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THE”

This is to certify that the

dissertation entitled

Humoral Factors in Sodium-Dependent Hypertension:
Characterization in Reduced Renal Mass Rats

presented by

Nancy Lapp Kanagy

has been accepted towards fulfillment
of the requirements for

 

 

Ph . D. degree in Pharmacology
and Toxicology

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Date 8/(5/92’

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

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HUMORAL FACTORS IN SODIUM-DEPENDENT HYPERTENSION:
CHARACTERIZATION IN REDUCED RENAL MASS RATS

By

Nancy Lapp Kanagy

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology

1992

ABSTRACT
HUMORAL FACTORS IN SODIUM-DEPENDENT HYPERTENSION:
CHARACTERIZATION IN REDUCED RENAL MASS RATS
By

Nancy Lapp Kanagy

There is a subset of the human population whose blood pressure is
sensitive to changes in dietary sodium. In these people, there is a direct
positive correlation between dietary sodium intake and their mean arterial
pressure (MAP) so that high sodium intake leads to hypertension. The
differences between sodium sensitive and sodium resistant individuals have
not been clearly defined. However, both clinical and animal studies suggest
that alterations in humoral substances are at least partly responsible. Two
circulating hormones that appear to be involved in this pathogenic response
are angiotensin II (ANG II) and aldosterone, both products of the renin
angiotensin system. The reduced renal mass (RRM) rat is an animal model
that has been used to study the mechanisms of sodium dependent
hypertension (SDH). This model of hypertension is very similar to the
clinical condition of chronic renal failure, a syndrome which almost
invariably leads to SDH. As in human SDH, MAP in RRM rats is very
sensitive to the effects of dietary sodium. Treatment with angiotensin

converting enzyme inhibitors (CEI), however, prevents a sodium-induced rise

in MAP implicating ANG II in the development of SDH in RRM. Because
of the lack of specificity of CEI, we used a more specific pharmacological
tool and found that the specific ANG II antagonist, losartan also prevents
RRM hypertension in rats. Further, we found that RRM rats are more
sensitive than intact rats to the hypertensive effects of circulating ANG II.
Together these findings suggest that an increased sensitivity to ANG II in

RRM rats contributes to their development of SDH.

iii

This dissertation is dedicated to my family and my husband who encouraged
me to work to my fullest potential and who have always believed in my

abilities.

iv

ACKNOWLEDGEMENTS

The first acknowledgement is to my mentor, Dr. Gregory Fink for the
knowledge, advise and assistance he gave so freely over the past five years.
His experience and expertise in the field of hypertension research are an
inspiration to me and a lifelong goal to strive toward. I feel fortunate to
count him as a friend and adviser. I also want to thank my thesis committee,
Dr. James Galligan, Dr. Keith Lookingland and Dr. William Spielman, for
their competent advice during the development and interpretation of this
dissertation. They have been patient and professional throughout these five
years.

I am grateful for the friendship and help of my fellow graduate
students, Dr. Corie Dahm, Dr. Luke Mortensen and Dr. Vyvian Gorbea,
whose sense of humor made the long recordings in the basement of the Life
Sciences Building a pleasant experience. The assistance and friendship of
Annette McLane, Douglas Shinover, John Meier and Renee Petit who helped
to set up, run and analyze these experiments is deeply appreciated. Their
commitment and diligence to quality work helped the lab run smoothly and
made many of these experiments possible.

Finally, I thank my husband, Kent, for his mental, emotional and

financial support throughout graduate school. His tolerance and forbearance
may have been stretched by the many demands on my time but he has

remained my best friend and staunchest supporter.

vi

TABLE OF CONTENTS

GENERAL INTRODUCTION .............................. l
I. Introduction ........................................ 2
A. Overview of the renin-angiotensin system .............. 2

B. Role of sodium in the development of hypertension ........ 4

l. Epidemiological evidence ..................... 4

2. Sodium intake intervention studies ............... 7

C. Characteristics of sodium-sensitive hypertension .......... 9

D. Potential mechanisms of sodium-sensitive hypertension ..... 14

1. Primary alteration of renal function .............. 17

2. Hormonal influences ......................... 21

a. hormone influences at the kidney ............ 21

b. extrarenal hormonal influences ............. 26

3. Alteration of the sympathetic nervous system ........ 34

a. renal sympathetic nervous system actions ...... 34

b. extrarenal sympathetic nervous system actions . . 36

vii

E. Animal models of hypertension ...................... 37

1. Genetic models ............................. 38
2. Hormone infusion models ..................... 40
a. mineralocorticoid infusion ................. 40
b. ANG II infusion ....................... 42

3. Reduced renal mass model of sodium-dependent

hypertension ............................ 44

a. physical and metabolic changes ............ 44

b. whole animal hemodynamic changes ......... 48

c. hormonal changes ...................... 52

1) angiotensin II ..................... 52

2) arginine vasopressin ................ 54

3) aldosterone ...................... 55

4) natriuretic factors .................. 57

d. sympathetic nervous system ............... 59

11. Methods and Materials ................................ 64
A. General experimental protocols ...................... 64
1. Surgical procedures .......................... 64

a. catheterization ......................... 64

viii

b. subcutaneous osmotic minipumps ........... 65

c. renal reduction ......................... 65

2. Hemodynamic measurements ................... 66

3. Analytical methods .......................... 67

a. sodium and potassium ................... 67

b. plasma osmolality ...................... 68

c. blood urea nitrogen ..................... 68

d. plasma aldosterone concentration ............ 68

e. plasma immunoreactive angiotensin II ........ 69

f. fluid homeostasis ....................... 70

4. Statistical methods .......................... 70

B. Specific protocols ............................... 70

1. Aldosterone infusion ......................... 71

2. PAC during chronic infusion of angiotensin II ....... 72

3. Aldosterone in reduced renal mass hypertension ...... 75
4. Angiotensin II antagonism in reduced renal mass

hypertension ............................ 76

a. chronic antagonist treatment ............... 76

b. acute antagonist treatment ................. 78

5. Angiotensin II infusion in reduced renal mass rats . . . . 78

ix

III. Results ........................................... 80

A. Plasma aldosterone .............................. 80
B. Chronic infusion of angiotensin II .................... 86
C. Plasma aldosterone in reduced renal mass hypertension ..... 92
D. Angiotensin II antagonism in reduced renal mass
hypertension ................................. 95
1. Chronic angiotensin II antagonist treatment ......... 95
2. Acute antagonist treatment ..................... 104
E. Angiotensin II infusion in reduced renal mass rats ......... 107
IV. Discussion ........................................ 114
A. Plasma aldosterone during angiotensin II infiIsion ......... 116
B. Plasma aldosterone in reduced renal mass hypertension ..... 125
C. Endogenous angiotensin II in reduced renal mass
hypertension ................................. 129
1. Chronic losartan in RRM rats ................... 129
2. Acute losartan treatment in hypertensive RRM rats . . . . 136
D. Changes in sensitivity to chronic angiotensin II in reduced

renal mass rats ............................... 137

LIST OF TABLES

Table 1: Plasma aldosterone following angiotensin II infusion ....... 81

xii

LIST OF FIGURES

Figure 1: Effect of aldosterone infusion on blood pressure, heart rate

and plasma aldosterone concentration. ....................

Figure 2: Water intake and urine output in intact rats receiving

aldosterone ......................................

Figure 3: Effect of aldosterone infusion on sodium and potassium

excretion in intact rats ..............................

Figure 4: Blood pressure, heart rate and plasma aldosterone in intact

rats receiving angiotensin II ...........................

Figure 5: Urine output, water intake, urinary sodium excretion and

urinary potassium excretion in rats receiving ANG II infusion . . .

Figure 6: Plasma sodium, potassium and osmolality during angiotensin

II infusion in rats on high sodium intake ..................

Figure 7: Blood pressure, heart rate and plasma aldosterone

concentration in rats receiving angiotensin II on normal sodium . .

Figure 8: Blood pressure, plasma aldosterone and heart rate in intact

and RM rats during changes in sodium intake .............

Figure 9: Blood urea nitrogen in intact and RRM rats during changes

in sodium intake ...................................

Figure 10: Water intake, urine output and water balance in intact and

xiii

82

87

88

90

91

93

94

RRM rats during changes in sodium intake ................ 96
Figure 11: Urinary electrolyte excretion and sodium balance in RRM

and intact rats during changes in sodium intake. ............. 97
Figure 12. Effect of losartan treatment on blood pressure and heart

rate in RRM and intact rats during changes in sodium intake. . . . 98
Figure 13. Correlation between blood pressure and BUN in RRM with

or without losartan treatment .......................... 99
Figure 14: Effect of losartan treatment on water intake, urine output

and water balance in RRM and intact rats during different

sodium intakes ..................................... 101
Figure 15: Effect of losartan treatment on urinary sodium excretion

and sodium balance in RRM and intact rats during different

sodium intakes ..................................... 102
Figure 16: Effect of losartan treatment on blood urea nitrogen in intact

and RM rats during changes in sodium intake. ............. 103
Figure 17: Acute blood pressure response to ANG II infusion in intact

and RM rats with and without losartan blockade. ........... 105
Figure 18: Blood pressure and heart rate response to acute losartan in

hypertensive RRM rats on high sodium intake ............. 106

Figure 19: Blood pressure and heart rate during angiotensin II infusion

xiv

in RRM and intact rats on normal sodium intake. ............ 108
Figure 20: Effect of angiotensin II infusion on water intake and urine

output in intact and RM rats on normal sodium intake. ....... 109
Figure 21: Effect of angiotensin II infusion of urinary electrolyte

excretion in RRM and intact rats on normal sodium intake. ..... 111
Figure 22: Plasma levels of immunoreactive angiotensin II in RM

and intact rats receiving angiotensin II infusion. ............. 112
Figure 23: Effect of angiotensin II infusion on BUN in RRM and

intact rats. ....................................... 1 13

XV

LIST OF ABBREVIATIONS

ACE - angiotensin converting enzyme
ANOVA - analysis of variance

ANG II - angiotensin II

ANP - atrial natriuretic peptide

AP - area postrema

AVP - arginine vasopressin

BV- blood volume

CEI - angiotensin converting enzyme inhibitor
CO - cardiac output

CI - cardiac index

DOCA - deoxycorticosterone

GFR - glomerular filtration rate

HR - heart rate

iv - intravenous

JG - juxtaglomerular

MAP - mean arterial pressure

mRNA - messenger ribonucleic acid

Na+/K+-ATPase - sodium potassium adenine triphosphate

xvi

transport enzyme
NTS - nucleus of the tractus solitarius
OLF - ouabain-like factor
PAC - plasma aldosterone concentration
PRA - plasma renin activity
RAS - renin angiotensin system
RRM - reduced renal mass
SAD - sinoaortic denervation
SEM - standard error of the mean
SDH - sodium dependent hypertension
SHR - spontaneously hypertensive rat
SNA - sympathetic nervous system activity
SNGFR - single nephron glomerular filtration rate
SNS - sympathetic nervous system
TPR - total peripheral resistance
UKV - urinary potassium excretion
UNaV - urinary sodium excretion
U0 - urine output

WI - water intake

xvii

GENERAL INTRODUCTION

The role of sodium chloride (i.e. salt) in the development of
hypertension has been the topic of research for a decade and is covered
extensively in the literature. The first section of the Introduction is a review
of some of the more recent work in this area.

The second section of the Introduction reviews common characteristics
of sodium-dependent hypertension (SDH) and the possible mechanisms they
suggest to explain the genesis of this form of the disease. This, too, is a very
extensive literature and the review covers only major advances providing an
overview of the specific humoral factors that may affect blood pressure in
SDH.

The final section describes the animal models of SDH. It includes a
discussion of the mechanisms which may lead to the development of
hypertension in these models and examines the evidence that humoral factors
are involved in the sodium-induced rise in blood pressure of reduced renal
mass rats.

The experiments which are described following the Introduction were
designed to clarify the role of specific humoral factors in reduced renal mass

hypertension and to apply those findings to the general condition of SDH.

I. Introduction

A. Overview of the renin-angiotensin system

The renin-angiotensin system (RAS) affects sodium homeostasis and
blood pressure in many ways. Thus a discussion of sodium-dependent
hypertension demands an understanding of the physiology of the RAS
synthesis cascade. The precursor molecule in the RAS is angiotensinogen, a
circulating polypeptide synthesized mainly in the liver. Production of
angiotensinogen increases during sodium deprivation and is inhibited by the
end product angiotensin (ANG) II. Angiotensinogen is released into the
bloodstream from the liver and is cleaved by renin to form ANG I, an
inactive decapeptide. Renin, the rate limiting enzyme, is produced primarily
in the kidneys by specialized smooth muscle cells called the juxtaglomerular
(JG) cells located in the afferent arterioles at the entrance to the glomerulus.
Renin is released from the kidney into the circulation where it acts to produce
ANG I. ANG I is further cleaved by angiotensin-converting enzyme (ACE)
to form the octapeptide ANG II also known as angiotensin (1-8). ACE is a
non-specific zinc-metaloproteinase found in the vascular endothelial cells of
many tissues including the lungs, brain and kidneys and degrades kinins in
addition to its action on ANG I. The peripheral cells with the highest ACE

activity are the endothelial cells of the bronchial arterioles where the majority

3

of circulating ANG II is produced and released into the systemic circulation.
In addition to ANG 11, there are several other peptide products of the
RAS with demonstrated physiological actions. These are designated
angiotensin (2-8) or ANG III, angiotensin (1-7), and angiotensin (3-8)
(Ferrario et a1. 1991). Circulating ANG II and ANG III bind to the same
membrane-bound receptors to induce Similar actions. Angiotensin (1-7)
produces very different actions, perhaps through a separate receptor (Diz and
Pirro 1991). There are two known ANG receptors, designated as AT, and
AT2 (Wong et al. 1990). ANG II and ANG 111 both act on membrane-bound
AT, receptors causing constriction of vascular smooth muscle cells, the
release of aldosterone from adrenal zona glomerulosa cells, and the absorption
of sodium at the renal proximal tubule cells. The intracellular response to
AT, receptor activation is mediated through a membrane bound guanine
nucleotide binding protein. The so-called G-protein stimulates inositol
triphosphate dependent calcium release in vascular smooth muscle cells and
adenylate cyclase generated cyclic AMP accumulation in renal proximal
tubular cells. The sensitivity of the adrenal AT, receptors appears to increase
during sodium depletion, a time of high ANG II levels, and decrease during
sodium excess (Aguilera and Catt 1978). The increased sensitivity reinforces

ANG II stimulated aldosterone release to contribute to restoration of sodium

4

balance. Tachyphylaxis of the vascular and renal responses occurs during
chronic elevations in circulating ANG II and there is a decrease in the
number of ANG 11 receptors during chronic sodium restriction and ANG II
excess (Schiffrin et al. 1983). In addition to the peripheral effects, both ANG
II and ANG III act centrally through AT, receptors to stimulate drinking and
to cause the release of arginine vasopressin (AVP) from hypothalamic nuclei.
The AT2 receptor has not been shown to mediate any action of the ANG
peptides and does not interact with guanine nucleotide binding proteins
although it is located in many tissues where ANG II acts such as the kidney,
adrenal gland and brain (Botarri et a]. 1991). Future studies are needed to
determine what role, if any, these receptors play in the systemic response to
ANG peptides.

In addition to the well characterized circulating RAS, it is speculated
that there are independent tissue RASs. ANG 11 produced by the tissue RAS
is proposed to act in an autocrine or paracrine fashion and independent of
circulating levels of renin or ANG II. In support of this proposal, mRNA
coding for angiotensinogen, renin and ACE has recently been found in a
multitude of tissues including the kidney (Dzau et a1. 1988), the adrenal
(Kohara et al. 1992) and the brain (Kohara et a1. 1992). Furthermore, it has

become increasingly clear that ANG II is not the only active component of

5

the RAS and other products of angiotensinogen appear to play important
physiological roles. Thus, circulating levels of ANG 11 may not be an
accurate measure of the activity of the RAS and activation of RAS may be
present even in the face of normal or suppressed plasma levels of the peptide.

B. Role of sodium in the development of hypertension

1. Epidemiological evidence

A direct relationship between salt intake and blood pressure has been
postulated for many years but has been difficult to prove. A landmark study
by Dahl combined the observations of many populations consuming a wide
range of sodium intakes, including primitive cultures with very low sodium
intakes and industrial societies with relatively high sodium intakes. He found
a significant tendency for blood pressure to be slightly higher in populations
with a higher intake of sodium (Dahl 1960). However, not all subsequent
studies have supported his findings and a direct relationship between salt
intake and blood pressure is neither clearly defined nor universally accepted.

The mechanisms through which salt intake can influence blood
pressure are undoubtedly related to the influence of sodium on fluid
homeostasis. Sodium is the major determinant of the osmolality of blood and
extracellular fluid. Dietary sodium intake, primarily in the form of sodium

chloride increases the amount of sodium in the body. If renal excretory

6

’ mechanisms do not completely eliminate the sodium ingested, it accumulates
to increase total body sodium content. When the amount of sodium in the
body changes, water volume also changes to maintain sodium concentration
and osmolality constant within a very narrow range. Sodium retention
therefore leads to body fluid volume expansion. This volume expansion can
in turn expand vascular volume causing increased venous return, elevated
cardiac output (CO) and a rise in blood pressure. Additionally, volume
expansion can result in edema of the vascular wall, thereby contributing to
increased vascular resistance. In both instances, there is an increase in blood
pressure.

In a recent review by Elliot of the accumulated epidemiological
evidence for an influence of dietary sodium on blood pressure, data from
sixteen independent observational studies were combined using meta analysis
(Elliot 1991). The data was derived from populations unscreened for
hypertension and covered a wide range of sodium intakes (Elliot 1991).
Although it was criticized for including populations with very different
cultural and physical attributes which may affect the relationship between
sodium and blood pressure, this study did find a significant positive
correlation between sodium intake and blood pressure.

Another large body of data was gathered in a huge international project

7
known as INTERSALT (Stammler et al. 1991). This study recorded blood

pressure and sodium intake in 10,049 patients at 52 separate clinics around
the world and was the most

comprehensive epidemiological examination of sodium intake‘s effect on
blood pressure ever performed. INTERSALT data revealed a significant
positive correlation between the average sodium intake and the average blood
pressure of the adult population within a geographical region. The incidence
of hypertension and the tendency for blood pressure to increase with age also
correlated positively with sodium intake. In this study, a change of 100
monay in sodium intake caused a 3.54 mm Hg change in arterial blood
pressure (Stammler et 01.1991). The authors suggest that essential
hypertension may not be a metabolic disorder or a disease as is commonly
perceived, but is instead a normal response to the modern diet with its sodium
intake far in excess of what is needed for health or survival. Indeed, in the
few regions of the world where sodium intake is less than 60 mmol/day
hypertension does not exist! However, excessive sodium intake alone cannot
be the sole contributor to the development of all essential hypertension since
there is a wide variation in blood pressure between individuals at any given
sodium intake. High sodium intake is therefore a risk factor for the

development of hypertension in the same way that obesity and smoking are

8

risk factors. It is, however, a very pervasive risk factor in industrialized

countries where sodium intake is typically in excess of 200 monay.

2. Sodium intake intervention studies

A weakness of epidemiological studies such as those cited above is that
there are many differences between populations with divergent sodium intakes
in addition to disparate dietary sodium. Intervention trials performed within a
single population can control those confounding variables. These studies alter
sodium intake within a group of individuals matched for age, weight and
other variables. The corresponding changes in blood pressure quantify the
dependence of blood pressure on sodium intake within individuals rather than
within an entire population. Intervention studies focus on the ability of
dietary modification to alter blood pressure and evaluate the role of sodium in
the development and management of hypertension.

A consistent observation in sodium intervention studies has been that
individuals within a population respond to changes in sodium intake with
both qualitatively and quantitatively different blood pressure responses
(Sakaguchi et al. 1988, Weinberger et a1. 1986, Weinberger 1991b, Mascioli
et 01.1991) so that even within a fairly homogeneous population some people

show an increase in blood pressure to a sodium load while others Show a

9

decrease. The average affect in most populations is a slight decrease
(Sullivan 1991). Thus, within the general pOpulation’ there is a subset of
persons whose blood pressure varies in direct correlation to sodium intake
and another subset whose blood pressure is very resistant to sodium-induced
alterations (Mascioli et al. 1991).

The trait of sodium-sensitive blood pressure appears to be a heritable
one (Weinberger 1991a, Weinberger et 01.1987) and occurs more frequently
in hypertensive than normotensive individuals (Weinberger 1991a, Sullivan
1991, Sakaguchi et al. 1988). In fact, it is estimated that anywhere from 20
to 80% of hypertensive patients experience a fall in blood pressure with
dietary sodium restriction while the frequency of sodium sensitivity in
norrnotensive individuals is between 5 to 20% (Cutler et al. 1991).

Sodium intervention trials have thus defined a subset of essential
hypertensive patients with “sodium-sensitive hypertension”, defined as
"chronically elevated blood pressure directly correlated to sodium intake”.
This can be evaluated clinically by measuring the blood pressure response to

sodium loading and/or to sodium restriction (Muntzel and Darke 1992).

C. Characteristics of sodium-sensitive hypertension

Within the general population, it is not possible to accurately identify

10

individuals with sodium sensitive blood pressure except through their
response to changes in sodium intake. It would be useful clinically to
identify sodium sensitive individuals through non-invasive observations and
experimentally to identify a set of characteristics which clarify the cause of
sodium-sensitivity. Many factors have been proposed to this end. Age, race,
insulin resistance, obesity, a positive family history for hypertension, activity
of the renin-angiotensin-aldosterone system, and circulating levels of
catecholamines are some of the factors proposed.

