DIABESITY CME Course Villa Camozzi 2015

Evaluation of renal function and renal risk in the

twenty-first century

Abstracts

GENE EXPRESSION PROFILE FROM BIOPSY SAMPLES OF MORBIDLY

OBESE PATIENTS WITH EARLY STAGES OF OBESITY RELATED

GLOMERULOPATHY.

M.Navarro-Díaz1, A. Serra

1, D.López

2, I.Bancu

1 and J. Bonet

1. Departments of Nephrology

1 and

Pathology2, Germans Trias i Pujol’s Hospital, Badalona, Spain.

INTRODUCTION: Kidney disease is an important cause of obesity-related comorbidity. A study

carried out by our group first showed the presence of early histological lesions (mostly podocyte

alterations) in obese patients lacking overt clinical renal symptoms. Several factors seem to be related to obesity-related glomerulopathy (ORG), although the pathogenic mechanisms are still unknown.

Genes related to inflammatory cytokines and lipid metabolism have been found to be expressed in

patients with ORG. Additionally, it is unknown whether these genes are expressed in the early stages of ORG. The major aim of our study is to investigate glomerular injury mechanisms in ORG by real

time PCR assessment of the expression of several genes (interleukine-6, vascular endothelial growth

factor, insulin-like growth factor-1, TNF-alpha, leptine receptor, glucose transporter-1, heart-type fatty

acid binding protein, CD44 and spleen-derived interleukine-10) in renal biopsies of patients with early stages of ORG and renal biopsies of healthy non-obese patients. The comparison of the gene

expression profiles found in the two renal biopsy groups will detect differences allowing the

establishment of a cause-effect relationship in ORG.

PATIENTS AND METHODS: Fifty morbidly obese patients (BMI≥ 40 Kg/m2) without history of

diabetes, without proteinuria and with normal renal function were selected. These patients underwent a

renal biopsy during bariatric surgery. The hystologic study showed the presence of early stages of ORG ( matrix mesangial incresase, mesangial cell proliferation, podocyt hypertrophy,

glomerulomegaly and focal and segmental glomerulosclerosis). We selected as the control group

patients with normal weight and renal function. They were non-diabetic, non-hypertensive, and they were undergoing nephrectomy or donating a kidney.After real time PCR assessment, we have used the

Livak Method to calculate the expression ratio or fold difference of our target genes. We have used

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene.

RESULTS: We show our preliminary results. We have selected 10 patients from the MO group and

10 patients from the control group.

Comparison between nephrectomy control group (n=6) and cadaveric kidney donor control group (n=4): Because of the presence of heterogeneity within the control group, we have made this

division. We have observed that cadaveric kidney donors’ group express more inflammatory gene

related (IL-6: LnCt=5,81; CD44: LnCt=1,38 and Glut-1: LnCt=3,06) than the nephrectomy

group ( IL-6: LnCt=2,09; CD44: LnCt=0,37 and Glut-1: LnCt =2,13), with p values 0,025, <0,0001 and 0,046 respectively. For this reason, we have choose the nephrectomy subgroup as the

control group for the next analyses. Comparison between morbid obese (MO) group and nephrectomy control group.We observed

that glucose transporter-1 gene and IL-6 gene are more expressed in MO group Glut-1: LnCt =3,93;

IL6: LnCt =7,51) than in control group (Glut-1: LnCt =2,13; IL-6: LnCt =2,09) with p values 0,025 and <0,0001 respectively. However, IGF-1 gene (an anti-inflammatory gene) is more expressed

in the control group (IGF-1: LnCt =6,83) than in MO group (IGF-1: LnCt = 4,99) with a p value of 0,046. The Livak method analyses are showed figure 1.

CONCLUSIONS: Although these are preliminary results, we have found that inflammatory genes

should be implicated in the pathogenesis of early stages of ORG.

ASSOCIATION OF APOLIPOPROTEIN E POLYMORPHISM WITH

DIABETIC NEPHROPATHY, OBESITY AND ADIPONECTIN.

