

adaptations that occur in pregnancies complicated by obesity. We used gold-standard

radiotelemetry to follow MAP and HR before and throughout pregnancy and found that the

rises of both MAP and HR were blunted towards term. We also adopted sophisticated

cardiac MRI technique to evaluate the cardiac function and structure at the pre-pregnancy

state and during late pregnancy. We found that obese dams failed to increase SV, CO and

LVM to the same magnitude as achieved by control mice at GA14, the time when cardiac

adaptations should be completed. To the best of my knowledge, these findings have not

been demonstrated in any animal model in the literature to date.


131

This study had several distinct advantages. Our mouse model of pre-pregnancy

obesity had profound diet-induced obesity (47% heavier) and significantly compromised

glucose metabolism prior to mating, reflecting its clinical relevance. In contrast, many studies

in the literature claimed they have used a model of maternal obesity. In fact, many of these

models were only fed a high fat diet for a few weeks, and many had very little rise in body

weight compared to controls, before conception.40,118,317,318 Further, the significant obesity

achieved prior to conception in our model allowed us to establish a profound cardiovascular

and renal phenotype in obese female mice including hypertension, tachycardia, cardiac

hypertrophy and fibrosis, elevated cardiac output and albuminuria, consistent with human

obesity.

Another key advantage of this study is that cardiovascular and renal profiles of control

and obese mice were well characterized before conception. This approach has been largely

absent in human studies where early first trimester or post-partum measurements are used

to calculate the change in cardiovascular and renal parameters during pregnancy.101,130,359 It

was only recently that a longitudinal study by Mahendru et al 236 prospectively and

comprehensively examined the maternal cardiovascular and renal function changes from

pre-pregnancy until 17 weeks post-partum. This study demonstrated that not all

cardiovascular parameters return to pre-pregnancy levels at the same time with stroke

volume reported as still significantly elevated at 17 weeks post-partum.236 Thus, this study

highlighted that the use of post-partum measurements as a surrogate for pre-pregnancy

values would likely confound the interpretation in the extent of the cardiovascular adaptations

during pregnancy. Surprisingly, no study to date has prospectively examined the adaptations

of the cardiovascular and renal system in obese women prior to and during pregnancy and

post-partum. The existing literature in women suggests the adaptations of heart rate, stroke

volume and cardiac output in pregnancies complicated by obesity are likely to be

compromised.1,96,159,354 Taken together with the data generated in this thesis, the findings

highlight the need for a prospective longitudinal study into the cardiovascular and renal

adaptations of obese women during pregnancy and post-partum. In addition, parity has been

shown to influence whether obese women experience the mid-pregnancy dip in arterial

pressure.355 Thus, parity should be considered in the design of future studies.

Our characterization of the intrauterine environment in obese dams was not without

limitations. One aspect beyond the scope of this thesis was the assessment of maternal

plasma profiles before and throughout pregnancy. Glucose metabolism was only assessed

pre-pregnancy but not during pregnancy. Although glucose metabolism was not examined

during pregnancy, this impaired glucose tolerance would likely persist into pregnancy given

mice were continually receiving the high fat diet throughout the pregnancy. Further,

assessment of other maternal circulating factors such as plasma insulin, leptin, adiponectin,


132

cholesterols and inflammatory makers might also be helpful to further characterize this model.

Given our results suggest that adequate volume expansion might not be achieved in obese

dams during pregnancy, future studies should measure hematocrit and plasma volume in

obese dams during pregnancy. Examination of the RAS including plasma renin activity and

aldosterone levels and tissue levels of the RAS and receptors during early pregnancy might

be beneficial in determining the contribution of this system to deficits in volume expansion in

obese dams.332,373 Further characterization of the maternal circulating profile would not only

be helpful in extending our understanding of the maternal and fetoplacental phenotypes of

our model but may also provide opportunities to identify possible therapeutics targets and

develop strategies to improve maternal and fetal outcomes in the future. Another piece of the

puzzle missing from our examination of the adaptation of renal system in Chapter 3 is how

GFR changes throughout pregnancy in obese dams. Future studies should measure GFR

before and at various stages throughout the pregnancy.111

Significant complications associated with obesity not only lead to poor maternal

outcomes during pregnancy but are also likely to have a prolonged impact in the mother.

Alarmingly, very little is documented in the literature regarding the long-term cardiovascular

and renal risks in this population of women post-partum. What we currently know is that

women who were obese before conception have a greater risk of mortality due to

cardiovascular events and these events occur earlier than women who had normal weight

before conception.223,384 Chapter 4 demonstrated that obesity-induced hypertension can be

exacerbated within a short period post-partum. Although renal function was largely preserved

at the time of assessment (4WPW), there was evidence of greater renal and glomerular

collagen deposition in obese primiparous mice. Although these mild renal and glomerular

injuries have not yet manifested in a detectable difference in albuminuria or GFR, they may

predispose these obese mice to early onset renal dysfunction and aggravated hypertension if

they were followed for a longer period or subjected to renal or cardiovascular insults. Again,

a longitudinal human study that examines the mother from pre-pregnancy, during gestation

and long after birth is desperately needed in improving our understanding of the impact of

pre-pregnancy obesity to a greater detail, therefore facilitating the development of

intervention for those women at risk.

6.3. THE IMPACT OF PRE-PREGNANCY OBESITY ON FETAL

HEALTH AND KIDNEY DEVELOPMENT

Pre-pregnancy obesity in humans is strongly associated with macrosomic infants.106,361

However, the literature also highlights that obesity in pregnancy is associated with greater

incidence of miscarriage, intrauterine growth restriction and particularly late gestational


133

stillbirth.14,67,270,293,316 Our model of pre-pregnancy is consistent with the later situation. We

demonstrated that pre-pregnancy obesity lead to higher fetal loss per litter and fetal growth

restriction in the remaining viable fetuses that manifest as lower body weight, shorter head

width and smaller kidneys. The greater fetal loss and growth restriction in the fetus of obese

dams might be a results of a blunted cardiovascular adaptation such as the limited rise in HR,

SV and CO we observed in later pregnancy. Although our study did not measure uterine

blood flow in pregnant mice, a study by Frias et al 121 showed that obesity in pregnancy led to

a reduced uterine blood flow in a non-human primate model of maternal obesity, contributing

to the high rate of stillbirth found in these dams. The fetal growth restriction found in our

model is also likely to be consequence of a compromised placental function in our model. We

found that placentas of obese dams had a greater incidence of placental thrombosis and a

reduced fetal/placental weight ratio, a surrogate measure of placental efficiency, specifically

in male fetuses. These findings are consistent with evidence in humans that high risk of

placental injury was associated with pre-pregnancy obesity.173

Kidney development, particularly a programmed low nephron endowment has been

associated with the development of hypertension and renal dysfunction in offspring.201 We

demonstrated that pre-pregnancy obesity leads to fetal renal and glomerular abnormalities,

and a nephron deficit in male fetuses at term. This novel finding was generated using the

gold-standard stereological analysis for estimating nephron number in developing kidneys.

Importantly, the fact that the nephron deficit found in male fetuses was independent of the

low fetal weight suggests that factors other than fetal weight must contribute to the significant

nephron deficit (25%). One possible explanation is that our model of pre-pregnancy obesity

may be complicated by an uncontrolled hyperglycemia. It has been shown that even a short

period of exposure to hyperglycemia during pregnancy could have significant impact on fetal

kidney development leading to a reduced nephron endowment.9 Assessment of glucose

metabolism during pregnancy would be helpful in determining whether our model of pre-

pregnancy obesity lead to hyperglycemia during pregnancy, therefore lead to nephron

deficiency in the offspring.167 Similarly, plasma level of corticosterone has also been shown

to be significantly elevated in obese rats during pregnancy.97 Numerous studies have shown

that elevated maternal plasma corticosterone programs low nephron number.263,272,277,341

Further evaluation of maternal plasma corticosterone level could provide possible

mechanism that how nephron deficiency might be programmed in our model.

Another factor that is likely to contribute greatly to this nephron deficit, but was not

fully elucidated in this thesis is placental dysfunction. Placental insufficiency has been shown

to cause nephron deficit in offspring. 24,262 Further both mouse 203 and human173 studies have

demonstrated that maternal obesity lead to placental injury and dysfunction. Consistent with

the literature,203 we found that there was a reduced placental efficiency restricted to male


134

fetuses of obese dams, which is likely to be associated with nephron deficit found in male

fetuses in our model. Further, similar to significant placental pathology observed in human

placentas collected at birth,173 we also observed a high incidence of placental thrombosis,

indicating these placental injuries might contribute to reduced placental efficiency. Gene

expression of pro-inflammatory cytokines had been found to be elevated in placentas of both

mouse models of maternal obesity203 and in humans,173 which may contribute to the placental

dysfunction. Unfortunately, placental pro-inflammatory cytokines expression was not

examined in our study. Histological analysis of placental morphology and evaluation in the

inflammatory status of the placenta are needed in assessing the extent of the placental

damage in this model.

6.4. THE RENAL FUNCTION IN NEPHRON DEFICIENT ANIMALS

In order to investigate the impact of reduced nephron endowment per se on renal

function and arterial pressure, it is important to choose a model in which other organ systems

that might influence renal function are less likely to be affected. We used GDNF HET mice as

this genetic mouse model of low nephron endowment allows us to examine the regulation of

renal function and arterial pressure without the confounding influence of globally

programmed cardiovascular dysfunction that often results from maternal undernutrition or

overnutrition, such as demonstrated in Chapter 3. Further, this model provides two distinct

levels of nephron deficit (30% and 65%) within one genotype and both GDNF HET-2K and

HET-1K mice have normal GFR and remain normotensive even through old age. Chapter 5

of this thesis examined whether NO, an important regulator of renal function and system

vasodilation, contributes to the maintenance of normal renal function and arterial pressure in

GDNF HET mice. To our surprise, our findings suggest NO might not be as critical as we

expected in regulating GFR and arterial pressure in the chronic setting. We found that GFR

of GDNF WT, HET-2K and HET-1K mice were not altered following 7-day of NOS inhibition

with L-NAME. And even more surprisingly, GDNF HET mice, and HET-1K in particular, had a

marked escape from L-NAME-induced hypertension. At the level of mRNA expression, we

showed that upregulation of AT1R gene expression during NO inhibition in WT mice was

absent in GDNF HET mice. This might explain the partial escape of hypertension and well-

maintained GFR in these nephron-deficient mice. Although our findings could not suggest

which compensatory mechanisms might be involved in mediating these effects, it certainly

warrants further investigations in the search for these mechanisms. Using microarray-based

gene expression profiling to examine renal tissues collected before and at various time points

following NO inhibition could be more efficient in narrowing down which pathways are likely

to be associated with the phenotypes observed in vivo.288


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6.5. LIMITATIONS AND FUTURE DIRECTIONS

Model of pre-pregnancy obesity

Human studies have demonstrated that pre-pregnancy obesity is strongly associated

with high prevalence of large for gestational age babies or macrosomic infants.106,361

However, it should be noted that majority of the babies born to obese mothers are not

macrosomic infants.221 In fact, pre-pregnancy obesity is also considered an independent

factor for miscarriage, IUGR babies and stillbirth.14,67,217,253,270,293,316 In the present study,

rather than resulting in large for gestation age fetuses as a common fetal outcome of pre-

pregnancy obesity in humans, fetus of obese dams in the model presented in Chapter 3

resembled the characteristics of IUGR fetus in humans. Further, our model also lead to

significant fetal loss and late reabsorption of fetus, consistent with increased incidence of

miscarriage217,253 and stillbirth14,67,316 associated with maternal obesity in humans. Thus it is

important to acknowledge that the findings from our model may be only relevant to proportion

of women who are at risk of adverse fetal outcome such as IUGR, miscarriage and stillbirth.

As mentioned above, it was not possible to measure the many important maternal

circulating factors such as glucose, insulin, leptin, maternal hormones (renin, aldosterone)

and inflammatory markers during pregnancy. Future studies should address these gaps in

order to further characterize this model. Further characterization of placental dysfunction in

our model may hold the clue to explain many of the fetal phenotypes in our models such as

nephron deficiency, IUGR fetuses and greater incidence of fetal loss. One aspect missing in

the characterization of cardiovascular system in our model post-partum was measurement of

cardiac structure and function using cine-MRI. Future studies should examine whether the

return of SV, CO and LV hypertrophy to pre-pregnancy levels post-partum were impacted by

pre-existing obesity in a chronic setting.

The impact of pre-pregnancy obesity on kidney development was only examined at

GA19 in the present thesis. It is known that mice complete nephrogenesis between postnatal

day 5-7.260 There is a possibility that the low nephron number observed in the present study

reflected a delay in nephrogenesis rather than a reduction, however the finding that the

nephron deficit in male fetuses was independent of fetal weight suggests this nephron deficit

is likely to persist post-birth until the completion of nephrogenesis. Future study should

examine nephron number and renal morphology at the end of nephrogenesis and later in

adulthood to further characterize the impact of pre-pregnancy obesity on kidney development.