Indications that sodium sensitivity increases with age came from
epidemiological studies. As discussed previously, the INTERSALT study
found that blood pressure increases with age in populations consuming a high
sodium intake. In this study, the statistical association of blood pressure with
sodium intake was stronger in older compared with younger patients
(Stammler et 01.1991). Some intervention studies have also shown an
increased incidence of sodium-sensitivity in older patients versus younger
patients (Weinberger et al. 1986, Zemel and Sowers 1988, Lqu et al. 1991),
but this association has not held true in every investigation (Umeda et al.
1988). In older patients as in younger patients, higher blood pressure
increases the probability of sodium sensitivity so that in studies of only

hypertensives, there is a much better correlation of sodium-sensitivity with

1 1
age (Umeda et 01.1988, Zemel and Sowers 1988). Older individuals have

also been shown to come into sodium balance more slowly than younger
individuals, an effect perhaps of decreased renal function or of increased
levels of naturally occurring antinatriuretic factors. This tendency may
contribute to their increased susceptibility to sodium-induced hypertension
(Lqu et 01.1991).

Racial differences have also been noted in the prevalence of sodium-
sensitive blood pressure. American blacks have a greater incidence of
sodium-sensitivity than either African blacks or American whites (Luft et
01.1991). It has been suggested that this apparent genetic alteration is the
result of natural selection brought about by the history of slavery which
created living conditions where the ability to conserve sodium was an
advantage (Wilson and Grim 1991). Similar to other groups at increased risk
for sodium-sensitivity, American blacks tend to excrete a sodium load more
slowly and are more responsive to diuretics than whites (Lufi et al. 1991,
Sowers et 01.1988). Furthermore, black hypertensives are particularly
responsive to diuretic therapy. This is generally accompanied by lower
plasma renin activity and increased levels of a circulating Na*/K+-ATPase

inhibitor, indications of volume expansion and sodium retention (Sowers et

al. 1988).

12

A third trait associated with sodium-sensitive hypertension is insulin
resistance. Patients and animal models of insulin-resistant diabetes exhibit a
greater incidence of sodium-induced alterations in blood pressure and slower
excretion of a sodium load (Rocchini e1 01.1989, Finch et al. 1990). A related
characteristic is obesity. Obesity is frequently a complicating factor in both
hypertension and diabetes and there is some evidence that it is independently
associated with increased sodium-sensitivity of blood pressure and
exaggerated sodium retention (Rocchini et 01.1989).

A positive family history for hypertension also increases the incidence
of sodium sensitivity (Weinberger et a1. 1986, Weinberger 1991a). The
tendency for this trait to follow family lines suggests a genetic abnormality
may be responsible for the altered response to sodium and for an overall
increased tendency to develop hypertension.

In addition to physical characteristics, certain hormonal alterations are
common in sodium-sensitive individuals. The first is an altered response of
the renin-angiotensin-aldosterone system (RAS) during changes in sodium
intake. In normal individuals, increased sodium intake leads to activation of
cardiopulmonary baroreceptors and concomitant decreases in sympathetic
activity, renin secretion, plasma ANG II concentration, and aldosterone

release (Creager et al. 1991). Among sodium-sensitive patients, there are two

l3

subgroups that exhibit abnormal RAS responses to changes in sodium intake.
One subgroup, termed ”non-modulators", fails to alter adrenal responsiveness
to ANG II during low sodium intake so that plasma levels of aldosterone do
not change during changes in sodium intake. A second characteristic of this
subgroup is failure of the renal blood flow to increase during high sodium
intake in response to lower ANG II levels. These changes could contribute to
sodium-sensitivity. The second subgroup with abnormal RAS responses are
”low-renin” hypertensives, a trait more common among black than white
hypertensives (Williams 1991, Keane et al. 1991). These patients have low
plasma renin levels and normal aldosterone levels. However, they exhibit
excessive aldosterone secretion in response to ANG 11. When exposed to
high sodium, therefore, these patients have suppressed renin and ANG II but
abnormally high aldosterone that may contribute to the sodium-sensitive
hypertension (reviewed Williams 1991, Sullivan 1991).

Additionally, observations of elevated plasma levels of other vasoactive
hormones have been made. These include: arginine vaSOpressin (AVP), a
potent neurohypophyseal vasoconstrictive and antidiuretic hormone (Mitch
and Wilcox 1981); atrial natriuretic peptide (ANP), a vasodilating and
natriuretic cardiac-secreted peptide (Sowers et al. 1988); endogenous ouabain-

like factor (OLF), a newly discovered natriuretic and perhaps vasoconstricting

14

compound (Keane et 01.1991); and insulin, an antinatriuretic and
sympathostimulatory compound in addition to its role in glucose homeostasis
(Tuck 1991). Finally, elevated plasma levels of catecholamines have also
been measured in sodium-sensitive patients suggesting that the expected
changes in sympathetic activity during alterations in sodium intake do not
occur in sodium-sensitive patients (Nishio et (11.1988).

There is not sufficient evidence to implicate any Specific factor as the
sole cause of sodium sensitivity of blood pressure. In fact, sodium-sensitive
blood pressure appears to be a heterogenous condition with multiple causes.
The characteristics can, however, be used as guidelines in the experimental

search for mechanisms which mediate this condition.

D. Potential mechanisms of sodium-sensitive hypertension

Mean arterial pressure (MAP) can be elevated in one of two ways,
through increased CO or increased total peripheral resistance (TPR). Under
most conditions, an increase in either of the two variables causes a reflex
decrease in the other variable; therefore blood pressure does not change.
However, in cases of sustained hypertension, the appropriate compensation
does not occur resulting in a persistent increase in one or both of these

variables. In almost all cases of hypertension including SDH, TPR is

15

increased.

One way TPR can be increased is through physical alterations of the
vascular bed. Remodeling of the vascular structure that reduces the number
of resistance vessels decreases the capacity of the vasculature and increases
the resistance to the same volume of blood. Similarly, decreased compliance
of blood vessels prevents volume induced distension of resistance vessels
during small changes in CO and increases blood pressure. Alternatively,
extrinsic neural or hormonal stimulation can cause vasoconstriction of
structurally normal resistance vessels leading to increased TPR. Increased
sympathetic nervous system activity (SNA) stimulates 0t,-adrenergic receptors
on small arterioles while vasoactive hormones such as ANG II or AVP act
directly on vascular receptors to stimulate vascular smooth muscle
contraction. The evidence for direct neural and hormonal vasoconstriction in
SDH is discussed below.

TPR may also be increased independent of structural changes to blood
vessels by tissue “autoregulation” in response to an initial elevation in CO.
The theory of autoregulation was first proposed by Borst and Borst-de-Geus
(Borst and Borst-de-Geus 1963) to explain the observed elevation of TPR
under conditions expected to produce elevated CO. According to this theory,

elevated CO increases the blood flow to peripheral vascular beds causing

l6

overperfusion of peripheral tissues. Intrinsic autoregulatory control systems
cause vasoconstriction of the overperfused beds normalizing tissue blood
flow. In cases of chronically elevated CO, sustained autoregulatory
vasoconstriction in overperfused beds should lead to a sustained elevation in
TPR.

The elevation in CO necessary to stimulate autoregulation can be
achieved in several ways. First, chronically expanded blood volume (BV)
elevates CO by overfilling the vasculature. Expanded BV due to inadequate
excretion and accumulation of water and sodium can be caused by a primary
renal defect; increased renal SNA leading to tubular sodium reabsorption and
the release of anti-natriuretic hormones (ANG II, aldosterone, eg.);
oversecretion of antinatriuretic hormones independent of SNA; or a
combination of the above. CO can also be elevated independent of BV
expansion by increased SNA to the heart causing increased strength and
fi'equency of contraction. This inotropic and chronotropic stimulation delivers
increased blood flow to the tissues resulting in autoregulatory
vasoconstriction. The primary stimuli that can lead to autoregulation induced
elevated TPR are therefore a primary renal defect, increased circulating
antinatriuretic hormones, or elevated SNA. The experimental evidence for

the participation of autoregulation in SDH is discussed below.

17

In summary, the mechanisms causing hypertension in any given
condition are complex and interrelated. Unraveling the mechanisms
responsible for sodium sensitivity of blood pressure is important, however, to
efficiently and effectively treat the hypertension which is a fi'equent result of
this condition. In SDH, increased sodium intake must cause an increase in
either CO or TPR to raise blood pressure. Normally, when sodium intake is
high enough to alter body sodium content, cardiopulmonary receptors are
stimulated to decrease SNA (lowering TPR and renal sodium retention), the
renin-angiotensin system is suppressed (Holtzman et al. 1989). Any increase
in CO by expanded blood volume is offset by a concurrent decrease in TPR
and blood pressure does not change. However, in SDH arterial pressure does
change so there must be either an insufficient suppression of the SNA, the
RAS or a markedly exaggerated increase in CO. One way CO could be
elevated is through excessive renal sodium and water retention.

1. Primary alteration of renal function

A primary role for renal function in the pathogenesis of essential
hypertension, especially sodium-sensitive hypertension, has been postulated
for several decades. Renal function and blood pressure are inexorably linked
through their common function of fluid homeostasis: the ability of the

kidneys to excrete water and sodium is dependent on renal perfusion pressure

l8

(systemic pressure), while perfusion pressure in turn is dependent on body
fluid volume, CO and thus blood pressure. A decrease in renal excretory rate
can thus increase body fluid volume and raise blood pressure. The elevated
blood pressure then restores renal excretion of water and sodium back to
initial levels. This relationship is frequently described as “pressure
natriuresis" and some investigators attribute the genesis of all hypertension to
an abnormal pressure natriuresis relationship (deWardener 1990 parts I & II,
Guyton et 01.1988).

According to this theory, any condition leading to impaired renal
excretion of sodium and water will cause increased blood volume and
ultimately, elevated blood pressure. Higher arterial pressure is seen as a
requirement to achieve fluid homeostasis in the face of impaired sodium
excretion by the kidney. Studies have shown that changes in renal function
do in fact, profoundly influence blood pressure (reviewed in deWardener
1990 parts I & II). A problem with this theory, however, is that it suggests
that hypertension should be associated with increased CO (due to expanded
blood volume); yet almost all hypertension, including sodium-sensitive
hypertension, is caused by increased vascular resistance (Sullivan 1991,
Muntzel 1992). To resolve this apparent contradiction, it has been proposed

that elevated CO rapidly brings about whole body autoregulation. As

19

described above, overperfusion of all vascular beds causes autoregulatory
vasoconstriction, increased total peripheral vascular resistance and a return of
CO to normal (Cowley 1990). This sequence of events could link reduced
renal sodium excretory capacity to a condition of chronic hypertension
characterized by increased TPR (Guyton 1990), without the need to invoke
any hormonal or neural abnormalities as etiologic factors. This theory
requires a residual increase in CO to maintain the elevated TPR but studies in
SDH have not revealed expanded plasma volume (Sullivan et al.1987) and
persons genetically predisposed to hypertension do not have increased body
sodium compared to normal subjects (Berretta-Piccoli 1990). This has
prevented definitive proof that decreases in renal function per se are the
primary cause of clinical hypertension. Furthermore, renal damage can also
be a consequence rather than an initiating factor of hypertension (Obatomi et
al. 1992).

Chronically elevated blood pressure leads to damage of glomerular
mesangial cells and to reduced glomerular filtration (Anderson et 01.1985,
Jackson and Johnston 1988). This is a primary symptom of renal damage in
clinical hypertension (Blythe and Maddux 1991). Damage to the glomerulus
leads to tissue hypertrophy and eventual necrosis (Ylitalo and Gross 1979)

which in turn increases the filtration load on the remaining glomeruli and a

20

vicious cycle of progressive renal deterioration leading to malignant
hypertension (Kincaid-Smith 1991). If renal arterioles are able to control
renal perfusion pressure at the glomerulus or if pharmacological agents are
used to selectively decrease glomerular perfusion pressure, damage to
glomeruli is prevented (Hostetter et al. 1981). In clinical hypertension
exhibiting renal damage, the amount of overt renal damage is directly
proportional to the duration and severity of the elevated blood pressure, and it
is difficult to determine if the renal damage or the hypertension was the
initiating condition (Blythe and Maddux 1991, Obatomi et al. 1992).

The possible importance of the kidney in human hypertension has been
highlighted by studies of renal transplant recipients. In these studies,
recipients of kidneys from patients with no family history of hypertension had
a decreased incidence of hypertension compared to transplant recipients with
kidneys from donors with a positive family history for hypertension (Guido et
al. 1985, Strandgaard and Hansen 1986). The protective effect of
"normotensive kidneys” was only seen, however, when the recipient was also
normotensive prior to the transplant surgery, so that in patients already
predisposed to hypertension, a kidney from a normotensive donor did not
alter the progression of the disease. It is possible, however, that in this latter

case the existing hypertension rapidly damaged otherwise normal kidneys,

2 1
leading to impaired renal function.

In some sodium-sensitive hypertensives there is evidence of decreased
renal excretory capacity as evidenced by acute excessive sodium retention
when compared to sodium-resistant patients (Sullivan 1991, Wu et 01.1990).
This could reflect an inherent (perhaps genetic in nature) defect in sodium
handling by the kidneys of these patients, or be the result of inappropriate
hormonal or neural influences on the kidney, as will be discussed in the
following section. Even though the majority of sodium-sensitive patients do
not have a clearly defined renal defect or gross sodium retention, most
researchers have sought to characterize sodium-sensitive hypertension in
terms of an intrinsic renal abnormality (Hall and Hungerford 1982). This
characterization seems unjustified in light of the multiple mechanisms in
addition to intrinsic renal abnormalities which also appear to be involved in
SDH.

2. Hormonal influences
a. hormone influences at the kidney

In the absence of structural or functional renal abnormalities, altered
renal function may still initiate SDH. Abnormal levels of circulating
hormones which affect renal function can act on a normal kidney to produce

hypertension.

22

First, excessive secretion of mineralocorticoids or infusion of
exogenous mineralocorticoids such as aldosterone act at the kidney to cause
transient sodium and water retention and produce a gradual elevation of
arterial pressure (Pan and Young 1982, Garwitz and Jones 1982). Although
excessive levels of mineralocorticoids are not typically found in the plasma of
sodium-sensitive hypertensives, the renin-angiotensin-aldosterone system
responds abnormally to changes in sodium intake in certain sodium sensitive
patients (Williams 1991).

The low-renin sodium-sensitive hypertensives described earlier provide
the best clinical example of how aldosterone acting at the kidney could
contribute to sodium sensitivity in apparent essential hypertension. In these
patients, adrenal secretion of aldosterone is abnormally sensitive to ANG 11 so
that blood levels of the mineralocorticoid are higher than normal for any
given level of ANG 11 (Williams 1991, Fraser and Padfield 1989). Studies in
animals given aldosterone infusion have shown that the mineralocorticoid acts
in the distal tubule of the kidney to cause sodium and water retention
(reviewed Brown et 01.1979). In addition, excess circulating aldosterone is
associated with increased glomerular filtration rate (GFR) and filtered sodium
load (Pan and Young 1982). These effects are accompanied by an initial

transient sodium retention and, when accompanied by high sodium intake, a

23

sustained increase in arterial pressure (Pan and Young 1982, Garwitz and
Jones 1982). In this way, chronic excess aldosterone could cause renally-
mediated SDH without apparent volume expansion. The absence of excessive
sodium and fluid retention during aldosterone excess is due to the sodium
escape phenomenon that has been described in cases of continued aldosterone
excess.

Evidence from angiotensin converting enzyme inhibitors (CEI) studies
suggests that ANG II can alter renal function in some cases of SDH. In the
group of sodium-sensitive patients termed non-modulators, CEI have been
shown to be very effective antihypertensive agents (Dluhy et a1. 1989). Also,
in hypertensive patients with chronic renal failure, CEI are very effective at
lowering blood pressure, reversing proteinuria and protecting against further
degradation of renal function (Brenner et al. 1985).

Within the kidney, ANG 11 may affect MAP by stimulating increased
sodium reabsorption and decreased renal blood flow (Mattson et al. 1991).
First, ANG 11 causes vasoconstriction of renal arterioles to decrease renal
blood flow and sodium delivery to the glomerulus. This is accompanied by
an ANG II receptor mediated decrease in the glomerular ultrafiltration
coefficient and a concomitant decrease in GFR (Navar et al. 1991). In the

face of elevated systemic pressure, preglomerular afferent renal arterioles

24

exhibit autoregulatory vasoconstriction further decreasing renal blood flow
(reviewed Carmines and Fleming 1990). In the proximal distal tubule, ANG
II stimulates NaVI-I+ exchange to directly increase sodium reabsorption and
enhances the sensitivity of tubuloglomerular feedback (Cogan 1990, reviewed
Ichikawa and Harris 1991). In the face of elevated ANG 11 delivery to the
kidney, these intrarenal actions of ANG 11 during sodium loading Should
combine to produce sodium retention and volume expansion leading to
increased CO, autoregulatory vasoconstriction and elevated blood pressure.
These actions at the kidney are independent of any intrinsic renal defect. It
has been proposed that increased activity of an entirely intrarenal RAS could
produce hypertension in the face of normal plasma levels of ANG 11 such as
those found in low-renin essential hypertension. The possible contribution of
an intrarenal RAS to SDH remains undetermined but the actions of
circulating ANG II are not confined to the kidney. Therefore the vascular
and neural actions of the peptide during chronic elevations in plasma ANG 11
may be more important in the maintenance of hypertension than the renal
effects and will be discussed in the next section.

Another circulating peptide that affects renal function is AVP, a
hormone produced and released in the anterior pituitary in response to

changes in plasma osmolality and by stimulation of the sympathetic nervous

25
system (Simon-Oppermann and Gunther 1990, Berecek and Swords 1990,

Simon et al. 1989). AVP acts on V,z receptors in the cortical collecting ducts
of the kidney to increase sodium and water reabsorption (Ando et al. 1989,
Jeffiies et 01.1991). Alterations in AVP have been noted in some cases of
SDH (Mitch and Maddux 1982) and systemic administration of the peptide
causes vasoconstriction, antidiuresis and antinatriuresis (Liard et al. 1987).
AVP differs from ANG 11, however, in its effects in the renal vasculature.
Systemic infusion of AVP does not alter either renal blood flow or GFR
suggesting that it does not constrict renal arterioles (reviewed Carmines and
Fleming 1991). Recent reports in isolated blood vessels show that AVP may
induce postglomerular vasoconstriction which contributes to the reabsorption
of water at the vasa recta but which does not decrease total renal blood flow
(Edwards et al. 1989). Consequently, the primary renal effect of AVP is
water, and to a lesser extent, sodium reabsorption. The lack of a strong anti-
natriuretic effect may be why chronic infusion of AVP only produces
sustained hypertension when renal perfusion pressure is servocontrolled to
decrease renal blood flow (Hall et al. 1987, Liard 1987). For this reason it is
unlikely that alterations in AVP actions on the kidney are the stimulus for
SDH.

Another alteration that could induce renally-mediated hypertension is

26

decreased delivery of natriuretic hormones to the kidney. However, no
decrease in atrial natriuretic peptide, a potent natriuretic peptide released from
the cardiac atria, has been found in SDH. In fact, some animal models of
SDH actually Show a slight increase in the peptide (Brandt et 01.1987). The
other primary natriuretic substance is an endogenous analog of the plant
product, ouabain. This factor has been found to be elevated rather than
reduced in the plasma of some essential hypertensive patients and to increase
during sodium loading (Haddy 1990, Graves et 01.1989). Therefore the renal
effect would be' to promote natriuresis and thus reduce blood pressure. A
contribution of this ouabain like factor (OLF) would therefore not be through
altered renal sodium handling but through a non-renal mechanism.
b. extrarenal hormonal influences

In addition to the renal actions outlined above, circulating ANG II
affects blood pressure through direct vasoconstriction of vascular smooth
muscle cells. This vasoconstrictor action is dependent on AT, type receptors
and is responsible for the acute increase in blood pressure following
intravenous ANG II administration (reviewed Timmerrnans et 01.1991).
Studies in both humans and laboratory animals have shown that during
chronic administration of ANG 11, there is an apparent decrease in both the

number of vascular receptors and the vasoconstrictor response (Pawloski

27
1990, Bruner and Fink 1986). Therefore the chronic condition of sodium-

sensitive hypertension isrunlikely to be mediated through direct
vasoconstriction by excess circulating ANG II.

An additional peripheral effect of circulating ANG II is enhanced
norepinephrine release fiom peripheral adrenergic terminals. This has been
demonstrated after acute direct administration of the peptide (Trachte 1988,
Ellis and Bumstock 1989) and after chronic ANG 11 treatment (Randall and
Zimmerman 1990). Chronic treatment with CEI has also been shown to
decrease norepinephrine release by sympathetic nerve stimulation (SNS)--an
effect reversed by ANG 11 replacement (Randall and Zimmerman 1990).
These studies suggest enhancement of SNA by ANG 11 could contribute to
chronic increases in TPR.

Circulating ANG II also acts at the adrenal medulla where it stimulates
epinephrine and norepinephrine release (F euerstein et al.1977) to augment
sympathetically mediated vasoconstriction. These direct and indirect
vasoconstricting actions of circulating ANG 11 may contribute to the increased
blood pressure of some sodium-sensitive hypertension. The possible
contribution of these actions have not been investigated because plasma levels
of ANG II are not elevated in sodium-sensitive hypertension and the

antihypertensive mechanism of CEI action in non-modulators and in

28

hypertensive patients with chronic renal failure has not been determined. If
the sensitivity to circulating ANG II is increased in these conditions, ANG II
interaction with the peripheral components of the SNA mediated
vasoconstriction could be responsible for the chronic elevation in TPR.

