Semmelweis University, School of PhD Studies Experimental Medical Sciences

PhD Thesis

NOVEL EXPERIMENTAL DATA IN BLOOD

PRESSURE REGULATION

Dr. Barta Péter

Supervisor: Prof. Dr. Losonczy György

Semmelweis University, Department of Pulmonology

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TABLE OF CONTENT I. P REFACE 3 I I . A GENE EXPRESSION ANALYSIS IN RAT K IDNEY FOLLOWING

H IGH AND LO W SALT IN T A K E 1. Introduction

1.1. Dietary salt intake and blood pressure 5 1.2. Renal function in salt-sensitive and resistant hypertension 9 1.3. Molecular mechanisms of sodium-related renal effects 14 1.4. Genomic approach in sodium homeostasis and renal research 20

2. Hypothesis and Aims 23 3. Methods

3.1. Animals 24 3.2 Tissue preparation and oligonucleotide microarray hybridisations 24 3.3. Data ana lysis 25 3.4. Expression verification 29 3.5. Statistics 30

4. Results 4.1. Oligonucleotide microarray and differentially regulated genes 31 4.2. Protein families of differentially regulated genes 34

4.3 Verification of the microarray results 36 5. Discussion and Conclusions 37 6. Summary 47

III. AT 1 RE C E P T O R AGONISTIC ANTIBODIES FR O M P REECLAMPTIC PATIENTS STIMULATE NADPH OXIDASE 1. Introduction

1.1. Preeclampsia and the renin-angiotensin system 48 1.2. AT1 receptor agonistic auto-antibodies in preeclampsia 50 1.3. ROS, NAD(P)H oxidase and NF-?B in preeclampsia 53

2. Hypothesis and Aims 57 3. Methods

3.1. Study subjects and autoantibody isolation 58 3.2. Cell culture 58 3.3. Intracellular redox state assay 59 3.4. Cytochrome C assay 60 3.5. Western blots, electrophoretic mobility shift assay,

immunohistochemistry, and immunofluorescence 60 3.6. TaqMan reverse transcription–polymerase chain reaction 61 3.7. Oxidative fluorescent microtopography 62 3.8. Statistics 62

4. Results 4.1. AT1-AA and Ang II induce ROS generation in VSMCs and trophoblasts via NADPH oxidase 63 4.2. Upregulation of NADPH oxidase by AT1-AA and Ang II 66 4.3. AT1-AA and Ang II cause increased NF-?B expression 66 4.4. Elevated ROS production, NADPH oxidase expression

and NF-?B activity in preeclamptic placentas 69 5. Discussion and Conclusions 73 6. Summary 81

IV. REFERENCES 82 V. AKNOWLEDGEMENTS 95

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PREFACE

Hypertension as a determinant of human morbidity and mortality was first

described more then 100 years ago. An association of elevated sodium

consumption, renin-angiotensin-aldosterone system alterations and renal

function disturbances with high blood pressure has been suggested ever since.

The renin-angiotensin system has been also long implicated in the human

pregnancy-specific form of hypertension, preeclampsia. Organ dysfunction in

preeclampsia and salt sensitivity hypertension are not confined merely to

blood pressure. Association of salt load with insulin resistance, accelerated

nephrosclerosis, increased cardiovascular damage or asthma suggests that

physiologic and pathophysiologic responses elicited by sodium-chloride are

systemic and rather complex. Preeclampsia is also a syndrome in that

virtually every organ system can be affected. Several different mechanisms

have been suggested, but the underlying cause of the disease is still unknown.

Despite extensive research we currently do not understand the molecular

mechanisms behind most of the sodium related phys iologic and

pathophysiologic effects and the genetic/genomic determinants of salt

sensitivity. Accordingly, preeclampsia has remained a disease of theories. Of

note, recent studies have indicated the importance of the local renin

angiotensin system, elevated oxidative stress, impaired endothelial function,

insulin resistance and vascular inflammation in the development of

hypertension and widespread organ damage both in preeclampsia and after

elevated salt intake.

This thesis introduces novel experimental data about the pathogenesis of

hypertension, focusing on salt-related renal effects and the pathogenesis of

preeclampsia syndrome. The first part discusses experiments investigating

sodium-chloride induced renal effects. We performed microrray gene

expression profiling on rat kidneys to explore novel genes and pathways in

renal adaptation after elevated sodium intake. We also aimed to show the

feasibility of this approach in renal, salt related research. The results of the

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experiments are summarized in conjuction with the summary of current

scientific evidence in this field. Some of the differentially expressed genes

regarding their established and possible physiologic significance are finally

discussed in detail.

The second part tests the novel hypothesis that angiotensin receptor 1

activation by agonsitic autoantibodies causes NADPH oxidase upregulation,

NF-?B transactivation and that this activation is a component of the

preeclampsia syndrome. Autoantibodies acting on angiotensin 1-receptors

may explain several pathophysiologic changes observed in preeclampsia. Our

current in vitro and in situ data corroborate this view indicating significance

for this activation cascade in preeclampsia.

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A GENE EXPRESSION ANALYSIS IN RAT KIDNEY

FOLLOWING HIGH AND LOW SALT INTAKE

INTRODUCTION

Dietary salt intake and blood pressure

The average sodium intake in the western societies is approximately 150 mmol

per day, which is equivalent to 3.5g of sodium or 8.7g of sodium chloride (salt). Sodium

excess is intimately involved in the pathogenesis of primary hypertension, playing

necessary but not sufficient role. Chloride, and not just sodium, may be involved in

causing hypertension. In rat models of sodium-dependent hypertension, hypertenion

could be induced with sodium chloride but not with sodium bicarbonate or ascorbate. In

people too, blood pressure raises more with NaCl than with nonchloride salts of sodium.

The issue, however, is largely academic, since chloride is tha major anion accompanying

sodium in the diet and in body fluids131. To avoid confusing the terms salt and sodium

(which is 40% of sodium chloride), only sodium will be used in this text (as it is on food

labels). This section focuses on sodium excess, but note that experimental data and

epidemiologic evidence support a close association between hypertension and a high ratio

of sodium to potassium intake132. However, most evidence favors a primary role for

sodium excess. Excess dietary sodium intake induces hypertension by increasing fluid

volume and preload, the reby increasing cardiac output, but sodium excess may increase

blood pressure in multiple other ways as well; it also effects vascular reac tivity and renal

function133. High sodium intake increases intracellular calcium level and plasma

catecholamines, causes worsening of insulin resistance and paradoxical rise in atrial

natriuretic peptide133. Additional damages may be associated with high sodium intake

that is not mediated by the effects of sodium on blood pressure. Both in animals and in

humans, a high intake of sodium increases the risk of stroke134, independent of the effect

on blood pressure. Other adverse effects of sodium excess include left ventricular

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hypertrophy and more rapid deterioration of renal function through hyperfiltration135.

Both osteoporosis and renal stones may accompany the increase in calcium excretion that

occurs with increased sodium excretion. As if these damages were no t enough, dietary

sodium intake is also correlated with stomach cancer mortality136 and asthma137

prevalence.

Guidelines1 recommend reducing the daily dietary sodium intake to 100 mmol

(equivalent to 2.3 g of sodium or 5.8 g of sodium chloride) or less. In the 1940s, Duke

University clinician Wallace Kempner demonstrated that he could successfully treat

hypertensive patients with a low-salt, rice-and-peaches diet. For years Kempner's

regimen was the only non-surgical treatment for severe hypertension. Whe ther it was low

salt that explained the diet's effect is still debatable, however. Kempner's regimen was

also extraordinarily low in calories and fat and high in potassium, factors that themselves

are now known to lower blood pressure (BP)2. The first most compelling evidence

against salt came from ecologic studies, identifying an association between salt intake and

blood pressure. The findings led researchers to speculate, that humans evolved in an

environment where salt was scarce, and so those who survived were those best adapted to

retaining salt. This trait would have been preserved even though we now live in an

environment of salt abundance. By this logic, the appropriate intake of salt is that of the

primitive societies - a few grams a day - and all industrialized societies eat far too much

and might pay for it in heart disease and stroke. However, the potentially fatal flaw in

ecologic studies was always the number of variables other than the amount of salt intake

that might differ between the populations and explain the relevant effect. With

recognition of the inherent weakness of ecological studies, attempts have been made to

relate sodium intake to blood pressure in epidemiological studies that compare

individuals of a more or less homogenous socioocological population3. The first large

intrapopulation study conducted was the Scottish Heart Health Study, launched in 1984.

Potassium seemed to have a beneficial effect on blood pressure however, sodium had no

effect. Intersalt study4 was designed specifically to resolve the contradiction between

ecologic and intrapopulation studies. Overall, no association between sodium intake and

blood pressure was identified by the Intersalt investigators in an analysis limited to the 48

centers consuming > 100 mmol sodium a day (Figure 1, left side). When the additional 4

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centers consuming 0.2 to 50 mmol sodium / 24 hours (“primitive” societies) were also

included, a significant positive association of sodium to blood pressure emerged (Figure

1, right side). Intersalt study estimated then cutting sodium intake by 70 mmol a day

would reduce the average rise in blood pressure between the ages of 25 and 55 by 9/4.5

mmHg.