Further, there is large body of work regarding the programming effect of pre-pregnancy

obesity on cardiovascular and renal outcomes of the offspring that could not be completed

within the scope of this thesis. Studies that characterize the blood pressure and renal

function in offspring of obese dams from weaning through to adulthood are needed to


136

investigate whether renal insufficiency programmed by pre-pregnancy obesity lead to

progressive renal dysfunction and hypertension in offspring.

Clearly, a longitudinal human study that prospectively and comprehensive examines

the maternal and fetal outcomes of pre-pregnancy obesity is desperately needed to gain

greater understanding of the pathophysiology associated with pre-pregnancy obesity. Future

studies in the field of research should focus on the understanding of the mechanisms

underpinning the adverse maternal and fetal outcomes of pre-pregnancy in order to identify

potential targets for intervention. The findings from this thesis suggest that interventions that

enhance the cardiovascular and renal adaptations in pregnancies complicated by obesity

may reduce the incidence of fetal loss, stillbirth and small for gestational age babies for

obese women, and minimize the long-term impact of fetal programming on the

cardiovascular and renal health of the offspring.

GDNF HET mice

Findings from Chapter 5 indicate that NO may play less important role in the

regulation of renal function and arterial pressure in nephron deficient GDNF HET mice.

However, as mentioned in Chapter 5 we only examined renal function at a single time point

following short-term (7 days) NO inhibition. Future studies should examine the impact of

acute and long-term (6-8 weeks) NOS inhibition on TGF sensitivity, GFR and renal excretory

function in nephron deficient GDNF HET mice. It is also important to acknowledge the fact

that we used creatinine clearance (HPLC) as a measure of GFR in Chapter 5. Although we

have demonstrated previously that measurement of creatinine using HPLC methods was

consistent with the results of inulin clearance, we could not fully ignore the possibility that

using creatinine clearance could overestimate GFR in mice due the contribution of tubular

secretion of creatinine.107 Future studies could adopt the newly developed transcutaneous

FITC-sinistrin clearance method (used in Chapter 4) to repeatedly examine GFR before and

over the course of NOS inhibition. Further, the study presented in Chapter 5 did not examine

NO bioavailability or NOS protein levels in the kidney of GDNF WT and HET mice at baseline

and during NOS inhibition. Future study should address this issue accordingly.

Taken together, future studies should continue to expand our understanding in the

protective mechanisms by which GDNF HET mice maintain a normal renal function through

to old age and the ability of these mice to escape the hypertension induced by NO deficiency.

If these protective mechanisms were identified, we may be able to develop intervention to

treat those individuals with a low nephron number who are at a greater risk of further

deterioration of renal function later in life.


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6.6. CONCLUDING REMARKS

The findings in this thesis demonstrated for the first time that pre-pregnancy obesity

compromises hemodynamic adaptation during pregnancy and leads to significant fetal loss,

growth restriction, and suboptimal fetal kidney development. Early awareness of the risks

involved and the development of interventions that enhance pregnancy-induced

cardiovascular and renal adaptations may reduce the detrimental impact of pre-pregnancy

obesity in the mother and offspring. This thesis also revealed that pregnancy could be

harmful to obese women in the long-term management of cardiovascular and renal health

post-birth. Moreover, the identification of the mechanisms involved in the surprising ability of

nephron deficient animal to avoid overt hypertension and preserve renal function in a nitric

oxide deficient state could be beneficial in managing cardiovascular and renal health in

individuals with a low nephron number. Taken together, our findings provide valuable insight

into the possible mechanisms that could be responsible for the programming of hypertension

and renal insufficiency in offspring by pre-pregnancy obesity. The understanding of the

impact of pre-pregnancy obesity during pregnancy and post-partum presented in this thesis

allows us to be one step closer towards developing effective interventions that may in the

long-term benefit the cardiovascular and renal health in obese women and their offspring.

References

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References

1. Abdullah A, Hoq S, Choudhary R, Laifer S, Zarich S. Cardiac performance is impaired in morbidly obese pregnant females. J. Obstet. Gynaecol. Res. 2012;38:258-265

2. ABS. Overweight and obesity. Australian Health Survey: Updated Results, 2011-2012. 2013;Cat. No: 4364.0.55.003

3. Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE. Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest. 2004;114:504-511

4. Aiello S, Noris M, Todeschini M, Zappella S, Foglieni C, Benigni A, Corna D, Zoja C, Cavallotti D, Remuzzi G. Renal and systemic nitric oxide synthesis in rats with renal mass reduction. Kidney Int. 1997;52:171-181

5. Alexander BT. Placental insufficiency leads to development of hypertension in growth-restricted offspring. Hypertension. 2003;41:457-462

6. Alexander BT, Hendon AE, Ferril G, Dwyer TM. Renal denervation abolishes hypertension in low-birth-weight offspring from pregnant rats with reduced uterine perfusion. Hypertension. 2005;45:754-758

7. Alsuwaida A, Mousa D, Al-Harbi A, Alghonaim M, Ghareeb S, Alrukhaimi MN. Impact of early chronic kidney disease on maternal and fetal outcomes of pregnancy. J Matern Fetal Neonatal Med. 2011;24:1432-1436

8. Amann K, Benz K. Structural renal changes in obesity and diabetes. Semin Nephrol. 2013;33:23-33

9. Amri K, Freund N, Vilar J, Merlet-Benichou C, Lelievre-Pegorier M. Adverse effects of hyperglycemia on kidney development in rats: In vivo and in vitro studies. Diabetes. 1999;48:2240-2245

10. Anderson NH, Sadler LC, Stewart AW, Fyfe EM, McCowan LM. Independent risk factors for infants who are small for gestational age by customised birthweight centiles in a multi-ethnic new zealand population. Aust. N. Z. J. Obstet. Gynaecol. 2013;53:136-142

11. Armas-Padilla MC, Armas-Hernandez MJ, Sosa-Canache B, Cammarata R, Pacheco B, Guerrero J, Carvajal AR, Hernandez-Hernandez R, Israili ZH, Valasco M. Nitric oxide and malondialdehyde in human hypertension. Am. J. Ther. 2007;14:172-176

12. Armitage JA, Lakasing L, Taylor PD, Balachandran AA, Jensen RI, Dekou V, Ashton N, Nyengaard JR, Poston L. Developmental programming of aortic and renal structure in offspring of rats fed fat-rich diets in pregnancy. J Physiol. 2005;565:171-184

13. Arranz C, Tomat A, Fellet A, Garcia J, Balaszczuk AM, de los Angeles Costa M. Renal and vascular nitric oxide system in reduced renal mass saline hypertension. Nephron Physiol. 2003;95:p36-42

14. Aune D, Saugstad OD, Henriksen T, Tonstad S. Maternal body mass index and the risk of fetal death, stillbirth, and infant death: A systematic review and meta-analysis. JAMA. 2014;311:1536-1546

15. Avelar E, Cloward TV, Walker JM, Farney RJ, Strong M, Pendleton RC, Segerson N, Adams TD, Gress RE, Hunt SC, Litwin SE. Left ventricular hypertrophy in severe obesity: Interactions among blood pressure, nocturnal hypoxemia, and body mass. Hypertension. 2007;49:34-39

16. Bachmann S, Bosse HM, Mundel P. Topography of nitric oxide synthesis by localizing constitutive no synthases in mammalian kidney. Am J Physiol. 1995;268:F885-898

17. Bailey RR, Rolleston GL. Kidney length and ureteric dilatation in the puerperium. J. Obstet. Gynaecol. Br. Commonw. 1971;78:55-61

References

139

18. Baker AM, Haeri S. Estimating risk factors and perinatal outcomes for gestational diabetes and impaired glucose tolerance in teen mothers. Diabetes. Metab. Res. Rev. 2012;28:688-691

19. Bamfo JE, Kametas NA, Chambers JB, Nicolaides KH. Maternal cardiac function in fetal growth-restricted and non-growth-restricted small-for-gestational age pregnancies. Ultrasound Obstet. Gynecol. 2007;29:51-57

20. Barker DJ, Osmond C. Infant mortality, childhood nutrition, and ischaemic heart disease in england and wales. Lancet. 1986;1:1077-1081

21. Barker DJ, Osmond C, Golding J, Kuh D, Wadsworth ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. BMJ. 1989;298:564-567

22. Barker DJ. The intrauterine environment and adult cardiovascular disease. Ciba Found. Symp. 1991;156:3-10; discussion 10-16

23. Barker DJ. The origins of the developmental origins theory. J. Intern. Med. 2007;261:412-417

24. Baserga M, Hale MA, Wang ZM, Yu X, Callaway CW, McKnight RA, Lane RH. Uteroplacental insufficiency alters nephrogenesis and downregulates cyclooxygenase-2 expression in a model of iugr with adult-onset hypertension. Am J Physiol Regul Integr Comp Physiol. 2007;292:R1943-1955

25. Battista MC, Oligny LL, St-Louis J, Brochu M. Intrauterine growth restriction in rats is associated with hypertension and renal dysfunction in adulthood. Am J Physiol Endocrinol Metab. 2002;283:E124-131

26. Bauer R, Walter B, Bauer K, Klupsch R, Patt S, Zwiener U. Intrauterine growth restriction reduces nephron number and renal excretory function in newborn piglets. Acta Physiol Scand. 2002;176:83-90

27. Baumann M, Schmaderer C, Kuznetsova T, Bartholome R, Smits JF, Richart T, Struijker-Boudier H, Staessen JA. Urinary nitric oxide metabolites and individual blood pressure progression to overt hypertension. Eur J Cardiovasc Prev Rehabil. 2011;18:656-663

28. Baylis C. The mechanism of the increase in glomerular filtration rate in the twelve-day pregnant rat. J Physiol. 1980;305:405-414

29. Baylis C, Rennke HG. Renal hemodynamics and glomerular morphology in repetitively pregnant aging rats. Kidney Int. 1985;28:140-145

30. Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest. 1992;90:278-281

31. Baylis C. Glomerular filtration and volume regulation in gravid animal models. Baillieres Clin. Obstet. Gynaecol. 1994;8:235-264

32. Baylis C. Impact of pregnancy on underlying renal disease. Adv. Ren. Replace. Ther. 2003;10:31-39

33. Baylis C. Nitric oxide deficiency in chronic kidney disease. Am J Physiol Renal Physiol. 2008;294:F1-9

34. Becerra JE, Khoury MJ, Cordero JF, Erickson JD. Diabetes mellitus during pregnancy and the risks for specific birth defects: A population-based case-control study. Pediatrics. 1990;85:1-9

35. Beharier O, Shoham-Vardi I, Pariente G, Sergienko R, Kessous R, Baumfeld Y, Szaingurten-Solodkin I, Sheiner E. Gestational diabetes mellitus is a significant risk factor for long-term maternal renal disease. J. Clin. Endocrinol. Metab. 2015;100:1412-1416

36. Belin de Chantemele EJ, Mintz JD, Rainey WE, Stepp DW. Impact of leptin-mediated sympatho-activation on cardiovascular function in obese mice. Hypertension. 2011;58:271-279

37. Bernardo BC, Weeks KL, Pretorius L, McMullen JR. Molecular distinction between physiological and pathological cardiac hypertrophy: Experimental findings and therapeutic strategies. Pharmacol Ther. 2010;128:191-227

References

140

38. Bertram JF, Douglas-Denton RN, Diouf B, Hughson MD, Hoy WE. Human nephron number: Implications for health and disease. Pediatr. Nephrol. 2011;26:1529-1533

39. Black RE, Allen LH, Bhutta ZA, Caulfield LE, de Onis M, Ezzati M, Mathers C, Rivera J, Maternal, Child Undernutrition Study G. Maternal and child undernutrition: Global and regional exposures and health consequences. Lancet. 2008;371:243-260

40. Blackmore HL, Niu Y, Fernandez-Twinn DS, Tarry-Adkins JL, Giussani DA, Ozanne SE. Maternal diet-induced obesity programs cardiovascular dysfunction in adult male mouse offspring independent of current body weight. Endocrinology. 2014;155:3970-3980

41. Bobadilla NA, Tapia E, Franco M, Lopez P, Mendoza S, Garcia-Torres R, Alvarado JA, Herrera-Acosta J. Role of nitric oxide in renal hemodynamic abnormalities of cyclosporin nephrotoxicity. Kidney Int. 1994;46:773-779

42. Bosio PM, McKenna PJ, Conroy R, O'Herlihy C. Maternal central hemodynamics in hypertensive disorders of pregnancy. Obstet. Gynecol. 1999;94:978-984

43. Boustany CM, Brown DR, Randall DC, Cassis LA. At1-receptor antagonism reverses the blood pressure elevation associated with diet-induced obesity. Am J Physiol Regul Integr Comp Physiol. 2005;289:R181-186