Within the central nervous system, ANG II alters efferent sympathetic
activity and therefore vascular tone. In the brain, specific ANG II binding
has been demonstrated in brainstem nuclei known to control cardiovascular
function (Gehlert et 01.1991, Wamsley et 01.1990) and central administration
of ANG II stimulates the release of norepinephrine and modulates the
function of the baroreflex (Xiong and Marshall 1990, Kannan et 01.1991).
These actions are all within the blood brain barrier and support a role for
centrally produced ANG II. This role is further substantiated by the finding
that direct infusion of ANG 11 into the lateral cerebral ventricles produces
sustained SDH (Fink et al. 1983, Bruner and Fink 1986).

However, intracerebroventricular administration of the ANG II
antagonist saralasin does not block the development of hypertension in
animals made hypertensive by chronic iv infusion of ANG II (Bruner and
Fink 1985). In contrast, lesion of the area postrema (AP) a circumventricular
organ with a high number of ANG 11 receptors (Ferguson 1990), prevents

development of hypertension dependent on circulating ANG II (Fink et

29
01.1987, Matsukawa and Ried 1990). The hypertension produced by

circulating ANG 11 therefore may be mediated through this brain structure
outside the blood brain barrier, independent of the separate actions of ANG 11
within the brain.

Finally, ANG II is a potent secretagogue of aldosterone acutely
(Williams 1991) but the effect of chronic ANG II excess on aldosterone
release is not clear. In dogs (Cowley and McCaa 1976) and sheep (Blair-
West et 01.1963), a chronic ANG II infusion did not maintain elevated PAC
whereas in humans (Oelkers and Schoneshofer 1983) and rats (Hanger et
01.1978) chronic elevations have been reported. The involvement of ANG II
in the alterations in PAC in some sodium-sensitive patients has not been
investigated and the effect of chronic excess ANG II on PAC during elevated
sodium intake is an important area that remains to be investigated.

Alternatively, the elevated PAC observed in some sodium-sensitive
hypertensive patients could be independent of the RAS and alone responsible
for the hypertension. Aldosterone excess was one of the first clinical
observations shown to produce SDH. Aldosterone acts on cytosolic receptors
stimulating increased mRNA transcription and subsequent protein synthesis.
The exact proteins induced are unknown but the direct effect is enhanced

Na+ +-ATPase synthesis and increased number of active Na+ channels

30

leading to increased renal sodium reabsorption accompanied by water
retention and potassium excretion ( Horisberger and Rossier 1992). In the
gastrointestinal tract, aldosterone causes sodium reabsorption and enhances
fecal potassium loss. Excess aldosterone is characterized by expanded total
body fluid volume, plasma hypokalemia, and systemic alkalosis. Increased
sodium intake exacerbates and accelerates the development of this condition
(Williams 1991, Mitch and Wilcox 1981).

The mineralocorticoid, deoxycorticosterone (DOCA), has been widely
used experimentally to establish a model of sodium-dependent, volume-
expanded hypertension. In this model, increased sodium intake causes
sodium and water retention and a gradual rise in blood pressure (Pan and
Young 1982). However, CO, a relative measure of expanded blood volume,
is not consistently elevated and the driving force of the hypertension appears
to be elevated peripheral resistance (Pan and Young 1982). Some have
suggested that mineralocorticoids act directly on vascular smooth muscle to
cause increased reactivity and enhance vasoconstriction (Garwitz and Jones
1982). It has also been suggested that autoregulation caused by increased
blood flow may be the mediator of the elevated TPR in DOCA-salt
hypertension (Hall et 01.1987). A recent study has reported a direct receptor-

mediated blunting of baroreceptor discharge by circulating aldosterone (Wang

31
et 01.1992).

Other investigators have suggested that aldosterone acts within the
central nervous system to alter the release of the vasoactive hormone AVP
(Janiak and Brody 1988) and the efferent activity of the sympathetic nervous
system (Gomez-Sanchez 1986). All of these actions may combine during
aldosterone excess to contribute to the resultant hypertension and be a part of
the etiology of sodium-sensitive essential hypertension (Williams 1991).

In addition to the abnormal renin-angiotensin-aldosterone response,
extrarenal actions of other humoral factors have been implicated in sodium-
sensitive hypertension. One of these is OLF, a blood-borne factor which
inhibits ATP dependent sodium-potassium exchange (Na+/K+-ATPase) and
which may be over-secreted in sodium-sensitive individuals. This factor is
known to elicit natriuresis, among other actions, and is often called the
natriuretic hormone. Evidence that the factor is involved in hypertension is
substantial but mostly indirect. The plasma of some volume-expanded
hypertensive patients inhibited Na+/K+-pump activity in vitro (Keane et a1.
1991, Nishio et al. 1988, Poston et al. 1981) and pump inhibition in
experimental animals induced natriuresis and vasoconstriction in viva
(Pamnani e1 01.1991). Plasma from patients with pregnancy-induced

hypertension (Kaminski and Rechsberger 1991), insulin-induced hypertension

32

(Graves et 01.1989), as well as sodium-sensitive essential hypertension (Keane
et 01.1991) has the ability to inhibit the Na“ + exchanger. In animal studies,
increased activity of a pump inhibitor has been demonstrated in volume
expanded dogs (Gruber 1980), and in reduced renal mass rats (Hout et al.
1983, Shima et al. 1988, Nakagawa et al.1990). Platelets fi'om hypertensive
individuals have inhibited ATP sensitive Na+ + transport and elevated levels
of intracellular calcium, presumably as a result of increased Na+/Ca+2
exchange in response to decreased Na+/K+ exchange (Poston et al. 1981,
Keane et 01.1991).

A candidate molecule for this factor has recently been isolated fiom
plasma and identified as a structural twin to the plant-derived cardiac
glycoside, ouabain (Ludens et al..l99l, Bova et al. 1991). Preliminary assays
of this cholesterol-derived steroid Show that the factor is elevated during
volume expansion (Nishio et al.1988, Kaminski et a1. 1991 and Keane et el.
1991), increases contractility in isolated human resistance arteries (Bova et al.
1991, Woolfson et al. 1990) and raises blood pressure in rats (Pamnani et
01.1991). Contradictory evidence has also been published in the rat, where
chronic infusion of ouabain did not elevate blood pressure (personal
observation and Yasujima et al. 1986).

The way in which such an inhibitor could theoretically contribute to

33

elevated TPR in sodium-sensitive hypertension is outlined below. Inhibition
of the Na+ +-ATPase elevates intracellular sodium levels stimulating sodium-
calcium exchange. This in turn increases intracellular calcium concentration
as sodium is exchanged for calcium. The electrical gradient across the cell
membrane is altered and the resultant decreased polarization in the membrane
potential leads to hyper-reactivity of the cell. Additionally, the increased
calcium content in smooth muscle cells causes increased force of contraction
(see Haddy 1990 for review).

In addition to effects on vascular smooth muscle cells, Na+/K+-ATPase
inhibitors affect contractility of cardiac myocytes and Na“/K+ exchange in
renal cortical collecting tubules (Pedrinelli et al. 1989, Haddy 1990). Chronic
inhibition of renal Na+/K+ exchange would promote natriuresis and diuresis to
prevent volume expansion but this would be offset by the chronic actions at
the heart to increase CO. Inappropriate expression of OLF in sodium-sensitive
individuals may thus contribute to the development of elevated TPR directly
and indirectly in SDH (Keane et al. 1991, Graves et al. 1989, Kaminski and
Rechsberger 1991, Nishio et al. 1988).

Another circulating hormone that has nonrenal cardiovascular actions
and may contribute to the development of sodium-sensitive hypertension is

the neurohypophysial peptide, AVP. Elevated plasma levels of this

34

vasoconstrictor have been measured in some sodium-sensitive hypertensives
(Mitch and Wilcox 1981). In addition to its renal effects discussed earlier,
circulating AVP acts on V, receptors on vascular smooth muscle to cause
vasoconstriction, in the central nervous system to increase sympathetic
outflow (Berecek and Swords 1990) and at unidentified peripheral sites to
alter baroreflex function (Pullan et al. 1980). Acutely, intravenous
administration of AVP causes a rapid increase in arterial pressure and a
significant decrease in water and sodium excretion (Pawloski et a1. 1989).
Although it has been reported that infusion of AVP into baroreceptor
denervated rabbits causes a sustained elevation in blood pressure (Ryuzaki et
01.1991), this is not the case in intact animals. In intact animals, prolonged
infusion of vasopressin for several days or weeks does not cause a sustained
rise in arterial pressure, even in the face of high sodium intake (Pawloski et
al. 1989, Liard 1987). Additionally, vasopressin antagonists fail to lower
blood pressure in models of SDH (Pawloski et 01.1989, Liard 1987) making it
difficult to contend that elevated circulating levels of this hormone contribute
to the chronic increase in peripheral resistance of SDH.

3. Alteration of the sympathetic nervous system

a. renal sympathetic nervous system actions

Altered activity of the sympathetic nervous system independent of

35

circulating hormones may contribute to sodium-sensitive hypertension.
Evidence of increased SNA in the periphery during SDH includes elevation
of plasma catecholamines (Campese et al. 1982, Takeshita et al. 1982, Nishio
et al. 1988), forearm vascular resistance (Fujita et al. 1980, Sullivan et al.
1987) and TPR (Mark et al. 1975). Additional support is found in the
abnormal hypotensive response to clonidine blockade (Dichtchenkenian et al.
1989) and the measurement of abnormally high norepinephrine levels in some
sodium-sensitive patients during. high sodium intake (Gill et 01.1988).
Normally, high sodium intake stimulates the cardiopulmonary baroreflex
which decreases sympathetic outflow. However, this response appears to be
altered in some sodium sensitive patients and the vascular response to
norepinephrine infusion during high sodium intake is elevated compared to
non-sodium sensitive patients (Sakaguchi et al. 1988, Creager et al. 1991,
Trimarco et al. 1991) suggesting that high sodium intake alters the post-
synaptic response to this catecholamine. Inappropriate SNA may contribute
to SDH through both renal and extrarenal actions. Indeed, there is evidence
of elevated total SNA (Anderson et al. 1989) and of selective renal SNA
(reviewed DiBona 1992) in some essential hypertensives.

The way increased renal SNA elevates blood pressure appears to be

through at least three separate mechanisms. First, selective stimulation of

36

efferent renal nerves causes sodium retention and vasoconstriction of renal
arterioles (Osborn et al. 1983). Secondly, direct application of norepinephrine
into the kidneys stimulates renin release and increases plasma renin activity
(Osborn 1987, Greenburg et al. 1991). The release of renin is mediated
through stimulation of [3,-adrenergic receptors on the granular cells of the
juxtaglomerular apparatus of the kidney and leads to increased plasma
concentration of ANG II (Osborn et al. 1983, Inagami et al. 1990).
Circulating ANG II, as described above, can cause hypertension.

A third renal effect of SNA is antinatriuresis independent of decreases
in renal blood flow and renin secretion. This antinatriuresis appears to be an
intrarenal response to (1, receptor stimulation (Osborn et al. 1983). Finally,
stimulation of efferent renal nerves decreases renal blood flow and therefore
sodium delivery to the glomerulus. The combined renal effect of SNA is
therefore decreased sodium excretion, increased renin release, decreased renal
blood flow and elevated systemic pressure. Increased sympathetic stimulation
at the kidney in SDH could therefore contribute to hypertension but should be
accompanied by sodium retention and elevated plasma levels of renin. These
are not seen in SDH so it is unlikely that purely renal effects of SNA are
responsible for the chronic elevation in blood pressure seen in SDH.

b. extrarenal sympathetic nervous system actions

37

The sympathetic nervous system innervates all tissues known to affect
blood pressure. In addition to the kidney, this includes the adrenal medulla
and cortex, arterial blood vessels, and the heart. The primary effects of the
SNS on systemic blood pressure include vasoconstriction of resistance vessels
through 0t, vascular receptors, elevated CO through [3, cardiac receptors, and
release of ANG 11. As described above, a sympathetically mediated elevation
in TPR or CO increases MAP. While circulating catecholamines affect
many physiological functions, the systemic cardiovascular effect is increased
heart rate and MAP so that elevated SNA is almost invariably associated with

elevated MAP (reviewed by DiBona 1992).

E. Animal models of hypertension

Because of the inherent ethical limitations of intervention in clinical
studies, animal models have been developed to more precisely examine the
ways alterations in renal function, hormone levels, the nervous system and
sodium intake interact to affect blood pressure.

The first animal model of chronic hypertension was developed by
Goldblatt in 1934. By partially constricting the renal artery and removing the
opposite kidney, dogs were made to develop persistent hypertension that was

exacerbated by elevated sodium intake (Goldblatt et al. 1934). Blood and

38

plasma from animals with Goldblatt hypertension cross-circulated into
anephric normal animals caused a sustained hypertension through a
circulating hormone subsequently identified as ANG II (Goldblatt 1968).
This model showed clearly for the first time that alterations in renal function
can cause chronic hypertension, in this case via a predominantly hormonal
mechanism.

Infusion of extracts from normal kidneys also elevated blood pressure
in rats. The substance in the renal extracts that increased pressure is renin, an
enzyme responsible for the formation of angiotensin II (reviewed Osborn
1991). Both of these early models revealed the important interaction between
circulating hormones and renal function in the genesis of SDH. Later models
of hypertension with specific correlates in clinical SDH have attempted to
further unravel the mechanisms of human SDH. Included among these
models are genetically selected animal strains which spontaneously develop
hypertension, surgical alterations of renal function which lead to sodium
dependent hypertension and hormone infusion models which artificially
increase vasoactive and/or antinatriuretic hormones to elevate blood pressure.

1. Genetic models
Since the majority of human hypertension, including SDH, is not

secondary to a specific physiological alteration but appears to be an heritable

39

condition of unknown etiology, animal strains which also have a genetic
predisposition to hypertension have been developed and used extensively in
hypertension research.

In 1962, Dahl and coworkers developed a genetic model of SDH.
They used selective breeding to develop two inbred strains of rats with
markedly different responses to increased sodium intake. On a diet of
moderately elevated sodium intake one strain developed a rapid increase in
blood pressure (Dahl-S) while the other showed complete resistance to
sodium-induced hypertension (Dahl-R) (Dahl 1960). In this model the kidney
is again implicated. When the kidney from a Dahl-S rat is transplanted into a
nephrectomized Dahl-R rat, the resistant rat develops SDH. This is not the
case when the Dahl-R rat receives a kidney from another Dahl-R rat.
However, Dahl-S rats receiving Dahl-R kidneys also develop hypertension,
suggesting that, while the kidney is important for the development of
hypertension in this strain, extrarenal factors also play a role (Morgan et
01.1990). In addition, cross-circulation of blood from a hypertensive Dahl-S
rat to a normotensive Dahl-R rat causes increased blood pressure (Dahl 1969).
This suggests that a circulating factor which is either released from the
kidney or which causes irreversible changes in the kidney, confers sodium-

sensitivity of blood pressure in this model. Interestingly, however, Dahl-S

40

rats do not retain more sodium than Dahl-R rats when sodium intake is
increased (Greene et al. 1990) showing that volume expansion is not the
primary mechanism maintaining hypertension in Dahl-S rats.

Another genetic model of hypertension, the spontaneously hypertensive
rat (SHR), is also sodium-sensitive and hypertension can again be transferred
by transplanting a kidney (Rettig et al. 1990, Rettig et a1. 1991). In these
rats, a high sodium intake enhances the renal SNA and antinatriuresis during
environmental stress suggesting that the apparent renal defect may be an
altered response to sympathetic stimulation (Koepke and DiBona 1985).

In genetic models of hypertension it is assumed that all genetic
alterations contribute to hypertension development. However, the inbreeding
necessary to develop such strains produces many strain-specific characteristics
that may be entirely independent of the hypertension. An alternative
approach in hypertension research has been to mimic hormonal alterations
observed in human SDH. By examining a specific alteration in isolation it
can be determined if such alterations indeed can initiate and maintain
hypertension.

2. Hormone infusion models
a. mineralocorticoid infusion

A widely studied model of SDH is mineralocorticoid excess

41

accompanied by high sodium intake. Deoxycorticosterone (DOCA), an
endogenous adrenal steroid, has been used more extensively than aldosterone
because of its solubility in water. However, findings with the two hormones
have been very similar and correlate well with observations in the human
condition of hyperaldosteronism. This rare form of hypertension is
characterized by elevated plasma levels of aldosterone, sodium retention,
hypokalemia, and chronically elevated TPR. In addition, there is increased
vascular smooth muscle reactivity to norepinephrine and metabolic alkalosis
(Fraser and Pudfield 1988).

The mechanisms responsible for mineralocorticoid hypertension have
not been clearly defined although many have been suggested. Excessive
sodium retention leading to blood volume expansion is a logical choice,
particularly since exogenous infusion of mineralocorticoids caused sodium
retention (Pan and Young 1982, Cox 1986). However, the increase in blood
pressure did not occur simultaneously with the sodium retention and blood
pressure continued to rise even after sodium balance was achieved (Pan and
Young 1982). It is possible that the initial volume expansion triggered an
autoregulatory increase in TPR as described above. This is in agreement with
the observation that the sustained phase of aldosterone-salt hypertension is

dependent on elevated vascular resistance (Hall et a1. 1987). Alternatively,

42

the elevated vascular resistance may be due to direct actions of the steroid on
vascular smooth muscle. Isolated blood vessels from DOCA-salt rats have an
increased response to pharmacological vasoconstrictors, apparently due to
altered ionic transport in vascular smooth muscle cells (Worcel and Moura
1987, Smith et al. 1988, Garwitz and Jones 1982) leading to the chronic
vasoconstriction and elevated TPR. However, administration of DOCA
without elevated sodium intake does not elevate MAP or increase vascular
reactivity in spite of altered sodium transport in vascular smooth muscle cells
(Cox 1986). In all cases, the hypertension following mineralocorticoid
infusion is sodium dependent and mediated chronically by elevated peripheral
vascular resistance. The contributions of volume-induced autoregulation,
direct vascular effects and centrally mediated elevated SNA to the
hypertension remain undefined.
b. ANG II infusion

Another widely used experimental model of SDH is ANG II infusion.
As described earlier, exogenous administration of low doses of ANG II in
combination with a moderate elevation in sodium intake produces a rapidly
reversible chronic elevation in blood pressure. Numerous investigators have
shown that ANG II-sodium hypertension is initially dependent on direct

vasoconstriction, but the chronic effect on MAP is due to actions at non-

43
vascular receptors (Brown et 01.1981, Pawloski 1990, Cox and Bishop 1991).

As discussed previously, ANG II has many non-vascular sites of action which
could contribute to hypertension including renal, adrenal and brain. It has
also been suggested that a trophic effect of ANG II on resistance vessels may
contribute to decreased compliance and hypertension (Simon 1992). More
evidence is available, however, to support the contention that the increased
TPR is due to either elevated SNA dependent on altered baroreflex, or
autoregulatory vasoconstriction dependent on decreased renal sodium
clearance. The evidence for a centrally mediated rise in SNA includes the
lesion studies cited earlier and the observation that the drop in MAP in
response to pharmacological ganglionic blockade during the chronic phase of
the hypertension is greater than during the initial phase (Pawloski 1990).. The
evidence of enhanced renal sodium retention has depended largely on in vitro
studies of ANG II affects on renal sodium handling. A recent study by
Krieger and Cowley found that sodium induced fluid volume expansion must
be present for ANG II to chronically elevate MAP (Krieger and Cowley
1990). However, in a similar study it was found that while volume expansion
was necessary for ANG II induced hypertension, volume expansion alone did
not increase MAP. Therefore volume expansion alone cannot initiate

hypertension and the mechanisms of ANG II hypertension are not yet clear.

44

The possible role of increased aldosterone secretion during elevated
circulating ANG II is also not resolved. The conflicting evidence from
different species suggests that rats and humans respond similarly with an
increase in PAC during chronic ANG II excess but the contribution of this
elevation in aldosterone to hypertension requires further investigation.

Finally, a role for ANG II in experimental models of hypertension not
marked by elevated plasma levels of the peptide is suggested by the ability of
CEI to lower blood pressure in SHR (Okumishi et al. 1991) and reduced
renal mass animals (Olson et al. 1982). The role of the peptide is in these
models needs further evaluation, perhaps through the use of the new specific
nonpeptide antagonists of ANG 11. Furthermore, similarities between
different models of hypertension that depend on ANG II to maintain the
elevated MAP Should clarify ways the peptide chronically increases MAP.

One animal model of SDH where ANG 11 appears to play a role is the
reduced renal mass model. This model directly correlates to the human
condition of chronic renal failure, a condition frequently associated with
hypertension (Blythe 1991) and has been used extensively in research of the
mechanisms in the progressive renal degeneration characteristic of chronic
renal failure. In addition, RRM-salt hypertension provides a method of

investigating the initiation of a form of SDH instead of observing only an

45
already established hypertensive condition.

3. Reduced renal mass model of sodium-dependent hypertension
a. physical and metabolic changes

In 1932 Chanutin and Ferris first introduced the model of reduced renal
mass (RRM) hypertension. In this model one kidney and both poles of the
other kidney are removed resulting in 66-75% ablation of the total renal mass.

The remaining renal tissue undergoes functional and anatomical adaptation to
maintain sufficient excretory function so that dialysis is not necessary. The
animals remain healthy and normotensive on normal or low sodium intake for
weeks to months. When the animals are placed on a high sodium intake,
however, hypertension rapidly develops (Ylitalo 1976) making this a good
model of SDH.