Roumeliotis S1, Tavridou A

2,Roumeliotis A

1, Theodoridis M

1, Panagoutsos S

1, Pasadakis P

1

1Department of Nephrology, Medical School, Democritus University of Thrace, Alexandroupolis,

Greece. 2Laboratory of Pharmacology, Medical School, Democritus University of Thrace,

Alexandroupolis, Greece

AIMS: Obesity is a worldwide health problem and is a significant risk factor for dyslipidemia, Type 2 Diabetes Mellitus (T2DM), CKD and atherosclerosis. Adipose tissue is currently considered to be not

only a reservoir for energy storage, but also an active endocrine tissue, producing several proactive

cytokines, the adipocytokines. Dysregulated adipose tissue as seen in cases of obesity raises the risk of morbidity from T2DM and atherosclerosis. Adiponectin is a adipose tissue specific adipocytokine that

has been noted as an important antiatherogenic, antidiabetic and anti-inflammatory protein. Obesity is

determined by several genetic and environmental factors. Apolipoprotein E (APOE) is a genetic determinant of coronary heart disease through its influence on the lipid profile and beyond. Since

APOE plays an important role in the metabolic fate of the APO containing lipoproteins, it appears

legitimate to verify whether APOE polymorphism could effect on obesity, atheroscleroris and CKD.

METHODS: Plasma concentrations of adiponectin and oxidized-LDL were quantitated by ELISA in

63 diabetic nephropathy patients (34 male and 29 female, mean age 67.4±9.2 years) with mean BMI

31.8 ± 5.5 kg/m2 and waist circumference 104.1±11.2 cm. Overhydration (OH) and fat mass were

estimated by BIA (Βioelectrical Ιmpedance Αnalysis) method with Body Composition Monitor

(Fresenius Medical Care Deutschland GmbH). The subjects were divided into 2 groups (obese/non-

obese) on the basis of their waist circumference (WC) and on the basis of their BMI according to

established classification standards (BMI>30 kg/m2, waist circumference: men>102 cm and

women>88 cm). APOE polymorphism was determined using PCR-RFLP. Measurements of cIMT

were performed using a high resolution, real-time B-mode ultrasonograph.

RESULTS: The percentage of obese patients according to WC cutoffs was significantly greater than

the percentage of obese subjects according to BMI criteria (74.6% compared to 65.1%, p<0.001- Chi

square test). Both BMI and WC were associated with total fat mass (p=0.002 and p<0.001 respectively, Spearman's test). The frequencies of the APOE alleles were consistent with previously

published estimates : E2 (n=5) 7.9%, E3 (n=48) 76.2% and E4 (n=10) 15.9%. APOE polymorphism

was significantly associated with obesity, total fat mass, overhydration and adiponectin levels (p=0.03,

p=0.03 and p=0.03 and p=0.007 respectively, Kruskal- Wallis test). Plasma adiponectin levels differed in the carriers of APOE gene alleles with the following order E2>E3>E4. Although APOE

polymorphism was not correlated with any of lipid profile parameters (Total cholesterol, LDL-

cholesterol, HDL-cholesterol, triglycerides and oxidized LDL) or atherosclerosis parameters (cIMT, presence of plaque), it was significantly associated with history of myocardial infarction and stroke in

the total sample population (p=0.025 and p=0.02 respectively, Chi-square test). APOE polymorphism

was associated with lowering eGFR and elevated albuminuria/ proteinuria (p<0.001, p=0.04 and p=0.02 respectively, Kruskal-Wallis test), with e2 allele conferring risk and e4 providing protection.

CONCLUSIONS: Apolipoprotein E may be considered as a prominent candidate gene for obesity in

T2DM. APOE genetic variations may contribute to progression of diabetic nephropathy. Obesity should be effectively managed in risk APOE genotypes to reduce cardiovascular risk and progression

to End Stage Renal Disease.

Table 1. Association of APOE polymorphism with obesity, lipid profile, diabetic nephropathy,

atherosclerosis and cardiovascular parameters.

ApoE polymorhism p ApoE polymorhism p

Obesity parameters Lipid profile parameters

Body Weight (kg) 0.31 Total cholesterol (mg/dl) 0.28

BMI (kg/m2) 0.02 LDL - cholesterol (mg/dl) 0.19

Waist circumference (cm) 0.04 HDL - cholesterol (mg/dl) 0.67

Obesity (BMI>30 kg/m2) 0.03 Triglycerides (mg/dl) 0.35

Total Fat Mass (kg) 0.03 Ox-LDL (U/l) 0.21

Overhydration (kg) 0.03

Plasma adiponectin (μg/ml) 0.007

Kidney Disease parameters Atherosclerosis parameters

Urea (mg/dL) 0.005 cIMT (mm) 0.99

Crea (mg/dL) 0.005 Presence of plaque 0.34

eGFR (ml/min/1.73m2) <0.001 History of cardiovascular events

Protein/creatinine in urine 0.02 Myocardial infraction 0.025

Albumin/creatinine in urine (mg/g) 0.04 Stroke 0.002

aP values of Chi-square or Kruskal-Wallis test for differences of variables among APOE genetic

variations.