FIGURE 1: Overall, no association between sodium intake and blood

pressure was identified by the Intersalt investigators in an analysis limited

to the 48 centers consuming >100 mmol/24 hours (left side). When the 4

centers consuming 0.2 to 50 mmol/24 hours were included, a significant

positive association of sodium to blood pressure emerged (right side).

A series of clinical trials followed. However, conduction of these studies proved

to be extremely difficult. Choosing low-salt foods, for instance, inevitably led to

changing other nutrients, as well, such as potassium, fiber, and calories. Results of these

clinical trials have been inconsistent, which led to a sequence of meta-analyses designed

to determine the most likely overall effect of dietary salt for a population. The most

rigorous meta-analyses are in general agreement5, showing a 3 to 5 mm Hg systolic and

approximately 1 mmHg diastolic change in pressure associated with a 75 to 100 mmol/24

hour difference in sodium intake among hypertensive and older subjects. The effect on

younger and normotensive subjects is less: 2 to 3 mmHg for systolic and < 1 mmHg for

diastolic blood pressure. Recent large trials also supported the association of salt intake

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and blood pressure in humans. The National Heart, Lung, and Blood Institute (NHLBI,

NIH, Bethesda, USA) funded Trials of Hypertension Prevention Phase II (TOHP II) was

published in March 1997. TOHP II, a 3-year clinical trial of 2400 people with "high

normal" blood pressure, coordinated by Hennekens et al. at Harvard Medical School,

found that a 70 mmol reduction in daily sodium intake correlated with a 2.9/1.6-mmHg

drop in blood pressure after 6 months2. Trial of Nonpharmacologic Interventions in the

Elderly (TONE) was a clinical trial of the efficacy of weight loss and/or sodium reduction

in controlling BP after withdrawal of drug therapy in patients with a BP < 145/85 mmHg

on 1 antihypertensive medication6. This study showed that antihypertensive medication

can be safely withdrawn in older persons without clinical evidence of cardiovascular

disease who do not have diastolic pressure greater than or equal to 150/90 mmHg at

withdrawal, providing that good BP control can be maintained with nonpharmacologic

therapy. The Dietary Approaches to Stop Hypertension (DASH) trial from 1997

demonstrated that a diet that emphasizes fruits, vegetables, and low-fat dairy products,

that includes whole grains, poultry, fish, and nuts, that contains only small amounts of red

meat, sweets, and sugar-containing beverages, and that contains decreased amounts of

total and saturated fat and cholesterol lowers blood pressure substantially both in people

with hypertension and those without hypertension, as compared with a typical diet in the

United States7. In 3 weeks, the diet reduced blood pressure by 5.5/3.0 mmHg in subjects

with mild hypertension and 11.4/5.5 mmHg in hypertensives, a benefit surpassing what

could be achieved by medication. Sacks et al. undertook this trial to address the question,

whether reducing the level of sodium from the average intake (150 mmol / day) in the

United States to below the currently recommended upper limit of 100 mmol per day lower

blood pressure more than reducing the sodium level only to the recommended limit.

Blood pressure could be lowered in the consumers of either a diet that is typical in

western societies by reducing the sodium intake from approximately 140 mmol per day to

an intermediate level of approximately 100 mmol per day (the cur rently recommended

upper limit) or from this level to a still lower level of 65 mmol per day.

A favorable effect of sodium restriction on various intermediate physiological

variables has been shown, particularly, but not exclusively, for blood pressure and other

hemodynamic characteristics. However, there is convincing evidence of adverse effects

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of a low-sodium diet on important physiological characteris tics, including the

sympathetic system and the renin-angiotensin system in particular8. Brief, severe

restriction raises plasma catecholamines, whereas moderate restriction lowers plasma

catecholamines. In addition, severe and short-term sodium restriction increases serum

LDL cholesterol and triglicerides and induce insulin resistance. Summary of these effects

on overall mortality and morbidity are not clear. Therefore several investigators have

been long opposing recommendations for general salt restrictio ns. To find out whether

dietary sodium is associated with mortality in a general population, Alderman et al.

examined the relation of sodium intake, measured in 1971–75, to all-cause and

cardiovascular-disease (CVD) mortality, up to mid-1992, among participants in the first

National Health and Nutrition Examination Survey (NHANES I). Surprisingly, their main

findings are that dietary sodium intake is inversely associated with all-cause and CVD

mortality, and that dietary sodium/calorie ratio is directly associated with mortality rates9.

These associations, although small, are significant and independent, both of each other

and of other factors known to influence mortality.

In summary, little controversy surrounds much of what is known about the effects

of dietary sodium on blood pressure. Few populations were found whose levels of sodium

intake were in the 50 to 100 mmol / day range, wherein the treshold for sodium effect on

blood pressure likely resides. Substantial variation in intake (75 to 100 mmol / 24 hours)

can produce measurable changes. The blood pressure reducing effect appears to be more

substantial in older subjects and in those with higher pressures. However, based on

genetic, behavioral and environmental heterogeneity different individuals may have

different optimum sodium intakes. Potential hazards of sodium restriction indicated by an

inverse association of sodium intake with mortality and blood pressure independent

effects of dietary sodium suggest that little is known about the widespread roles of

sodium in homeostasis regulation.

Renal function in salt-sensitive and resistant hypertension

In the early 1900s Starling clarified the concept that volume homeostasis and

blood pressure regulation are closely linked and emphasized the importance of renal fluid

retention in maintaining arterial pressure in circumstances associated with circulatory

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depression, such as heart failure 10. The role of renal sodium excretion in regulating

arterial pressure remained rather vague, however, until the 1960s when Guyton11 and

Burst and Borst-deGeus working independently clearly articulated the idea that long-term

blood pressure regulation is inextricably link ed to renal excretory function (Figure 2).

FIGURE 2. Basic renal-body fluid feedback mechanisms for long-term

regulation of blood pressure and body fluid volumes. (Adapted from ref.

10)

This concept was expressed quantitatively by Guyton and Coleman12. Hall et al.10

quantified the importance of pressure natriuresis in hypertension by comparing the

chronic blood pressure and renal effects of various antinatriuretic and vasoconstricting

hormones in dogs in which renal perfusion pressure was either permitted to increase or

servo-controlled at the normal level to prevent pressure natriuresis. A shift of renal-

pressure natriuresis initiated and sustained the hypertension even after chronic

FIGURE 3. Long-term effects of a powerful vasoconstrictor that has a

relatively weak effect in impairing pressure natriuresis. The normal curve

(solid line) is compared with the vasoconstrictor curve (dashed line).

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Initially the vasoconstrictor raises blood pressure (from point A to point

B) above the renal set point for sodium balance. Increased arterial

pressure, however, causes a transient natriuresis and decreases

extracellular fluid volume until arterial pressure eventually stabilizes at a

level (point C) at which sodium intake and output are balanced. (Adapted

from ref. 10)

administration of „primary” vasoconstrictors such as vasopressin and norepinephrine

(Figure 3). These experiments demonstrated causative role for altered pressure-

natriuresis relationship in the development of various hypertension forms. Series of renal

cross transplantation studies between spontaneously hypertensive rats (SHR), Wistar-

Kyoto rats (WKY), Milan and Dahl rats gave further evidence for the role of kidneys in

hypertension13. In hypertensive, chronically dialyzed patients successful kidney

transplantation from normotensive, healthy donor often leads to the disappearance of

hypertens ion.

Renal mechanisms determining sensitivity of blood pressure to excess sodium

have been in focus of interest since decades. Human subjects are classified as salt-

sensitive or salt-resistant based on their blood pressure response to changes in NaCl

intake14. Approximately 40% of patients with essential hypertension seem to be of the

salt-sensitive type, when salt sensitivity is defined as a blood pressure increase of at least

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10 mmHg during a 250 mol/day NaCl diet, compared with a diet containing only 10–30

mol/day of salt. Salt-sensitivity of blood pressure has been recently shown to be a major

cardiovascular risk factor independent of blood pressure, and it predicts higher mortality

in normotensive and hypertensive subjects15. Kidney transplantation exper iments between

salt-sensitive and salt-resistant rats suggested that salt sensitivity of blood pressure is due

to local or humoral renal factors that impairs either natriuresis or vasodilatory adaptive

responses to volume overload16. Kimura and Brenner17 have extended this approach and

described the various pressure–natriuresis curves in sodium-sensitive and sodium-

resistant forms of secondary hypertension. They proposed three major renal mechanisms

leading to the development of hypertension: an increased pre-glomerular vascular

resistance, a decrease in whole kidney ultrafiltration, and an increase in tubular sodium

reabsorption. In addition, they suggest that pre-glomerular vasoconstriction leads to a

salt-resistant hypertension whereas a reduced nephron mass and alterations of renal

sodium handling result in the development of salt-sensitive forms of hypertension. A

parallel, rightward shift of the pressure-natriuresis relationship is characteristic for a salt-

resistant form of hypertension. A decreased slope in the pressure-natriuresis relationship

indicates the presence of a salt-sensitive form of hypertension. The underlying

abnormality in sodium excretion may be either intrinsic or extrinsic to the kidneys. In

hypertensive Dahl salt-sensitive rats, blunted pressure-natriuresis relationships exist and

are intrinsic to the kidneys themselves 18. In contrast, in DOCA-salt hypertensive Sabra

(SBH/y) rats the decreased slope of the pressure-diuresis-natriuresis relationship appears

more a factor maintaining the hypertension rather than an induction mechanism19.