A longitudinal study of the evolution of RRM hypertension in rats
(Koletsky and Goodsitt 1960) documented the renal and hemodynamic
changes that occur over a ten month period following 75% renal ablation.
When RRM rats were given tap water to drink and normal laboratory rat
chow ad libitum, they remained healthy and gained weight throughout the
observation period. While on tap water, only 16% became hypertensive
within the first week and only 56% were hypertensive by the end of the ten

month period. Blood urea nitrogen (BUN) was essentially normal

46

immediately following ablation then rose steadily to terminal values of 46-
105 mg%. In the same study, a second group of RRM rats were given 1%
sodium chloride in their drinking water. They failed to gain weight and had a
much shorter lifespan. One hundred per cent of these rats were hypertensive
within one week and blood pressures were much higher than in the water
drinking rats. BUN was similar in the two groups (49 - 108 mg%)
suggesting that all rats had similar reductions in renal mass before the
divergence in sodium intake. Vascular, renal and cardiac lesions appeared in
both groups but were much more prevalent in the saline drinking rats. The
lesions typically appeared coincident with or following the development of

i the hypertension and were proportional to the severity of the hypertension.
Morphological examination of the kidney stump after several months of
RRM-salt hypertension revealed normal glomeruli; diseased, sclerotic and
non-functioning glomeruli; and hypertrophic glomeruli. Over time, the
proportion of sclerotic glomeruli increased and it was hypothesized that the
increased blood pressure at the glomerulus of the hypertrophic nephrons
damaged the glomerular membranes causing the sclerosis (Hostetter 1981,
Brenner 1985, Kaufman 1975 ). When glomerular perfusion pressure was
controlled, the hypertrophy and resultant tissue damage was prevented (Meyer

1988) supporting the conclusion that elevated perfusion pressure was

47

responsible for the initiation of the lesions. Further evidence of the
detrimental effect of elevated systemic pressure was documented through the
use of antihypertensive treatment in this model: lowering blood pressure with
either calcium channel blockers (Brunner 1989) or CEI (Brenner 1985,
Jackson 1988) prevented the vascular lesions. However, CEI were especially
effective at preventing renal damage and were efficacious at lower doses
(Tolins 1990, Anderson 1985).

Dramatic hypertrophy of the renal stump occurs due to rapid structural
and metabolic changes and at least partially offsets the loss of renal tissue.
Hypertensive RRM rats have a compensatory increase in Single nephron
glomerular filtration rate (SNGF R) proportional to the initial decrease in renal
mass, removing 75% of total renal tissue causes a 150% increase in SNGFR.
Renal blood flow increases even more while filtration fraction decreases
slightly (Kaufman et al. 1975). In Spite of the increased SNGFR, excretion
of urea and other filtered waste products decreases causing a gradual build up
of urea in the plasma. The elevated plasma level of urea increases plasma
osmolality, stimulates AVP release and induces polydipsia and polyuria
(Jackson 1988).

In addition to decreased excretory capacity (ie. fewer nephrons), there

is also a decrease in the number of renal secretory cells. In the kidney, the

48

primary endocrine cells are the granular cells of the JG apparatus which
synthesize and secrete renin (Gomez et al. 1990). In RRM rats, increased
renin production by JG cells and elevated renin content per nephron has been
measured (Rosenberg et al. 1991) explaining the previously observed normal
PRA following extensive renal ablation ( Anderson et al. 1985, Bouby et al.
1990) and supporting the hypothesis that the RAS is stimulated in RRM
hypertension in spite of a decreased number of renin synthesizing cells.

Muirhead and others have proposed that renomedullary interstitial cells
also secrete a circulating hormone, medullipin I, which is activated in the
liver to form medullipin II. Medullipin II is purported to be a vasodilator
that suppresses sympathetic tone, causes natriuresis and suppresses the central
nervous system (Muirhead 1991). Increased renal perfusion pressure
apparently stimulates secretion of medullipin I and formation of medullipin II
which is proposed to act in concert with ANG II as a double feedback system
to control blood pressure and renal sodium excretion (Muirhead et al. 1991).
RRM decreases the number of renal interstitial cells, and decreased plasma
levels of medullipin 11 may contribute to the vasoconstriction observed in
RRM-salt hypertension.

b. whole animal hemodynamic changes

The comparison of hemodynamic variables between studies is

49
somewhat difficult. In the majority of studies of RRM hypertension, MAP is

recorded at weekly intervals or only once afier months of hypertension.
.Since RRM-salt hypertension is a progressive disease, the duration of the
hypertension is an important variable and the time course of the events that
follow ablation is important to deduce their cause. Several recent studies
measured MAP acutely, recording the animals for days or weeks instead of
months of sodium loading. Since acute and chronic measurements have not
been made in the same animals or under the exact same conditions it is
difficult to discern which acute changes lead to the chronic condition of
elevated TPR. However, by combining observations fiom several studies,
three stages of hypertension can be recognized. The first is a short transition
phase with rapidly rising MAP accompanied by sodium and volume
expansion (Lombard et al. 1989). This is followed by a long stable phase
with little change in MAP, little or no volume expansion but increased body
sodium. Finally, if the study is carried out long enough, there is a terminal
phase of uremia, gross edema and very high MAP that ends with total renal
failure and death (Koletsky and Goodsitt 1960).

Following renal reduction, the first phase of hypertension is initiated by
increased sodium intake and was only detected when MAP was recorded

continuously over the first hours or days of sodium and volume loading. The

50

initial rise in MAP is accompanied by expanded sodium space (Hall et al.
1992) and elevated cardiac index (CI), a measure of CO normalized to body
weight (Cowley 1980, Lombard et al. 1989). In three studies which
measured the early volume expansion, plasma volume returned to normal
within 24 hours. TPR rose more slowly but remained elevated. MAP also
increased in this initial phase and appeared to parallel the TPR response. In
several studies, high salt intake increased sodium space and CI to a similar
degree in intact and RRM rats (Ando et al. 1990, Lombard et al. 1989). In
this initial phase, PRA and PAC were both depressed by the high sodium
intake and sodium balance was achieved by the second day of sodium
infusion (Hall et al. 1992).

The initial period appears to be an equilibration period in both intact
and RRM rats. The rapid increase in plasma volume by intravenous infirsion
of sodium increased sodium excretion but plasma sodium remained slightly
elevated until sodium balance was again achieved. The role that volume
expansion plays in the increased pressure is unclear. The authors in the
studies cited above suggest that the elevated CO caused overperfusion leading
to autoregulatory vasoconstriction. While it is possible that autoregulation
caused the increase in TPR, it is difficult to understand why equivalent

overperfusion did not elevate MAP in the control animals and why MAP

51

continued to rise at a time when CO was declining. More studies of this
initial period in RRM-salt hypertension need to be conducted to clarify the
role sodium retention and volume expansion play.

The second, chronic phase of RRM-salt hypertension is characterized
by elevated but relatively stable MAP, slowly rising BUN, increased SNGFR,
and elevated proteinuria. The length of this phase is inversely correlated to
the sodium intake and the degree of initial renal reduction. The mechanisms
maintaining elevated MAP in this phase may be identical to those in the
initial phase or may be different. Both the rapid sodium induced rise in
blood pressure and the chronic condition of hypertension were prevented by
administration of CEI (Meyer et al. 1985, Brooks et a1. 1989) and by
administration of [Sar‘,Ala8]ANG II (saralasin), a non-specific ANG II
antagonist (Pelayo et al. 1990). However, established RRM-salt hypertension
was not reversed by treatment with saralasin so that the involvement of ANG
II in the sodium-induced hypertension has not been resolved. It has been
contended that the antihypertensive effect of ANG II blockade was a non-
specific vasodilator effect that reversed the autoregulatory vasoconstriction.
The role of ANG II in this model of hypertension therefore remains
unresolved.

In addition to a slow increase in systemic blood pressure, RRM rats

52

also develop continued renal deterioration during the stable phase of
hypertension (Koletsky and Goodsitt 1969, Ylitalo and Gross 1979). ANG II
blockade prevented this progressive decrease in renal function (Meyer et al.
1985). Based on these observations, intrarenal actions of ANG 11 may have
caused the adaptive changes in renal function (ie. hyperfiltration with elevated
SNGFR and increased glomerular perfusion pressure) and systemic
hypertension secondary to these renal changes. However, saralasin blockade
of ANG 11 did not alter SNGFR or glomerular pressure in spite of decreased
proteinuria and normalization of MAP (Pelayo et al. 1990). Therefore, the
mechanisms responsible for the renal changes and the hemodynamic changes
during this phase appear to be different.
c. hormonal changes
1) angiotensin 11

Plasma levels of ANG II and plasma renin activity (PRA) in RRM rats
are consistently suppressed (Anderson et al. 1985, Ylitalo et al.1976). In
spite of this, and as noted earlier, administration of CEI prevented the
sodium-induced increase in blood pressure in RRM rats and reversed
established hypertension as outlined above (Brunner et al. 1989). CEI were
uniquely able to prevent both the hypertension and the renal damage that

typically develops in RRM rats and in patients with chronic renal failure

53
(Frohlich 1989). The effects of CEI were not due to their vasodilator

properties since treatment with calcium channel blockers also lowered blood
pressure but did not prevent deterioration of renal function in RRM rats
(Jackson and Johnston 1988, Tolins and Raij 1989, Brunner et al. 1989).

This implies that lowering MAP is not sufficient to alter the fundamental
disease process but merely alleviates one symptom. Preventing the formation
of ANG 11, however, prevented the initiation of the hypertension and slowed
the progression of the disease, implying that ANG II was an important
contributor to the initiation of RRM-salt hypertension and chronic renal
failure in humans.

It is difficult to conclude that increased activity of the RAS is
responsible for the pathogenesis of RRM-salt hypertension, since neither PRA
(Ylitalo et al.1979, Bouby et al.1990, Anderson et a1. 1985) nor circulating
levels of ANG II (Kuczera et al.1991) are elevated. It has been suggested
that CEI therapy acts instead through a non-angiotensin mechanism. One
proposed mechanism is increased activity of the renomedullary vasodilator
medullipin II which depends on kallikrein-kinin synthesis (Karlstrom et
01.1990). Increased levels of this vasodilator were found in renal extracts
from CEI treated rats'and demedullation deceased the effectiveness of CEI

therapy, suggesting that decreased degradation of this vasodilator may

54

mediate the protective effects of CEI therapy. Another proposed mechanism
is inhibition of an endogenous vascular RAS. (Kuczera et al. 1991). While
endogenous formation of ANG II in isolated perfused hind-limb in intact and
RRM rats was similar, ANG II formation during exogenous renin infusion
was slightly greater in RRM rats. The authors concluded that RRM rats had
increased activity of the vascular renin-angiotensin system and that CEI
lowered MAP through actions on this vascular system.

The effect of circulating ANG II on sodium excretion and blood
pressure in hypertensive RRM dogs is apparently enhanced (Hall et al. 1992).
RRM dogs treated with CEI then exposed to increased sodium intake showed
a modest increase in MAP. When renal perfusion pressure in these dogs was
servo-controlled through an elegant computer system blood pressure rose
slightly more. In RRM dogs with controlled perfusion pressure, replacement
of endogenous levels of ANG II by iv infusion at 3 ng/min, caused blood
pressure to rise steeply (Hall et al. 1992). This study showed that decreased
circulating ANG 11 during RRM sodium-loading is very important to control
blood pressure and sodium excretion. Even very small alterations in
circulating ANG 11 concentrations therefore, would be expected to have a
very profound effect on both blood pressure and sodium excretion in this

model of hypertension.

55

The existing evidence reviewed here and previously suggests that ANG
II is importantly involved in the pathogenesis of RRM-salt hypertension. But
the exact role of the peptide, the mechanisms of action, and the time course
of its involvement remain to be clarified.

2) arginine vasopressin

During the chronic phase of renal insufficiency hypertension, an
osmotically induced elevation in AVP has been documented in both clinical
cases of chronic renal failure (Mitch and Wilcox 1981) and RRM
hypertension (Bouby 1990). In RRM rabbits, plasma levels of AVP were
twofold higher compared to intact rabbits. This increase was significantly
correlated with an increase in plasma osmolality (Bouby 1990). The remnant
kidney appeared relatively insensitive to AVP and polyuria was exhibited
even in the presence of elevated AVP (Bouby 1990). The effect of elevated
AVP on blood pressure in RRM animals was addressed indirectly by giving
RRM rats a hypo-osmotic water load to decrease plasma AVP levels. The
hypovolemic animals had a slower rise in blood pressure but the change in
plasma AVP was not significant. Other investigations in rats discussed
previously have shown that chronically elevated plasma AVP concentrations
do not cause hypertension and AVP antagonists do not lower blood pressure

(Pawloski 1990). Finally, treatment with an AVP antagonist in hypertensive

56

RRM rats lowered blood pressure after an acute volume expansion but only
produced a minimal decrease during the chronic phase of hypertension
(Gavras 1982). Therefore it is unlikely that the modest elevations of plasma
AVP in this model contribute to the sodium-induced hypertension.

3) aldosterone

Another hormone implicated in RRM-salt hypertension is the
mineralocorticoid, aldosterone. Plasma levels of aldosterone in RRM rats
drinking saline are elevated compared to sham rats drinking saline and RM
rats drinking water (Chi et al.1986). RRM rabbits drinking saline (Vehaskari
and Hemdon 1991) and humans with chronic renal failure (Mitch and Wilcox
(1982) also have elevated levels of aldosterone. Since it has been shown that
chronically elevated PAC causes hypertension (Pan and Young 1982, Cowley
and McCaa 1976, Garwitz and Jones 1982), especially in combination with
high sodium intake, it is possible that high levels of the mineralocorticoid
may contribute to RRM-salt hypertension.

Elevated PAC in RRM rats was measured following seven weeks of
hypertension (Chi et al. 1986). Since the decrease in renal clearance of
potassium causes RRM rats to develop elevated plasma levels of potassium
(Chi et al. 1986) which stimulates aldosterone release (reviewed Williams

1991), it is possible that the observed increase in PAC was a result of the

57
hyperkalemia and not a cause of the hypertension. If RRM-salt hypertension

is due in part to mineralocorticoid excess, the time course of the increased
PAC should precede or parallel the development of the hypertension.
Furthermore, if aldosterone excess is contributing to RRM—salt hypertension
and is not stimulated by the changes in plasma potassium due to decreased
glomerular filtration, the electrolyte imbalances and increased reactivity of the
vasculature seen in mineralocorticoid hypertension (Worcel and Moura 1987)
should also be present in RRM rats. This has not been investigated, however,
and the role of aldosterone in early stages of RRM-salt hypertension has not
been defined.
4) natriuretic factors

Atrial natriuretic peptide (ANP) is released in response to atrial stretch
and volume loading is a potent stimulus for the release of ANP (Lang et al.
1985). The volume expansion in hypertensive RRM rats (Chi et al. 1986)
leads, as expected, to elevated plasma levels of this peptide. Jackson
measured the plasma levels of the peptide during the development of sodium-
induced hypertension in RRM rats and found that the plasma levels rose
progressively following the first week of sodium-induced hypertension while
atrial content fell suggesting increased secretion rather than production was

responsible for the change (Jackson et 01.1988). Other investigators have also

58

found an increase in ANP during sodium-induced hypertension in RRM but
the changes were very slight and followed rather than accompanied the
development of the hypertension (Brandt et 01.1989, Smith et al. 1988).

Because of the alterations in ANP levels during RRM hypertension,
ANP may modulate the hypertensive response to increased sodium intake in
these animals. ANP has been shown to affect blood pressure by direct
vasodilation (Winquist et a1. 1984), direct natriuresis (DeBold et 01.1981) and
by down-regulating the renin-angiotensin system (Maack et 01.1984) to
cumulatively promote diuresis, natriuresis and vasodilation (Jackson et a1.
1988). However, the two-fold increases in ANP measured in RRM rats are
not sufficient to effect blood pressure and it is unlikely that this hormone
exerts cardiovascular control during RRM-salt hypertension.

RRM alone (Hall et 01.1992) and with sodium-loading (Hout et
01.1983, Ylitalo et al. 1979, Hall et al.1992) is associated with extracellular
fluid volume expansion and increased body sodium space. The volume
expansion and sodium retention may release the endogenous ouabain-like
factor (OLF) discussed above. Hout et al. performed an extensive
examination of the activity of the Na”/K+ pump in hypertensive RRM rats and
found that the RRM rats had decreased pump activity in vascular smooth

muscle and in cardiac microsomes. In addition, boiled plasma extracts caused

59

in vitro inhibition of Na‘L/K+ pump when applied to tail arteries of normal
animals. Specific hypothalamic lesions in RRM rats prevented both the
development of sodium-induced hypertension and an increase in circulating
pump inhibitor (Hout et al. 1983). Others have also found increased
circulating levels of a Na‘lK“ pump inhibitor that cross-reacts with a digitalis
antibody and which increases parallel to the rise in blood pressure (Nakagawa
et 01.1990, Wauquier et 01.1986).

A direct study of the contribution of this endogenous inhibitor to
hypertensive states has been impossible because there is no known antagonist.
However, a recently developed assay has shown that other forms of
hypertension associated with volume expansion do have increased levels of
OLF (Kaminski and Rechsberger 1991). Contrary to what would be expected
if OLF can influence blood pressure, infusion of plant ouabain into rats did
not consistently cause hypertension (Yasujima et a1. 1986). Therefore it is
still unclear if the natural compound would be able to contribute to SDH.

d. sympathetic nervous system

There have been several reports of elevated SNA in RRM-salt
hypertension. Initially it was observed that RRM rats on high sodium intake
had elevated plasma levels of norepinephrine (NE) and responded to

epinephrine synthesis blockade with a fall in MAP. Moreover, when the

60

inhibitor used was unable to cross the blood brain barrier, there was no
decrease in MAP (Dipette et al 1982). It has also been reported that lesion of
the anteroventral third ventricle (AV3V) prevented a sodium-induced rise in
MAP in RRM rats (Hout et al. 1983). These observations suggest that
sodium-loading during RRM may cause increased epinephrine synthesis
within the CNS leading to elevated sympathetic outflow and the observed
hypertension (Gavras 1982).

An alternative role for the SNS is increased release of NE mediated by
a circulating factor such as ANG II aldosterone acting either in the CNS or at
peripheral sites. Exogenous application of NE to isolated blood vessels
produced a greater response in RRM vessels compared to vessels from intact
rats (Chi et al. 1986, Shima et a1. 1988) while plasma from RRM-salt rats
increased NE overflow during electrical stimulation (Shima et a1. 1988).
Furthermore, in situ recording of membrane potentials in resistance vessels in
the mesenteric vascular bed found lower potentials (ie. depolarization) in
hypertensive RRM rats compared to sodium loaded intact rats (Stekiel et al.
1991). The basis of the increased reactivity of the blood vessels may be
elevated levels of the Na+/K+-ATPase inhibitor, OLF, or through another
circulating factor that interacts with the SNS such as ANG II. The identity of

this factor is not known but the in situ depolarization to ouabain application

61

was identical between RRM and sham rats suggesting that there is not
endogenous blockade of these receptors in RRM hypertension (Stekiel et al.
1991)

In spite of the evidence of both humoral and neurological changes in
RRM-salt hypertension, the mechanisms responsible for the condition are not
clearly defined. There appear to be multiple factors altered during the
sodium-induced rise in MAP in RRM animals and the possible interactions
between circulating factors and the SNS make interpretation of data difficult.
Finally, the mechanisms responsible for the initial rise in pressure and the
sustained hypertension may not be the same. Therefore additional
experiments which closely examine alterations in hormone levels throughout
the development of the hypertension are needed to define which changes
initiate the hypertensive response and which changes occur in response to the

hypertension.

62
Statement of Purpose

Sodium sensitive hypertension (SDH) is defined by the blood pressure
response to sodium intake. It is not defined by a homogeneous set of
individuals, but is a condition common to several different forms of
hypertension. In spite of the different etiologies of the condition, there are
several common characteristics of SDH. The purpose of the experiments
described here was to characterize one of these features, alterations in
humoral factors, and to determine what changes in humoral factors occur in
one form of SDH, RRM-salt hypertension.

Humoral factors known to influence both blood pressure and sodium
homeostasis have been implicated in previous studies of SDH and in RRM-
hypertension in particular. Angiotensin II, aldosterone and an endogenous
ouabain-like factor have all been suggested as contributors to this
hypertension and these hormones were the ones investigated.

Initially, we sought to establish the parameters under which each of
these factors initiates the development of SDH and to characterize the
changes that occur as the increase in blood pressure develops. Because there
are interactions between these hormones and between the hormones and the

sympathetic nervous system, it was necessary to determine how the hormones

63

alone affect blood pressure and sodium homeostasis.

After characterization in normal rats, the RRM model was used to
determine if any of these hormones might be playing a role in the
development of the sodium-induced increase in arterial pressure. The RRM
model was chosen because of its relevance to the human condition of chronic
renal failure, a condition associated with a high incidence of SDH, and
because it is not associated with any genetic alterations in blood pressure
control that might be independent of the sodium-induced hypertension.
Furthermore, characteristics of this model have been shown to apply directly
to human hypertension associated with chronic renal failure and presumably

the more general condition of SDH .