Figure 1. Mean values of eGFR in the three different APOE isoforms.

Genetic Variants of APOE polymorphism

• E2=e2e3 • E3=e3e3

• E4= e3/e4 and e4/e4

• e2e4 carriers(n=2) were excluded from the analysis

Roumeliotis S : st_roumeliotis@hotmail.com

Tavridou A: annatavr@gmail.com

Roumeliotis A: a_roumeliotis@hotmail.com

Theodoridis M: cymarth@otenet.gr

Panagoutsos S: spanagou@med.duth.gr

Pasadakis P: ploumis@med.duth.gr

THE ROLE OF PPARΓ IN ENDOPLASMIC RETICULUM STRESS IN

PODOCYTE INJURY INDUCED BY PALMITIC ACID

Adriana Izquierdo-Lahuerta (1) Esther Paniagua (1), Feng Zeng (1, 2), Patricia Corrales (1), Ángela M. Valverde (3), Richard Coward (4), Gema Medina-Gómez (1)

(1) Universidad Rey Juan Carlos, Área de Bioquímica, y Genética Molecular, Dpto. Ciencias Básicas de la Salud. Avda. de Atenas s/n. 28922, Alcorcón, Madrid, España.(2) Huadong Medical Institute of

Biotechniques, 293 East Zhongshan Road, Nanjing 210002, P.R.China.(3) Instituto de Investigaciones

Biomédicas Alberto Sols (CSIC/UAM), 28029 Madrid, Spain.(4) School of Clinical Sciences, Dorothy Hodgkin Building, University of Bristol, Bristol BS1 3NY, UK

Changes in lifestyle and dietary habits have raised the overall incidence of obesity. Obese patients

often suffer other comorbidities such as hypertension, heart disease, dyslipidaemia, type 2

diabetes and renal disease. Recently, evidences suggest that renal lipid accumulation leads to

glomerular damage and more specifically, whether this accumulation produces podocyte

dysfunction. An imbalance between protein load and folding capacity is referred to as

endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible

chaperons, as glucose-regulated proteins (GRPs). Recently, the subfamily of the nuclear receptor

transcription factors peroxisome proliferator activated receptors (PPARs) has been increasingly

recognized as a key player in the pathogenesis of Metabolic Syndrome (MetS) and its renal

complications. The aim of this study was to analyze the role of PPARγ isoform in the maintenance

of the integrity of the podocyte and the involvement of ER stress in podocyte injury induced by

palmitic acid (PA) and/or High Glucose (HG). We have used two lines of mouse podocytes (WT

and PPARγ knockdown) treated with PA (500μM) and/or HG (25mM) with/without the

thiazolidinedione Pioglitazone (Pio) (0.1 μm) for 24h. PA treatment produced oxidative stress and

ER stress through PERK pathway with increased expression of CCAAT-enhancer-binding protein

homologous protein (CHOP). Furthermore rearrangements of the actin cytoskeleton by PA was

observed in the WT podocyte, but these effects were significant greater in podocytes PPARγ

knockdown. Pio prevented PA-induced injury in WT podocyte, but this injury did not ameliorated

in podocytes PPARγ knockdown. This study suggests a crucial role PPARγ in the protection

against ER stress in the podocyte injury induced by Palmitic acid. Acknowledgements: Fundación

de la SEEN, MINECO (BFU2012- 33594), CAM (S2010/BMD-2423) y Ayudas a la Movilidad

2012 URJC.

THE WEIGHT OF OBESITY AND METABOLIC SYNDROME ON RENAL

FUNCTION

Mangione E., Vigo V., Salvetti G., Santini F., Donadio C. Dept. Clinical Experimental Medicine, University of Pisa, Italy

Background. It is widely accepted that metabolic syndrome, a cluster of cardiovascular risk factors often related with obesity, is associated with an increased risk of chronic kidney disease (CKD).