44. Brawley L, Itoh S, Torrens C, Barker A, Bertram C, Poston L, Hanson M. Dietary protein restriction in pregnancy induces hypertension and vascular defects in rat male offspring. Pediatr Res. 2003;54:83-90

45. Brennan KA, Kaufman S, Reynolds SW, McCook BT, Kan G, Christiaens I, Symonds ME, Olson DM. Differential effects of maternal nutrient restriction through pregnancy on kidney development and later blood pressure control in the resulting offspring. Am J Physiol Regul Integr Comp Physiol. 2008;295:R197-205

46. Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens. 1988;1:335-347

47. Brown R, Ollerstam A, Persson AE. Neuronal nitric oxide synthase inhibition sensitizes the tubuloglomerular feedback mechanism after volume expansion. Kidney Int. 2004;65:1349-1356

48. Burke SD, Barrette VF, Bianco J, Thorne JG, Yamada AT, Pang SC, Adams MA, Croy BA. Spiral arterial remodeling is not essential for normal blood pressure regulation in pregnant mice. Hypertension. 2010;55:729-737

49. Burke SL, Prior LJ, Lukoshkova EV, Lim K, Barzel B, Davern PJ, Armitage JA, Head GA. Reduced preprandial dipping accounts for rapid elevation of blood pressure and renal sympathetic nerve activity in rabbits fed a high-fat diet. Chronobiol. Int. 2013;30:726-738

50. Burton GJ, Jauniaux E, Watson AL. Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: The boyd collection revisited. Am J Obstet Gynecol. 1999;181:718-724

51. Burton GJ, Yung HW, Cindrova-Davies T, Charnock-Jones DS. Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia. Placenta. 2009;30 Suppl A:S43-48

52. Butz GM, Davisson RL. Long-term telemetric measurement of cardiovascular parameters in awake mice: A physiological genomics tool. Physiol Genomics. 2001;5:89-97

53. Cadnapaphornchai MA, Ohara M, Morris KG, Jr., Knotek M, Rogachev B, Ladtkow T, Carter EP, Schrier RW. Chronic nos inhibition reverses systemic vasodilation and glomerular hyperfiltration in pregnancy. Am J Physiol Renal Physiol. 2001;280:F592-598

54. Calkin AC, Giunti S, Jandeleit-Dahm KA, Allen TJ, Cooper ME, Thomas MC. Ppar-alpha and -gamma agonists attenuate diabetic kidney disease in the apolipoprotein e knockout mouse. Nephrol Dial Transplant. 2006;21:2399-2405

References

141

55. Carey LC, Rose JC. The midgestational maternal blood pressure decline is absent in mice lacking expression of the angiotensin ii at2 receptor. Journal of the renin-angiotensin-aldosterone system : JRAAS. 2011;12:29-35

56. Carlstrom M, Brown RD, Edlund J, Sallstrom J, Larsson E, Teerlink T, Palm F, Wahlin N, Persson AE. Role of nitric oxide deficiency in the development of hypertension in hydronephrotic animals. Am J Physiol Renal Physiol. 2008;294:F362-370

57. Cazorla O, Le Guennec JY, White E. Length-tension relationships of sub-epicardial and sub-endocardial single ventricular myocytes from rat and ferret hearts. J. Mol. Cell. Cardiol. 2000;32:735-744

58. Chagnac A, Weinstein T, Korzets A, Ramadan E, Hirsch J, Gafter U. Glomerular hemodynamics in severe obesity. Am J Physiol Renal Physiol. 2000;278:F817-822

59. Challier JC, Basu S, Bintein T, Minium J, Hotmire K, Catalano PM, Hauguel-de Mouzon S. Obesity in pregnancy stimulates macrophage accumulation and inflammation in the placenta. Placenta. 2008;29:274-281

60. Chapman AB, Abraham WT, Zamudio S, Coffin C, Merouani A, Young D, Johnson A, Osorio F, Goldberg C, Moore LG, Dahms T, Schrier RW. Temporal relationships between hormonal and hemodynamic changes in early human pregnancy. Kidney Int. 1998;54:2056-2063

61. Chen CW, Jaffe IZ, Karumanchi SA. Pre-eclampsia and cardiovascular disease. Cardiovasc. Res. 2014;101:579-586

62. Cheong Y, Sadek KH, Bruce KD, Macklon N, Cagampang FR. Diet-induced maternal obesity alters ovarian morphology and gene expression in the adult mouse offspring. Fertil Steril. 2014;102:899-907

63. Cheung KL, Lafayette RA. Renal physiology of pregnancy. Advances in chronic kidney disease. 2013;20:209-214

64. Christensen BM, Wang W, Frokiaer J, Nielsen S. Axial heterogeneity in basolateral aqp2 localization in rat kidney: Effect of vasopressin. Am J Physiol Renal Physiol. 2003;284:F701-717

65. Christensen T, Klebe JG, Bertelsen V, Hansen HE. Changes in renal volume during normal pregnancy. Acta Obstet Gynecol Scand. 1989;68:541-543

66. Christiansen T, Stodkilde-Jorgensen H, Klebe JG, Flyvbjerg A. Changes in kidney volume during pregnancy in non-diabetic and diabetic rats measured by magnetic resonance imaging. Exp. Nephrol. 1998;6:302-307

67. Chu SY, Kim SY, Lau J, Schmid CH, Dietz PM, Callaghan WM, Curtis KM. Maternal obesity and risk of stillbirth: A metaanalysis. Am J Obstet Gynecol. 2007;197:223-228

68. Chu SY, Callaghan WM, Bish CL, D'Angelo D. Gestational weight gain by body mass index among us women delivering live births, 2004-2005: Fueling future obesity. Am J Obstet Gynecol. 2009;200:271 e271-277

69. Chu SY, Kim SY, Bish CL. Prepregnancy obesity prevalence in the united states, 2004-2005. Matern Child Health J. 2009;13:614-620

70. Clapp JF, 3rd, Capeless E. Cardiovascular function before, during, and after the first and subsequent pregnancies. Am. J. Cardiol. 1997;80:1469-1473

71. Clark NR, Reichek N, Bergey P, Hoffman EA, Brownson D, Palmon L, Axel L. Circumferential myocardial shortening in the normal human left ventricle. Assessment by magnetic resonance imaging using spatial modulation of magnetization. Circulation. 1991;84:67-74

72. Collis T, Devereux RB, Roman MJ, de Simone G, Yeh J, Howard BV, Fabsitz RR, Welty TK. Relations of stroke volume and cardiac output to body composition: The strong heart study. Circulation. 2001;103:820-825

73. Conrad KP. Renal hemodynamics during pregnancy in chronically catheterized, conscious rats. Kidney Int. 1984;26:24-29

74. Conrad KP, Debrah DO, Novak J, Danielson LA, Shroff SG. Relaxin modifies systemic arterial resistance and compliance in conscious, nonpregnant rats. Endocrinology. 2004;145:3289-3296

References

142

75. Conrad KP. Maternal vasodilation in pregnancy: The emerging role of relaxin. Am J Physiol Regul Integr Comp Physiol. 2011;301:R267-275

76. Conrad KP, Davison JM. The renal circulation in normal pregnancy and preeclampsia: Is there a place for relaxin? Am J Physiol Renal Physiol. 2014;306:F1121-1135

77. Corstius HB, Zimanyi MA, Maka N, Herath T, Thomas W, van der Laarse A, Wreford NG, Black MJ. Effect of intrauterine growth restriction on the number of cardiomyocytes in rat hearts. Pediatr Res. 2005;57:796-800

78. Costa MA. The endocrine function of human placenta: An overview. Reproductive biomedicine online. 2016;32:14-43

79. Crane JM, Murphy P, Burrage L, Hutchens D. Maternal and perinatal outcomes of extreme obesity in pregnancy. JOGC. 2013;35:606-611

80. Cullen-McEwen LA, Drago J, Bertram JF. Nephron endowment in glial cell line-derived neurotrophic factor (gdnf) heterozygous mice. Kidney Int. 2001;60:31-36

81. Cullen-McEwen LA, Armitage JA, Nyengaard JR, Moritz KM, Bertram JF. A design-based method for estimating glomerular number in the developing kidney. Am J Physiol Renal Physiol. 2011;300:F1448-1453

82. Cullen-McEwen LA, Armitage JA, Nyengaard JR, Bertram JF. Estimating nephron number in the developing kidney using the physical disector/fractionator combination. Methods Mol. Biol. 2012;886:109-119

83. Cullen-McEwen LA, Douglas-Denton RN, Bertram JF. Estimating total nephron number in the adult kidney using the physical disector/fractionator combination. Methods Mol. Biol. 2012;886:333-350

84. Cunningham CE, Teale GR. A profile of body mass index in a large rural victorian obstetric cohort. Med. J. Aust. 2013;198:39-42

85. Cuspidi C, Rescaldani M, Sala C, Grassi G. Left-ventricular hypertrophy and obesity: A systematic review and meta-analysis of echocardiographic studies. J Hypertens. 2014;32:16-25

86. Dagan A, Kwon HM, Dwarakanath V, Baum M. Effect of renal denervation on prenatal programming of hypertension and renal tubular transporter abundance. Am J Physiol Renal Physiol. 2008;295:F29-34

87. Dagan A, Habib S, Gattineni J, Dwarakanath V, Baum M. Prenatal programming of rat thick ascending limb chloride transport by low-protein diet and dexamethasone. Am J Physiol Regul Integr Comp Physiol. 2009;297:R93-99

88. Dagan A, Gattineni J, Habib S, Baum M. Effect of prenatal dexamethasone on postnatal serum and urinary angiotensin ii levels. Am J Hypertens. 2010;23:420-424

89. Davis EM, Peck JD, Thompson D, Wild RA, Langlois P. Maternal diabetes and renal agenesis/dysgenesis. Birth defects research. Part A, Clinical and molecular teratology. 2010;88:722-727

90. Davison JM, Noble MC. Serial changes in 24 hour creatinine clearance during normal menstrual cycles and the first trimester of pregnancy. Br. J. Obstet. Gynaecol. 1981;88:10-17

91. de Paula RB, da Silva AA, Hall JE. Aldosterone antagonism attenuates obesity-induced hypertension and glomerular hyperfiltration. Hypertension. 2004;43:41-47

92. Debrah DO, Conrad KP, Danielson LA, Shroff SG. Effects of relaxin on systemic arterial hemodynamics and mechanical properties in conscious rats: Sex dependency and dose response. J Appl Physiol. 2005;98:1013-1020

93. Debrah DO, Conrad KP, Jeyabalan A, Danielson LA, Shroff SG. Relaxin increases cardiac output and reduces systemic arterial load in hypertensive rats. Hypertension. 2005;46:745-750

94. Debrah DO, Novak J, Matthews JE, Ramirez RJ, Shroff SG, Conrad KP. Relaxin is essential for systemic vasodilation and increased global arterial compliance during early pregnancy in conscious rats. Endocrinology. 2006;147:5126-5131

95. Deji N, Kume S, Araki S, Soumura M, Sugimoto T, Isshiki K, Chin-Kanasaki M, Sakaguchi M, Koya D, Haneda M, Kashiwagi A, Uzu T. Structural and functional

References

143

changes in the kidneys of high-fat diet-induced obese mice. Am J Physiol Renal Physiol. 2009;296:F118-126

96. Dennis AT, Castro JM, Ong M, Carr C. Haemodynamics in obese pregnant women. International journal of obstetric anesthesia. 2012;21:129-134

97. Desai M, Jellyman JK, Han G, Beall M, Lane RH, Ross MG. Maternal obesity and high-fat diet program offspring metabolic syndrome. Am J Obstet Gynecol. 2014;211:237 e231-237 e213

98. do Carmo JM, Tallam LS, Roberts JV, Brandon EL, Biglane J, da Silva AA, Hall JE. Impact of obesity on renal structure and function in the presence and absence of hypertension: Evidence from melanocortin-4 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2009;297:R803-812

99. Douglas-Denton R, Moritz KM, Bertram JF, Wintour EM. Compensatory renal growth after unilateral nephrectomy in the ovine fetus. J Am Soc Nephrol. 2002;13:406-410

100. Douglas-Denton RN, McNamara BJ, Hoy WE, Hughson MD, Bertram JF. Does nephron number matter in the development of kidney disease? Ethn. Dis. 2006;16:S2-40-45

101. Ducas RA, Elliott JE, Melnyk SF, Premecz S, daSilva M, Cleverley K, Wtorek P, Mackenzie GS, Helewa ME, Jassal DS. Cardiovascular magnetic resonance in pregnancy: Insights from the cardiac hemodynamic imaging and remodeling in pregnancy (chirp) study. Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance. 2014;16:1