Contrary to salt resistants, in salt-sensitive rats a decrease of RBF and GFR20, elevations

in sympathetic nerve activity and increased vasopressin levels21 have also been observed

after NaCl- loading.

In humans, abundant evidence from a variety of studies suggests a renal

abnormality or multiple alterations in renal function in hypertension and also in first

degree relatives of patients with essential hypertension22. Studies conducted in

hypertensive men demonstrated racial differences in the response of glomerular filtration

rate to dietary sodium loading. Even though no significant differences in blood pressure

response to the salt load were seen when black and white subjects were compared, the

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former had a significant increase in glomerular filtration, suggesting hyperfiltration23. It

should be emphasized that these indirect reports did not describe differences in blood

pressure response to sodium in the subjects studied. Another group found that the salt-

sensit ive subjects - nearly all blacks - demonstrated a decrease in renal blood flow in

response to the high-salt diet, whereas the salt-resistant group showed an increase in renal

blood flow24. In a study of 22 Italian hypertensive patients during the high-salt diet renal

plasma flow decreased, filtration fraction and intraglomerular pressure increased in the

salt-sensitive but not in the salt-resistant group. Furthermore, salt sensitive animals

exhibited significantly greater albumin excretion25. The investigators of both studies

interpreted these findings as indicative of a selective increase in glomerular capillary

pressure with high-salt intake, which is specific to the salt-sensitive subjects.

Proximal renal sodium handling - independent of changes in renal hemodynamics

- is an important determinant of the alteration in the pressure-natriuresis relationship that

occurs in patients with salt-sensitive hypertension. On high-salt intake, fractional

proximal sodium reabsorption is significantly reduced. However, when subjects are

classified into 3 groups according to tertiles of blood pressure response to altered sodium

intake, those whose blood pressure increase most on high-sodium intake are the ones who

have the least reduction or even a paradoxical increase in fractional proximal sodium

reabsorption25.

Over the last two decades, genetic studies have provided important clues about the

nature of inherited functional defects in renal sodium handling that cause an increase in

blood pressure. Monogenic forms of hypertension have been described that are caused by

well-characterized mutations, most often associated with major alterations in the rate of

renal tubular sodium chloride reabsorption26. In humans, mutations in alpha-adducin,

beta2-adrenergic receptor, beta-subunit of amilorid-sensitive epithelial sodium channel,

11-beta-hydroxy steroid dehydrogenase 2 (11-bHSD2) have been implicated in salt-

sensitive form of hypertension. Most of these mutations affect the distal tubules, and

together probably account for less than 1% of the prevalence of human hypertension.

Aside monogenic forms of hypertension, a number of relatively common genetic variants

appear to be associated with higher blood pressures and increased susceptibility to

hypertension. Although these alterations seem to account for a still small portion of blood

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pressure variability in the population; large majority of these genes encode for proteins

that are either directly involved with sodium transport through the renal tubular epithelia

or with the endocrine/paracrine regulation of renal tubular sodium handling. In contrast to

the monogenic forms affecting the distal tubule, revealed genetic variants cause salt

sensitivity by an increased rate of sodium reabsorption in the proximal tubule. The

Gly460Trp variant of the alpha-adducin gene is associated with an enhanced sodium–

potassium–adenosine tr iphosphatase activity caused by a gain-of- function interaction

between the mutated alpha-adducin molecule and the sodium-potassium pump27. An

increased prevalence of hypertension, enhanced proximal sodium reabsorbition have also

been described in individuals carrying a functional mutation of the glucagon receptor

(GCGR) gene 28. The Arg40Ser variant has recently been found in 3.8% of an unselected

sample of Italian male adult population (n=970), only in the heterozygous condition. This

is associated with reduced receptor affinity for glucagon in liver cells and, in turn, with a

lower secretory rate of its intracellular messenger cAMP. Very recently, single nucleotide

polymorphisms of a G protein–coupled receptor kinase (GRK4) have been associated

with higher activity resulting increased receptor phosphorylation and uncoupling of the

dopamine-1 receptor from its G protein/effector enzyme complex in renal proximal

tubular cells29. Transgenic mice expressing the polymorphic variant develop salt-sensitive

hypertension. Another very interesting candidate gene for salt-sensitive hypertension is

serum-glucocorticoid regulated kinase 1 (SGK1), which stimulates the expression of

epithelial Na+ channels on binding of aldosterone to its own receptor, thus promoting

sodium chloride reabsorption. Two polymorphic variants of the SGK1 gene have been

reported to be associated with higher blood pressures. On the other hand, SGK1-knockout

mice appear to have an impaired ability to decrease urinary sodium excretion on dietary

sodium chloride restriction and display a tendency to lower blood pressure 30.

Molecular mechanisms of sodium related renal effects

Apart from the effects on blood pressure regulation, elevated sodium intake was

associated with cardiovascular and renal changes lead ing to end organ damage 31. Rats on

4% dietary NaCl for 10 weeks display numerous and advanced lesions of the glomeruli,

as well as significant matrix deposition throughout the cortex and interstitial fibrosis. The

glomerular lesions display capillary loop collapse, mesangial matrix expansion,

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sometimes adhesion to Bowman’s capsule and glomerular membrane thickening32.

Several mechanisms has been explored which may be - directly or indirectly - explain

these findings. During the production of concentrated urine, cells of the renal medulla are

subjected to high concentrations of solutes, particularly of NaCl and urea. Hyperosmolal

NaCl and urea are both able to cause DNA breaks, to inhibit cellular protein synthesis

and to induce apoptosis in a dose-dependent ma nner33. However, the underlying

mechanisms appear different and many effects are exerted exclusively by NaCl. Exposure

of a cell to hyperosmolal salt (hypertonicity) results in an immediate increase in ionic

strength inside the cell and double-stranded DNA breaks. Extreme acute hypertonicity

induced by NaCl causes a mitochondrial dysfunction which may also be involved in the

initiation of apoptosis. As a result, the classical response to double-stranded DNA breaks

is induced: apoptosis on one hand, and activation of p53, which in turn opposes apoptosis

and arrests cell cycle progression, on the other hand. The balance of the two opposing

pathways is determined by the degree of hypertonicity. In contrast, hyperosmolar urea

does not increase cellular ionic strength nor causes double-stranded DNA breaks34. The

cells, both in vivo and in cell culture, adapt to these adverse conditions by a number of

mechanisms, including accumulation of a variety of organic osmolytes (betaine, taurine,

sorbitol and myo- inositol)35 and induction of heat shock proteins36. The cellular

accumulation of compatible osmolytes is orchestrated in large part by a transcription

factor named tonicity-responsive enhancer binding protein (TonEBP, also called

NFAT5). TonEBP is stimulated by hypertonicity and, in turn, stimulates transcription of

genes that encode the Na+-myo- inositol cotransporter (SMIT), the Na+-Cl- - betaine

cotransporter (BGT1), and aldose reductase (AR), which are responsible for the cellular

accumulation of myo- inositol, betaine, and sorbitol, respectively. Emerging data suggest

that hypertonicity is also a signal for tissue-specific gene expression. The vasopressin-

regulated urea transporter (UT-A) is exclusively expressed in the renal medulla via

TonEBP37 and plays a key role in accumulation of urea. Moreover, in medullary cell

lines, high NaCl and/or urea induce expression of GADD-45 and GADD-153 (GADD:

„growth arrest and DNA-damage- inducible ”), proteins believed to be involved in DNA

damage repair38. Thus, hypertonicity induces a specific set of gene expression that

determines the phenotype of the renal medulla and allows cells to overcome the stress of

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hypertonicity. Signaling pathways for stimulation of gene transcription in response to

hypertonicity are quite diverse, involving a variety of protein and lipid kinases. Activation

of the p38 MAPK seems necessary for the rapid activation of G2 arrest after acute

hypertonic stress in renal inner medullary epithelial cells, which protects cells from DNA

breaks caused by aberrant mitosis entry. Tyrosine kinases and phosphatidylinositol 3-

kinase also play significant role39. How sodium chloride exerts all these cellular effects is

currently unknown.

Salt intake and salt sensitivity has been associated with increased oxidative

stress40. Increased superoxide production in both vasculature and kidney was extensively

reported in various forms of hypertension in experimental models and humans.