11. Materials and Methods

A. General experimental protocols
1. Surgical procedures
a. catheterization

Catheters were constructed of polyethylene (Tygon‘) tubing attached to
silicon (SilastinR) tubing. Arterial and venous catheters were surgically
implanted under pentobarbital anesthesia (40 mg/kg plus 0.4 mg atropine
sulfate). One silicon-rubber/polyvinyl catheter was placed into the abdominal
aorta via the external iliac artery. This catheter was used to record arterial
pressure and heart rate and to draw blood samples from conscious, freely-
moving animals. Another silicon-rubber/polyvinyl catheter was inserted into
posterior vena cava via the external iliac vein. This catheter was used to
infuse drugs and sodium. Both catheters were tunneled subcutaneously to the
top of the head where they were exteriorized and fed through a stainless steel
spring to protect the tubing fi'om the rat. One end of the metal spring was
attached to the skull with dental acrylic and jewelers screws, while the other
end was attached to a plastic swivel mounted above the cage allowing the rat
to move freely within the cage. The venous line was attached to a syringe-

type Harvard infusion pump (Boston, MA) via the swivel to allow continuous

64

65

24 hour infusions. Arterial lines were filled with a heparin-sucrose solution
and occluded when not in use. After the animal awoke following surgery, it
was placed in a metabolism cage with the swivel suspended over the cage.
This allowed the rats freedom of movement within the cage and free access
to the venous and arterial catheters from outside of the cage. Rats were
allowed at least three days recovery after surgery before measurements were

begun.

b. subcutaneous osmotic minipumps
Alzet osmotic minipumps (Alza, CA ) were filled with a sterile

solution of the drug to be administered. The concentration of the drug was
calculated on the basis that the pumps deliver either 1.0 or 2.0 til/hr over a 24
hour period. Rats were anesthetized with methohexital (40 mg/kg ip or 25
mg/kg iv) and a small incision was made in the fold of skin just behind the
head. The pumps were slipped under the skin to deliver drugs
subcutaneously. The incision was closed with silk sutures and the animals
were given 20,000 units penicillin G and 25 mg streptomycin, 0.1 ml
CombioticR ip (Pfizer New York, NY) to prevent infection.

0. renal reduction

Rats were pretreated with atropine sulfate (0.40 mg ip) and

66
anesthetized with sodium pentobarbital (40 mg/kg; ip) for all surgical

procedures. Surgical renal reduction was performed in two stages. Initially,
a left mid-flank incision was made and the left kidney exteriorized. The renal
artery was temporarily occluded and both poles of the kidney (2/3 of the
mass) excised with a scalpel. The clamp was removed and bleeding
controlled with Thrombostat (Parke-Davis, Ann Arbor, MI) and pressure
before the kidney stump was returned to the peritoneum. The incision was
sutured and the rat allowed one week recovery after which the right kidney
was exposed, the right renal artery, vein and ureter ligated with silk, and the
entire kidney removed. Rats were allowed one to two weeks recovery to

return to pre-surgery weight after the second procedure before catheterization

surgery.

2. Hemodynamic measurements
Blood pressure was measured by connecting the exposed arterial line to
a Gould P50 Statham pressure transducer (Oxnard, CA) attached to a Grass
polygraph (Quincy, MA). The signal was fed into a Stiemke digital output
device (New Orleans, LA) which displays the average mean arterial blood
pressure on a digital display. Heart rate was read directly from the tracing on

the recording chart. For animals with sinoaortic denervation, the pressure

67

signal was fed into an Apple computer which sampled blood pressure and
heart rate every 30 seconds over a twenty minute period. The mean values
and standard deviations were calculated by a program written by Dr. Gregory
Fink and displayed on the screen.

All blood samples were taken following daily blood pressure recording.
Blood was drawn into syringes containing either 0.05 ml 3M EDTA or 0.05
ml 10,000 IU/ml heparin from the exposed arterial line. Samples were
immediately spun in 4°C refrigerated centrifuge, the plasma drawn off and
frozen at -70°C until assays were performed. In animals with repeated blood
samples, red cells were returned suspended in physiological saline equal to
the volume of the sample. In other animals, saline equal to the volume of the
sample was given. Blood samples were not drawn more fiequently than

every 4 days in any of the protocols.

3. Analytical methods
a. sodium and potassium
Sodium and potassium concentrations in urine and plasma were
measured with an Instrumentation Laboratory IL943 flame photometer
(Lexington, MA). Within assay variability was 4%. The appropriate

standards were run daily before analysis so that between assay variability was

68

less than 10%. Electrolyte excretion was calculated by multiplying urine
concentration times 24 hour urine volume. All plasma samples from each rat
were analyzed on the same day to control for interassay variability.
b. plasma osmolality
Plasma osmolality was measured by freezing point depression using a
Precision Systems, Inc. Model 5004 p. Osmette (Natick, MA). Three
determinations were made for each sample and the mean of the three values
recorded. Appropriate standards were run daily before each determination
and interassay variability was 2-3%.
c. blood urea nitrogen
Blood urea nitrogen (BUN) was determined by a colorometric assay
using a prepared assay kit, No. 640, from Sigma Chemical (St. Louis, MO).
All samples from each rat were run in the same assay and a new standard
curve was calculated for each assay. Standard curves between assays were
virtually identical so that interassay variability was negligible.
(1. plasma aldosterone concentration
Plasma aldosterone concentration was measured with a
radioimmunoassay kit (Coat-a-Count) fiom Diagnostic Products (Los Angeles,
CA). 1 ml of blood was drawn using heparin treated syringes to obtain 500

pl of plasma. Two 200 [11 plasma samples were assayed and the average of

69
the two values recorded. The sensitivity limit of the assay is 16 pg/ml. The

intraassay coefficient of variation averages 5% and interassay coefficient of
variation is 8%. All samples from one animal were run in the same'assay to
eliminate interassay variability.
e. plasma immunoreactive angiotensin II

1 ml of blood was drawn into an EDTA treated syringe. 400 pl of
plasma was extracted twice with 100 % ethanol, the supematants combined
and dried under a stream of air. The residue was stored in the freezer until
assayed. At the time of the assay, samples were reconstituted with 200 p1
assay buffer solution (0.30% bovine serum albumin, 0.50M Trizrna base,
0.20% neomycin sulphate, pH 7.4) and the concentration determined by
radioirnmunoassay. Recovery of angiotensin II was 95% after extraction. ”’1
angiotensin II was purchased from New England Nuclear (Boston MA) and
the antibody was purchased fi'om Amel laboratories (New York, NY). The
crossreactivity of the antibody has recently been evaluated by Navar et al.
They found that angiotensin III crossreacts 100% with the antibody while
angiotensin I was only detected in concentrations >100 fold higher than
angiotensin II {Navar 1992). The sensitivity of the assay allowed
measurement down to about 20 pg/ml. The intraassay coefficient of variation

was approximately 4% and the "interassay coefficient of variation was 9%.

70

All samples fi'om one animal were run in the same assay.
f. fluid homeostasis
Daily water intake was determined from a calibrated drinking tube and
daily urine output was determined by collecting 24 hour urine samples in a
calibrated urine cup. Water balance was calculated by subtracting urine
volume from the sum of water intake volume plus 5 ml/day iv infusion

volume.

4. Statistical methods

A mixed-design analysis of variance (ANOVA) blocked for between
animal variation was used to evaluate within groups differences in daily
measurements. A one-way ANOVA was used to evaluate differences
between groups at individual time points. The Student-Newman-Keuls' test
of significance was used for comparisons between groups and least significant
difference t-test was used to test for the significance of changes over time.
Paired t-tests were used to evaluate differences when only two measurements
were made. A p-value of less than 0.05 was considered statistically

significant.

B. Specific protocols

71

1. Aldosterone infusion

Chronic elevation of aldosterone causes increased blood pressure in
uninephrectomized rats on high sodium intake and aldosterone excess may
play a role in other forms of SDH. It has been reported that aldosterone is
elevated in hypertensive RRM rats(Chi et al. 1986) and that infusion of ANG
11 causes increases in plasma aldosterone concentration (PAC) (Cowley and
McCaa 1976). The first study was performed to establish what level of PAC
will alone cause hypertension during a moderately elevated sodium intake to
determine the relevance of any increases in PAC seen in later protocols. To
accomplish this, the following experiment was performed.

Arterial and venous catheters were surgically implanted in fifteen rats
as described above. Immediately following surgery, intravenous infusion
delivering six meq/day sodium was begun. Animals were allowed access ad
Iibitum to sodium deficient rat chow and tap water. Beginning three days
after surgery, daily determinations of MAP, HR, WI, UO, urinary potassium
excretion and urinary sodium excretion were made. After three days of
control measurements, the rats were briefly anesthetized with sodium
methohexital, BrevitalR (Lilly, Indianapolis, IN) and a small incision was
made at the back of the neck. An osmotic minipump, AlzetR model 2002

(Alza, CA), filled to deliver one of three doses of aldosterone was positioned

72

subcutaneously and the incision sutured. The minipump was replaced after
twelve days with a new model 2001 Alzet“ minipump filled with aldosterone.
The second pump was removed four days later following the daily MAP and
HR measurements. All pumps were prepared according the manufacturer's
directions to deliver either 0.10, 0.25, or 0.50 ug/hr of aldosterone. PAC was
monitored by drawing 1.2 ml arterial blood samples into heparin containing
syringes approximately every four days in the morning just after recording
MAP and HR. PAC was determined as described under analytical methods.
Plasma samples were also used to determine plasma osmolality, potassium

and sodium.

2. PAC during chronic infusion of angiotensin II

This study was performed to determine the involvement of aldosterone
during chronic infusion of ANG II. First, infusion of ANG 11 over hours to
days causes increased secretion of aldosterone (Aguilera and Catt 1978,
Hanger at al. 1978, Komer and Muller 1979). Second, chronic elevations in
PAC cause an increase in arterial pressure both in clinical and animal studies
(Garwitz and Jones 1982, Oelkers and Schoneshofer 1983) as demonstrated in

the previous study. Finally, chronic exposure of the adrenal gland to ANG II

73

causes an increase in adrenal ANG II receptor number and increased
sensitivity of receptors to ANG 11 both in rats and humans (Aguelera et al.
1980, Naruse et al. 1987). This last point suggests that chronically elevated
plasma levels of ANG II in these species could lead to progressively
increasing plasma levels of aldosterone. The aims of the present study were
to: 1) measure changes in PAC occurring in response to chronic low dose
infirsions of ANG II in rats under conditions where the peptide does (high
sodium intake) or does not (normal sodium intake) cause hypertension; and 2)
determine from the above results whether changes in PAC could make an
independent contribution to the development of hypertension caused by
chronic low dose ANG II infusion. These findings would be expected to
apply to any situation of prolonged elevation of plasma levels of ANG II. To
test these hypotheses, the‘following two experiments were performed.
Chronic ANG II infusion 1: Fifteen rats had venous and arterial
catheters surgically implanted. Ten rats were randomly placed in an ANG 11
group and five in a control group. Following surgery, rats were allowed only
sodium deficient rat chow and received 6 meq/day sodium iv infused
continuously over twenty-four hours. The rats were maintained on high
sodium intake for the remainder of the protocol. The protocol included three

control days of sodium infusion alone, sixteen ANG 11 plus sodium infusion

74

days and four recovery days when again, only sodium was infused. In the
ANG 11 group, ANG II was added daily to the infirsate during the ANG II
infusion period in concentrations to produce a constant infusion rate of 10
ng/min. The control rats continued toreceive only the 6 meq/day sodium
solution during this period. WI, UO, urinary sodium excretion (UNaV), and
urinary potassium (UKV) excretion were measured daily. MAP, and HR
were recorded every other day and plasma sodium, plasma potassium, and
PAC were measured every fourth day from blood samples drawn into
syringes containing EDTA immediately after the daily MAP and HR
recording. One sample was taken on the second day of the control period,
four during the ANG II infusion period and one on the last day of recovery.
Care was taken to draw samples at approximately the same time each day to
control for diurnal variation in aldosterone secretion (Quinn and Williams
1988).

Chronic ANG II infusion 2: Catheters were surgically implanted in ten
animals as described above and divided into one group of five control
animals and one group of five ANG 11 animals. Following surgery, animals
were allowed only sodium deficient rat chow and were placed on a
continuous 2 meq/day sodium iv infusion. The experimental protocol

consisted of three control days, twenty-eight ANG II infusion days and four

75
recovery days. The ANG 11 group received 10 ng/min ANG 11 added to the

saline infusion during the ANG II infusion period. WI, UO, urinary sodium
and urinary potassium excretion were measured daily. MAP and HR were
measured every other day and plasma sodium, plasma potassium and PAC
measured every fourth day from blood samples taken as described above,

beginning on day two of the control period.

3. Aldosterone in reduced renal mass hypertension

Reduced renal mass rats have been shown to have elevated plasma
levels of aldosterone during the established phase of sodium-induced
hypertension. It is possible that this increase causes at least a portion of the
blood pressure elevation. If the increase in PAC is of sufficient magnitude
and occurs during the initiation of the hypertension would support the
hypothesis that RRM-salt hypertension is dependent on the elevated PAC.
This hypothesis was tested with the following experiment.

Nineteen rats underwent renal reduction and catheterization as
described above. 5 additional intact control rats were catheterized. After
recovery from surgery, all animals were given normal sodium intake (2
meq/day) for 5 days, high sodium intake (6 meq/day) for 10 days followed by

5 more days normal sodium intake. MAP, HR, WI, UO, UNaV, UKV were

76

recorded daily. Blood samples were drawn into heparinized syringes every 4

days to measure BUN, plasma sodium, plasma potassium, plasma osmolality

and PAC.

4. Angiotensin II antagonism in reduced renal mass

hypertension

Plasma levels in RRM-sodium hypertension in rats in normal or even
slightly suppressed. However treatment with CEI prevents the, development
of hypertension and can reverse the hypertension once it is established. This
may be due to an action of CEI independent of the renin-angiotensin system
or may be due to actions of normal circulating levels of ANG II. If it is due
to ANG II actions, treatment with the specific ANG II antagonist losartan
should mimic both the chronic and acute CEI results. If the antagonist results
are different from the CEI results, it is unlikely that circulating ANG II is

involved.

a. chronic antagonist treatment
4 groups of rats were used. Rats in groups 1 and 2 underwent 2-stage
renal reduction followed by recovery before catheterization. All rats were

catheterized and, following surgical recovery on normal sodium intake,

77
maintained for 5 days on normal sodium followed by 7 days of high sodium.

Group 1 (n = 9) received daily bolus intravenous injections of 3 mg/kg
losartan during high sodium intake. Group 2 RRM rats (n = 7) received
vehicle during the high sodium period. Group 3 sham operated rats (n = 8)
received losartan and group 4 sham controls (n = 8) received vehicle. The
RRM groups had approximately 80% renal ablation while the sham groups
had no reduction. Losartan injections were given once daily as an
intravenous bolus in dextrose (3 mg/ml) after the daily recording of MAP and
HR beginning on day 5 of normal sodium intake. Losartan treatment began
simultaneously with the change fi'om normal to high sodium. MAP, HR, WI,
UO, UNaV, and UKV were measured daily in all groups. Plasma samples
were drawn into heparinized syringes on day l of normal sodium to measure
initial BUN and on day 7 of high sodium to measure final BUN. Graded 8
minute infusions of ANG II (3, 10 and 30 ng/min) were given on day 3 of
normal sodium and on day 5 of high sodium to measure the acute pressor
response to ANG II. In animals receiving losartan, the ANG II infusions
were given before the daily losartan bolus. After a stabilization period of 20
minutes, a baseline MAP was recorded and the 3 ng/min infusion was started.
After 8 minutes, the MAP and HR were recorded and the change in MAP and

HR calculated. The infusion rate was increased to 10 ng/min and after 8

78

minutes, the change in MAP at the new rate was calculated from the initial
baseline value. After the third step increase, the change in AP and HR were

again calculated and the infusionwas stopped.

b. acute antagonist treatment

Six RRM rats were catheterized and maintained on high sodium intake
(6 meq/day). After 3 days recovery fi'om surgery, MAP, HR, WI, UO,
UNAV and UKV were recorded daily. On day 5 of high sodium, rats were
given a bolus of 3 mg/kg losartan intravenously. MAP and HR were
recorded 5, 15, 30, 60, 120, and 360 minutes after the losartan challenge. 5
days after the first challenge, a second challenge was given and measurements
were again made at 5, 15, 30, 60, 120, and 360 minutes . MAP and HR were
recorded daily on the 3 days following the second losartan challenge. UNAV

and water balance were calculated daily.

5. Angiotensin II infusion in reduced renal mass rats
Another test of the involvement of ANG II in RRM-sodium
hypertension was to examine the sensitivity of normotensive RRM rats to
elevations in circulating ANG II. It is possible that renal reduction increases

the response to ANG 11 so that compared to intact rats, plasma levels of the

79

peptide appear normal. If RRM rats are more responsive to ANG II
hypertension, then they will become hypertensive at plasma levels that do not
produce hypertension in intact rats just as they would become normotensive
to antagonist treatment even though plasma levels are not elevated.
Thirty-eight rats underwent renal reduction followed by catheterization
as described above. Eleven animals were randomly chosen as controls and
the other 27 animals received 10 ng/min ANG II. An additional control
group of 7 rats with both kidneys intact received 10 ng/min ANG II infusion.
Following catheterization surgery, animals were allowed only sodium
deficient rat chow and received 2 meq Na/day in 5 ml water by continuous iv
infusion. The experimental protocol consisted of 3 control days, 10 ANG II
infusion days and 4 recovery days. The RRM control group received sodium
infusion during the ANG II infusion period. MAP, HR, WI, UO, UNaV and
UKV were measured daily in all three groups. Plasma sodium concentration,
plasma potassium concentration, plasma osmolality and plasma ANG 11
concentration were determined from blood samples taken as described above.
Samples were taken once during control, twice during ANG II infusion and

once during recovery.

8 0
III. Results

A. Plasma aldosterone concentration in intact rats

Acute ANG II Infusion- The data in Table 1 is from previous work in
our laboratory and illustrates the MAP and PAC of rats receiving one hour
infusions of ANG II at 10, 30 and 60 ng/min; the infusion rate of 10 ng/min
produced plasma levels of ANG 11 similar to those measured during
renoprival hypertension (Kuczera 1991) was used for the remainder of the
studies. The 10 ng/min infusion of ANG 11 caused MAP to increase
significantly from 105.6 i: 2.5 to 128.8 i 5.1 mm Hg while PAC increased
significantly from 73.5 :t 23.8 to 168.6 :t 36.5 pg/ml. Higher infusion rates
of ANG II caused higher PACS and correspondingly greater pressor
responses.

Chronic Aldosterone Infusion- The MAP, PAC and HR of the three
groups of rats receiving subcutaneous aldosterone infusions are shown in
Figure 1. Only the group receiving the highest rate of aldosterone (0.50
pg/hr) had a sustained hypertension during the aldosterone infusion. The
MAP in this group tended to increase slowly over time, reaching statistical
significance on day 10 and the highest MAP level on day 16 of the infusion.

PAC in the rats receiving the highest aldosterone infusion rate reached

81

 

10 ng/min 30 ng/min 60 ng/min

MAP PAC MAP PAC MAP PAC

 

pre- 105.6 73.5 i 101.2 81.2 2!: 101.7 32.3 i

infusion :t 2.5 23.8 :i: 1.5 74.9 a: 4.6 9.6

 

post- * *
* It * It
infusion 486.6 762.0
128.8 168.6 137.6 147.2
:t a:
:1: 5.1 :i: 36.5 a: 6.1 d: 6.2
125.6 198.9

 

 

 

 

 

 

 

 

 

Table 1: Plasma aldosterone and blood pressure following angiotensin II
infusion. Mean arterial pressure (MAP) is listed as mm Hg and plasma
aldosterone concentration (PAC) is listed as pg/ml in rats receiving ANG II
infused iv at either 10, 30, or 60 ng/min. MAP was recorded and blood
samples drawn prior to ANG II infusion and 60 minutes after the infusion
was started. MAP and PAC were both significantly elevated at all three
infusion rates (p s 0.05). Values are the mean of 7 (10 ng/min) or 5 (30 and
60 ng/min) animals :1: standard error of the mean (SEM).

82

 

3>
Meon Arterial Pressure (mm Hg)

 

 

 

   

 

 

 

 

 

 

 

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Figure 1: Effect of aldosterone infusion on blood pressure, heart rate
and plasma aldosterone concentration. A. shows mean arterial pressure, B.
shows plasma aldosterone and C. shows heart rate in 3 groups of rats
receiving subcutaneous aldosterone infusion at 0.10 (circles and open bars),
0.25 (triangles and hatched bars) or 0.50 (squares and cross-hatched bars)
rig/hr. The first 3 days were control (no aldosterone), the 16 days in the
shaded area were aldosterone treatment and the final 4 days were recovery.
Each point represents the mean of five rats. Statistically significant
differences between control and infusion periods within a group for p s 0.05
are marked with *. Error bars represent standard error of the mean (SEM).

83

five to eight times the control level and was significantly increased by day
four of the infusion. The two lower rates of aldosterone infusion caused a
smaller increase in PAC and did not cause a sustained increase in MAP over
the time course of this experiment. The PAC on day 16 during the 0.25
ug/hr infusion was significantly elevated due to very high PAC'S in 2 of the 5
rats. The high levels could have been due to an error in preparing the
replacement pumps since they were not consistent with the data from the
other infusion rates. Also the MAP was much higher in these two animals.
These results indicate that the minimum increase in PAC required to Show a
statistically measurable elevation fiom control levels was 45 to 100 pg/ml
(depending on the variation within the group); and that a PAC of at least 250-
300 pg/ml was required to elicit hypertension in rats on a 6 meq/day sodium
intake and that an infusion rate of 0.50 ug/hr was required to produce this
PAC. WI and U0 were unaltered by the two lower infusion rates but were
significantly elevated in the group receiving 0.50 ug/hr (Figure 2). There was
no difference in water balance in any of the groups. The 0.50 pg/hr group
also had a significant decrease in urinary sodium excretion (Figure 3). There

were no other differences in the electrolyte excretion in the three groups.

84

 

100 -
O 0.10 ug/hr
' A 0.25 ug/hr
80 h I 0.50 ug/hr

Water Intake (ml/day)

 

 

 

 

 

 

 

 

 

 

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Figure 2: Water intake and urine output in intact rats receiving aldosterone
subcutaneously either 0.10, 0.25, or 0.50 pg/hr aldosterone subcutaneously.
The aldosterone infusion period is indicated by the hatched area. Statistically
significant differences for p s 0.05 are indicated by *. Error bars represent
the SEM. All points represent the mean of measurements made in 5 rats.