102. Dunn SR, Qi Z, Bottinger EP, Breyer MD, Sharma K. Utility of endogenous creatinine clearance as a measure of renal function in mice. Kidney Int. 2004;65:1959-1967

103. Duvekot JJ, Cheriex EC, Pieters FA, Peeters LL. Severely impaired fetal growth is preceded by maternal hemodynamic maladaptation in very early pregnancy. Acta Obstet Gynecol Scand. 1995;74:693-697

104. Ece I, Uner A, Balli S, Kibar AE, Oflaz MB, Kurdoglu M. The effects of pre-pregnancy obesity on fetal cardiac functions. Pediatr. Cardiol. 2014;35:838-843

105. Eghbali M, Deva R, Alioua A, Minosyan TY, Ruan H, Wang Y, Toro L, Stefani E. Molecular and functional signature of heart hypertrophy during pregnancy. Circ. Res. 2005;96:1208-1216

106. Ehrenberg HM, Mercer BM, Catalano PM. The influence of obesity and diabetes on the prevalence of macrosomia. Am J Obstet Gynecol. 2004;191:964-968

107. Eisner C, Faulhaber-Walter R, Wang Y, Leelahavanichkul A, Yuen PS, Mizel D, Star RA, Briggs JP, Levine M, Schnermann J. Major contribution of tubular secretion to creatinine clearance in mice. Kidney Int. 2010;77:519-526

108. Elahi MM, Cagampang FR, Anthony FW, Curzen N, Ohri SK, Hanson MA. Statin treatment in hypercholesterolemic pregnant mice reduces cardiovascular risk factors in their offspring. Hypertension. 2008;51:939-944

109. Elahi MM, Cagampang FR, Mukhtar D, Anthony FW, Ohri SK, Hanson MA. Long-term maternal high-fat feeding from weaning through pregnancy and lactation predisposes offspring to hypertension, raised plasma lipids and fatty liver in mice. Br. J. Nutr. 2009;102:514-519

110. Elahi MM, Cagampang FR, Ohri SK, Hanson MA. Long-term statin administration to dams on high-fat diet protects not only them but also their offspring from cardiovascular risk. Ann. Nutr. Metab. 2013;62:250-256

111. Ellery SJ, Cai X, Walker DD, Dickinson H, Kett MM. Transcutaneous measurement of glomerular filtration rate in small rodents: Through the skin for the win? Nephrology (Carlton). 2015;20:117-123

112. Endres LK, Straub H, McKinney C, Plunkett B, Minkovitz CS, Schetter CD, Ramey S, Wang C, Hobel C, Raju T, Shalowitz MU, Community Child Health Network of the Eunice Kennedy Shriver National Institute of Child H, Human D. Postpartum weight retention risk factors and relationship to obesity at 1 year. Obstet. Gynecol. 2015;125:144-152

References

144

113. Erdely A, Wagner L, Muller V, Szabo A, Baylis C. Protection of wistar furth rats from chronic renal disease is associated with maintained renal nitric oxide synthase. J Am Soc Nephrol. 2003;14:2526-2533

114. Erdely A, Freshour G, Baylis C. Resistance to renal damage by chronic nitric oxide synthase inhibition in the wistar-furth rat. Am J Physiol Regul Integr Comp Physiol. 2006;290:R66-72

115. Esposito C, He CJ, Striker GE, Zalups RK, Striker LJ. Nature and severity of the glomerular response to nephron reduction is strain-dependent in mice. Am J Pathol. 1999;154:891-897

116. Fan L, Lindsley SR, Comstock SM, Takahashi DL, Evans AE, He GW, Thornburg KL, Grove KL. Maternal high-fat diet impacts endothelial function in nonhuman primate offspring. Int J Obes (Lond). 2013;37:254-262

117. Fernandez-Twinn DS, Ekizoglou S, Wayman A, Petry CJ, Ozanne SE. Maternal low-protein diet programs cardiac beta-adrenergic response and signaling in 3-mo-old male offspring. Am J Physiol Regul Integr Comp Physiol. 2006;291:R429-436

118. Fernandez-Twinn DS, Blackmore HL, Siggens L, Giussani DA, Cross CM, Foo R, Ozanne SE. The programming of cardiac hypertrophy in the offspring by maternal obesity is associated with hyperinsulinemia, akt, erk, and mtor activation. Endocrinology. 2012

119. Flynn ER, Alexander BT, Lee J, Hutchens ZM, Jr., Maric-Bilkan C. High-fat/fructose feeding during prenatal and postnatal development in female rats increases susceptibility to renal and metabolic injury later in life. Am J Physiol Regul Integr Comp Physiol. 2013;304:R278-285

120. Franco M, Bell PD, Navar LG. Evaluation of prostaglandins as mediators of tubuloglomerular feedback. Am J Physiol. 1988;254:F642-649

121. Frias AE, Morgan TK, Evans AE, Rasanen J, Oh KY, Thornburg KL, Grove KL. Maternal high-fat diet disturbs uteroplacental hemodynamics and increases the frequency of stillbirth in a nonhuman primate model of excess nutrition. Endocrinology. 2011;152:2456-2464

122. Gademan MG, van Eijsden M, Roseboom TJ, van der Post JA, Stronks K, Vrijkotte TG. Maternal prepregnancy body mass index and their children's blood pressure and resting cardiac autonomic balance at age 5 to 6 years. Hypertension. 2013;62:641-647

123. Gaillard R, Steegers EA, Hofman A, Jaddoe VW. Associations of maternal obesity with blood pressure and the risks of gestational hypertensive disorders. The generation r study. J Hypertens. 2011;29:937-944

124. Gaillard R, Steegers EA, Duijts L, Felix JF, Hofman A, Franco OH, Jaddoe VW. Childhood cardiometabolic outcomes of maternal obesity during pregnancy: The generation r study. Hypertension. 2014;63:683-691

125. Gallop PM, Paz MA. Posttranslational protein modifications, with special attention to collagen and elastin. Physiol. Rev. 1975;55:418-487

126. Gant NF, Worley RJ, Everett RB, MacDonald PC. Control of vascular responsiveness during human pregnancy. Kidney Int. 1980;18:253-258

127. Gaspari T, Brdar M, Lee HW, Spizzo I, Hu Y, Widdop RE, Simpson RW, Dear AE. Molecular and cellular mechanisms of glucagon-like peptide-1 receptor agonist-mediated attenuation of cardiac fibrosis. Diabetes & vascular disease research. 2015

128. Gelson E, Curry R, Gatzoulis MA, Swan L, Lupton M, Steer P, Johnson M. Effect of maternal heart disease on fetal growth. Obstet. Gynecol. 2011;117:886-891

129. Gilson GJ, Mosher MD, Conrad KP. Systemic hemodynamics and oxygen transport during pregnancy in chronically instrumented, conscious rats. Am J Physiol. 1992;263:H1911-1918

130. Gilson GJ, Samaan S, Crawford MH, Qualls CR, Curet LB. Changes in hemodynamics, ventricular remodeling, and ventricular contractility during normal pregnancy: A longitudinal study. Obstet. Gynecol. 1997;89:957-962

References

145

131. Goel A, Maski MR, Bajracharya S, Wenger JB, Zhang D, Salahuddin S, Shahul SS, Thadhani R, Seely EW, Karumanchi SA, Rana S. Epidemiology and mechanisms of de novo and persistent hypertension in the postpartum period. Circulation. 2015;132:1726-1733

132. Grassi G, Seravalle G, Dell'Oro R, Trevano FQ, Bombelli M, Scopelliti F, Facchini A, Mancia G, Study C. Comparative effects of candesartan and hydrochlorothiazide on blood pressure, insulin sensitivity, and sympathetic drive in obese hypertensive individuals: Results of the cross study. J Hypertens. 2003;21:1761-1769

133. Gray SP, Kenna K, Bertram JF, Hoy WE, Yan EB, Bocking AD, Brien JF, Walker DW, Harding R, Moritz KM. Repeated ethanol exposure during late gestation decreases nephron endowment in fetal sheep. Am J Physiol Regul Integr Comp Physiol. 2008;295:R568-574

134. Gray SP, Denton KM, Cullen-McEwen L, Bertram JF, Moritz KM. Prenatal exposure to alcohol reduces nephron number and raises blood pressure in progeny. J Am Soc Nephrol. 2010;21:1891-1902

135. Greenwood JP, Scott EM, Stoker JB, Walker JJ, Mary DA. Sympathetic neural mechanisms in normal and hypertensive pregnancy in humans. Circulation. 2001;104:2200-2204

136. Griffin KA, Kramer H, Bidani AK. Adverse renal consequences of obesity. Am J Physiol Renal Physiol. 2008;294:F685-696

137. Grigore D, Ojeda NB, Robertson EB, Dawson AS, Huffman CA, Bourassa EA, Speth RC, Brosnihan KB, Alexander BT. Placental insufficiency results in temporal alterations in the renin angiotensin system in male hypertensive growth restricted offspring. Am J Physiol Regul Integr Comp Physiol. 2007;293:R804-811

138. Gross V, Obst M, Kiss E, Janke J, Mazak I, Shagdarsuren E, Muller DN, Langenickel TH, Grone HJ, Luft FC. Cardiac hypertrophy and fibrosis in chronic l-name-treated at2 receptor-deficient mice. J Hypertens. 2004;22:997-1005

139. Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;56:56-64

140. Guidi E, Menghetti D, Milani S, Montagnino G, Palazzi P, Bianchi G. Hypertension may be transplanted with the kidney in humans: A long-term historical prospective follow-up of recipients grafted with kidneys coming from donors with or without hypertension in their families. J Am Soc Nephrol. 1996;7:1131-1138

141. Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, et al. The new stereological tools: Disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS. 1988;96:857-881

142. Gunderson EP, Abrams B, Selvin S. Does the pattern of postpartum weight change differ according to pregravid body size? Int. J. Obes. Relat. Metab. Disord. 2001;25:853-862

143. Gunderson EP, Chiang V, Lewis CE, Catov J, Quesenberry CP, Jr., Sidney S, Wei GS, Ness R. Long-term blood pressure changes measured from before to after pregnancy relative to nonparous women. Obstet. Gynecol. 2008;112:1294-1302

144. Gupte M, Thatcher SE, Boustany-Kari CM, Shoemaker R, Yiannikouris F, Zhang X, Karounos M, Cassis LA. Angiotensin converting enzyme 2 contributes to sex differences in the development of obesity hypertension in c57bl/6 mice. Arterioscler. Thromb. Vac. Biol. 2012;32:1392-1399

145. Guron G, Friberg P. An intact renin-angiotensin system is a prerequisite for normal renal development. J Hypertens. 2000;18:123-137

146. Gurusinghe S, Brown RD, Cai X, Samuel CS, Ricardo SD, Thomas MC, Kett MM. Does a nephron deficit exacerbate the renal and cardiovascular effects of obesity? PLoS One. 2013;8:e73095

147. Guyton AC. Dominant role of the kidneys and accessory role of whole-body autoregulation in the pathogenesis of hypertension. Am J Hypertens. 1989;2:575-585

References

146

148. Hadden DR. Congenital anomalies in diabetic pregnancy: An important confirmation. Diabetologia. 2012;55:870-872

149. Hall JE, Brands MW, Dixon WN, Smith MJ, Jr. Obesity-induced hypertension. Renal function and systemic hemodynamics. Hypertension. 1993;22:292-299

150. Hall JE. Mechanisms of abnormal renal sodium handling in obesity hypertension. Am J Hypertens. 1997;10:49S-55S

151. Hall JE, do Carmo JM, da Silva AA, Wang Z, Hall ME. Obesity-induced hypertension: Interaction of neurohumoral and renal mechanisms. Circ. Res. 2015;116:991-1006

152. Hall ME, George EM, Granger JP. [the heart during pregnancy]. Rev. Esp. Cardiol. 2011;64:1045-1050

153. Hancke K, Gundelach T, Hay B, Sander S, Reister F, Weiss JM. Pre-pregnancy obesity compromises obstetric and neonatal outcomes. J. Perinat. Med. 2015;43:141-146

154. Hastie R, Lappas M. The effect of pre-existing maternal obesity and diabetes on placental mitochondrial content and electron transport chain activity. Placenta. 2014;35:673-683

155. Heaton JM, Turner DR. Persistent renal damage following pre-eclampsia: A renal biopsy study of 13 patients. J. Pathol. 1985;147:121-126

156. Heiberg E, Sjogren J, Ugander M, Carlsson M, Engblom H, Arheden H. Design and validation of segment--freely available software for cardiovascular image analysis. BMC medical imaging. 2010;10:1

157. Heida KY, Franx A, van Rijn BB, Eijkemans MJ, Boer JM, Verschuren MW, Oudijk MA, Bots ML, van der Schouw YT. Earlier age of onset of chronic hypertension and type 2 diabetes mellitus after a hypertensive disorder of pregnancy or gestational diabetes mellitus. Hypertension. 2015