Moreover, a recent report connects excess sodium intake with oxidative stress and

nephrosclerosis in Dahl-sensitive hypertensive rats41. The direct effect of sodium on

oxidative stress in rat, however, was first investigated only in 2003 by Dobrian et al.42.

Their results showed that the ability of thoracic aortic rings to generate superoxide anions

is double in rats on both 2% and 4% dietary NaCl vs. rats on regular sodium diet,

indicating an increase in oxidative stress in the large vessels of sodium- loaded obese

animals. Immunohistochemistry with monoclonal antibodies against nonenal protein

adducts (indicative of oxidative damage in the tissues) in the kidney cortex after high-

sodium intake showed increased free radical production.

Not surprisingly, sodium effects also the major renal autacoids mechanisms

involved in sodium-water homeostasis and blood pressure regulation. Eicosanoid,

nitrogene monoxide (NO), bradykinin, and endothelin production, expression of

cyclooxiganse (COX), disturbed function of the renin-angiotensin and endogen sodium

pump ligand system, altered renal effects of dopamine, ANP and uroguanylin have been

all linked to salt-sensitivity of hypertension. I give a brief summary of the recent

experimental evidence on this field :

The renin-angiotensin system and pressure-natriuresis relationship are closely

coordinated mechanisms that are crucial for maintaining sodium balance and systemic

blood pressure. In mice, loss of angiotensin 1A receptor (AT1A -/-) results in lower blood

pressure, decreased ability to conserve sodium, and an inability to appropriately

concentrate the urine. Recent micropuncture studies indicate that AT1A receptor-deficient

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mice have a complete absence of tubuloglomerular feedback response, a major mediator

of sodium homeostasis and renal blood- flow autoregulation43. While a pressure-

natriuresis relationship is maintained in AT1A-deficient mice, the tight coupling between

changes in blood pressure and changes in sodium excretion is significantly altered. Blood

pressure is regulated in a salt-sensitive manner in the absence of the AT1A receptor. The

leftward shift in the pressure-natriuresis relationship demonstrated by knock-out mice

shows that they are more natriuretic at lower blood pressures than (+/+) mice and they

require a larger change in blood pressure to excrete additional sodium compared with

wild -type controls44.

Bradykinin, the major effector of the kallikrein-kinin system, acts through at least

two receptors in the kidney. The bradykinin type2 (B2) receptor is believed to mediate

most of the physiological functions, including vasodilatation, the natriuresis-diuresis

relationship, and effects on cardiovascular structure. Kininogen-deficient Brown Norway

Katholiek rats45 and mutant mice lacking the B2 receptor exhibit salt-sensitive

hypertension46. On the other hand, transgenic mice overexpressing the human B2 receptor

are hypotensive47,48. Luft et al. investigated the pressure-diuresis-natriuresis mechanism

in B2 receptor knockout mice given a usual and a high-salt diet. However, they found no

differences in blood pressure, pressure-diuresis-natriuresis relationships between the two

strains49.

In human subjects, Higashi and colleagues50 demonstrated that the renal vascular

endothelium of salt-sensitive hypertensives produced lesser amounts of NO compared to

that of salt-resistant hypertensives. In addition, in black hypertensives and salt-sensitive

patients, the plasma NO metabolite levels are decreased under conditions of high-sodium

intake51. This would suggest that impaired NO metabolism is involved in the

pathogenesis of salt-sensitivity.

Renal endothelin (ET) system has been also implicated in salt sensitive and

resistant regulation of blood pressure. Considerable evidence indicates that endothelin B

(ETB) receptors located on renal tubular epithelium inhibit sodium and water

reabsorption, whereas those located on vascular endothelium mediate vasodilation52.

Vassileva et al. have recently proposed that ETB receptors facilitate pressure natriuresis

through increases in medullary blood flow53. Both of these mechanisms appear to account

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for the natriuretic and diuretic actions attributed to ET-1 and the ETB receptor. ETB

receptors within the kidney are upregulated in the DOCA-salt–treated rat model of high-

sodium hypertension54, and renal ET-1 production, as assessed by urinary ET-1 excretion,

is increased during dietary sodium excess. Furthermore, long-term ETB receptor blockade

results in hypertension that is exacerbated by high-sodium intake50.

Several lines of evidence indicate that atrial natriuretic peptide (ANP) is involved

in the pathogenesis of salt-sensitive hypertension55. Chronic blockade of endogenous

ANP with a monoclonal antibody accelerates the development and exacerbates the

severity of hypertension in stroke-prone, spontaneously hypertensive rats (SHR-SP) and

DOCA-salt hypertensive rats56. Dietary sodium chloride supplementation in normotensive

salt-resistant rats is associated with increased plasma ANP levels 57, whereas salt-sensitive

SHR do not increase plasma ANP levels appropriately in response to dietary NaCl

supplementation, thus failing to mount a natriuretic response and normalize blood

pressure in the presence of dietary sodium stress. Furthermore, administration of either

exogenous ANP or the pharmacological inhibition of the endogenous ANP hydrolysis

prevents salt-sensitive hypertension in SHR. Thus, impaired induction of the ANP is

involved in the salt-sensitive blood pressure elevation, at least in SHRs.

Arachidon acid metabolites are essential regulators of tubuloglomerular feedback,

medullary and cortical blood flow, natriuresis and blood pressure. Lo w-salt diet enhances

the expression of COX-2 in the macula densa cells. COX-2-derived prostanoids in the

macula densa may directly mediate the effect of NaCl intake on renin secretion and renin

gene expression. Prostaglandin E2 and I2 (PGE2 and PGI2) have stimulatory effect on

juxtaglomerular epithelioid cells. Sodium exerts direct influence on the renal expression

of different P450 enzymes and their products. Upregulation of thromboxane-prostanoid

receptors (TP-Rs) and thromboxane A2 synthase (TxA2-S) in the kidney cortex during

high sodium intake 58 may contribute to the prohypertensive role of vasoconstrictor PGs in

salt-dependent forms of hypertension, such as Ang II-salt, DOCA-salt, or the Dahl salt-

sensitive rat model of hypertension. In turn, TP-R mimetic causes salt-sensitive

hypertension and renal afferent arteriolar vasoconstriction59. High NaCl intake potentates

increase in blood pressure that occurs during a prolonged infusion of a TP-R mimetic60

19

and can the increase the afferent arteriolar vasoconstriction during local microperfusion

of a TP-R mimetic.

Studies revealed that renal CYP450 omega-hydroxylase and epoxygenase activity

are differentially modified by sodium chloride. Regio- and stereoisomeric

epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are

the major arachidonic acid products of cytochrome P450 monooxygenase metabolism in

the kidney. Epoxygenase metabolites, EETs, make important contributions to integrate

kidney function by directly affecting tubular transport processes, vascular tone, and

cellular proliferation. Renal microvessels dilate in response to 11,12-EET and 14,15-

EET61. In contrast, 20-HETE – an endothelium-derived vasoconstricting factor - is a

potent constrictor of renal arterioles. Additionally, 20-HETE exerts natriuretic action via

direct inhibition of tubular sodium reabsorbtion. Epoxidation of arachidonic acid has been

attributed to members of the CYP2C and CYP2J families, whereas production of 20-

HETE is catalyzed by CYP4A and CYP4F isoforms62. Sodium excess downregulates the

renal expression of CYP4A isoform63, while the renal expression of CYP2C11 and

CYP2C23 are increased by a high-salt diet 64. Although studies in Sprague-Dawley rats

suggest that a sodium- inducible renal epoxygenase has antihypertensive properties,

kidney EET production is inappropriately low during the development of salt-sensitive

hypertension65. Increased CYP expression and 20-HETE synthesis, with exaggerated 20-

HETE vasoconstriction and impairment of renal hemodynamic adjustments to a sodium-

load, participate in the hypertension of SHR and of mice with genetically altered ratios of

CYP4A isoforms60. In contrast, a deficit in the inhibitory effects of 20-HETE on renal

sodium transport contributes to the salt-dependent hypertension of Dahl rats. These

animals have diminished CYP4A protein and 20-HETE contents in the outer renal

medulla66, with increased medullary thick ascending limb chloride transport, a shift in

pressure natriuresis. In addition, the CYP 4A2 genotype cosegregates with salt-sensitive

hypertension in an F2 cross between Dahl salt-sensitive (SS) rats and normotensive Lewis

rats, and also with salt- induced hypertension but not spontaneous hypertension in an F2

cross between SHR and normotensive Brown-Norway rats67. 20-HETE excretion has

been shown to be affected by NaCl balance in humans as well, with values 66% higher

during NaCl-loading than during sodium depletion. Furthermore, this study68 showed that

20

the relationship between 20-HETE and sodium excretion is different between the salt-

sensitive and resistant groups, suggesting that abnormalities in the actions of 20-HETE on

sodium excretion may be a major factor responsible for the salt-sensitive blood pressure

regulation.