Obesity itself has been found to be a strong independent risk factor for end stage renal disease even

after adjustment for other major risk factors. The aim of this study is to investigate whether, besides metabolically abnormal obesity (MAO), also

metabolically healthy obesity (MHO) phenotype is associated with increased risk of renal dysfunction.

Patients and methods. Obese patients (80 females, 21 males; BMI 34-68 kg/m2, average 48; mean

age 46 years; serum creatinine 0.3-2.3 mg/dl), admitted to the Department of Endocrinology of Pisa

Hospital between 2009 and 2012. Patients were classified as MHO (n=40) or MAO (n=61) according to the

absence or presence of metabolic syndrome (Adult Treatment Panel-III criteria); clinical and biochemical

parameters were then compared (Student’s T test) in the two groups. Results were presented as

mean+SD. A p value<0.05 was considered significant. A multiple regression model was used to investigate the predictors of decreased GFR (CKD-EPI-Cr equation).

Results. The prevalence of metabolic syndrome was 60.4% (n=61, males=19) (Table). No significant differences were observed in age and BMI between the two groups, whereas MAO phenotype was

associated with significantly higher values of waist circumference and waist-hip ratio. As expected,

also other metabolic parameters (systolic blood pressure, serum glucose, glycated hemoglobin, serum

triglycerides, HDL and LDL and even serum uric acid) were higher in MAO phenotype. Serum creatinine

and urea, markers of renal function, were significantly lower in MHO patients. No significant differences

were observed in eGFR (CKD-EPI-Cr), proteinuria and albuminuria between the two groups.

Multivariate analysis demonstrated that older age (p<0.0001), higher serum uric acid (p=0.0002), lower body surface area (p<0.0001) and higher urine albumin-creatinine ratio (p=0.0037) were

independent risk factors for lower eGFR.

MHO n=40

MAO n=61 p

Male/Female 2/38 19/42 0.001

Age (years) 44+10 48+11 NS

Systolic Blood Pressure (mmHg) 127+15 133+13 0.044

Diastolic Blood Pressure (mmHg) 81+9 83+8 NS

Fasting blood glucose (mg/dl) 86.7+10.6 123.4+61.6 0.0003

Glycated hemoglobin (%) 5.7+0.4 7.7+2.2 0.0001

BMI (kg/m2) 46+7 49+7 NS

Waist circumference (cm) 130+17 137+15 0.018

Hip circumference (cm) 138+14 138+16 NS

Waist/Hip Ratio 0.95+0.12 1.00+0.09 0.009

Serum uric acid (mg/dl) 5.3+1.2 6.1+1.6 0.0064

Triglycerides (mg/dl) 110+38 186+108 0.0001

Total Cholesterol (mg/dl) 186+30 197+40 NS

LDL-Cholesterol (mg/dl) 114+24 127+35 0.049

HDL-Cholesterol (mg/dl) 52+12 42+11 0.00002

Serum Creatinine (mg/dl) 0.68+0.16 0.79+0.29 0.043

Blood urea (mg/dl) 27.8+7.8 34.4+15.2 0.017

CKD-EPI-Cr (ml/min/1.73 m2) 104.8+16.4 98.3+20.5 NS

Proteinuria 24h (mg) 262.0+223.3 376.8+612.9 NS

Albuminuria (mg/g creatininuria) 11.2+19.5 44.3+142.5 NS

ERRORS IN THE ESTIMATION OF RENAL FUNCTION DECLINE IN

CLINICAL RESEARCH.

Aldea-Perona A

1, Luis-Lima S

2, González A

1, Rinne F

2, Delgado P

1, Miquel R

1, Negrín N

2, Pérez-

Carreño E1, Cervino M

1, Lorenzo V

1 and Porrini E

2

1Hospital Universitario de Canarias, Tenerife, Spain; 2University of La Laguna, Tenerife, Spain; Background: Creatinine- and/or cystatin-C based formulas are neither accurate nor precise in the

prediction of renal function. In clinical trials in which renal function decline over time is the main

outcome the use of formulas can be misleading.

Aim: To compare the decline of renal function using eGFR (estimated glomerular filtration rate) and

mGFR (measured glomerular filtration rate).