158. Heida KY, Franx A, van Rijn BB, Eijkemans MJ, Boer JM, Verschuren MW, Oudijk MA, Bots ML, van der Schouw YT. Earlier age of onset of chronic hypertension and type 2 diabetes mellitus after a hypertensive disorder of pregnancy or gestational diabetes mellitus. Hypertension. 2015;66:1116-1122

159. Helmreich RJ, Hundley V, Varvel P. The effect of obesity on heart rate (heart period) and physiologic parameters during pregnancy. Biological research for nursing. 2008;10:63-78

160. Henegar JR, Zhang Y, De Rama R, Hata C, Hall ME, Hall JE. Catheter-based radiorefrequency renal denervation lowers blood pressure in obese hypertensive dogs. Am J Hypertens. 2014;27:1285-1292

161. Heslehurst N, Simpson H, Ells LJ, Rankin J, Wilkinson J, Lang R, Brown TJ, Summerbell CD. The impact of maternal bmi status on pregnancy outcomes with immediate short-term obstetric resource implications: A meta-analysis. Obes Rev. 2008;9:635-683

162. Heslehurst N, Rankin J, Wilkinson JR, Summerbell CD. A nationally representative study of maternal obesity in england, uk: Trends in incidence and demographic inequalities in 619 323 births, 1989-2007. Int J Obes (Lond). 2010;34:420-428

163. Hink E, Bolte AC. Pregnancy outcomes in women with heart disease: Experience of a tertiary center in the netherlands. Pregnancy hypertension. 2015;5:165-170

164. Hinkle SN, Sharma AJ, Kim SY, Park S, Dalenius K, Brindley PL, Grummer-Strawn LM. Prepregnancy obesity trends among low-income women, united states, 1999-2008. Matern Child Health J. 2011

165. Hladunewich MA, Myers BD, Derby GC, Blouch KL, Druzin ML, Deen WM, Naimark DM, Lafayette RA. Course of preeclamptic glomerular injury after delivery. Am J Physiol Renal Physiol. 2008;294:F614-620

166. Hochner H, Friedlander Y, Calderon-Margalit R, Meiner V, Sagy Y, Avgil-Tsadok M, Burger A, Savitsky B, Siscovick DS, Manor O. Associations of maternal prepregnancy body mass index and gestational weight gain with adult offspring cardiometabolic risk factors: The jerusalem perinatal family follow-up study. Circulation. 2012;125:1381-1389

References

147

167. Hokke SN, Armitage JA, Puelles VG, Short KM, Jones L, Smyth IM, Bertram JF, Cullen-McEwen LA. Altered ureteric branching morphogenesis and nephron endowment in offspring of diabetic and insulin-treated pregnancy. PLoS One. 2013;8:e58243

168. Hoppe CC, Evans RG, Bertram JF, Moritz KM. Effects of dietary protein restriction on nephron number in the mouse. Am J Physiol Regul Integr Comp Physiol. 2007;292:R1768-1774

169. Hoy WE, Douglas-Denton RN, Hughson MD, Cass A, Johnson K, Bertram JF. A stereological study of glomerular number and volume: Preliminary findings in a multiracial study of kidneys at autopsy. Kidney Int Suppl. 2003:S31-37

170. Hoy WE, Hughson MD, Singh GR, Douglas-Denton R, Bertram JF. Reduced nephron number and glomerulomegaly in australian aborigines: A group at high risk for renal disease and hypertension. Kidney Int. 2006;70:104-110

171. Hoy WE, Kondalsamy-Chennakesavan S, Wang Z, Briganti E, Shaw J, Polkinghorne K, Chadban S, AusDiab Study G. Quantifying the excess risk for proteinuria, hypertension and diabetes in australian aborigines: Comparison of profiles in three remote communities in the northern territory with those in the ausdiab study. Aust. N. Z. J. Public Health. 2007;31:177-183

172. Hoy WE, Kincaid-Smith P, Hughson MD, Fogo AB, Sinniah R, Dowling J, Samuel T, Mott SA, Douglas-Denton RN, Bertram JF. Ckd in aboriginal australians. Am J Kidney Dis. 2010;56:983-993

173. Huang L, Liu J, Feng L, Chen Y, Zhang J, Wang W. Maternal prepregnancy obesity is associated with higher risk of placental pathological lesions. Placenta. 2014;35:563-569

174. Huang Y, Yan X, Zhao JX, Zhu MJ, McCormick RJ, Ford SP, Nathanielsz PW, Ren J, Du M. Maternal obesity induces fibrosis in fetal myocardium of sheep. Am J Physiol Endocrinol Metab. 2010;299:E968-975

175. Hughson M, Farris AB, 3rd, Douglas-Denton R, Hoy WE, Bertram JF. Glomerular number and size in autopsy kidneys: The relationship to birth weight. Kidney Int. 2003;63:2113-2122

176. Hughson MD, Douglas-Denton R, Bertram JF, Hoy WE. Hypertension, glomerular number, and birth weight in african americans and white subjects in the southeastern united states. Kidney Int. 2006;69:671-678

177. Hughson MD, Gobe GC, Hoy WE, Manning RD, Jr., Douglas-Denton R, Bertram JF. Associations of glomerular number and birth weight with clinicopathological features of african americans and whites. Am J Kidney Dis. 2008;52:18-28

178. Hughson MD, Puelles VG, Hoy WE, Douglas-Denton RN, Mott SA, Bertram JF. Hypertension, glomerular hypertrophy and nephrosclerosis: The effect of race. Nephrol Dial Transplant. 2014;29:1399-1409

179. Hunter S, Robson SC. Adaptation of the maternal heart in pregnancy. Br. Heart J. 1992;68:540-543

180. Huuskes BM, Wise AF, Cox AJ, Lim EX, Payne NL, Kelly DJ, Samuel CS, Ricardo SD. Combination therapy of mesenchymal stem cells and serelaxin effectively attenuates renal fibrosis in obstructive nephropathy. FASEB J. 2015;29:540-553

181. Huxley R, Neil A, Collins R. Unravelling the fetal origins hypothesis: Is there really an inverse association between birthweight and subsequent blood pressure? Lancet. 2002;360:659-665

182. Huxley RR, Neil HA. Does maternal nutrition in pregnancy and birth weight influence levels of chd risk factors in adult life? Br J Nutr. 2004;91:459-468

183. Hyndman KA, Boesen EI, Elmarakby AA, Brands MW, Huang P, Kohan DE, Pollock DM, Pollock JS. Renal collecting duct nos1 maintains fluid-electrolyte homeostasis and blood pressure. Hypertension. 2013;62:91-98

184. Hytten FE, Paintin DB. Increase in plasma volume during normal pregnancy. The Journal of obstetrics and gynaecology of the British Empire. 1963;70:402-407

References

148

185. Iliescu R, Tudorancea I, Irwin ED, Lohmeier TE. Chronic baroreflex activation restores spontaneous baroreflex control and variability of heart rate in obesity-induced hypertension. Am J Physiol Heart Circ Physiol. 2013;305:H1080-1088

186. Ingul CB, Loras L, Tegnander E, Eik-Nes SH, Brantberg A. Maternal obesity affects foetal myocardial function already in first trimester. Ultrasound Obstet. Gynecol. 2015

187. Jackson CM, Alexander BT, Roach L, Haggerty D, Marbury DC, Hutchens ZM, Flynn ER, Maric-Bilkan C. Exposure to maternal overnutrition and a high fat diet during early postnatal development increases susceptibility to renal and metabolic injury later in life. Am J Physiol Renal Physiol. 2011

188. Jarvie E, Ramsay JE. Obstetric management of obesity in pregnancy. Semin Fetal Neonatal Med. 2010;15:83-88

189. Jiang T, Wang Z, Proctor G, Moskowitz S, Liebman SE, Rogers T, Lucia MS, Li J, Levi M. Diet-induced obesity in c57bl/6j mice causes increased renal lipid accumulation and glomerulosclerosis via a sterol regulatory element-binding protein-1c-dependent pathway. J Biol Chem. 2005;280:32317-32325

190. Jover B, Herizi A, Ventre F, Dupont M, Mimran A. Sodium and angiotensin in hypertension induced by long-term nitric oxide blockade. Hypertension. 1993;21:944-948

191. Kambham N, Markowitz GS, Valeri AM, Lin J, D'Agati VD. Obesity-related glomerulopathy: An emerging epidemic. Kidney Int. 2001;59:1498-1509

192. Kandasamy Y, Smith R, Wright IM, Lumbers ER. Reduced nephron endowment in the neonates of indigenous australian peoples. Journal of developmental origins of health and disease. 2014;5:31-35

193. Kang IS, Pyun WB, Shin J, Kim JH, Kim SG, Shin GJ. Association between central obesity and circadian parameters of blood pressure from the korean ambulatory blood pressure monitoring registry: Kor-abp registry. J. Korean Med. Sci. 2013;28:1461-1467

194. Kanno A, Kikuya M, Asayama K, Satoh M, Inoue R, Hosaka M, Metoki H, Obara T, Hoshi H, Totsune K, Sato T, Taguma Y, Sato H, Imai Y, Ohkubo T. Night-time blood pressure is associated with the development of chronic kidney disease in a general population: The ohasama study. J Hypertens. 2013;31:2410-2417

195. Kashiwagi M, Shinozaki M, Hirakata H, Tamaki K, Hirano T, Tokumoto M, Goto H, Okuda S, Fujishima M. Locally activated renin-angiotensin system associated with tgf-beta1 as a major factor for renal injury induced by chronic inhibition of nitric oxide synthase in rats. J Am Soc Nephrol. 2000;11:616-624

196. Kasiske BL, Umen AJ. The influence of age, sex, race, and body habitus on kidney weight in humans. Arch. Pathol. Lab. Med. 1986;110:55-60

197. Katkhuda R, Peterson ES, Roghair RD, Norris AW, Scholz TD, Segar JL. Sex-specific programming of hypertension in offspring of late-gestation diabetic rats. Pediatr Res. 2012;72:352-361

198. Katz R, Karliner JS, Resnik R. Effects of a natural volume overload state (pregnancy) on left ventricular performance in normal human subjects. Circulation. 1978;58:434-441

199. Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: Analysis of worldwide data. Lancet. 2005;365:217-223

200. Keller G, Zimmer G, Mall G, Ritz E, Amann K. Nephron number in patients with primary hypertension. N. Engl. J. Med. 2003;348:101-108

201. Kett MM, Denton KM. Renal programming: Cause for concern? Am J Physiol Regul Integr Comp Physiol. 2010

202. Khouri MG, Peshock RM, Ayers CR, de Lemos JA, Drazner MH. A 4-tiered classification of left ventricular hypertrophy based on left ventricular geometry: The dallas heart study. Circulation. Cardiovascular imaging. 2010;3:164-171

203. Kim DW, Young SL, Grattan DR, Jasoni CL. Obesity during pregnancy disrupts placental morphology, cell proliferation, and inflammation in a sex-specific manner across gestation in the mouse. Biol. Reprod. 2014;90:130

References

149

204. King V, Dakin RS, Liu L, Hadoke PW, Walker BR, Seckl JR, Norman JE, Drake AJ. Maternal obesity has little effect on the immediate offspring but impacts on the next generation. Endocrinology. 2013;154:2514-2524

205. Kirk SL, Samuelsson AM, Argenton M, Dhonye H, Kalamatianos T, Poston L, Taylor PD, Coen CW. Maternal obesity induced by diet in rats permanently influences central processes regulating food intake in offspring. PLoS One. 2009;4:e5870

206. Kober F, Iltis I, Cozzone PJ, Bernard M. Cine-mri assessment of cardiac function in mice anesthetized with ketamine/xylazine and isoflurane. MAGMA. 2004;17:157-161

207. Kone BC, Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol. 1997;272:F561-578

208. Kotsis V, Stabouli S, Bouldin M, Low A, Toumanidis S, Zakopoulos N. Impact of obesity on 24-hour ambulatory blood pressure and hypertension. Hypertension. 2005;45:602-607

209. Koual M, Abbou H, Carbonnel M, Picone O, Ayoubi JM. Short-term outcome of patients with preeclampsia. Vascular health and risk management. 2013;9:143-148

210. Kovo M, Zion-Saukhanov E, Schreiber L, Mevorach N, Divon M, Ben-Haroush A, Bar J. The effect of maternal obesity on pregnancy outcome in correlation with placental pathology. Reproductive sciences. 2015

211. Kramer SP, Powell DK, Haggerty CM, Binkley CM, Mattingly AC, Cassis LA, Epstein FH, Fornwalt BK. Obesity reduces left ventricular strains, torsion, and synchrony in mouse models: A cine displacement encoding with stimulated echoes (dense) cardiovascular magnetic resonance study. Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance. 2013;15:109