Genomic approach in sodium homeostasis and renal research

The successful mapping of many determinants of complex cardiovascular

function was reported for the male F2 rats in Science, November 200169. In this study, all

rats were maintained on a low-salt (0.4%) diet until 5 week of age to allow for normal

development and then placed on a lower salt diet (0.1% NaCl) until 9 wk of age, followed

by 3 week of high (8%) salt diet. Variety of physiological parameters and a number of

morphometric measurements were determined. The result – a cardiovascular genetic map

- has provided the first rough approximation of the regions of the genome that are linked

to the mechanisms of the cardiovascular and renal function related to the homeostatic

control of sodium and water excretion and arterial pressure. Many of the quantitative trait

loci (QTL) involved in determining blood pressure were found to aggregate within broad

regions of specific chromosomes. Specifically, six or more QTL with overlapping 95%

statistical confidence intervals were found on rat chromosomes 1, 2, 7, and 18 of the male

F2 rats. One or more QTL for blood pressure exists on rat chromosome 1, in the vicinity

of the Sa gene. Examination of SHR, WKY and the congenic SHR strain harboring WKY

chromosome 1 has recently revealed70 that the rat chromosome 1 blood pressure QTL

region also influences pressure-natriuresis relationship, salt sensitivity, and most probably

sympathetic activation following salt loading. An other interesting particular aggregate of

QTL on chromosome 18 could be divided into three functional groups: blood pressure

salt sensitivity, plasma lipid concentrations, and renal function71,72. Blood pressure salt

sensitivity within this aggregate of traits accounted for 17% of the overall variance in salt

sensitivity, indicating that other chromosomal regions also contribute importantly to this

trait. This collective profile of phenotypes on chromosome 18 is particularly interesting

because it resembles Syndrome X or the "metabolic syndrome" in humans.

In addition to linkage analysis, techniques of chromosomal sub stitution to inbreed

strains (consomic and congenic rats) have been utilized. Using chromosome substitution

21

technique it became evident, that chromosomes 13, 16, and 18 contain genes of interest to

blood pressure salt sensitivity68. Substitution of chromosome 18 from the Brown-Norway

(BN/Mcw) strain into the salt sensitive (SS/Mcw) genomic background significantly

reduced the level of hypertension and proteinuria achieved in these rats when fed a high

(4%)-salt diet compared with the parental SS/Mcw strains. It is also evident that

chromosomes were revealed that were not apparent in the linkage analysis.

Potential utility of microarray technology in nephrology is currently limited to a

few reports. Microarray has been very recently applied, successfully, to identify new

gene candidates for salt sensitivity and acute rejection. SS.BN13 rats harbor the

chromosome 13 of Brown-Norway rats in the genetic background of SS/Mcw. They

exhibit reduced hypertension, renal interstitial fibrosis, and glomerular sclerosis compared

with the SS/Mcw strain. Renal medulla has been studied to identify differentially

expressed genes between SS.BN13 and SS/Mcw rats induced by a high-salt (4%) diet73.

These microarray studies identified a set of genes (nearly 50) that were expressed

different ially in response to excess ditary sodium intake. Interestingly, it was found that

the differences in the gene expression patterns could be largely attributed to a lack of

response in the salt-sensitive SS/Mcw strain, whereas the genes of the SS.BN13

consistently changed (either increased or decreased) their expression in response to the

high-salt intake. The failure of these genes to respond in the SS/Mcw rats points toward

failed response elements of pathways that account in part for the severe salt- induced

hypertension and renal dysfunction in this strain. On the basis of what is currently known

about these differentially expressed genes, ~30% of them were involved in ion transport,

endothelial and smooth muscle function, belong to hormone/paracrine receptors, and

variously related signal transduction pathways that would be expected to influence

vascular resistance and arterial pressure. Interestingly, another 30% of the genes were

related to pathways of cell growth, proliferation, apoptosis, formation of extracellular

matrix, and cellular stress. In both instances, the directional changes in the expression

were consistent with reduced arterial pressure salt sensitivity, the absence of medullary

interstitial fibrosis and reduced urinary protein excretion seen in the SS.BN13 consomic

rat fed a high-salt diet. The phenotypic impact of the remaining third of the differentially

expressed genes was less apparent, and these results provide stratum for the discovery of

22

novel pathways that may be involved in these events. Intriguing however, that in contrast

to “logical” expectations, only a handful of these differentially expressed genes mapped

to chromosome 13. This indicates that extensive interchromosomal gene interactions

occur that are probably secondarily related to the effects of high-salt intake (e.g., changes

in renin-angiotensin, sympathetic activity, vasopressin, volume changes, etc.). It needs

also to be recognized that there were only 2,000 cDNAs printed on this array and more

than 30,000 genes went undetected in this study.

The use of microarray technology has been introduced in addition in other fields

of nephrology as well. A recent report contains speculations about the application of

microarray technology on peripheral blood lymphocytes in finding more sensitive

markers for acute rejection74. Akalin et al. applied microarray technology on single

biopsy specimens comparing seven patients with acute rejection type II with three

patients without any signs of renal disease75. Among the novel identified genes that were

consistently upregulated in all acute rejection samples was HuMig, the human homologue

of murine Mig. This molecule binds to the CXCR3 receptor, which is present in IL-2-

stimulated T cells, and the authors point out that application of exogenous antiserum

against Mig in mice leads to prolonged skin allograft survival.

23

HYPOTHESIS AND AIMS

The search to elucidate the controllers of sodium and water homeostasis has long

been the focus of physiologists. The tools are becoming available that enable

physiologists to begin moving forward with the task of defining homeostasis at the

genomic level.

In our study, we hypothesized that oligonucleotide microarray (Affymetrix

GeneChip) has utility in identifying novel, une xpected gene targets in rat kidneys in

response to altered salt intake.

24

METHODS

Animals

We elected to rely on Sprague–Dawley (SD) rats rather than a specifically genetic

hypertensive strain since these animals are so ubiquitously and commonly used in animal

studies. Eight male rats weighing 200–220 g were used at 6 weeks of age. The animals

were given two different diets for 7 days. Four rats received a 6% salt diet by weight,

while four rats received a 0.3% salt diet by weight. All other constituents of the diet were

identical. Blood pressure and salt excretion were not measured since this level of salt

intake does not influence blood pressure in rats and since rats achieve salt balance in a

matter of hours76. The rats were sacrificed by cervical dislocation, and the left kidney was

removed. The organ was immediately snap-frozen in liquid nitrogen and stored at -80°C.

Animal experiments were conducted in accordance with institutional (Max Delbrück

Center for Molecular Medicine, Berlin-Buch, Germany) guidelines for the care and use of

laboratory animals.

Tissue preparation and oligonucleotide microarray hybridizations

We analyzed total RNA from four rats in each group independently. Parallel

analysis of gene expression was carried out with commercial rat gene-probe arrays. The

Affymetrix GeneChip RGU34A was derived from the rat UNIGENE collection

representing 8800 genes and expressed sequence tags. The probes have the capacity to

display transcript levels of approximately 8800 rat genes, and expressed sequence tags

(RGU34; Affymetrix, Woodburn Green, UK). Sample labeling and processing was

performed according to the supplier's instructions 77,78: 20 µg total RNA were extracted

and reverse-transcribed to cDNA using oligo dT24 primers containing a T7 RNA

polymerase promoter (Roche Inc., Mannheim, Germany). In vitro transcription was

performed in the presence of biotin- labeled CTP and UTP (Enzo Diagnostics,

Framingdale, New York, USA) on double -stranded cDNA, resulting in labeled cRNA

that was used as the target in the hybridization process. Hybridizations were performed

overnight; gene chips were washed and stained with streptavidin–phycoerythrin. Data

25

were collected by a laser scanning technique and pixel levels were analyzed with

commercial software (AffymetrixT M). For each transcript, an absolute expression level

was calculated depending on its expression.

Data analysis

Detailed protocols for data analysis of Affymetrix microarrays, and extensive

documentation of the sensitivity and quantitative aspects of the method, have been

described elsewhere77,78 . Briefly, a GeneChip probe array is a tool used to monitor gene

expression for thousands of transcripts. A transcript is represented as a probe set. A probe

set is made up of probe pairs comprised of Perfect Match (PM ) and Mismatch (MM)

probe cells. The intensities of each probe pair are the key ingredients used to make an

expression measurement. This measurement is calculated for each probe set and is

described in the form of qualitative and quantitative values. Additionally, the expression

measurements of a baseline and experimental array can be compared to understand the

relative change in abundance of a transcript.

In our experiment single array analyses were performed to build databases of gene

expression profiles in each experimental group (low and high-salt). A threshold of 40

arbitrary fluorescence units was assigned to any gene with a calculated transcript level

below 40, because discrimination of mRNA levels at this low range could not be

performed with high confidence. The analyses of these experimental databases provided

the initial data required to perform comparisons between high-salt and low-salt arrays.