Method: NEFROVID clinical trial is a study aimed at evaluating the impact of calcifediol or

paricalcitol or standard therapy in reducing proteinuria in patients with CKD. In a sub-group of

patients, GFR was measured every three months with the plasma clearance of iohexol. Simultaneously

with the iohexol plasma clearance, creatinine and cistatin-c were measured and formulas representative formulas were calculated. GFR decline over time was determined for mGFR and eGFR

(formulas) in all patients with at least three measurements.

Results: 65 GFR measures were conducted in 22 patients in the clinical trial NEFROVID until 31

th

october 2015. Only 12 patients had at least three measures. The table above shows GFR decline

(ml/min/year) using the gold standard precedure i.e. iohexol (mGFR) and 11 formulas using creatinine and or cystatin-c. Relevant differences in GFR decline are highlighted

Creatinine formulas Cystatin-C formulas Cystatin-Creatinine formulas

Patient

mGFR aMDRD CKD-EPI C-G Rule Effersoe CKD-EPI Rule Hoek CKD-EPI Stevens Ma

1 -5,90

-5,01

-5,34

-7,73

-4,03

-5,22

-10,32

-8,52

-10,53

-6,36

-7,03

-8,09

2 -9,09

-10,61

-11,07

-13,08

-11,03

-11,31

-0,09

0,01

0,07

-4,45

-5,98

-6,26

3 -3,56

-5,41

-5,83

-3,43

-7,50

-5,55

-1,99

-1,65

-1,84

-3,27

-3,92

-4,29

4 -0,64

1,40

1,32

1,46

1,02

1,63

1,82

1,72

2,25

1,33

1,62

1,88

5 -8,39

-7,93

-8,46

-10,53

-5,93

-8,20

-2,73

-2,20

-3,07

-4,86

-6,16

-6,59

6 -3,11

-1,18

-1,26

-3,19

-1,14

-1,25

-1,12

-0,91

-1,27

-0,98

-1,18

-1,31

7 -4,32

-4,11

-4,54

-6,09

-3,50

-3,80

-1,14

-0,80

-1,02

-2,58

-3,26

-3,41

8 -4,38

0,24

0,21

-1,47

1,16

0,35

13,41

11,14

12,46

6,09

5,78

7,18

9 -1,98 -5,49 -5,53 -5,36 -4,72 -6,05 -0,17 -0,10 -0,12 -2,18 -3,03 -3,17

10 -8,85

-13,62 -14,27 -17,68 -12,15 -14,50 -4,83 -4,07 -5,12 -8,51 -10,61 -11,54

11 -27,17 -35,67 -39,74

-30,90 -52,81 -35,27 -51,55 -43,51 -40,56 -40,88 -42,79 -49,70

12 -0,15

-26,70

-29,07

-32,20

-42,29 -27,17 -23,21 -19,55 -19,99 -23,21 -25,95 -29,34

Conclusions: Formulas have a significant error in reflecting real GFR decline. This error can be severe and even indicate false slow, fast or even improvement in renal function over time. Formulas

cannot be used in clinical research.

Funding: Fondos FEDER; Ministerio de Sanidad, Servicios Sociales e Igualdad. ISCIII EC10/248.

THE MEASUREMENT OF BODY CELL MASS ALLOWS TO PREDICT 24-

HOUR URINARY CREATININE EXCRETION AND CREATININE

CLEARANCE IN SEVERELY OBESE PATIENTS

Carlo Donadio, Diego Moriconi, Michele Marchini, Marco Anselmino, Rossana Berta

Post-graduate School of Nephrology, University of Pisa; Bariatric Surgery, AOUP, Pisa, Italy.

In obese patients the accuracy of prediction of renal function by means of different formulas based on

serum creatinine (PCr) and anthropometric data is quite low. The inaccuracy in the prediction of

urinary creatinine excretion (UCr) is probably the major cause of error of the prediction formulas. In particular, Cockcroft & Gault (CG) formula overestimates UCr and hence creatinine clearance (CCr)

in obese patients.

The aim of this study was to evaluate if the value of body cell mass (BCM), which is the body

compartment more strictly correlated with muscle mass, allows to predict UCr and then CCr by

means of a formula based on BCM and Pcr. The increased volume of fat mass, which in severely

obese patients affects the relationship between body weight (BW) and UCr, should not influence this formula.