212. Kren S, Hostetter TH. The course of the remnant kidney model in mice. Kidney Int. 1999;56:333-337

213. Kulandavelu S, Qu D, Adamson SL. Cardiovascular function in mice during normal pregnancy and in the absence of endothelial no synthase. Hypertension. 2006;47:1175-1182

214. Lager S, Samulesson AM, Taylor PD, Poston L, Powell TL, Jansson T. Diet-induced obesity in mice reduces placental efficiency and inhibits placental mtor signaling. Physiological reports. 2014;2:e00242

215. Langley-Evans SC, Phillips GJ, Jackson AA. In utero exposure to maternal low protein diets induces hypertension in weanling rats, independently of maternal blood pressure changes. Clin Nutr. 1994;13:319-324

216. Lankadeva YR, Singh RR, Moritz KM, Parkington HC, Denton KM, Tare M. Renal dysfunction is associated with a reduced contribution of nitric oxide and enhanced vasoconstriction after a congenital renal mass reduction in sheep. Circulation. 2015;131:280-288

217. Lashen H, Fear K, Sturdee DW. Obesity is associated with increased risk of first trimester and recurrent miscarriage: Matched case-control study. Hum. Reprod. 2004;19:1644-1646

218. Laura HC, Menezes AB, Noal RB, Hallal PC, Araujo CL. Maternal anthropometric characteristics in pregnancy and blood pressure among adolescents: 1993 live birth cohort, pelotas, southern brazil. BMC Public Health. 2010;10:434

219. Lavie CJ, Milani RV, Ventura HO, Cardenas GA, Mehra MR, Messerli FH. Disparate effects of left ventricular geometry and obesity on mortality in patients with preserved left ventricular ejection fraction. Am. J. Cardiol. 2007;100:1460-1464

220. Lawlor DA, Najman JM, Sterne J, Williams GM, Ebrahim S, Davey Smith G. Associations of parental, birth, and early life characteristics with systolic blood pressure at 5 years of age: Findings from the mater-university study of pregnancy and its outcomes. Circulation. 2004;110:2417-2423

221. Leddy MA, Power ML, Schulkin J. The impact of maternal obesity on maternal and fetal health. Reviews in obstetrics & gynecology. 2008;1:170-178

References

150

222. Lee H, Jang HC, Park HK, Cho NH. Early manifestation of cardiovascular disease risk factors in offspring of mothers with previous history of gestational diabetes mellitus. Diabetes Res. Clin. Pract. 2007;78:238-245

223. Lee KK, Raja EA, Lee AJ, Bhattacharya S, Bhattacharya S, Norman JE, Reynolds RM. Maternal obesity during pregnancy associates with premature mortality and major cardiovascular events in later life. Hypertension. 2015

224. Leelahavanichkul A, Yan Q, Hu X, Eisner C, Huang Y, Chen R, Mizel D, Zhou H, Wright EC, Kopp JB, Schnermann J, Yuen PS, Star RA. Angiotensin ii overcomes strain-dependent resistance of rapid ckd progression in a new remnant kidney mouse model. Kidney Int. 2010;78:1136-1153

225. Lemmer B, Arraj M. Effect of no synthase inhibition on cardiovascular circadian rhythms in wild-type and enos-knock-out mice. Chronobiol. Int. 2008;25:501-510

226. Lifestyles Statistics Team HaSCIC. Statistics on obesity, physical activity and diet: England. March 03, 2015

227. Lim K, Zimanyi MA, Black MJ. Effect of maternal protein restriction in rats on cardiac fibrosis and capillarization in adulthood. Pediatr Res. 2006;60:83-87

228. Lim SS, Vos T, Flaxman AD, Danaei G, Shibuya K, Adair-Rohani H, al e. A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: A systematic analysis for the global burden of disease study 2010. Lancet. 2012;380:2224-2260

229. Lohmeier TE, Iliescu R, Liu B, Henegar JR, Maric-Bilkan C, Irwin ED. Systemic and renal-specific sympathoinhibition in obesity hypertension. Hypertension. 2012;59:331-338

230. Lu D, Fu Y, Lopez-Ruiz A, Zhang R, Juncos R, Liu H, Manning RD, Jr., Juncos LA, Liu R. Salt-sensitive splice variant of nnos expressed in the macula densa cells. Am J Physiol Renal Physiol. 2010;298:F1465-1471

231. Lumbers ER, Pringle KG. Roles of the circulating renin-angiotensin-aldosterone system in human pregnancy. Am J Physiol Regul Integr Comp Physiol. 2014;306:R91-101

232. Lutsiv O, Mah J, Beyene J, McDonald SD. The effects of morbid obesity on maternal and neonatal health outcomes: A systematic review and meta-analyses. Obes Rev. 2015;16:531-546

233. Lyamina NP, Lyamina SV, Senchiknin VN, Mallet RT, Downey HF, Manukhina EB. Normobaric hypoxia conditioning reduces blood pressure and normalizes nitric oxide synthesis in patients with arterial hypertension. J Hypertens. 2011;29:2265-2272

234. Ma LJ, Fogo AB. Model of robust induction of glomerulosclerosis in mice: Importance of genetic background. Kidney Int. 2003;64:350-355

235. Mahendru AA, Everett TR, Wilkinson IB, Lees CC, McEniery CM. Maternal cardiovascular changes from pre-pregnancy to very early pregnancy. J Hypertens. 2012;30:2168-2172

236. Mahendru AA, Everett TR, Wilkinson IB, Lees CC, McEniery CM. A longitudinal study of maternal cardiovascular function from preconception to the postpartum period. J Hypertens. 2014;32:849-856

237. Majid DS, Navar LG. Nitric oxide in the control of renal hemodynamics and excretory function. Am J Hypertens. 2001;14:74S-82S

238. Malassine A, Frendo JL, Evain-Brion D. A comparison of placental development and endocrine functions between the human and mouse model. Hum. Reprod. Update. 2003;9:531-539

239. Mallamaci F, Leonardis D, Bellizzi V, Zoccali C. Does high salt intake cause hyperfiltration in patients with essential hypertension? J. Hum. Hypertens. 1996;10:157-161

240. Mannisto T, Mendola P, Vaarasmaki M, Jarvelin MR, Hartikainen AL, Pouta A, Suvanto E. Elevated blood pressure in pregnancy and subsequent chronic disease risk. Circulation. 2013;127:681-690

References

151

241. Martin JE, Hure AJ, Macdonald-Wicks L, Smith R, Collins CE. Predictors of post-partum weight retention in a prospective longitudinal study. Maternal & child nutrition. 2014;10:496-509

242. Mattson DL, Wu F. Nitric oxide synthase activity and isoforms in rat renal vasculature. Hypertension. 2000;35:337-341

243. McGuinness OP, Ayala JE, Laughlin MR, Wasserman DH. Nih experiment in centralized mouse phenotyping: The vanderbilt experience and recommendations for evaluating glucose homeostasis in the mouse. Am J Physiol Endocrinol Metab. 2009;297:E849-855

244. McIntyre HD, Gibbons KS, Flenady VJ, Callaway LK. Overweight and obesity in australian mothers: Epidemic or endemic? Med. J. Aust. 2012;196:184-188

245. McKeating A, Maguire PJ, Farren M, Daly N, Sheehan SR, Turner MJ. The clinical outcomes of unplanned pregnancy in severely obese women. J Matern Fetal Neonatal Med. 2015:1-5

246. McMullen JR, Shioi T, Huang WY, Zhang L, Tarnavski O, Bisping E, Schinke M, Kong S, Sherwood MC, Brown J, Riggi L, Kang PM, Izumo S. The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway. J Biol Chem. 2004;279:4782-4793

247. McNamara BJ, Diouf B, Hughson MD, Douglas-Denton RN, Hoy WE, Bertram JF. Renal pathology, glomerular number and volume in a west african urban community. Nephrol Dial Transplant. 2008;23:2576-2585

248. McNamara BJ, Diouf B, Douglas-Denton RN, Hughson MD, Hoy WE, Bertram JF. A comparison of nephron number, glomerular volume and kidney weight in senegalese africans and african americans. Nephrol Dial Transplant. 2010;25:1514-1520

249. Meran S, Steadman R. Fibroblasts and myofibroblasts in renal fibrosis. Int. J. Exp. Pathol. 2011;92:158-167

250. Messerli FH. Cardiovascular effects of obesity and hypertension. Lancet. 1982;1:1165-1168

251. Messerli FH, Sundgaard-Riise K, Reisin E, Dreslinski G, Dunn FG, Frohlich E. Disparate cardiovascular effects of obesity and arterial hypertension. Am J Med. 1983;74:808-812

252. Messerli FH, Sundgaard-Riise K, Reisin ED, Dreslinski GR, Ventura HO, Oigman W, Frohlich ED, Dunn FG. Dimorphic cardiac adaptation to obesity and arterial hypertension. Ann. Intern. Med. 1983;99:757-761

253. Metwally M, Ong KJ, Ledger WL, Li TC. Does high body mass index increase the risk of miscarriage after spontaneous and assisted conception? A meta-analysis of the evidence. Fertil Steril. 2008;90:714-726

254. Mills JL, Troendle J, Conley MR, Carter T, Druschel CM. Maternal obesity and congenital heart defects: A population-based study. Am. J. Clin. Nutr. 2010;91:1543-1549

255. Mills PA, Huetteman DA, Brockway BP, Zwiers LM, Gelsema AJ, Schwartz RS, Kramer K. A new method for measurement of blood pressure, heart rate, and activity in the mouse by radiotelemetry. J Appl Physiol. 2000;88:1537-1544

256. Mirabito KM, Hilliard LM, Wei Z, Tikellis C, Widdop RE, Vinh A, Denton KM. Role of inflammation and the angiotensin type 2 receptor in the regulation of arterial pressure during pregnancy in mice. Hypertension. 2014;64:626-631

257. Moeller HB, Knepper MA, Fenton RA. Serine 269 phosphorylated aquaporin-2 is targeted to the apical membrane of collecting duct principal cells. Kidney Int. 2009;75:295-303

258. Molenaar EA, Hwang SJ, Vasan RS, Grobbee DE, Meigs JB, D'Agostino RB, Sr., Levy D, Fox CS. Burden and rates of treatment and control of cardiovascular disease risk factors in obesity: The framingham heart study. Diabetes Care. 2008;31:1367-1372

References

152

259. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A. Renal and neuronal abnormalities in mice lacking gdnf. Nature. 1996;382:76-79

260. Moritz KM, Wintour EM. Functional development of the meso- and metanephros. Pediatr. Nephrol. 1999;13:171-178

261. Moritz KM, Wintour EM, Dodic M. Fetal uninephrectomy leads to postnatal hypertension and compromised renal function. Hypertension. 2002;39:1071-1076

262. Moritz KM, Mazzuca MQ, Siebel AL, Mibus A, Arena D, Tare M, Owens JA, Wlodek ME. Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats. J Physiol. 2009;587:2635-2646

263. Moritz KM, De Matteo R, Dodic M, Jefferies AJ, Arena D, Wintour EM, Probyn ME, Bertram JF, Singh RR, Zanini S, Evans RG. Prenatal glucocorticoid exposure in the sheep alters renal development in utero: Implications for adult renal function and blood pressure control. Am J Physiol Regul Integr Comp Physiol. 2011;301:R500-509

264. Mount PF, Power DA. Nitric oxide in the kidney: Functions and regulation of synthesis. Acta Physiol (Oxf). 2006;187:433-446

265. Muller V, Tain YL, Croker B, Baylis C. Chronic nitric oxide deficiency and progression of kidney disease after renal mass reduction in the c57bl6 mouse. Am. J. Nephrol. 2010;32:575-580

266. Muller-Suur R, Norlen BJ, Persson AE. Resetting of tubuloglomerular feedback in rat kidneys after unilateral nephrectomy. Kidney Int. 1980;18:48-57

267. Napoli C, Glass CK, Witztum JL, Deutsch R, D'Armiento FP, Palinski W. Influence of maternal hypercholesterolaemia during pregnancy on progression of early atherosclerotic lesions in childhood: Fate of early lesions in children (felic) study. Lancet. 1999;354:1234-1241

268. Nenov VD, Taal MW, Sakharova OV, Brenner BM. Multi-hit nature of chronic renal disease. Curr Opin Nephrol Hypertens. 2000;9:85-97

269. Nielsen GL, Norgard B, Puho E, Rothman KJ, Sorensen HT, Czeizel AE. Risk of specific congenital abnormalities in offspring of women with diabetes. Diabet. Med. 2005;22:693-696

270. Nohr EA, Bech BH, Davies MJ, Frydenberg M, Henriksen TB, Olsen J. Prepregnancy obesity and fetal death: A study within the danish national birth cohort. Obstet. Gynecol. 2005;106:250-259