Principles of the Single Array Analysis and Comparison Analysis will be discussed

below:

Single array analysis

This analysis generated a Detection p-value which is evaluated against the default

cut-offs to determine the Detection call. This call indicated whether a transcript is reliably

detected (Present) or not detected (Absent). Additionally, a signal value is calculated

which assigns a relative measure of abundance to the transcript. The Detection algorithm

uses probe pair intensities to generate a Detection p-value and assign a Present, Marginal

or Absent call. Each probe pair in a probe set is considered as having a potential vote in

determining whether the measured transcript is detected (Present) or not detected

26

(Absent). The vote is described by a value called the Discrimination score [R]. The score

is calculated for each probe pair and is compared to a predefined threshold Tau. Probe

pairs with scores higher than Tau vote for the presence of the transcript. Probe pairs with

scores lower than Tau vote for the absence of the transcript. The voting result is

summarized as a p-value. The higher the discrimination scores are above Tau, the smaller

the p-value and the more likely the transcript will be Present. The lower the

discrimination score below Tau, the larger the p-value and the more likely the transcript

will be Absent. The p-value associated with this test reflects the confidence of the

Detection call. A two-step procedure determines the Detection p-value for a given probe

set. First, calculation of the Discrimination score [R] for each probe pair. Second, test the

Discrimination scores against the user-definable threshold Tau. The Discrimination score

is a basic property of a probe pair that describes its ability to detect its intended target. It

measures the target-specific intensity difference of the probe pair (PM-MM) relative to its

overall hybridization intensity (PM+MM)

?R = (PM - MM) / (PM + MM)?

The user-modifiable Detection p-value cut-offs, Alpha 1 (? 1 ) and Alpha 2 (? 2 ), provide

boundaries for defining Present, Marginal or Absent calls. At the default settings,

determined for probe sets with 15-20 probe pairs (defaults ? 1 = 0.04 and ? 2 = 0.06), any

p-value that falls below ? 1 is assigned a Present call, and above ? 2 is assigned a n

Absent call. Marginal calls are given to probe sets which have p-values between ? 1 and

? 2. In summary, the Detection Algorithm assesses probe pair saturation, calculates a

Detection p-value and assigns a Present, Marginal or Absent call.

Signal is a quantitative metric calculated for each probe set, which represents the

relative level of expression of a transcript. Signal is calculated using the One -Step

Tukey’s Biweight Estimate which yields a robust weighted mean that is relatively

insensitive to outliers, even when extreme. Similar to the Detection algorithm, each probe

pair in a probe set is considered as having a potential vote in determining the Signal

value. The vote, in this case, is defined as an estimate of the real signal due to

hybridization of the target. The mismatch intensity is used to estimate stray signal. The

real signal is estimated by taking the log of the Perfect Match intensity after subtracting

the stray signal estimate (CT). The probe pair vote is weighted more strongly if this probe

27

pair signal value is closer to the median value for a probe set. Once the weight of each

probe pair is determined, the mean of the weighted intensity values for a probe set is

identified. This mean value is the quantitative metric Signal. When the Mismatch

intensity is lower than the Perfect Match intensity, then the Mismatch is informative and

provides an estimate of the stray signal. Rules are employed in the Signal algorithm to

ensure that negative signal values are not calculated. Negative values do not make

physiological sense and make further data processing, such as log transformations

difficult. Mismatch values can be higher than Perfect Match values for a number of

reasons such as cross hybridization. If the Mismatch is higher than the Perfect Match, the

Mismatch provides no additional information about the estimate of stray signal.

Therefore, an imputed value called Change Threshold (CT) is used instead of the

uninformative Mismatch. The following rules are applied in this process, according to

Affymetrix Genechip technology: Rule 1: If the Mismatch value is less than the Perfect

Match value, then the Mismatch value is considered informative and the intensity value is

used directly as an estimate of stray signal. Rule 2: If the Mismatch probe cells are

generally informative across the probe set except for a few Mismatches, an adjusted

Mismatch value is used for uninformative Mismatches based on the bi-weight mean of

the Perfect Match and Mismatch ratio. Rule 3: If the Mismatch probe cells are generally

uninformative, the uninformative Mismatches are replaced with a value that is slightly

smaller than the Perfect Match. These probe sets are generally called Absent by the

Detection algorithm.

Comparison Analysis (Experiment versus Baseline arrays)

In a Comparison Analysis, two samples, hybridized to two GeneChip probe arrays

of the same type, are compared against each other in order to detect and quantify changes

in gene expression. One array is designated as the baseline and the another as an

experiment. The analysis compares the difference values (PM-MM) of each probe pair in

the baseline array to its matching probe pair on the experiment array. Two sets of

algorithms are used to generate change significance and change quantity metrics for

every probe set. A change algorithm generates a Change p-value and an associated

Change. A second algorithm produces a quantitative estimate of the change in gene

expression in the form of Signal Log Ratio. Before comparing two arrays, scaling or

28

normalization methods must be applied. Scaling and normalization correct for variations

between two arrays. Two primary sources of variation in array experiments are biological

and technical differences. Normalization and scaling techniques were applied by using

data from all probe sets. An additional normalization factor - defined as Robust

Normalization (not user modifiable) accounted for unique probe set characteristics due to

sequence dependent factors such as affinity of the target to the probe and linearity of

hybridization of each probe pair in the probe set. More specifically, this approach

addresses the inevitable error of using an average intensity of the majority of probes (or

selected probes) on the array as the normalization factor for every probe set on the array.

The noise from this error, if unattenuated, would result in many false positives in

expression level changes between the two arrays being compared. The perturbation value

directly affects the subsequent p-value calculation. Of the p-values that result from

applying the calculated normalization factor and its two perturbed variants, the one that is

most conservative is use to estimate whether any change in level is justified by the data.

A default established at 1.1 based on calls made from the Latin Square data set by the

manufacturer was used.

Change algorithm: As in the Single Array Analysis, the Wilcoxon’s signed-rank

test is used in Comparison Analysis to derive biologically meaningful results from the

raw probe cell intensities on expression arrays. During a Comparison Analysis, each

probe set on the experiment array is compared to its counterpart on the baseline array,

and a Change p-value is calculated indicating an increase, decrease or no change in gene

expression. Default cut-offs (gammas) were applied to generate discrete Change calls

(Increase, Marginal Increase, No Change, Decrease, or Marginal Decrease).

The Wilcoxon’s signed rank test uses the differences between Perfect Match and

Mismatch intensities, as well as the differences between Perfect Match intensities and

background to compute each Change p-value. From Wilcoxon’s signed rank test, a total

of three, one-sided p-values are computed for each probe set. These are combined to give

one final p-value which is provided in the data analysis output (.CHP file). The p-value

ranges in scale from 0.0 to 1.0, and provides a measure of the likelihood of change and

direction. Values close to 0.0 indicate likelihood for an increase in transcript expression

level in the experiment array compared to the baseline, whereas values close to 1.0

29

indicate likelihood for a decrease in transcript expression level. Values near 0.5 indicate a

weak likelihood for change in either direction. Hence, the p-value scale is used to

generate discrete change calls using thresholds. The final Change p-value described

above is categorized by cutoff values called gamma1 (?1) and gamma2 (?2). These cut-

offs provide boundaries for the Change calls: Increase (I), Marginal Increase (MI), No

Change (NC), Marginal Decrease (MD), or Decrease (D). Gamma1 (?1) and ?2 each

derived from two adjustable parameters, ?L and ?H. Defaults for probe sets with 15-20

probe pairs were used (?1L= 0.0025, ?1H= 0.0025 and ?2L= 0.003, ?2H= 0.003), which

define the lower and upper boundaries for ?1 and ?2. Gammas are computed as a linear

interpolation of ?L and ?H.

In summary, the Change algorithm assesses probe pair saturation, calculates a

Change p-value and assigns an Increase, Marginal Increase, No Change, Decrease, or

Marginal Decrease call.

Finally, we used Student's t test to calculate the quantitative estimate of the

change in gene expression between low-salt and high- salt groups.

Expression verification

We confirmed gene expression levels for the renin gene and the gene for the B7-1

antigen by an independent method using a real-time polymerase chain reaction (PCR).

We used the same RNA (four salt-loaded and four normal Sprague–Dawley rats) for both

microarray and quantitative PCR analysis. Two micrograms of DNA-free total RNA was

reverse-transcribed with oligo(dT) primers (Invitrogen, Karlsruhe, Germany), Superscript

II reverse transcriptase (Gibco-BRL), and deoxynucleoside triphosphate (dNTP;

Invitrogen) in 40 µl reaction buffer supplied by the manufacturer.

Renin (accession number S60054): forward, 5'-gcttt ggacgaatcttgctca-3'; reverse,

5'-ctactccccgctcctccag-3'; probe, 5'-6-FAM-aaaatgccctcggtccgggaaa-TAMRA-3';

B7 antigen (accession number X76697): forward, 5'-gatccaggatgaacaccttcct-3';

reverse, 5'-cccaagtctcctgtttg gatct-3'; probe, 5'-6-FAM-

cctcaccatcagtctcctggttgtggt-TAMRA-3'.