85

 

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A 0.25 ug/hr
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Figure 3: Effect of aldosterone infusion on sodium and potassium excretion
in intact rats receiving 0.10, 0.25, or 0.50 rig/hr aldosterone infusion. The
aldosterone infusion period is in the shaded area bracketed by 3 control and 4
recovery days. The solid line in the top figure represents the daily sodium
intake. Significant differences within groups are marked with a * for p <
0.05. Each point represents the mean of 5 rats and error bars are SEM.

86

B. Chronic infusion of angiotensin II in intact rats

Chronic ANG II Infusion (high sodium)- Figure 4 illustrates the MAP,
HR and PAC for the control, ANG II infusion and recovery periods of rats
receiving 6 meq/day sodium. MAP was significantly increased above
controlby day two of the ANG II infusion (from control values of 97 i 4 and
101 d: 4 to 130 at 7) and reached a peak level (155 i 9 mm Hg) on the eighth
day of the ANG II infusion. The MAP returned to control levels when the
ANG II infusion was stopped. No change in HR occurred during ANG II
infusion. There was an upward trend in PAC from a control value of 32.1 :1:
10.1 pg/ml to a high of 109 i 39.5 pg/ml on day eight of ANG II and 96 :1:
35.2 pg/ml on day sixteen of ANG 11. However, the latter PACS were not
significantly different from the control value and did not reach the level
found necessary to produce an independent hypertensive response (250-300
pg/ml). Unlike the rats receiving the highest dose of aldosterone, urinary
electrolyte excretion was not altered by the ANG II infirsion. Water balance,
sodium excretion and potassium excretion were measured daily and did not
differ between control and infusion periods or between the saline infused and
the ANG II infused groups. Urinary electrolyte excretion and water balance
(Figure 5) was identical within both groups throughout the protocol.

Although others have reported sodium and water retention in rats chronically

87

 

 

150 -
2"
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E 140 - Cl Control (5)
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Time (days)

 

 

 

Figure 4: Blood pressure, heart rate and plasma aldosterone in intact
rats receiving angiotensin II . Control rats are shown in open squares and
bars while ANG II rats are depicted by solid squares and bars. The ANG II
infusion period (10 ng/min iv) is inside the shaded area. Each point
represents the mean of 10 (ANG II) or 5 (control) rats. Statistically
significant differences between control and infusion periods for p s 0.05 are
marked with ‘. Bars represent SEM.

88

 

 

 

 

 

 

 

 

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Potassium Excretion

 

 

Time (days)

 

 

 

Figure 5: Urine output, water intake, urinary sodium excretion and
urinary potassium excretion in rats receiving ANG II infusion. Control rats
are depicted by the open squares and ANG 11 rats by the solid squares. All
rats were on a fixed 6 meq/day sodium intake with sodium fi'ee chow and
water ad Iibitum. ANG II infusion period (10 ng/min) is inside the shaded
area. Points represent the mean of 10 (ANG II) or 5 (control) rats. There
were no statistically significant differences between control and infusion
periods for p s 0.05. Bars represent SEM..

89

receiving ANG II, no such retention was observed here and the hypertension
developed without changes in water or sodium balance. Furthermore, plasma
potassium and sodium levels and plasma osmolality were also unchanged by
the infusion (Figure 6) further revealing no evidence of volume expansion.

Chronic ANG II Infusion (normal sodium)- As shown in Figure 7,
MAP was significantly increased above control only on days four and
eighteen of the 28 day ANG II infusion period in the rats receiving 2 meq
sodium/day. Figure 7 also reveals that, although the control level for PAC
was higher in the normal sodium group than in the high sodium group, ANG
11 did not cause a further increase--even after 28 days of continuous 10
ng/min ANG II infusion. The HR was unchanged fi'om control in either
group of animals. The water and electrolyte data Showed no differences
between the two groups (data not shown). Plasma osmolality, plasma sodium
and plasma potassium were also unchanged by ANG II infusion .

In summary, aldosterone infusion elevates plasma levels of aldosterone
(PAC) and MAP in a dose-dependent manner so that a 3-fold elevation in
PAC caused a significant elevation in blood pressure during high salt intake.
Infusion of ANG II acutely elevates plasma levels of aldosterone to
hypertensive levels and elevates MAP in a dose-dependent relationship.

However, infusion of 10 ng/min ANG II for 2 to 28 days

90

 

Control (5)
ANG II (10)

Plasma Sodium (meq/I)

 

 

%

Plasma Potassium

 

rt

 

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Figure 6: Plasma sodium, potassium and osmolality during angiotensin II
infirsion in rats on high sodium intake. Control rats are depicted by the solid
bars and ANG 11 rats by the open bars The ANG II infusion period (10
ng/min iv) is inside the shaded area. Bars represent the mean of 10 (ANG II)
or 5 (control) rats. Blood samples were drawn approximately every 4 days
and blood volume replaced with physiological saline. Statistically significant
differences between control and infusion periods for p s 0.05 are marked
with *. Eror bars represent SEM.

91

 

175 1
i I Control (n=5)
El Ang ll (n=5)

MAP (mm Hg)

 

 

 

 

 

 

 

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Plosmo Aldosterone (pg/ml)

 

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Figure 7: Blood pressure, heart rate and plasma aldosterone concentration in
rats receiving angiotensin II on normal sodium. Control rats are depicted in
open squares and bars while ANG 11 rats are depicted by the solid squares
and bars during a fixed 2 meq/day sodium intake. The ANG II infusion
period (10 ng/min iv) is in the shaded area. Points represent the mean of 5
rats. Statistically significant differences between control and infusion periods
for p s 0.05 are marked with *. Error bars represent SEM.

92

not cause a sustained elevation in PAC in rats on either high sodium intake or
normal sodium intake. Therefore, the elevated MAP during the ANG II
infusion combined with high sodium intake was not dependent on elevated
PAC. Furthermore, infusion of ANG II at either sodium intake does not
alter sodium or water balance or cause changes in plasma electrolyte

concentrations such as would be expected during PAC excess.

C. Plasma aldosterone in reduced renal mass hypertension

RRM rats (average BUN = 28 i 1.8 mg%; n = 19) consistently
exhibited a significant increase in MAP when changed from normal to high
sodium intake. MAP returned toward control values after rats were returned
to normal sodium intake. Intact rats (average BUN = 14.4 i 1.1 mg%; n =
5) did not Show an increase in MAP during high sodium intake. PAC was
not different between groups and did not change when the sodium intake
increased (Figure 8). BUN was significantly higher in the RM rats
compared to intact rats at all time points but did not change significantly
withingroups. There was a tendency for BUN to increase in the RM rats
over time (Figure 9). There was a significant positive correlation (r = 0.43)
between the mean BUN value and MAP on day 10 of high sodium in RRM

rats. In RRM rats WI and U0 were both significantly higher throughout the

93

 

160 -

140

120

100

Arterial Pressure (mmHg)

 

 

80

 

500

400

 

- Int t 5
y/ 3 Ram)

300 ~

200

Plasma Aldosterone pg/ml

 

 

 

100 -
Lhfl
O

600 —

. I intact (5)
500 — / a RRM (19)
400 L . " “/4 :21
r .‘ ' , .

DoyO DoyS Doyl O Doyl 5 Doy2O

 

Heart Rate (beats/min)

 

 

Time (days)

 

 

 

Figure 8: Blood pressure, plasma aldosterone and heart rate in intact and
RM rats during changes in sodium intake. Intact--2 kidney--rats are
depicted in the solid circles and bars and while RRM --5/6 nephrectomy rats
are depicted in open circles and bars during a normal sodium intake period (2
meq/day) a high sodium intake period (6 meq/day) and another normal
sodium intake period. The high sodium period is inside the hatched box.
Points represent the mean of 19 (RM) or 5 (intact) rats. Statistically
significant differences between control and infusion periods for p s 0.05 are
marked with *. Bars represent SEM.

94

 

 

60 F

_RRM n=
E::j Intoci n ==

// ' l

30 e

9
4

 

 

 

 

20 e

 

BUN (mg percent)

 

 

 

 

 

 

 

 

 

O 5 l0 15 20

Time (days)

 

 

 

Figure 9: Blood urea nitrogen in intact and RRM rats during changes in
sodium intake. Intact rats are depicted by the open bars and RRM rats by the
solid bars during a normal sodium intake period (2 meq/day), a high sodium
intake period (6 meq/day) and another normal sodium intake period. The high
sodium period is inside the hatched box. Points represent the mean of 9
(RRM) or 4 (intact) rats. There were no statistically significant differences
between control and infusion periods for p s 0.05. Bars represent SEM.

95

protocol but did not change when sodium intake was increased. Water
balance, the difference between water intake and urine output, was not
different between the two groups and was not altered by the change in
sodium intake (Figure 10). UNaV increased in all groups during highsodium
intake and, although there was apparent sodium retention in all groups during
high sodium intake, the differences were not statistically significant because
of the large within groups variance. Potassium excretion was not different
between any of the groups (Figure 11). Plasma electrolytes did not change in

any of the groups (data not shown).

D. Angiotensin II antagonism in reduced renal mass hypertension
1. Chronic angiotensin II antagonist treatment

Control RRM rats (average initial BUN = 34.7 i 3.5 mg%; n = 7) had
a significant increase in MAP when changed from normal to high sodium
intake. Losartan treated RRM rats (average BUN = 38.4 i 2.4 mg%; n = 9)
did not Show an increase in MAP during high sodium intake. Both treated
(average BUN = 15.9 i 1.8, n = 7) and control (average BUN = 18.2 i 1.8,
n = 7) intact rats had no change in MAP. Heart rate did not change in any
group over the 12 day experimental period and was not different between

groups. These data are shown in Figure 12. There was a significant positive

96

 

100 ~

75— Cl Intact (5)
I RRM (19)

Water Intake mI/doy

 

 

Urine Output ml/doy

 

 

Water Balance mI/doy

 

 

 

 

 

Time (days)

 

 

 

Figure 10: Water intake, urine output and water balance in intact and '
RRM rats during changes in sodium intake. Intact rats are depicted by the
open squares and RRM rats by the solid squares during a normal sodium
intake period (2 meq/day), a high sodium intake period (6 meq/day) and
another normal sodium intake period. The high sodium period is inside the
hatched box. Points represent the mean of 19 (RM) or 5 (intact) rats.
There were no statistically significant within group differences in WI or U0
and no statistically significant between group differences in WB. Bars
represent SEM.

97

 

8“ :1 RRM n=9
{1 - Intact n=4

 

 

Sodium Excretion meq/day

 

 

 

 

 

 

 

 

Normal Na

Sodium Balance (meq/day)

 

 

Potassium Excretion
in

 

 

Time (days)

 

 

 

Figure 11: Urinary electrolyte excretion and sodium balance in RM
and intact rats during changes in sodium intake. Daily sodium excretion in
the top figure represents the average of 19 RRM rats (open bars) and 5 intact
rats (solid bars). The horizontal lines represent the daily sodium intake
during normal (2 meq/day) or high (6 meq/day) sodium intake. The middle
figure illustrates daily sodium balance, calculated by subtracting urinary
sodium excretion from sodium intake, during the same three periods. The
hatched box is the high sodium intake period. The bottom figure illustrates
the daily urinary potassium excretion in the two groups of rats. There are no
error bars on the middle figure because differences were very large. There
were no statistically significant differences between groups for any of the
variables. Error bars represent SEM.

98

 

U RRM Control, 7
I RRM Losorton. 9
A Intact Control. 8

140 — A Intact Losartan. 8

120*

MAP (mm Hg)

100 —

 
 
 
 

 

 

 

 

 

 

...I T .

 

Heart Rate (bpm)

 

 

 

 

n

 

 

5 6 7 8 9 10 11 12 13

Time (days)

 

Figure 12. Effect of losartan treatment on blood pressure and heart rate in
RM and intact rats during changes in sodium intake. 4 groups of rats are
depicted during normal sodium intake (2 meq/day) and high sodium intake (6
meq/day). The two control groups received no drug treatment. The two
losartan groups received 3 mg/kg losartan as an iv bolus once a day following
measurement of blood pressure during the high sodium period only. The

high sodium (and losartan treatment) period is in the hatched box. Error bars
represent SEM. Statistically significant differences within groups for p s 0.05

are marked with *.

 

99

 

 

 

 

180 I I I l l 1
Control ,

160 — (r = 77)/__,..-«-" _

E

.2

8 140

(f)

_C

.9“

£ 120

{\

>

O

3 100

CL

<1:

2
80 — -
6O 1 1 1 L l 1

 

 

O 10 2O 3O 4O 50 6O 7O
BUN (day 7 high sodium)

 

 

 

Figure 13. Correlation between blood pressure and BUN in RRM with or
without losartan treatment on day 7 of high sodium intake for each of the
RRM rats. The lines of regression for the 7 untreated control rats (open
circles, broken line) and 6 losartan treated rats (solid circles, solid line) are
Shown. There is a significant correlation between the two variables in both
groups but the lines of regression are different.

100

correlation (r = 0.77 for control and r = 0.58 for treated) between BUN and
MAP on day 7 of high salt in both groups of RM rats. The scatter plot is
Shown in Figure 13.

Figure 14 Shows water intake and urine output in the 4 groups. The
change in sodium intake did not cause a change in water intake or urine
output in any of the rats except for the control RRM rats which had an
increase in both WI and U0 on two of the high sodium days. Water balance
(the difference between WI and U0) was unchanged over time in all groups.

Sodium excretion and sodium balance are shown in Figure 15. Urinary
sodium excretion was equivalent between groups with sodium retention
apparent in. all groups during the first 3 days after the increase in sodium
intake from 2 to 6 meq/day. There was no difference between groups in the
time course or amount of sodium retention. Furthermore, sodium retention
did not correlate with MAP. Urinary potassium excretion (data not shown)
was not different within or between any of the groups throughout the
protocol.

The BUN values of the 4 groups are illustrated in Figure 16. BUN
was significantly greater in both RRM groups than in the intact groups during
the entire protocol. The initial BUN values were not different between the

two intact groups or between the two RRM groups although BUN was

 

. [I RRM Control, 7
I RRM Losartan. 9
80 ~ A Intact Control, 8
A Intact Losartan. B

i V r i ’12., , £3.45/
Normal Sodium ‘2. e /

o l l

 

Water Intake (ml/day)

 

 

 

Ur ne Output (ml/day)
a at an

o o o

fir I I

 

 

 

 

Water Balance (WI — U0)

 

 

 

 

Time (days)

 

 

 

Figure 14: Effect of losartan treatment on water intake, urine output and
water balance in RRM and intact rats during different sodium intakes. The
two control groups did not receive any drug treatment. The two losartan
groups were untreated during the normal sodium period (2 meq/day Na) and
received 3 mg/kg/day losartan iv during the high sodium period (6 meq/day)
only. Losartan treatment and high sodium intake both started immediately
following daily recordings on day 5 of normal sodium, indicated by the
shaded area. The losartan bolus was given each day after measurements were
made. Error bars represent SEM. Statistically significant differences within
group for p < 0.05 are marked with a *.

102

 

High Sodltlm + Losartan

 

_RPM Control. 7
[:RRM Losartan. 9
_ Shalom Control. 8
_lnlact Losartan, 8

UNaV (meq/day)

 

 

 

2 Norrmol Somali l
0
0 l 2 3 4 5 6 7 8 9 10 ll 12 I3
6 i
4 i Normal Sodium High Sodium

    
   

No Balance (meq/day)

   

 

'l'l'lll'l‘
t O O o O O 0 I
""""

- O O O O

 

Time (days)

 

 

 

Figure 15: Effect of losartan treatment on urinary sodium excretion and
sodium balance in RRM and intact rats during different sodium intakes. The
two control groups did not receive any drug treatment. The two losartan
groups were untreated during the normal sodium period (2 meq/day Na) and
received 3 mg/kg/day losartan iv during the high sodium period (6 meq/day)
only. Losartan treatment and high sodium intake (the shaded area) both
started immediately following daily recordings on day 5 of normal sodium,
indicated by the shaded area. The losartan bolus was given each day
following measurement of water intake and urine output. The horizontal lines
represent daily water intake. Error bars represent SEM. Statistically
significant differences within group for p < 0.05 are marked with a *. There
were no significant differences between groups.

103

 

 

 

 

 

 

 

 

60 _
* - initial BUN
a? |:I Final BUN
O‘
E
C
8. 40 ~
9
."Z:
O
93
3
8
9 20 _
a)
I
I
O
RRM Cont. RRM Losart. Intact Cont. Intact Losart.
(n=7) (n29) (n=8) (n=8)

 

 

 

Figure 16: Effect of losartan treatment on blood urea nitrogen in intact and
RM rats during changes in sodium intake. The initial and final BUN in 2
groups of RM rats and 2 groups of intact rats maintained for 5 days on
normal sodium followed by 7 days on high sodium are depicted in the ifgure
above. The solid bars represent the mean of each group on the first day of
normal sodium intake (2 meq/day Na). The open bars represent the mean of
each group on the last day of high sodium intake (6 meq/day). Control
groups did not receive any drug treatment. The two losartan groups were
untreated during the normal sodium period and received 3 mg/kg/day losartan
iv during the high sodium period. Error bars represent SEM. Significant
differences within group are marked with a *. Significant differences between
intact and RM groups are marked with +. Differences were considered
statistically significant for p s 0.05.

104

significantly higher in RRM than intact rats at both time points. Final BUN
was significantly elevated above the initial value in control RRM but not in
the treated RRM rats. BUN did not change over time in either of the intact
groups.

Acute ANG II infusion caused a dose dependent increase in MAP in
all rats during the normal sodium period (no antagonist treatment in any
group) and in the untreated groups during the high sodium period (Figure 17).
Losartan treatment completely prevented a rise in blood pressure to ANG 11
during the high sodium period. There were no differences between intact and
RRM groups on either normal or high sodium intake and the elevated MAP

in the untreated RRM rats did not affect the acute response to ANG II.

2. Acute antagonist treatment
Bolus iv injections of 3 mg/kg losartan into RRM rats with a modest
sodium-induced hypertension did not significantly lower MAP on the fifth
day of high sodium intake. (Figure 18). There was a modest decrease in
MAP on the tenth day of high sodium intake but the decrease was only
significant at 2 and 6 hours following losartan treatment. Losartan did not

alter WI or UO in this group of rats (data not shown).

105

 

 

_RRM Control, 7
[:IRRM Losartan. 9

_Intact Control, 8
Mintact Losartan, 8

 

 

50—

 

v
4‘

'01.:

0.

Normal Sodium High Sodium

+ Losartan

4oe

V
A

{91010191010

0

 

V
A

O

O

v
A

Change in MAP (mm Hg)
0

0:0:

'
A

 
    

O

       
     

v
A

O
1- +

' III/IIIIIIIA

V
A

O

  

.'
*4-
*+

v
A

 

 

 

III/IIIIIIIIIIIIIIIIIA

t
\
\
\
\
\
\
N.
\n
\
\
\
\
\
\
t
\

 

 

 

//////////i—I

   

o
VIII/IA

I
0:
I

 

 

3 ng 10 ng 30 ng 3 ng 10 ng 30 ng

Dose Ang II (infusion/min)

 

 

 

Figure 17: Acute blood pressure response to ANG II infusion in intact and
RM rats with and without losartan blockade. The change in MAP was
recorded 5 minute after the start of ANG II infusion at 3, 10 or 30 ng/min iv
in each of 4 groups of rats. The bars on the left side of the figure represent
the average response of each group while on normal sodium intake (2
meq/day Na). None of the groups were receiving drug treatment during this
time. The bars on the right side of the figure represent the average response
of each group during high sodium intake (6 meq/day Na). Both losartan
groups were receiving a daily iv 3 mg/kg bolus of losartan during high
sodium intake. RRM control rats were hypertensive during the high sodium
period. Error bars represent SEM. Significant differences within group are
marked with a *. Significant differences between losartan and control groups
at any time point are marked with +. There were no differences between
RRM and intact. Differences were considered statistically significant for p s
0.05. '

106

 

 

 

 

 

 

 

 

 

 

 

 

 

* T /AA///

* Nil. WM/NW/
ml. 0%

ml. nan/M
it. and

1 Or/// /
maritfl /////

 

 

 

                

  

g
%
w/fl/If/xé
7%?

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552923922

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140 r

130 I-
20
10
100

0:86 2:305 9005 coo:

I Losartan

I Losartan

Time

 

.IWIEEIIZIIIIIZII
.l'flflllflalznfllflr.

 

)Cflflfl//VUC//

//////////// /

MUCK//UUfiA/VUC/

 

 

 

Iflfllzflaflzflfll'flflf

.//////////l

I Losartan

 

g

.21
.I/l/l/l/l/da

/UC//VUhV/VUHK//

 

 

 

/

rJvV/UCK/VUK/Jfll/y

///////4////

TL /VUCC///UUC/

 

 

 

 

     

.////////
.lfi/l/l/l/lé
ill/lg
W/AI/l
7////////

 

I Losartan

    

 

 

500 ~

Ac_E\2oeov 83.. too...

Time

 

 

Figure 18: Blood pressure and heart rate response to acute losartan in

10 of

measurements. The open bars are measurements following losartan challenge

at 5, 15, 30, 60, 120, and 360 minutes. Each bar represents the mean of 7

following an iv bolus of 3 mg/kg losartan is shown. The solid bars are daily
animals. Error bars are SEM. Losartan was given on day 5 and day

high sodium intake. The only significant differences (p s 0.05) were at 120
and 360 minutes following the second losartan challenge. MAP was not

hypertensive RRM rats on high sodium intake (6 meq/day). MAP before and

significantly different from control 24 hours after the second challenge butdid
not reach normal levels (the shaded area) at any time. HR did not change

throughout and was in the normal range (the shaded area).