Patients and methods Fifty-one patients (35 women), aged 26-66 years (mean 48.0), severely obese (BW 92-205 kg, m

127.0; BMI 35.4-73.4 kg/m2, m 45.5),with normal values of PCr (0.58-1.06 mg/dl, m 0.77). All

patients were scheduled for bariatric surgery. The examined parameters were: PCr; UCr (length of

urine collection 2 hrs); CCr measured (m-CCr) with the standard formula as UCr x V / PCr,; CCr was also predicted by means of CG formula (CG-CCr) and by means of Salazar and Corcoran formula

(S&C-CCr). GFR was predicted using simplified MDRD formula (IDMS) and with CKD-EPI

formula. In the mean time the value of BCM was measured using a single frequency tetrapolar impedance analyzer (Akern).

Results 24h-UCr was between 1080 and 3038 mg, m 1823. BCM value was 25-74 kg (m 37.7 kg men, 32 kg

women). A strict linear correlation was found between 24h-UCr and BCM (r=0.800), which was

closer than that between 24h-UCr and BW (r=0.654); no significant correlation was found between

24h-UCr and FM. 24h-UCr and CCr were predicted from the individual values of BCM (BCM-CCr) (Donadio C, et al. Creatinine clearance predicted from body cell mass is a good indicator of renal

function. Kidney International 63: S166-S168, 1997).

The results of the various measures, reported in the following table, indicate that the difference

between BCM-CCr and m-CCr is insignificant, while quite relevant differences were found with the

results of the other prediction formulas. Furthermore, BCM-CCr values showed a good correlation with m-CCr (r=0.717, p<0.0001) and the concordance between the two measures resulted similar for

all explored values of renal function.

ml/min m CCr BCM CCr C&G CCr S&C CCr MDRD GFR CKD-EPI GFR

mean ± SD 166±44 170±40 195±64 147±41 121±29 131±29

mean difference = +4.2 + 29.0 -19.2 -44.1 -34.6

statistical

significance

= NS <0.0001 <0.0001 <0.0001 <0.0001

In conclusion, in severely obese patients urinary creatinine excretion and creatinine clearance can be

accurately predicted from the measurement of body cell mass combined with serum creatinine.

IOHEXOL PLASMA CLEARANCE, A SIMPLIFIED AND RELIABLE

METHOD TO MEASURE RENAL FUNCTION IN CONSCIOUS MICE

Luis-Lima S

1, Rodríguez-Rodríguez AE

2, Salido E

3, Martin-Higueras C

3, Sierra-Ramos C

4, Alvarez de

la Rosa D4, Porrini E

1.

1 Centre for Biomedical Research of the Canary Islands (CIBICAN). University of La Laguna,

Tenerife, Spain. 2 Research Unit, University Hospital of the Canary Islands, Tenerife, Spain.

3 Department of Pathology, Centre for Biomedical Research on Rare Diseases (CIBERER)

4 Department of Physiology and Institute of Biomedical Technologies, University of La Laguna,

Tenerife, Spain (D.A.D.l.R.)

Introduction Mice models are useful to study the pathogenesis of renal disease. Currently, glomerular filtration rate

(GFR), the best index to evaluate renal function, is estimated in mice by creatinine clearance (CrCl) or

measured by clearance of radioactive markers in blood. However, creatinine determination is neither accurate nor precise, leading an overestimation of true creatinine (1-2). Aditionally, renal secretion of

creatinine in rodents implies an additional inaccuracy to estimate GFR (3). On the other hand, the use

of radiactive labeled markers (3H or

14C inulin) is cumbersome and implies to manage animals under

anaesthesia, which can affect GFR in an unpredictable manner. Other markers (51

Cr-EDTA and

[125

I]iothalamate) have been utilized in conscious mice, but it requires safety regulations related on the

management of radioactivity. Thus, a more simple and practical method to measure renal function in

mice is urgently needed. We aimed to set up the iohexol plasma clearance technique by a simplified method and test its reliability in mice with moderate and severe CKD.