271. O'Regan D, Kenyon CJ, Seckl JR, Holmes MC. Prenatal dexamethasone 'programmes' hypotension, but stress-induced hypertension in adult offspring. J. Endocrinol. 2008;196:343-352

272. O'Sullivan L, Cuffe JS, Koning A, Singh RR, Paravicini TM, Moritz KM. Excess prenatal corticosterone exposure results in albuminuria, sex-specific hypotension, and altered heart rate responses to restraint stress in aged adult mice. Am J Physiol Renal Physiol. 2015;308:F1065-1073

273. Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of childhood and adult obesity in the united states, 2011-2012. JAMA. 2014;311:806-814

274. Ollerstam A, Pittner J, Persson AE, Thorup C. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J Clin Invest. 1997;99:2212-2218

275. Organization WH. Obesity and overweight. Fact Sheet No.311. 2015;2015 276. Ortiz LA, Quan A, Weinberg A, Baum M. Effect of prenatal dexamethasone on rat

renal development. Kidney Int. 2001;59:1663-1669 277. Ortiz LA, Quan A, Zarzar F, Weinberg A, Baum M. Prenatal dexamethasone

programs hypertension and renal injury in the rat. Hypertension. 2003;41:328-334 278. Osmond C, Barker DJ, Winter PD, Fall CH, Simmonds SJ. Early growth and death

from cardiovascular disease in women. BMJ. 1993;307:1519-1524 279. Ouzounian JG, Elkayam U. Physiologic changes during normal pregnancy and

delivery. Cardiol. Clin. 2012;30:317-329

References

153

280. Ozaki T, Nishina H, Hanson MA, Poston L. Dietary restriction in pregnant rats causes gender-related hypertension and vascular dysfunction in offspring. J Physiol. 2001;530:141-152

281. Palatini P. Heart rate as an independent risk factor for cardiovascular disease: Current evidence and basic mechanisms. Drugs. 2007;67 Suppl 2:3-13

282. Peco-Antic A, Paripovic D, Kotur-Stevuljevic J, Stefanovic A, Scekic G, Milosevski-Lomic G. Renal functional reserve in children with apparently normal congenital solitary functioning kidney. Clin. Biochem. 2012;45:1173-1177

283. Penn N, Oteng-Ntim E, Oakley LL, Doyle P. Ethnic variation in stillbirth risk and the role of maternal obesity: Analysis of routine data from a london maternity unit. BMC pregnancy and childbirth. 2014;14:404

284. Persson AE, Ollerstam A, Liu R, Brown R. Mechanisms for macula densa cell release of renin. Acta Physiol Scand. 2004;181:471-474

285. Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. Gdnf is required for kidney development and enteric innervation. Cold Spring Harb Symp Quant Biol. 1996;61:445-457

286. Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. Defects in enteric innervation and kidney development in mice lacking gdnf. Nature. 1996;382:73-76

287. Pladys P, Lahaie I, Cambonie G, Thibault G, Le NL, Abran D, Nuyt AM. Role of brain and peripheral angiotensin ii in hypertension and altered arterial baroreflex programmed during fetal life in rat. Pediatr Res. 2004;55:1042-1049

288. Pradervand S, Zuber Mercier A, Centeno G, Bonny O, Firsov D. A comprehensive analysis of gene expression profiles in distal parts of the mouse renal tubule. Pflugers Arch. 2010;460:925-952

289. Premaratne E, Verma S, Ekinci EI, Theverkalam G, Jerums G, MacIsaac RJ. The impact of hyperfiltration on the diabetic kidney. Diabetes Metab. 2015;41:5-17

290. Prior LJ, Davern PJ, Burke SL, Lim K, Armitage JA, Head GA. Exposure to a high-fat diet during development alters leptin and ghrelin sensitivity and elevates renal sympathetic nerve activity and arterial pressure in rabbits. Hypertension. 2014;63:338-345

291. Puelles VG, Hoy WE, Hughson MD, Diouf B, Douglas-Denton RN, Bertram JF. Glomerular number and size variability and risk for kidney disease. Curr Opin Nephrol Hypertens. 2011;20:7-15

292. Quiroz Y, Pons H, Gordon KL, Rincon J, Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, Johnson RJ, Rodriguez-Iturbe B. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition. Am J Physiol Renal Physiol. 2001;281:F38-47

293. Radulescu L, Munteanu O, Popa F, Cirstoiu M. The implications and consequences of maternal obesity on fetal intrauterine growth restriction. Journal of medicine and life. 2013;6:292-298

294. Rahman MM, Abe SK, Kanda M, Narita S, Rahman MS, Bilano V, Ota E, Gilmour S, Shibuya K. Maternal body mass index and risk of birth and maternal health outcomes in low- and middle-income countries: A systematic review and meta-analysis. Obes Rev. 2015;16:758-770

295. Regazzoni BM, Genton N, Pelet J, Drukker A, Guignard JP. Long-term followup of renal functional reserve capacity after unilateral nephrectomy in childhood. J. Urol. 1998;160:844-848

296. Reisin E, Messerli FG, Ventura HO, Frohlich ED. Renal haemodynamic studies in obesity hypertension. J Hypertens. 1987;5:397-400

297. Reisin E, Weir MR, Falkner B, Hutchinson HG, Anzalone DA, Tuck ML. Lisinopril versus hydrochlorothiazide in obese hypertensive patients: A multicenter placebo-controlled trial. Treatment in obese patients with hypertension (trophy) study group. Hypertension. 1997;30:140-145

References

154

298. Reynolds CM, Vickers MH, Harrison CJ, Segovia SA, Gray C. Maternal high fat and/or salt consumption induces sex-specific inflammatory and nutrient transport in the rat placenta. Physiological reports. 2015;3

299. Reynolds RM, Allan KM, Raja EA, Bhattacharya S, McNeill G, Hannaford PC, Sarwar N, Lee AJ, Bhattacharya S, Norman JE. Maternal obesity during pregnancy and premature mortality from cardiovascular event in adult offspring: Follow-up of 1 323 275 person years. BMJ. 2013;347:f4539

300. Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension. 1992;20:298-303

301. Ribstein J, du Cailar G, Mimran A. Combined renal effects of overweight and hypertension. Hypertension. 1995;26:610-615

302. Rider OJ, Lewandowski A, Nethononda R, Petersen SE, Francis JM, Pitcher A, Holloway CJ, Dass S, Banerjee R, Byrne JP, Leeson P, Neubauer S. Gender-specific differences in left ventricular remodelling in obesity: Insights from cardiovascular magnetic resonance imaging. Eur. Heart J. 2013;34:292-299

303. Rincon J, Correia D, Arcaya JL, Finol E, Fernandez A, Perez M, Yaguas K, Talavera E, Chavez M, Summer R, Romero F. Role of angiotensin ii type 1 receptor on renal nad(p)h oxidase, oxidative stress and inflammation in nitric oxide inhibition induced-hypertension. Life Sci. 2015;124:81-90

304. Roberts KA, Riley SC, Reynolds RM, Barr S, Evans M, Statham A, Hor K, Jabbour HN, Norman JE, Denison FC. Placental structure and inflammation in pregnancies associated with obesity. Placenta. 2011;32:247-254

305. Roberts M, Lindheimer MD, Davison JM. Altered glomerular permselectivity to neutral dextrans and heteroporous membrane modeling in human pregnancy. Am J Physiol. 1996;270:F338-343

306. Roberts VH, Pound LD, Thorn SR, Gillingham MB, Thornburg KL, Friedman JE, Frias AE, Grove KL. Beneficial and cautionary outcomes of resveratrol supplementation in pregnant nonhuman primates. FASEB J. 2014;28:2466-2477

307. Robles RG, Villa E, Santirso R, Martinez J, Ruilope LM, Cuesta C, Sancho JM. Effects of captopril on sympathetic activity, lipid and carbohydrate metabolism in a model of obesity-induced hypertension in dogs. Am J Hypertens. 1993;6:1009-1015

308. Robson SC, Hunter S, Moore M, Dunlop W. Haemodynamic changes during the puerperium: A doppler and m-mode echocardiographic study. Br. J. Obstet. Gynaecol. 1987;94:1028-1039

309. Robson SC, Hunter S, Boys RJ, Dunlop W. Serial study of factors influencing changes in cardiac output during human pregnancy. Am J Physiol. 1989;256:H1060-1065

310. Rowlands I, Graves N, de Jersey S, McIntyre HD, Callaway L. Obesity in pregnancy: Outcomes and economics. Semin Fetal Neonatal Med. 2010;15:94-99

311. Rumantir MS, Vaz M, Jennings GL, Collier G, Kaye DM, Seals DR, Wiesner GH, Brunner-La Rocca HP, Esler MD. Neural mechanisms in human obesity-related hypertension. J Hypertens. 1999;17:1125-1133

312. Ruta LA, Dickinson H, Thomas MC, Denton KM, Anderson WP, Kett MM. High-salt diet reveals the hypertensive and renal effects of reduced nephron endowment. Am J Physiol Renal Physiol. 2010;298:F1384-1392

313. Saben J, Lindsey F, Zhong Y, Thakali K, Badger TM, Andres A, Gomez-Acevedo H, Shankar K. Maternal obesity is associated with a lipotoxic placental environment. Placenta. 2014;35:171-177

314. Saetun P, Semangoen T, Thongboonkerd V. Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses. Am J Physiol Renal Physiol. 2009;296:F1346-1354

315. Sahajpal V, Ashton N. Renal function and angiotensin at1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci (Lond). 2003;104:607-614

References

155

316. Salihu HM. Maternal obesity and stillbirth. Semin. Perinatol. 2011;35:340-344 317. Samuelsson AM, Matthews PA, Argenton M, Christie MR, McConnell JM, Jansen EH,

Piersma AH, Ozanne SE, Twinn DF, Remacle C, Rowlerson A, Poston L, Taylor PD. Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: A novel murine model of developmental programming. Hypertension. 2008;51:383-392

318. Samuelsson AM, Morris A, Igosheva N, Kirk SL, Pombo JM, Coen CW, Poston L, Taylor PD. Evidence for sympathetic origins of hypertension in juvenile offspring of obese rats. Hypertension. 2010;55:76-82

319. Samuelsson AM, Clark J, Rudyk O, Shattock MJ, Bae SE, South T, Pombo J, Redington K, Uppal E, Coen CW, Poston L, Taylor PD. Experimental hyperleptinemia in neonatal rats leads to selective leptin responsiveness, hypertension, and altered myocardial function. Hypertension. 2013;62:627-633

320. Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M. Renal agenesis and the absence of enteric neurons in mice lacking gdnf. Nature. 1996;382:70-73

321. Sanna-Cherchi S, Ravani P, Corbani V, Parodi S, Haupt R, Piaggio G, Innocenti ML, Somenzi D, Trivelli A, Caridi G, Izzi C, Scolari F, Mattioli G, Allegri L, Ghiggeri GM. Renal outcome in patients with congenital anomalies of the kidney and urinary tract. Kidney Int. 2009;76:528-533

322. Schannwell CM, Zimmermann T, Schneppenheim M, Plehn G, Marx R, Strauer BE. Left ventricular hypertrophy and diastolic dysfunction in healthy pregnant women. Cardiology. 2002;97:73-78

323. Schmittgen TD, Livak KJ. Analyzing real-time pcr data by the comparative c(t) method. Nature protocols. 2008;3:1101-1108

324. Schneider MP, Ge Y, Pollock DM, Pollock JS, Kohan DE. Collecting duct-derived endothelin regulates arterial pressure and na excretion via nitric oxide. Hypertension. 2008;51:1605-1610

325. Schnermann J, Briggs JP. Restoration of tubuloglomerular feedback in volume-expanded rats by angiotensin ii. Am J Physiol. 1990;259:F565-572

326. Schock-Kusch D, Sadick M, Henninger N, Kraenzlin B, Claus G, Kloetzer HM, Weiss C, Pill J, Gretz N. Transcutaneous measurement of glomerular filtration rate using fitc-sinistrin in rats. Nephrol Dial Transplant. 2009;24:2997-3001

327. Schock-Kusch D, Geraci S, Ermeling E, Shulhevich Y, Sticht C, Hesser J, Stsepankou D, Neudecker S, Pill J, Schmitt R, Melk A. Reliability of transcutaneous measurement of renal function in various strains of conscious mice. PLoS One. 2013;8:e71519

328. Schreiber A, Shulhevich Y, Geraci S, Hesser J, Stsepankou D, Neudecker S, Koenig S, Heinrich R, Hoecklin F, Pill J, Friedemann J, Schweda F, Gretz N, Schock-Kusch D. Transcutaneous measurement of renal function in conscious mice. Am J Physiol Renal Physiol. 2012;303:F783-788

329. Schreuder MF, Langemeijer ME, Bokenkamp A, Delemarre-Van de Waal HA, Van Wijk JA. Hypertension and microalbuminuria in children with congenital solitary kidneys. J. Paediatr. Child Health. 2008;44:363-368