107

In summary, RRM rats switched from normal sodium to high sodium
intake developed the expected increase in MAP while RRM rats treated with
losartan did not. MAP in intact rats was not affected by the change in
sodium intake or by losartan treatment. Furthermore, neither losartan
treatment nor renal reduction affected the time required to achieve sodium
and water balance following the change in sodium intake. Losartan
prevented a sodium-induced increase in BUN in the losartan treated RRM
rats. Acute losartan treatment in hypertensive RRM rats did not lower MAP
after 5 days on high salt but did cause a small decrease after 10 days of high

sodium treatment.

E. Angiotensin II infusion in reduced renal mass rats

Figure 19 shows the daily blood pressure and heart rate in three groups
of rats maintained on normal sodium intake: intact rats receiving 10 ng/min
ANG II (n = 7), RRM rats receiving 10 ng/min ANG II (n = 27) and control
RRM rats (n = 11). Both intact rats and RM rats receiving ANG II had a
significant increase in MAP during the first 4 days of ANG II infusion.
However, RRM rats had a larger increase in MAP and their MAP remained

elevated throughout the ANG II infusion period. MAP in intact rats returned

108

 

A RRM Ang. n=27
A RRM con. n=11
0 Intact Ang. n=7

     
 

\.

.25; .Ԥ\\ . m

Arterlal Pressure (mmHg)

 

 

 

50° _ —Angiotensin II Infusion (lOng/min)—

 

 

A \
E 450 I
E
e . .
E . -— .
.9 A.’
~: 400 — ‘“ - 5A, kid
‘I ‘ Vi.
a? V > c
r:
8
I 350 —
300 I

 

 

 

 

Tlme (days)

 

 

 

Figure 19: Blood pressure and heart rate during angiotensin II infusion in
RRM and intact rats on normal sodium intake. Daily MAP and HR in each of
3 groups of rats: RRM receiving ANG II, intact rats receiving ANG II, and
RM controls. The ANG II infusion period (10 ng/min), indicated by the
shaded area, was preceded by 3 control days and followed by 4 recovery
days. All rats received a fixed sodium intake of 2 meq/day. Error bars
represent SEM. Significant differences within group are marked with a *.
Significant differences between RRM and intact at any time point are marked
with +. Differences were considered statistically significant for p s 0.05.

109

 

BO

60

40

Water Intake (ml/day)

20

80

60

4O

Urlne Output (ml/day)

20

 

Ag ’5‘“ \ ‘ ‘ .

  
   

 

 

 

\\

A RRM Ang. n=27
A RRM con. n=ll
0 lntact Ang. n=7

A\

 

«V*V3‘\‘&\\\

 

~Angiotensin II Infusion (10ng/min)—

§

 

 

 

s\\

 

5 1O
Tlme (days)

 

 

Figure 20: Effect of angiotensin II infusion on water intake and urine output
in intact and RRM rats on normal sodium intake. Daily water intake and
urine output in each of 3 groups of rats is depicted: RRM receiving ANG II,
intact rats receiving ANG II, and RRM controls. The ANG II infusion
period (10 ng/min), indicated by the shaded area, was preceded by 3 control
days and followed by 4 recovery days. All rats received a fixed sodium
intake of 2 meq/day. Error bars represent SEM. There were no differences
between groups or within any group.

110

to control levels by day 5 of ANG II infusion. From day 6 of ANG II
infusion through day 3 of recovery, MAP in RRM rats receiving ANG II was
significantly higher than in intact rats receiving ANG II. There was a
positive correlation (r = 0.34) between mean BUN and MAP on day 10 of
ANG II infusion in all rats receiving ANG 11; however, the correlation was
not statistically significant (p = 0.11). Control RRM rats exhibited a gradual
increase in MAP throughout the protocol. Heart rate tended to increase in all
three groups of rats over the 17 day experimental period but was not different
between groups.

Figure 20 shows water intake and urine output in the 3 groups of rats.
ANG II infusion did not cause a change in water intake or urine output
ineither group of rats. Water balance was unchanged over time in all three
groups.

Urinary electrolyte excretion is illustrated in Figure 21. Urinary
sodium excretion was equivalent in all three groups. Urinary potassium
excretion was significantly higher in the intact rats than in the RRM rats
throughout the protocol.

Plasma immunoreactive ANG II concentrations ([ANG Illir) in the
three groups are depicted in Figure 22. All three groups had similar [ANG

lI]ir during the control period, although the level in the control RRM group

 

A RRM ANG II. n=27
A RRM con. nstt
0 Intact ANG II. n=7

\s‘

 

i}

'l ‘

/

-Anglotensln II Infusion (10 ng/min)-

Ne Excretion (meq/day)
I
\

 

 

 

K Exeretlen (meq/day)

 

 

 

Tlme (days)

 

 

 

Figure 21: Effect of angiotensin II infusion of urinary electrolyte excretion in
RRM and intact rats on normal sodium intake. Daily sodium and potassium
excretion in each of 3 groups of rats is depicted: RRM receiving ANG II,
intact rats receiving ANG II, and RM controls. The ANG II infusion
period (10 ng/min) is indicated by the shaded area, preceded by 3 control
days and 4 recovery days. All rats received a fixed sodium intake of 2
meq/day. Error bars represent SEM. There were no differences in sodium
intake either within groups or between groups. Potassium excretion in intact
rats was significantly higher than either of the two RRM groups through the
protocol but there was no difference between the 2 RRM groups. Differences
were considered statistically significant for p _<_ 0.05.

112

 

 

 

 

 

 

 

 

 

300 -
m RRM. no Ang
\ [mm] RRM + Ang
A g Intact + Ang
E
\ 200 r-
U
D:
V
0.
F1
=
M 100 r-
g "a.
<
I——l
0

 

 

     

a 12 16
Time (days)

 

 

 

Figure 22: Plasma levels of immunoreactive angiotensin II in RM and
intact rats receiving angiotensin II infusion. Plasma levels of ANG II in each
of 3 groups of rats is depicted: intact receiving ANG II, RRM receiving
ANG II and RM control. The 10 day ANG II infusion period (10 ng/min)
is indicated by the shaded area, preceded by 3 control days and 4 recovery
days. Plasma samples were taken on day 3 control, days 5 and 10 of ANG II
infusion and on day 4 of recovery. All rats received a fixed sodium intake of
2 meq/day. Error bars represent SEM. Significant differences within group
are marked with a *. Significant differences between RRM and intact at any

time point are marked with +. Differences were considered statistically
significant for p S 0.05.

113

 

m Intact Ang II (n=6)
- RRM Ang II (n: 27)
I: RRM con (n=1t)

 

, // ,

40 e / ~7~ Ang/1Mpfus;on*

auu (mg/dl)

 

 

 

 

 

 

 

 

   

 

 

 

Figure 23: Effect of angiotensin II infusion on BUN in RM and intact rats.
BUN in each of 3 groups of rats is depicted: intact receiving ANG II, RRM
receiving ANG II and RRM control. The 10 day ANG II infusion period (10
ng/min) is indicated by the shaded area, preceded by 3 control days and 4
recovery days. Plasma samples were taken on day 3 control, days 5 and 10
of ANG II infusion and on day 4 of recovery. All rats received a fixed
sodium intake of 2 meq/day. Error bars represent SEM. Significant
differences between RM and intact at any time point are marked with +.
There were no significant differences over time within any of the groups
Differences were considered statistically significant for p S 0.05.

114

slightly but significantly higher than that of intact rats. ANG II infusion
produced similar increases in [ANG 11],, in intact and RRM rats. In control
RRM rats, [ANG II]it decreased somewhat over the course of the protocol but
the changes were not statistically significant and the three groups had
equivalent levels during recovery.

The BUN values of the 3 groups are illustrated in Figure 23. BUN
was significantly greater in both RRM groups than in the intact group during
the entire protocol. BUN values in the RRM groups were not different fi'om
each other and did not change significantly over time in any group.

In summary, chronic infusion of 10 ng/min ANG 11 into normotensive
RRM rats on normal sodium intake caused a significant and sustained
increase in MAP compared to either intact rats receiving the same dose ANG
II or with control RRM rats (no ANG II). Infusion of ANG 11 into intact rats
on normal sodium intake caused an initial increasein MAP but MAP returned
to control levels during the last 6 days of the 10 day infusion while MAP
remained elevated throughout the infusion in RRM rats. The hypertension in
the RRM rats was not accompanied by changes in water or sodium balance

and MAP returned to control levels during the recovery period.

IV. Discussion

In the face of different causes, the basic hemodynamic abnormality
underlying the development and maintenance of hypertension is, almost
without exception, increased peripheral vascular resistance. Two major
factors lead to increased vascular resistance--elevated activity of the
sympathetic nervous system and altered levels of vasoactive hormones.
These two systems interact with each other so that vascular resistance is the
product of the combined actions of circulating hormones and sympathetic
outflow.

Both clinical and experimental cases of SDH are indeed characterized
by elevated TPR. The cause of the increased vasoconstriction is not clearly
defined but there are indications that it is due to increases in one or more
transferable circulating factors (Posten et al. 1981, Nishio er a1. 1988) and
increased activity of the sympathetic nervous system (Mark et al. 1975, Gill
er 01.1988). Clinical studies of sodium-sensitivity have demonstrated
alterations in the activity of the renin-angiotensin system (Weinberger 1991,
Sullivan 1991), plasma levels of aldosterone (Weinberger 1991), and plasma
levels of an endogenous ouabain-like factor (Nishio et al. 1988). These same
changes are also seen in animal models of sodium-induced hypertension

(Nakagawa et al. 1990, Wauquier et al. 1986). The focus of this dissertation

114

115

is on the changes in humoral factors that occur during the initiation of SDH
and how those changes influence resting arterial pressure.

In a review article on the role of the kidney and salt in the aetiology of
hypertension, deWardener proposed a theory to explain how alterations in
sodium intake could initiate hypertension. The theory proposes that the basic
alteration is a renal defect restricting the excretion of sodium. This inability
to excrete a sodium load leads to sodium retention during increased sodium
intake. Water follows sodium and the animal becomes volume expanded.
This triggers so-called "volume controlled mechanisms" in an attempt to re-
establish sodium and water balance. The volume controlled mechanisms
include elevated sympathetic output, decreased secretion of antinatriuretic
hormones such as aldosterone, angiotensin II and vasopressin, and increased
secretion of natriuretic factors such as atrial natriuretic peptide and the
endogenous Na“ +-A'I‘Pase inhibitor. It is the sustained activity of the
volume controlled mechanisms that maintain hypertension (deWardener 1990
Parts I & II).

It is difficult to distinguish what the initiating factor is in SDH. In
clinical cases, hypertension is in the established phase when it is diagnosed
and the observed hormone imbalances (Sullivan 1991) and renal damage

(Kincaid-Smith 1991) could be either a cause or a consequence of the

116

hypertension. In experimental genetic models, alterations in renal fimction
may be extraneous to the genetic alterations that caused the hypertension.

The alteration which precipitates the rise in blood pressure can only be
defined with certainty in models of hypertension where the initiating event is
controlled and defined by the investigator. A useful model for studying SDH
with such control is the reduced renal mass(RRM)-salt model of hypertension.
In this model there is an obvious decrease in the total capacity of the kidney
to excrete excess sodium and water but blood pressure does not rise until
sodium intake is elevated. As predicted by deWardener‘s theory, the initiation
of sodium-induced hypertension is associated with a transient sodium and
water retention leading to a temporary elevation of CO. After the initial
increase in CO, sodium and water balance are re-established and the
hypertension is maintained by chronically elevated TPR (Lombard et al.
1989). The elevated peripheral resistance is accompanied by increased
sympathetic activity, elevated aldosterone and OLF, and altered activity of
ANG 11. Our studies addressed possible contributions of these circulating

hormones in this and other sodium dependent models of hypertension.

A. Plasma aldosterone during angiotensin II infusion

As addressed in the Introduction, rats with established RRM-salt

117

hypertension have alterations in both the renin-angiotensin system and in
PAC (Ylitalo and Gross 1979, Chi and Foreman 1986, Jackson et al. 1988).
Furthermore, elevations in circulating ANG II affect the secretion rate of
aldosterone so that a chronic increase in PAC could be attributed to an
increase in circulating ANG 11. Our first study examined the possibility that
increases in circulating ANG 11 may increase PAC and that an increase in
PAC might contribute to an apparent ANG II induced hypertension. In this
study PAC was measured in intact rats receiving chronic ANG II infusion.

The question posed by this study was: What role does aldosterone
play in ang-II induced hypertension? A contribution of aldosterone to ANG
II-induced hypertension was proposed based on the following observations: 1)
acute ANG II infusion in rats caused an increase in plasma levels of
aldosterone, 2) elevated PAC in rats lead to sustained hypertension and 3)
chronic exposure of rat adrenals to elevated plasma ANG [I caused an
increase in the number of adrenal ANG 11 receptors and an accompanying
increase in plasma levels of aldosterone.

The ability of ANG II to increase the secretion rate and plasma level of
aldosterone in rats has been previously demonstrated by several investigators.
For example, a study with rats in 1978 by Aguilera and Catt reported

increased plasma ANG II levels and aldosterone secretion in response to

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sodium deprivation. The increase in both hormones was prevented by the
concomitant administration of angiotensin converting enzyme (ACE)
inhibitors (Aguilera and Catt 1978). Since both ANG II and PAC were
decreased by the ACE inhibitors, the elevated PAC was apparently caused by
elevated ANG II levels. Hanger et al. in 1978 found that 50 ng/min ANG II,
given to rats on uncontrolled, normal sodium intake produced a sustained
elevation in PAC and a significant increase in adrenal ANG II receptor
number (Hanger et al. 1978). A more recent study by Lqu et al. measured
urinary aldosterone excretion in rats given ANG II (76 ng/min sc) for ten
days. Their study found a sustained elevation in urinary aldosterone
excretion with a seven-fold elevation in the daily excretion level by day seven
(Luff et al. 1989).

Table 1 illustrates the increase in PAC we observed following a one
hour infirsion of 10, 30, and 60 ng/min ANG 11. These data support the
earlier findings in rats (Hanger et al. 1978, Luft et a1. 1989) and in humans
(Oelkers and Schbneshofer 1974) that ANG II is a potent secretagogue of
aldosterone and that a one hour infusion of even a low dose of ANG 11
produces an increase in PAC.

The second premise of the current study was that elevated PAC alone

in intact rats leads to sustained hypertension. Our data (Figure 1) illustrate

119

that this is a slowly developing hypertension. The animals with sufficiently
elevated PACs became hypertensive only afier ten days of hormone
administration. Mineralocorticoid hypertension can be produced more rapidly
by subjecting uninephrectomized rats to a much higher sodium intake
(Garwitz and Jones 1982). However, the current study shows that increased
PAC in combination with moderate increases in sodium intake also produces
hypertension in intact rats. This is consistent with findings in other animals,
including primary aldosteronism in humans (Conn and Louis 1955, Darke et
a1. 1977, Pan and Young 1982).

The third observation in support of our hypothesis was first made by
Aguilera, er al. (Catt et al. 1987, Aguilera and Catt 1978). This group
reported an increased number of adrenal ANG 11 receptors and increased
aldosterone secretion rates after chronic exposure of isolated adrenals to ANG
II. This observation implies that chronically elevated plasma ANG 11 may
lead to progressively higher PAC. This is also suggested by the progressive
increase in urinary aldosterone excretion seen in Lnfi's study (Lufi er al.
1989). The current study directly examined the relationship between systemic
ANG 11, PAC and blood pressure during a persistent elevation in plasma
ANG II levels produced by iv hormone infusion.

The first chronic ANG II experiment investigated ANG 11's ability to

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increase PAC and MAP in rats on a moderately high sodium intake (6
meq/day). Both ANG II and aldosterone induce hypertension more rapidly in
animals on high sodium intakes (Hall et al. 1984, Fink er al. 1987, Young et
al. 1982). We postulated that if aldosterone contributes to the development
of ANG II hypertension, increases in PAC of at least five times the control
levels should precede the increase in arterial pressure during ANG II
administration and that a time course of sixteen days should be sufficient for
both the hypertension and the elevation in PAC to be expressed.

Chronic ANG II infusion appeared to cause a slight increase in PAC
that lagged behind the increase in arterial pressure but there was no indication
of a progressive increase in PAC over time. Also, at no time during ANG II
infirsion was the PAC significantly higher than control levels, although rapid
and substantial increases in MAP occurred. During the ANG II infirsion,
PAC remained lower than that found necessary to cause a sustained increase
in MAP with chronic aldosterone infusion (i.e. 250 - 300 pg/ml) (Figure 2).
Hauger's study in rats (Hanger et al. 1978) found the increase in PAC to be
maintained for at least six days during chronic infusion of 50 ng/min ANG II,
and Luft's study in rats (Luft et al. 1989) found the elevation in urinary
aldosterone to be maintained for nine days with 76 ng/min ANG II. This

implies that the high infusion rates used in their study maintain adrenal

121

stimulation over a period of days. In the current study, the dose of ANG II
was 10 ng/min, a dose found previously to cause an acute increase in PAC
(Table 1) and a sustained elevation in MAP (Pawloski 1990). The differences
in the doses and the resultant aldosterone responses suggest that high doses of
ANG II can cause sustained adrenal stimulation, but that a lower dose of
ANG 11, although acutely stimulatory to the rat adrenal gland, does not
produce a significant chronic elevation in PAC. It is evident, in any case,
that PAC is not sufficiently elevated in rats receiving 10 ng/min ANG II to
contribute to the observed hypertension.

The lack of change in urinary and plasma electrolytes illustrated in
Figure 3 supports previous findings that ANG II hypertension in rats is not
associated with sodium and water retention, or expanded fluid volume (Fink
et al. 1987, Brown et al. 1979). This lack of change further strengthens the
conclusion that aldosterone did not contribute to the development of
hypertension in these animals, since aldosterone-induced hypertension is
accompanied by polydipsia, hypokalemia and transient sodium retention
(Conn and Louis 1955, Darke et al. 1979, Pan and Young 1982). No
changes in plasma levels of either sodium or potassium occurred, (Figure 4)
revealing neither hypernatremia nor hypokalemia occurred during the ANG II

infusion.

122

The second chronic ANG II infusion experiment was performed to
investigate the possibility that chronic ANG II stimulation of the adrenal in
rats on a normal sodium intake would lead to a progressive increase in PAC,
and ultimately an aldosterone-dependent hypertension. This reasoning was
based on the known ability of high sodium intake to suppress both PAC and
the aldosterone response to ANG II (Holtzman et al. 1989).

In animals on normal sodium intake, PAC did not increase significantly
or progressively with time of infusion as expected. These data and the blood
pressure data are depicted in figure 5 and reveal that administration of ANG
II at 10 ng/min in rats on a normal sodium intake did not cause a sustained
rise in MAP. The results of these experiments illustrate three points about
the relationship between sodium intake, ANG 11, PAC and arterial pressure
regulation in rats. First, it is apparent that the hypertension produced by low
dose ANG II infusion in intact rats is dependent on the level of sodium
intake. Although others have reported hypertension development during
ANG II infusion in rats on a normal (1 - 2 mEq) daily sodium intake (see
Young et al. 1982 for references), generally much higher doses of ANG II
were employed than used here. Experiments in dogs also support a dose-
related dependence of ANG II-induced hypertension on the level of sodium

intake (Cowley and McCaa 1976). ANG II infirsion in rats, unlike in dogs,

123

does not cause measurable sodium or water retention as measured in this
study and in previous studies (Textor et al. 1981).

Second, it is clear that increased PAC is not required for hypertension
development during chronic ANG II infusion in rats on increased sodium
intake. This finding supports conclusions drawn fi'om earlier experiments in
dogs (Cowley and McCaa 1976). Studies involving exogenous aldosterone
infusion in rats on increased sodium intake (6 mEq/day) showed that a PAC
of 250 - 300 pg/ml was required to increase blood pressure significantly. Yet
PAC never achieved this range in rats receiving ANG II on a chronic basis.
However, this result does not rule out the possibility that a low blood
concentration of aldosterone is necessary to support ANG II-induced
hypertension. Adrenal ablation experiments would be necessary to test this
point. A synergistic effect of ANG II and aldosterone also is a possibility,
but studies conducted in dogs do not support this idea (Cowley et al. 1986).

The third point raised by these results is the seeming inability of
circulating ANG II to chronically affect aldosterone secretion by the adrenal.
The role of ANG II as a chronic modulator of aldosterone secretion in
sodium-depleted animals is well established (Aguilera and Catt 1978,
Holtzman er al. 1989). Sodium depletion also enhances the adrenal response

to changes in circulating ANG 11 concentrations (Cowley and McCaa 1976).

124

The ability of ANG II to stimulate aldosterone secretion in a potent and
prolonged fashion in sodium-depleted animals may be due in part to removal
of the inhibitory effect of ANP on ANG II-induced aldosterone release
(Chartier et al. 1984, Naruse et al. 1987. The chronic influence of circulating
ANG II on PAC in animals on normal or high sodium intake is more
controversial. A number of previous studies in rats (KOmer and Mfiller 1979,
Marieb and Mnlrow 1965), sheep (Blair-West et al. 1963) and dogs (Cowley
and McCaa 1976) indicated that prolonged infusion of ANG II in animals on
normal or high sodium intakes produced only a transient (hours) increase in
PAC. Other investigators, however, have reported a sustained elevation in
PAC during ANG II infusion in rats (Hanger et a1. 1978), dogs (Hall and
Hungerford 1982), and humans (Oelkers and Schoneshofer 1983). The
reasons for this disparity of results is not clear, but could be related to
differences in the dose of ANG II employed, dietary potassium content, or
other factors which also influence aldosterone secretion (Quinn and Williams
1988). In the current study, a dose of ANG II was employed which is known
to cause hypertension in rats on a 6 meq daily intake of sodium (Fink er al.
1987), since ANG II-induced hypertension was the central focus of the work.
It is possible that this relatively low dose of ANG 11 caused chronic

increments in PAC below our ability to detect (50 - 100 pg/ml), and that

125

higher amounts of ANG 11 would have brought about a measurable and
maintained increase in PAC. It should be pointed out, however, that the rate
of ANG II administration used here has been shown to increase plasma ANG
11 concentration to a level equivalent to that observed in benign, two-kidney
renovascular hypertension (Mann et al. 1980, Brown et al. 1981). Thus, the
concentrations of ANG 11 produced in these experiments were presumably
within a relevant pathophysiological range.