Material And Methods We studied two groups of mice. i.- testing group: 14 C57BL/6J mice (7 males and 7 female), ii.-

validation group: 14 C57BL/6J mice (7 males and 7 female) both six weeks old. For clearance

measurements, 6,47 mg iohexol (corresponding to 100 µl Omipaque 300 solution, diluted 1:10, GE

Healthcare) were injected intravenously into the mouse tail vein. The mice were slightly anesthetized with isoflurane (2.5%). Five uL of blood samples were collected by a heparinized capillary tube and

analysed by HPLC analysis. For the testing group, based on Sallstrom approach (4), iohexol

concentrations were fitted by a nonlinear regression according to a 2-compartment model (reference method) at 1, 3, 7, 10, 15, 35, 55, and 75 minutes after iohexol injection (figure 1). The plasma

concentrations of iohexol were recalculated from blood levels using the formula: Cplasma= Cblood/1-

Hct where Hct is hematocrit of mouse. Iohexol reference clearance (CL2) was determined as Dose/AUC, where AUC is the area under the plasma-time concentration curve. For the validation

group, iohexol concentrations were fitted by a slope-intercept method according to a one-

compartment model (simplified method) during the elimination phase at 15, 35, 55 and 75 minutes

(figure 2). This AUC underestimates the true area value, which leads to an overestimated clearance (CL1). So, a correction factor (CF) represented by the mean ratio between CL1 and CL2 was

calculated for the testing group, obtaining the true clearance value. Experimental animals: To

evaluate the reliability of the method in reflecting changes in renal function, two groups of animals with CKD were studied: i.- 8 hyperoxaluric male mice (Agxt-/-) twelve weeks old fed 1%

hydroxyproline diet during 10 days to induce nephrocalcinosis. ii.- 5 heminephrectomized male

mice (C57BL6/J) eight weeks old. The iohexol plasma clearance was performed 2-w surgery.

Table 1: Correction factor calculated as the ratio between clearance values determined by 1-

compartment (simplified method) and 2-compartment model (reference method) approaches.

Results. For most of cases, the GFR was greater when estimated using the 1-compartment model method than the two-compartment method (table 1). The CF averaged 0.87 for male and 0.91 for

female (table 1). When GFR in the validation group (one-compartment model) were recalculated by

using the CF yielded corrected GFR values similar to the reference clearance CL2 (table 1). So, the correction factor was proven to be reliable and confirm the suitability of the simplified method and the

absence of systematic errors after the introduction of the CF. Control group showed a GFR of 13.6±2.6

ml/min/gr (mean±SD) for male, and 10.6±1.6 for female, in line with previous studies (4). Heminephrectomized group showed a GFR of 5,0±1,5 ml/min/gr and hiperoxaluric 3,7±1,5 ml/min/gr.

(figure 3)

Conclusions. We set up a simple and reliable approach to measure GFR in conscious mice. This method is useful for studies where renal function is an important outcome in mice models, such as

ERC, DM or obesity. Moreover, it makes possible to evaluate renal function by repeated measurement

of GFR, which allows the assessment of renal function changes over time.

(1) Meyer MH, Meyer RA Jr, Gray RW, Irwin RL. Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography. Anal

Biochem. 1985;144(1):285-90.

(2) Dunn SR1, 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(5):1959-67. (3) 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(6):519-26. (4) Sällström J, Fridén M. Simultaneous determination of renal plasma flow and glomerular filtration

rate in conscious mice using dual bolus injection. J Pharmacol Toxicol Methods. 2013;67(3):187-93.

Mouse (sex)

Weight (gr)

Reference method (ul/min)

Simplified method (ul/min)

Reference method

(ul/min/g)

Simplified method

(ul/min/g)

CF Simplified corrected (ul/min/g)

1 (M) 20 220 230 11 11.5 0.96 10

2(M) 22 244 270 11.1 12.3 0.97 10.6

3(M) 22 361 400 16.4 18.2 0.97 15.8

4(M) 22 299 370 13.6 16.8 0.81 14.6

5(M) 18 162 180 9 10 0.9 8.7

6(M) 18 213 270 11.8 15 0.79 13

7(M) 22 330 410 15 18.6 0.8 16.1

Mean ±SD 20.6±2 261.3±71 304.3±89.4 12.6±2.6 14.6±3.4 0.87 12.7±3

1 (F) 18 142 160 7.9 8.9 0.89 8.1

2(F) 18 169 200 9.4 11.1 0.85 10.1

3(F) 18 228 260 12.7 14.4 0.88 13.1

4(F) 16 189 200 11.8 12.5 0.95 11.4

5(F) 18 226 250 12.6 13.9 0.9 12.6

6(F) 18 197 200 10.9 11.1 0.99 10.1

7(F) 18 183 200 10.2 11.1 0.92 10.8

Mean ±SD 17.7±0.7 190.8±37.0 214.0±41.0 10.9±2,1 12.2±2.2 0.91 10.8±1.7