330. Schrier RW, Briner VA. Peripheral arterial vasodilation hypothesis of sodium and water retention in pregnancy: Implications for pathogenesis of preeclampsia-eclampsia. Obstet. Gynecol. 1991;77:632-639

331. Schrier RW, Ohara M. Dilemmas in human and rat pregnancy: Proposed mechanisms relating to arterial vasodilation. J. Neuroendocrinol. 2010;22:400-406

332. Sealey JE, Itskovitz-Eldor J, Rubattu S, James GD, August P, Thaler I, Levron J, Laragh JH. Estradiol- and progesterone-related increases in the renin-aldosterone system: Studies during ovarian stimulation and early pregnancy. J. Clin. Endocrinol. Metab. 1994;79:258-264

333. Segar EM, Norris AW, Yao JR, Hu S, Koppenhafer SL, Roghair RD, Segar JL, Scholz TD. Programming of growth, insulin resistance and vascular dysfunction in offspring of late gestation diabetic rats. Clin Sci (Lond). 2009;117:129-138

References

156

334. Sherwood OD. Relaxin's physiological roles and other diverse actions. Endocr. Rev. 2004;25:205-234

335. Shirley DG, Walter SJ. Acute and chronic changes in renal function following unilateral nephrectomy. Kidney Int. 1991;40:62-68

336. Shweta A, Cullen-McEwen LA, Kett MM, Evans RG, Denton KM, Fitzgerald SM, Anderson WP, Bertram JF. Glomerular surface area is normalized in mice born with a nephron deficit: No role for at1 receptors. Am J Physiol Renal Physiol. 2009;296:F583-589

337. Siega-Riz AM, Herring AH, Carrier K, Evenson KR, Dole N, Deierlein A. Sociodemographic, perinatal, behavioral, and psychosocial predictors of weight retention at 3 and 12 months postpartum. Obesity (Silver Spring). 2010;18:1996-2003

338. Sigmon DH, Gonzalez-Feldman E, Cavasin MA, Potter DL, Beierwaltes WH. Role of nitric oxide in the renal hemodynamic response to unilateral nephrectomy. J Am Soc Nephrol. 2004;15:1413-1420

339. Simmons LA, Gillin AG, Jeremy RW. Structural and functional changes in left ventricle during normotensive and preeclamptic pregnancy. Am J Physiol Heart Circ Physiol. 2002;283:H1627-1633

340. Simonds SE, Pryor JT, Ravussin E, Greenway FL, Dileone R, Allen AM, Bassi J, Elmquist JK, Keogh JM, Henning E, Myers MG, Jr., Licinio J, Brown RD, Enriori PJ, O'Rahilly S, Sternson SM, Grove KL, Spanswick DC, Farooqi IS, Cowley MA. Leptin mediates the increase in blood pressure associated with obesity. Cell. 2014;159:1404-1416

341. Singh RR, Cullen-McEwen LA, Kett MM, Boon WM, Dowling J, Bertram JF, Moritz KM. Prenatal corticosterone exposure results in altered at1/at2, nephron deficit and hypertension in the rat offspring. J Physiol. 2007;579:503-513

342. Slangen BF, Out IC, Verkeste CM, Peeters LL. Hemodynamic changes in early pregnancy in chronically instrumented, conscious rats. Am J Physiol. 1996;270:H1779-1784

343. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta. 1999;1411:217-230

344. Stypmann J, Engelen MA, Troatz C, Rothenburger M, Eckardt L, Tiemann K. Echocardiographic assessment of global left ventricular function in mice. Lab. Anim. 2009;43:127-137

345. Suzuki H, Watanabe Y, Arima H, Kobayashi K, Ohno Y, Kanno Y. Short- and long-term prognosis of blood pressure and kidney disease in women with a past history of preeclampsia. Clin Exp Nephrol. 2008;12:102-109

346. Szabo AJ, Wagner L, Erdely A, Lau K, Baylis C. Renal neuronal nitric oxide synthase protein expression as a marker of renal injury. Kidney Int. 2003;64:1765-1771

347. Tain YL, Freshour G, Dikalova A, Griendling K, Baylis C. Vitamin e reduces glomerulosclerosis, restores renal neuronal nos, and suppresses oxidative stress in the 5/6 nephrectomized rat. Am J Physiol Renal Physiol. 2007;292:F1404-1410

348. Tain YL, Huang LT, Hsu CN, Lee CT. Melatonin therapy prevents programmed hypertension and nitric oxide deficiency in offspring exposed to maternal caloric restriction. Oxid Med Cell Longev. 2014;2014:283180

349. Takata K, Hirano H. Mechanism of glucose transport across the human and rat placental barrier: A review. Microsc. Res. Tech. 1997;38:145-152

350. Talmor A, Dunphy B. Female obesity and infertility. Best practice & research. Clinical obstetrics & gynaecology. 2015;29:498-506

351. Tchernof A, Despres JP. Pathophysiology of human visceral obesity: An update. Physiol. Rev. 2013;93:359-404

352. Thompson ML, Williams MA, Miller RS. Modelling the association of blood pressure during pregnancy with gestational age and body mass index. Paediatr. Perinat. Epidemiol. 2009;23:254-263

353. Thorup C, Erik A, Persson G. Macula densa derived nitric oxide in regulation of glomerular capillary pressure. Kidney Int. 1996;49:430-436

References

157

354. Tomoda S, Tamura T, Sudo Y, Ogita S. Effects of obesity on pregnant women: Maternal hemodynamic change. Am. J. Perinatol. 1996;13:73-78

355. Tomoda S, Tamura T, Sudo Y, Ogita S. Effects of obesity on pregnant women: Maternal hemodynamic change. Am. J. Perinatol. 1996;13:73-78

356. Ujiie K, Yuen J, Hogarth L, Danziger R, Star RA. Localization and regulation of endothelial no synthase mrna expression in rat kidney. Am J Physiol. 1994;267:F296-302

357. Valdivielso JM, Perez-Barriocanal F, Garcia-Estan J, Lopez-Novoa JM. Role of nitric oxide in the early renal hemodynamic response after unilateral nephrectomy. Am J Physiol. 1999;276:R1718-1723

358. Vallon V. Tubuloglomerular feedback in the kidney: Insights from gene-targeted mice. Pflugers Arch. 2003;445:470-476

359. van Oppen AC, van der Tweel I, Alsbach GP, Heethaar RM, Bruinse HW. A longitudinal study of maternal hemodynamics during normal pregnancy. Obstet. Gynecol. 1996;88:40-46

360. Vasapollo B, Valensise H, Novelli GP, Larciprete G, Di Pierro G, Altomare F, Casalino B, Galante A, Arduini D. Abnormal maternal cardiac function and morphology in pregnancies complicated by intrauterine fetal growth restriction. Ultrasound Obstet. Gynecol. 2002;20:452-457

361. Vinturache AE, Chaput KH, Tough SC. Pre-pregnancy body mass index (bmi) and macrosomia in a canadian birth cohort. J Matern Fetal Neonatal Med. 2016:1-28

362. Vinyoles E, de la Sierra A, Roso A, de la Cruz JJ, Gorostidi M, Segura J, Banegas JR, Martell-Claros N, Ruilope LM, Spanish ARi. Night-time heart rate cut-off point definition by resting office tachycardia in untreated hypertensive patients: Data of the spanish abpm registry. J Hypertens. 2014;32:1016-1024

363. Wadhwa PD, Buss C, Entringer S, Swanson JM. Developmental origins of health and disease: Brief history of the approach and current focus on epigenetic mechanisms. Semin Reprod Med. 2009;27:358-368

364. Waki M, Kral JG, Mazariegos M, Wang J, Pierson RN, Jr., Heymsfield SB. Relative expansion of extracellular fluid in obese vs. Nonobese women. Am J Physiol. 1991;261:E199-203

365. Walker KA, Cai X, Caruana G, Thomas MC, Bertram JF, Kett MM. High nephron endowment protects against salt-induced hypertension. Am J Physiol Renal Physiol. 2012;303:F253-258

366. Wang A, Ziyadeh FN, Lee EY, Pyagay PE, Sung SH, Sheardown SA, Laping NJ, Chen S. Interference with tgf-beta signaling by smad3-knockout in mice limits diabetic glomerulosclerosis without affecting albuminuria. Am J Physiol Renal Physiol. 2007;293:F1657-1665

367. Wang J, Ma H, Tong C, Zhang H, Lawlis GB, Li Y, Zang M, Ren J, Nijland MJ, Ford SP, Nathanielsz PW, Li J. Overnutrition and maternal obesity in sheep pregnancy alter the jnk-irs-1 signaling cascades and cardiac function in the fetal heart. FASEB J. 2010;24:2066-2076

368. Wang Y, Shoemaker R, Thatcher SE, Batifoulier-Yiannikouris F, English VL, Cassis LA. Administration of 17beta-estradiol to ovariectomized obese female mice reverses obesity-hypertension through an ace2-dependent mechanism. Am J Physiol Endocrinol Metab. 2015;308:E1066-1075

369. West CA, Verlander JW, Wall SM, Baylis C. The chloride-bicarbonate exchanger pendrin is increased in the kidney of the pregnant rat. Exp. Physiol. 2015;100:1177-1186

370. Wilcox CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, Schmidt HH. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci U S A. 1992;89:11993-11997

371. Wilcox CS. Role of macula densa nos in tubuloglomerular feedback. Curr Opin Nephrol Hypertens. 1998;7:443-449

References

158

372. Wilson BJ, Watson MS, Prescott GJ, Sunderland S, Campbell DM, Hannaford P, Smith WC. Hypertensive diseases of pregnancy and risk of hypertension and stroke in later life: Results from cohort study. BMJ. 2003;326:845

373. Wilson M, Morganti AA, Zervoudakis I, Letcher RL, Romney BM, Von Oeyon P, Papera S, Sealey JE, Laragh JH. Blood pressure, the renin-aldosterone system and sex steroids throughout normal pregnancy. Am J Med. 1980;68:97-104

374. Wintour EM, Moritz KM, Johnson K, Ricardo S, Samuel CS, Dodic M. Reduced nephron number in adult sheep, hypertensive as a result of prenatal glucocorticoid treatment. J Physiol. 2003;549:929-935

375. Wlodek ME, Westcott K, Siebel AL, Owens JA, Moritz KM. Growth restriction before or after birth reduces nephron number and increases blood pressure in male rats. Kidney Int. 2008;74:187-195

376. Woodiwiss AJ, Libhaber CD, Majane OH, Libhaber E, Maseko M, Norton GR. Obesity promotes left ventricular concentric rather than eccentric geometric remodeling and hypertrophy independent of blood pressure. Am J Hypertens. 2008;21:1144-1151

377. Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res. 2001;49:460-467

378. Woods LL, Weeks DA, Rasch R. Hypertension after neonatal uninephrectomy in rats precedes glomerular damage. Hypertension. 2001;38:337-342

379. Woods LL, Weeks DA, Rasch R. Programming of adult blood pressure by maternal protein restriction: Role of nephrogenesis. Kidney Int. 2004;65:1339-1348

380. Wu F, Park F, Cowley AW, Jr., Mattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol. 1999;276:F874-881

381. Xie L, Hoffert JD, Chou CL, Yu MJ, Pisitkun T, Knepper MA, Fenton RA. Quantitative analysis of aquaporin-2 phosphorylation. Am J Physiol Renal Physiol. 2010;298:F1018-1023

382. Xiong XQ, Chen WW, Han Y, Zhou YB, Zhang F, Gao XY, Zhu GQ. Enhanced adipose afferent reflex contributes to sympathetic activation in diet-induced obesity hypertension. Hypertension. 2012;60:1280-1286

383. Yan J, Li X, Su R, Zhang K, Yang H. Long-term effects of maternal diabetes on blood pressure and renal function in rat male offspring. PLoS One. 2014;9:e88269

384. Yaniv-Salem S, Shoham-Vardi I, Kessous R, Pariente G, Sergienko R, Sheiner E. Obesity in pregnancy: What's next? Long-term cardiovascular morbidity in a follow-up period of more than a decade. J Matern Fetal Neonatal Med. 2015:1-5

385. Zhang X, Qu X, Sun YB, Caruana G, Bertram JF, Nikolic-Paterson DJ, Li J. Resolvin d1 protects podocytes in adriamycin-induced nephropathy through modulation of 14-3-3beta acetylation. PLoS One. 2013;8:e67471

386. Zhou Y, Blustein J, Li H, Ye R, Zhu L, Liu J. Maternal obesity, caesarean delivery and caesarean delivery on maternal request: A cohort analysis from china. Paediatr. Perinat. Epidemiol. 2015;29:232-240

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Maternal and Fetal Outcomes of Pregnancies Complicated by ... · nephron endowment is associated with adult cardiovascular and renal disease, many models of reduced nephron endowment