30

TaqMan analysis was carried out according to the manufacturer's instructio ns using an

Applied Biosystems 7700 Sequence detector (Perkin Elmer, Weiterstadt, Germany).

Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) expression using the 2- ??Ct or standard curve method. Expression values were

calculated relative to animals receiving the low-salt diet.

Statistics

Genes with P < 0.05 and a change in expression level of at least two- fold were

considered to be differentially expressed between rats receiving a high-salt diet and a

low-salt diet. The differential expression according to these criteria is highly reproducible

by TaqMan analysis79.

31

RESULTS

Oligonucleotide microarray experiments and differentially regulated genes

The reproducibility of expression values in different individual animals was high. We

verified our findings by repeating the experiment in quadruplicate. The results were

invariably the same. The mean correlation coefficient between individual animals within

the two groups was 0.978; between animals of different groups it was 0.977. Figure 4

shows the comparison of high-salt rats versus low-salt rats. Each point on the scatter plot

represents the transcription value of one transcript in the two groups. Differences in

transcription levels control are apparent.

FIGURE 4. Scatter plot of gene expression levels. For each gene, mean

mRNA expression levels averaged from low-salt and high-salt groups

were plotted on a double- logarithmic scale. Genes identified as

differentially expressed between low-salt and high-salt groups are

indicated in bold. A threshold of 40 arbitrary fluorescence units was

10

100

1000

10000

100000

10 100 1000 10000 100000high salt group

low salt

gro

up

32

U50412 PI 3 kinase p50 subunitAI138070 SH-PTP2 homolog AA866455 mouse HIPK3 homologAB010154 PKN serin/threonine kinase D64047 PI 3 kinase p55 subunit U57502 Tyrosine phosphatase delta AA891302 MEKK homolog AA859827 Uck2-pending homologAJ006855 Synaptojanin

-4.9-2.5-2.3

-2.02.2

2.22.32.5

assigned to any gene with a calculated transcript level below 40, because

discrimination of mRNA levels at this low range could not be performed

with high confidence. Ordinate and abscissa are a rbitrary fluorescent units.

Figure 5 shows differentially expressed genes between rats receiving a low-salt

diet and those receiving a high-salt diet. Thirty-five genes were upregulated and 30 genes

were downregulated in high-salt rats, compared with low-salt rats. Our positive control is

the renin gene. This gene showed 2.7- fold downregulation, comparing high salt intake

with low salt intake.

FIGURE 5: Differentially expressed genes between rats receiving a low-

salt and a high-salt diet. Gene expression pa tterns in rats receiving low and

high salt were generated from four independent rats per group. Bars

represent the fold change of transcript levels of a particular gene between

rats from low-salt and high-salt groups (mean of four experiments per

group). Positive values indicate that the transcript was more abundant in

the high-salt group compared with the low-salt group; negative values

indicate the opposite. Abbreviations for gene names are given along with

Genbank accession numbers. The figure represents all genes that were

s

c

o

r

e

d

as differentially expressed in the experiment.

kinases and phosphatases

5.3

33

-2.9

-2.5-2.4

3.23.9

D17469 TRH receptorAA900380 similar to TNF I receptor M99418 Brain cholecystokinin receptorS37461 AT1B receptorM88751 Calcium channel beta subunit III

receptors and ion channels

M18363 P450(M-1)AA963449 Lanosterol demethyl.homologM29853 P450(IVB2)

-2.42.7

2.9

cytochrome P450 family

M31725 Axonal glycoproteinJ00738 Submaxil.gl. alpha-2u globulinAA893495 CbgL12025 Tage4L140001 Polymeric Ig receptor

X15551 Beta-2 glycoprotein IX86086 Annexin VI M10934 RBPX60661 RYD5AI169612 ALBP homolog

-4.5-3.7

-2.4-2.3

-3.2-3.1

-2.52.0

4.1

immunglobulins and globulins

lipid and phospholipid binding proteins

4.2

AA892659 AP4 homologAF039832 rPtx2U29147 DRG11

-3.22.2

2.5

homeobox and Zink finger proteins

AI639352 cDNA

S81025 1,4-galactosyltransferase K02814 MAP-alpha1M17526 G-alpha-0AA800782 cDNAA858605 cDNAAA955950 cDNAS60054 Renin AI639141 cDNAAI639125 cDNAAI179027 GRASP65AA893275 cDNAU17253 NAB1AA875665 similar to LOC57333AI009682 Psi-DRS1 homologAA893035 HP33 homologAI639253 cDNAAI639028 cDNAX55286 HMG-CoA reductase AI639313 cDNAM58308 HistidaseAI638958 cDNAAA892895 Rps15 homologY10019 DRM proteinAA860039 cDNAX76697 B7 antigenAA894119 KIAA0660 homologAI639212 cDNAAA892800 ZnBP homologAI102031 Amphiphysin homologAA925529 cDNAAI176460 28S rRNA homologL41684 cDNA

-9.3

-4.4

-4.2 -4.0

-3.8

-2.7

-2.6

-2.1 -2.1

-2.0 2.0

2.1

2.1 2.1

2.1

2.2

2.3

2.3 2.4

2.6

2.7 2.7

2.9

3.0

3.1 4.0

4.2

5.9 6.9

-3.6

-2.1

-2.8

4.3

other genes

34

Protein families of differentially regulated genes

Figure 6 shows the number of up and down regulated genes by salt according to encoded protein families (Swiss prot).

FIGURE 6. Number of the up and down regulated genes by salt according

to encoded protein families.

0

1

2

3

4

5

amphi

physin

s

histida

ses

ionic c

hann

els

memb

rane p

roteins

NAB f

amily

peptida

ses

plasm

a glyc

oprote

ins

reduct

ases

ribosom

al prote

ins

RNA b

inding

prote

in fam

ily

serpin

family

synthe

tases

transfe

rases

zinc b

inding

protein

s

zinc fi

nger

protein

s

G - pro

teins

homeob

ox pro

teins

cytoch

rome P

450 fa

mily

phosph

atases

immun

oglob

ulins /

globu

lins

other

protein

s

recep

tors

lipid bin

ding p

roteins kin

ases

Nu

mb

er o

f reg

ula

ted

gen

es

Upregulated genes Downregulated genes

down

up Fold Regulation

35

Figure 7 shows the relative percentage of encoded protein families of the

differentially regulated genes in (A) the high and low salt groups, (B) high salt group, (C)

in the low-salt group.

FIGURE 7 (below). Relative percentage of encoded protein families of the

differentially regulated genes in (A) the high and low salt groups, (B) high

salt group, (C) in the low-salt group.

FIGURE 7/A

Verification of the microarray results

Independent TaqMan verification analyses on four rats in each group confirmed

downregulation of the renin gene by high salt, compared with low salt, by a factor of 2.38

reductases 2%

plasma glycoproteins2%peptidases

2%

NAB family2%

ionic channels2%

membrane proteins2%

histidases2%

amphiphysins2%

ribosomal proteins 2%RNA binding protein

family 2%

serpin family2%

synthetases2%

transferases2%

zinc binding proteins2%

zinc finger proteins2%

G - proteins4%

homeobox proteins4%

cytochrome P450 family 6%

kinases13%

lipid binding proteins10%

receptors8%

other proteins8%

immunoglobulins / globulins

8%phosphatases

6%

36

normalized to GAPDH. This value corresponded to the value of 2.72 obtained by the

microarray. On the contrary, the B7 antigen gene was upregulated by a factor of 2.01

normalized to GAPDH. These results were similar to the 2.9- fold upregulation

determined by the microarray. Both results are in relatively good agreement.

37

histidases4%

ionic channels4%

reductases4%

ribosomal proteins4%

RNA binding protein family

4%

zinc binding proteins4%

G - proteins4%

synthetases4%

immunoglobulins / globulins

4%

homeobox proteins7%

cytochrome P450 family

7%

phosphatases7%

receptors7%

lipid binding proteins7%

kinases11%

other proteins15%

amphiphysins4%

plasma glycoproteins6%

serpin family6%

transferases6%

zinc finger proteins6%

G - proteins6%

phosphatases6%

immunoglobulins / globulins

16%

receptors10%

lipid binding proteins16%

kinases16%

cytochrome P450 family 5%

FIGURE 7/B

FIGURE 7/C

38

DISCUSSION

The important finding in this study was that oligonucleotide arrays have utility in

identifying unexpected gene targets in rat kidneys in response to altered salt intake. We

found 35 genes that were upregulated by a high salt intake, while 30 were downregulated.

We verified our findings by repeating the experiment in quadruplicate. The results were

invariably the same. We examined a positive control, the renin gene. This gene was

downregulated by a factor of 2.7 by a high-salt diet. Plasma renin activity decreases more

than two- fold in response to a 10-fold difference in salt intake2. However, the renin gene

is primarily expressed in the juxtaglomerular apparatus that makes up less than 1% of the

total renal mass. Thus, we believe a 2.7-fold downregulation is a convincing result.

