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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
2
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
3
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
4
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.
5
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
6
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
7
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
8
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
9
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
10
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).
11
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
12
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
13
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
14
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,
15
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
16
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
17
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
18
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
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