Genes encoding channel proteins, signaling molecules, transcription factors, and enzymes

were represented in both the downregulated and upregulated gene groups. Few have been

associated with the effects of salt intake previously and, for most, the ultimate function is

not clear. I have listed the genes (Figure 5) and discuss several that caught our particular

interest.

We were particularly interested in the behavior of phosphatidyl-inositol 3-kinase

(PI3 kinase). Three genes responsible for the catalytic subunits and three encoding

regulatory subunits encode the enzyme complex. Two splicing variants80 of the gene

encoding the p85alfa regulatory subunit of PI3 kinase, with different insulin–receptor–

substrate (IRS) binding affinity81, are among the regulated genes. The p55 subunit of the

enzyme complex was upregulated, while the p50 subunit was downregulated. IRS

binding affinity is different among splice variants: p50 has the highest ability, followed

by p85 and p55 82. Hyperglycemic, obese mice (strain ob/ob) with insulin resistance have

reduced p85 expression in the liver83, whereas p55 and p50 are upregulated. Mice lacking

the p85 isoform (p85-/-) show upregulation of p50 and p55 84. This is associated with

hypoglycemia and increased insulin sensitivity due to enhanced signaling through the p50

subunit and increased surface translocation of glucose transporter 4 (GLUT-4). Thus,

upregulation of p55 subunit and downregulation of p50 subunit by high sodium intake

may result in reduced insulin signaling and insulin resistance. Indeed, a recent

39

observation clearly showed that high-salt fed rats develop hyperglycemia,

hyperinsulinemia, and systemic insulin resistance85. The PI3 kinase enzyme complex is

an essential regulator of epithelial sodium channel opening, aldosterone, and insulin-

stimulated sodium reabsorption, and sodium–proton exchange 86. Therefore, differential

regulation of PI3 kinase regulatory subunits by salt may have direct – yet undiscribed –

effects on renal tubular sodium handling. Results of a recent experiment suggest that

augmented natrium-proton exchanger activity contributes to the development of salt-

sensitive blood pressure elevation in Wistar fatty rats87. Serum-glucocorticoid kinase

(SGK) is a recently described serin-threonin kinase, with close similarity to Akt and

under PI3 kinase regulation. It responses rapidly to mineralocorticoid and insulin and

seems to be responsible for elevated apical Na+ transport within 30 minutes after

mineralocorticoid activation88. Two polymorphic variants of the SGK1 gene have been

reported to be associated with higher blood pressures by Luft et al.89. On the other hand,

SGK1-knockout mice appear to have an impaired ability to decrease urinary sodium

excretion on dietary sodium chloride restriction and display a tendency to lower blood

pressure90. Renal expression of SGK1 has been recently shown to be regulated by a ltered

dietary salt intake in Dahl rats91. Thus, SGK1 is an important gene candidate for salt

sensitivity. It is intriguing to hypothesize, that different splice variants of PI3 kinase –

similar to IRS – may have altered binding affinity and activation capacity for SGK1.

Moreover, involvement of PI3 kinase – Akt activation in GLUT-4 surface translocation,

regulated exocytosis and endocytosis and their pivotal role in survival signaling and

nuclear factor-?B activation may arise further speculation about the physiologic

importance of our findings.

Amphiphysin (Amph2) - a brain-enriched protein implicated in clathrin-mediated

endocytosis - was upregulated by high dietary salt intake. Amphiphysins has been shown

to inhibit endocytosis in vivo via enhanced dynamin ring dissesambly 92. Synaptojanin 1

(Synj1) a nerve terminal protein with a relative molecular mass of 145 kDa appears to

participate with dynamin in synaptic vesicle recycling 93. It was also among the

upregulated genes. The role of these genes in kidney is currently unknown. Their

hypothetic link to renal physiology would be a role in dynamin-dependent endocytosis

40

via clathrin-coated vesicles, which has been shown to be the major mechanism for

internalization of angiotensin 1 receptor, apical Na(+) channel and Na(+)/Ca(+)

exchanger. This possibility remains to be elucidated.

The gene homolog of the fatty acid binding protein 4 (FABP4, also called ALBP:

adipocyte lipid binding protein) was upregulated by excess salt intake. A current

observation suggests tha t FABP acts as a cytosolic gateway for PPAR-? and PPAR-?

agonists and plays a central role in trafficking of fatty acids within adipocytes94. The

recently revealed central role for FABP in obesity-insulin coupling comes from studies

on FABP-deficient mice 95. Mice lacking this gene showed an increased weight gain but

failed to develop insulin resistance or diabetes on a high fat, high caloric diet compared to

the wild type animals. Its role, if any, in renal sodium regulation remains obscure.

However, our results indicate that FABP and PI3 kinase are both possible candidate

genes for linking sodium intake to insulin resistance and obesity. It is also worth to note

that two other FABP protein family member showed salt-induced regulation in our

experiment. First, the corticosteroid -binding-protein (CBG) was downregulated. In the

kidney high concentration of CBG-binding sites accumulate in the extravascular region

of papilla- inner medulla region96. One study showed negative correlation between CBG

and mean arterial pressure in normotensive, but not in hypertensive patients97. Second, rat

retinol-binding protein was upregulated. The retinol-binding proteins (RBPI, II, and

recently described III) are the only members of the FABP family that process

intracellular retinol. Its single known function is to deliver retinol to tissues. However,

RBP knock-out mice and transgenic rats show no detectable phenotype alterations under

efficient vitamin A supply98.

Analogue of phosphotyrosine phosphatases 2C gene was downregulated in high

salt group compared to low salt group. The SH2-domain-containing phosphotyrosine

phosphatase 2 (PTP2) is a positive signal transducer for several receptor tyrosine kinases

and cytokine receptors. PTP2C is widely expressed in human tissues with particular

abundance in the heart, brain, and skeletal muscle 99. PTP2C is required for growth factor,

insulin (PTP2C dephosphorilates IRS1) and MAPK signaling. In the kidney, PTPs are

involved in the regulation of the activity of small conductance K(+) channels of rat

cortical conducting ducts and inhibition of PTPs may facilitate the internalization of the

41

channel100. PTP2B has also been shown to regulate single chloride-channel conductance

in renal epithelium 101. Altered expression of protein-tyrosine phosphatase 2C in renal

cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase

activation102. Homozygous knock-out Ptp2 (-/-) mice die at midgestation with multiple

defects in mesodermal patterning. In contrary, Ptp1b (-/-) mice remain alive and show

resistance against both diabetes and obesity103.

Rat brain cholecystochinin (CCK) receptor was downregulated by excess salt

intake. CCK-B receptor transcripts can be detected in the kidney, most abundant in

tubules, followed by glomeruli and interstit ium. A study using RT-PCR revealed CCK-B

receptor transcripts in proximal tubules and in mesangium104. A long-term administration

of high dose of loxiglumide, a CCK receptor antagonist, decreases pancreatic enzyme

output and causes insulin resistance105. The function of CCK receptors in the kidneys is

currently unknown.

Four CYP enzymes were differentially regulated by altered dietary NaCl intake.

We would like to emphasize that the rate-limiting and another enzyme in the cholesterol

biosynthesis pathway (HMG-CoA reductase, lanosterol 14-demethylase) were

upregulated by high sodium intake. First, HMG-CoA reductase was upregulated by high-

salt ingestion. This key and rate-limiting enzyme in cholesterol biosynthesis is of

particular influence in vascular disease and obesity. Animal models of obesity,

hypertension and insulin resistance display differences with respect to salt sensitivity.

Salt sensitivity and obesity is coupled in Zucker rats? obese (but not lean) rats are

sensitive to dietary salt excess and exhibit salt sensitive hypertension106. A QTL region

with collective profile of phenotypes resembling Syndrome X or the "metabolic

syndrome" in humans has been recently described on rat chromosome 18. Interestingly,

blood pressure salt sensitivity within this aggregate of traits accounted for 17% of the

overall variance in salt sensitivity71,72. An interesting study by Dobrian et al. from 2003

indicates that high-salt intake is able to induce hypertrophy of adipocytes107. The

adipocyte morphometry and number was significantly different between the groups

placed on low vs. high salt diets. 2% NaCl diet induced an increase by 12-20% in cell

volume and 12-15% in cell size with the highest effect on obesity prone adipocytes. Also,

42

a decrease in adipocyte number was measured on 2% vs. 0.8% NaCl diet. It remains to be

elucidated, whether salt- induced upregulation of HMG-CoA reductase is limited to the

kidneys. The observed upregulation of HMG-CoA reductase might explain the sodium-

induced hypertrophy of adypocytes described by Dobrian et al. Moreover, HMG-CoA-

reductase inhibitors are antiproliferative in epithelial tubular cells, partly by inhibition of

p21ras-activated and AP-1-dependent mitogenic cascades108. In addition, HMG-CoA

reductase inhibitors induce