In summary, long-term intravenous infusion of 10 ng/min ANG 11
causes sustained hypertension in rats on a 6 meq/day sodium intake, but not
in rats on a 2 meq/day sodium intake. In rats on the higher sodium intake,
raising PAC to 250 - 300 pg/ml will also cause a slowly-developing
hypertension. Administration of ANG II at 10 ng/min does not, however,
cause PAC to increase to this level under either of the sodium intake
conditions tested. We conclude that, in intact rats, increased PAC is not

necessary for chronic ANG II-induced hypertension in rats.

B. Plasma aldosterone in reduced renal mass hypertension
The next experiment addressed the possibility that elevations in PAC
play a role in RRM hypertension independent of ANG II. The rationale for

the investigation was based on the following two points. First, as established

126

in the introduction, it has been reported that RRM rats have elevated plasma
levels of aldosterone (Chi et al. 1986). Second, the previous experiment
demonstrated that chronic elevation of PAC in combination with a sodium
intake of 6 meq/day will cause hypertension in intact rats. If PAC is
sufficiently elevated during RRM hypertension, it would be expected to
contribute to the elevated blood pressure.

In the previous reports of elevated aldosterone, the measurements of
PAC were made afler weeks or months of hypertension (Chi and Foreman
1979). In this stage of established RRM-salt hypertension, plasma levels of
potassium are increased. Plasma potassium increases because of the impaired
ability of the remnant kidney to secrete potassium. Measurements of plasma
potassium in RRM rats reveal elevations that are directly proportional to the
degree of renal impairment. This is expected because renal secretion is the
primary route of potassium clearance during steady-state conditions (reviewed
in Mitch 1982). The increase in plasma potassium of RM rats presumably
stimulates aldosterone secretion since plasma potassium is a potent
secretagogue of the mineralocorticoid (Brown et al. 1979). Therefore the
elevated PAC could either cause the hypertension, if it increased before the
blood pressure rose, or it could be a result of the hypertension if it occurs

only during the later stages of hypertension.

127

Salt-induced hypertension in RRM rats is a progressive condition with
three stages. When animals are initially exposed to high sodium intake, there
is a rapid rise in blood pressure and a transient sodium retention. Within
hours, blood pressure stabilizes and remains relatively stable with only a
slight increase over the next few weeks to months (Lombard er al. 1989).

The duration of the stable period is dependent on sodium intake and on the
original amount of renal tissue destroyed. Even during this plateau phase,
progressive renal deterioration continues (Meyer and Rennke 1988).
Eventually, enough nephrons are destroyed so that the remaining nephrons are
unable to maintain renal function. The animal becomes uremic and goes into
end-stage renal failure. This is characterized by a rapid rise in blood pressure
to very high levels followed by a fall in blood pressure to hypotensive levels
just before there is total renal and cardiovascular collapse (Ylitalo and Gross
1979, Kaufman et al. 1974).

It is expected that the mechanisms responsible for maintaining blood
pressure are different during the different stages of the hypertension. It is the
mechanisms responsible for the stable phase in pressure that this study
addresses and which are most relevant to the general question of what
maintains sodium-induced hypertension.

In the study reported here, the sodium induced rise in MAP was not

128

associated with changes in PAC (Figure 7). The failure of PAC to increase
during the development of the hypertension does not support our hypothesis
that elevated PAC contributes to the early stages of hypertension but suggests
that elevations in PAC reported previously in RRM rats were due to
potassium stimulated secretion after the onset of hypertension. In those
animals, it is assumed that chronic renal insufficiency decreased potassium
excretion and lead to elevated plasma levels of potassium which stimulated
aldosterone release (Brown et a1. 1979). Plasma levels of potassium were not
reported in those previous studies so it is impossible to determine exactly
why differences were observed. In the current study, plasma electrolytes
were not altered during the alterations in sodium intake. Therefore if
potassium stimulation was responsible for the eventual elevation in PAC,
elevated PAC would not be expected in these animals. Additionally, there
was no apparent deterioration of renal function in the RRM rats in this study
as measured by blood urea nitrogen (BUN), which did not increase during the
twenty days of the protocol as shown in figure 8. This is indicative that these
rats did not progress from the stable phase to end-stage renal failure.

During the initial phase of RRM hypertension, CO is not elevated
beyond the first 24 hours of sodium excess. Rather, elevations in TPR cause

the increased arterial pressure (Lombard et al. 1989). This also supports our

129

conclusion that aldosterone excess is not responsible for the initial rise in
blood pressure in RRM rats, since mineralocorticoid excess is initially
associated with sodium and water retention, and increased CO. Furthermore,
although the stimulus for the elevated peripheral resistance is not apparent

from this study, it does not appear to be dependent on elevated PAC.

C. Endogenous angiotensin II in reduced renal mass hypertension
1. Chronic losartan in RRM rats

As outlined in the Introduction, RRM-salt hypertension in rats can be
prevented with CEI (Jackson and Johnston 1988, Meyer er al. 1985).
Furthermore, it has been shown that CEI are uniquely able to prevent both the
hypertension and the deterioration of renal function when compared to other
antihypertensive treatments (Tolins and Raij 1990, Brunner er a1. 1989). In
addition, both ANG II-induced hypertension and RRM-salt hypertension are
due to increased vasoconstriction. Therefore it is possible that ANG II is
responsible for the initial salt-induced rise in blood pressure in RRM rats.

Neither ANG 11 nor PRA are elevated in RRM animals (Rosenberg et
a1. 1991, Jackson and Johnston 1988. Chi et a1. 1986). However, there is
evidence that chronic angiotensin-dependent hypertension does not require

large elevations in plasma levels of the peptide. Brown et al. infused ANG II

130

at the rate of 20 ng/kg/min into rats for seven days. They found that plasma
levels were barely elevated at this infusion rate (Brown et al. 1981). Blood
pressure was not elevated acutely by this dose but rose gradually throughout
the week of infusion. Furthermore, plasma levels of ANG 11 did not change
during the infusion so that at the time of maximal blood pressure response,
plasma ANG II levels were equal to acutely subpressor levels. Other reports
of ANG II-induced hypertension report the same slow pressor response that
does not require a large change in plasma levels of the peptide (Cox and
Bishop 1991, Pawloski 1990). In addition, CEI and losartan are both able to
reverse hypertension in SHR rats, a model that also does not have increased
plasma levels of ANG II (Bunkenburg et al. 1991, Okunishi et al. 1991, Li
and Jackson 1987). Additionally, SHR rats appear to be hyper-responsive to
the slow-presser effect of ANG 11 (Li and Jackson 1987) so that CEI
apparently protect SHR through an ANG II-dependent mechanism in spite of
normal PRA. CEI may act through an analogous ANG II mechanism in
RRM rats even though plasma levels of the peptide are not elevated.
Alternatively, it has been shown that CEI affect several cardiovascular
control systems in addition to their effects on ANG II formation. CEI
treatment increases plasma levels of kinins (Fenoy, et al. 1991), increases the

synthesis of vasodilatory prostaglandins (Zucker et al. 1991) and causes the

131

release of an endothelium dependent vasodilator (Gilst et al. 1988). It is
therefore possible that the antihypertensive affect of CEI in RRM rats is
independent of alterations in circulating ANG II.

There is some evidence that CEI lower blood pressure in RRM rats by
increasing kinin levels (Karlstrom et al. 1990). It has also been suggested
CEI protection in RRM hypertension is due to inhibition of a local ANG II
system rather than effects on circulating levels of the peptide (Jackson and
Johnston 1988, Kuczera et a1. 1991). Therefore the exact mechanism of CEI
protection in this model is not clear and this study tested the hypothesis that
ANG II is necessary for the initiation of RRM hypertension. This was done
by administering an ANG 11 specific antagonist, losartan, concurrent with
high sodium intake.

Losartan is a recently developed antagonist specific for the ANG II
AT, receptor. The AT, and AT2 receptors are the two known ANG II
receptor subtypes with the AT, subtype apparently mediating all the
cardiovascular (reviewed Timmerrnans et a1. 1991), adrenal (Hajnoczky et al.
1992) and renal (Edwards er al. 1991) actions of ANG 11. At concentrations
up to 10" M, losartan does not affect the responses to potassium chloride,
norepinephrine, isoproterenol, AVP, bradykinin, acetylcholine, histamine or

serotonin (Wong et al. 1990a, Wong et al. 1990b). Also unlike the ANG II

132

partial agonist saralasin, losartan is a pure antagonist lowering blood pressure
in renal hypertension (Wong et a1. 1991, Bunkenburg et a1. 1991) with a
binding affinity of 1.9 X 10'8 M and a pA2 of 8.48. At the dose of 3 mg/kg
iv, losartan lowers blood pressure in SHR, renal hypertensive rats (Wong et
al. 1990a), and ANG II infused rats (Smits et al. 1991) but not in their
normotensive controls. Furthermore, although the antagonist crosses the
blood brain barrier and inhibits ANG II binding in circumventricular organs,
and in select nuclei within the CNS (Song et 01.1991, Gehlert et al. 1990)
central administration of losartan does not reverse the hypertension in SHR
while peripheral antagonism does (Mangiopane et al. 1991). The specific
antagonist should therefore selectively block only ANG II dependent changes
in blood pressure in RRM rats clarifying the mechanism of CEI
antihypertensive effect in rats without elevated plasma levels of ANG II.

In the current study losartan prevented salt-induced hypertension in
RRM rats. The same sodium intake caused a significant elevation in blood
pressure in untreated RRM rats (illustrated in figure 11). This is analogous to
the antihypertensive effect of CEI in RRM rats and supports our hypothesis
that the salt-induced rise in blood pressure in this model is dependent on the
actions of angiotensin II at AT, receptors.

Plasma levels of ANG II were not measured in RRM rats in this study

133

but previous reports have shown that circulating ANG II levels in RRM rats
are no different from the intact rats. In spite of assumed similar plasma
levels of ANG II in RM and control rats, the antagonist lowered blood
pressure only in the RRM rats. This suggests that blood pressure in RRM
rats is not normally dependent on the actions of circulating ANG II, but that
the rise in MAP in RRM rats that follows increased salt intake is dependent
on the peptide's action at AT, receptors.

In addition, losartan appears to have a renoprotective effect in RRM
rats. Previous studies have established that even one week on high salt intake
can cause progressive deterioration of renal function in RRM rats (Brandt et
al. 1989, Ylitalo and Gross 1979) as evidenced by the elevated BUN levels of
the untreated rats in this study (figure 15). The losartan treatment prevented
an increase in BUN and the treated rats appeared healthier than the untreated
rats. This supports the additional earlier finding that CEI afford renal
protection in this model (Anderson 1985, Meyer et al. 1985, Brooks et a1.
1989)

The renoprotection by CEI in remnant kidneys produced dramatic
decreases renal damage in humans (Sanchez et a1. 1991) and animal studies
(Brenner 1985). The protection was apparently due to a decrease in intrarenal

ANG 11 production resulting in decreased intraglomerular pressure (Raij er al.

134
1989, Meyer 1985, Tolins and Raij 1990). This renoprotection may not

contribute to the antihypertensive effect of the drugs. If losartan prevented
hypertension by inhibiting renal deterioration, the correlation between MAP
and BUN should be identical in treated and untreated animals. That is,
losartan should lower blood pressure by improving renal function, not alter
blood pressure at a given level of renal function. In the current study,
losartan treatment shifted the relationship between MAP and BUN (see figure
12) so that for any given BUN, MAP was lower in the treated animals. This
suggests that losartan's antihypertensive effect is independent of its ability to
slow renal deterioration.

One possible mechanism by which normal concentrations of circulating
ANG 11 could cause RRM hypertension is if RRM causes an increase in
sensitivity of the vascular AT, receptors. If the mechanism of action of ANG
II in RRM hypertension involves increased sensitivity of vascular receptors,
there should be an increased vascular response to a bolus of ANG 11 during
hypertension. As depicted in figure 16, the acute pressor response to ANG
II was not different in RRM and intact rats during either normal or high
sodium intake. Therefore renal reduction does not cause an increased
vascular response to ANG 11. Furthermore, the pressor response to ANG II

was not altered in either group during elevated sodium intake so that the

135

increase in blood pressure could not be due to a sodium-induced change in
sensitivity of the vascular ANG 11 receptors. Since the acute response was
totally blocked by losartan treatment, the acute pressor response was due only
to ANG II actions at AT, receptors.

Another effect of ANG II that could contribute to salt-induced
hypertension in RRM rats is sodium and water retention at the kidney. It is
possible that blockade of renal ANG 11 receptors by losartan, which have
been shown to be of the AT, subtype (Herblin er a1. 1990, Edwards er al.
1991), prevented sodium and water retention and the initiation of a volume-
dependent hypertension.

The RRM rats in this study did have a higher water intake than the
intact rats but they also had a higher urine output so that the water balance
was not increased in the RRM animals. Losartan treatment did not affect
either water or sodium balance in either the intact or the RRM rats so that the
antihypertensive effect was independent of alterations in water and sodium
balance (figures 13 and 14).

In summary, salt-induced hypertension and progressive renal
deterioration in RRM rats is apparently due to actions of ANG II on AT,
receptors. The antihypertensive and renoprotective effects appear to be

independent and are not accompanied by changes in sensitivity of vascular

 

136

ANG 11 receptors or changes in water and sodium balance.

2. Acute losartan treatment in hypertensive RRM rats

Hypertension produced by angiotensin II infusion is characterized by
two phases. Initially, blood pressure rises dose-dependently and appears to be
dependent on activation of vascular ANG 11 receptors since the increase can
be acutely reversed by the ANG II antagonist saralasin (Fink et al. 1987, Cox
and Bishop 1991). This early phase gradually diminishes and a chronic
increase in blood pressure dependent on the integrity of the area postrema
develops (Fink et al. 1987, Cox and Bishop 1991). During the chronic phase,
acute vascular receptor blockade only partially reverse the hypertension while
ganglionic blockade causes a much greater fall in pressure (Cox and Bishop
1991, Pawloski 1990). Thus it is possible to differentiate between vascular
and central ANG 11 effects on blood pressure.

Although chronic treatment of RRM rats with losartan revealed a role
for ANG II in salt-induced hypertension in this model, it is impossible to
separate the acute and chronic actions of the treatment. Acute blockade of
ANG 11 receptors in RRM animals with established hypertension should help
to differentiate between vascular and centrally mediated actions. If RRM

hypertension is entirely dependent on ANG II actions at vascular receptors,

 

137

acute blockade of those receptors should cause a rapid reversal of the
hypertension. Conversely, if the hypertension is dependent on less accessible
receptors, antagonism should only slowly reverse the hypertension.

Acute losartan treatment in RRM rats on high salt intake did not
appear to cause either an acute or a delayed decrease in blood pressure. This
first challenge was made after 5 days of high salt treatment and blood
pressure was only slightly elevated in some of the animals. Therefore the
ANG II-dependent mechanisms may not have been operating at this time. A
second challenge 5 days later did cause a significant fall in pressure, but only
after 2 - 6 hours. These results are only preliminary but firrther support the
previous conclusion that the antihypertensive effect of losartan is due to

blockade of non-vascular ANG 11 receptors.

D. Changes in sensitivity to chronic angiotensin II in reduced renal
mass rats

The goal of this investigation was to examine the sensitivity of blood
pressure in RRM rats to chronic administration of exogenous ANG II. It was
established in the previous experiment that the acute vascular pressor
response to ANG II is not increased in RRM rats. Furthermore, ANG II

dependent hypertension develops during infusion at doses that are not acutely

 

138

pressor and appears to depend on activation of central rather than peripheral
pathways (Fink er 01.1987, Cox and Bishop 1991, Brown et al. 1979).

In the Introduction, it was established that circulating levels of ANG II
are normal or even suppressed in RRM rats. Therefore, if the chronic
hypertension during salt-loading in RRM rats is due to increased activity of
the circulating renin-angiotensin system, it would require an increased
response to the blood-bome peptide.

In the current experiment, RRM rats on normal sodium intake
developed a sustained hypertension throughout the infusion of 10 ng/min
ANG II. Intact rats on normal sodium intake receiving the same dose of
ANG II had an initial small increase in MAP but MAP returned to control
levels and stayed normal during the last 6 days of the 10 day ANG II
infusion. Thus this study demonstrated that RRM rats have increased
responsiveness to the chronic pressor effect of circulating ANG II.

An obvious explanation for the increased pressor response to ANG II
in RRM rats would be decreased clearance of the hormone. The same
infirsion rate would then produce higher plasma concentrations in RRM
compared to intact rats. Measurements of the plasma concentration of
immunoreactive ANG 11, however, revealed similar levels in intact and RRM

throughout the ANG II infusion period. (Figure 22). Thus, RRM rats

139

developed hypertension at lower circulating levels of the hormone, which are
not chronically pressor in intact rats.

Rats with a sufficient impairment in renal firnction (ie. BUN > 40
mg%) are hypertensive even while on a normal salt intake (Hostetter et al.
1981, Hallet al. 1985 and unpublished observations). It is possible then that
the angiotensin infusion caused a firrther deterioration in renal function in
RRM rats sufficient to produce accelerated renoprival hypertension even
while the rats were maintained on a normal salt intake since some
investigators have shown that ANG II infusion causes renal damage (Johnson
et 01.1992). This is not supported by our measurements of BUN in RRM rats
receiving ANG 11 since BUN did not increase. Further, the observation that
neither water intake nor urine output increased during the development of
hypertension lends additional support. However, it is possible that more
sensitive measures of renal function might have detected subtle ANG II-
induced changes that contributed to the increased sensitivity to the chronic
pressor actions of the peptide.

One such change could be altered proximal tubular sodium
reabsorption. Previous studies demonstrated that ANG II acutely stimulates
Na reabsorption and decreases urinary sodium excretion in both normal

(Navar et al. 1991) and remnant (Pelayo et al. 1990) rat kidneys. Previous

140

chronic experiments using this dose of ANG II in intact rats have not shown
sodium retention (Pawloski 1990), yet the remnant kidneys of RRM rats
could respond to chronic infusion of ANG II in a quantitatively different
fashion than intact kidneys. An ANG II-induced decrease in urinary sodium
excretion in RRM rats could then have produced a ”volume-dependent”
hypertension (Hall 1986, Hall et a1. 1984). In the current experiments,
however, neither intact nor RRM rats exhibited significant sodium retention
during the ANG II infusion period. Water balance also was unchanged by
the ANG II infusion in both groups of rats. Therefore, no evidence was
found for disparate effects of ANG II on renal sodium and water handling
between normal and RRM rats under the conditions of this experiment.

A quite different explanation for the increased chronic pressor
responsiveness to ANG II in RRM rats would be an increased sensitivity of a
non-renal target tissue for ANG 11. Although many such target tissues are
known, previous experiments fiom this laboratory suggest that the brain plays
a particularly important role in the rat model of chronic ANG II-induced
hypertension utilized here. As discussed earlier, ablation of the area postrema
prevented sustained hypertension in rats receiving chronic intravenous
infusions of ANG II (Fink et al. 1981, Matsukawa and Ried 1990).

Physiological changes resulting from a chronic loss of functioning nephrons

 

141

in RRM rats might increase the sensitivity of this brainstem cardiovascular
control system to ANG 11. One such physiological change could be
extracellular fluid volume expansion, since it is well established that a high
salt intake increases both extracellular fluid volume and the chronic pressor
response to ANG II (Krieger er 01.1989, Ando et al. 1990, Cowley and
McCaa 1976). High salt intake also potentiates the chronic pressor response
to ANG II delivered selectively into the brain via the ventricles (Bruner et al.
1985). Further, acute activation of the area postrema-dependent pathway with
intravascular infusions of ANG II in dogs was prevented by placing the
animals on a low salt intake (Szilagyi and Ferrario 1987). The importance of
body fluid volume expansion to the chronic pressor effect of Ang II was
convincingly demonstrated in a recent study by Krieger and Cowley. When
extracellular fluid accumulation was prevented in dogs on a high salt intake,
the chronic hypertensive response to ANG II infusion was completely
inhibited (Krieger and Cowley 1990). In the current study and in previous
investigations, the acute pressor response to ANG II infirsion was identical in
both the intact and the RRM rats but the sustained hypertension, the area
postrema dependent phase (Szilagyi and Ferrario 1987), was seen only in the
RRM rats. This supports the hypothesis that renal reduction sensitizes rats to

the slow pressor phase of ANG II-induced hypertension.

 

142

Previous studies have shown that RRM rats have significantly
increased extracellular fluid volumes (Ylitalo and Gross 1979), even while on
normal salt intake. It is probable that volume expansion would be greater in
rats with more severe impairment of renal function (higher BUN values)
although extracellular fluid volume was not measured in the current
experiments. Accordingly, a positive correlation should exist between BUN
and the MAP response to ANG 11. Such a positive correlation was found
here, although the correlation did not reach statistical significance. We
hypothesize, therefore, that extracellular fluid volume expansion in RRM rats
on a normal salt intake led to the increased chronic pressor responsiveness to
ANG 11 observed in these animals. This enhanced responsiveness may reflect
increased sensitivity of the brainstem cardiovascular control system activated
by blood-borne ANG II in RRM rats--but this remains to be proven. This
hypothesis can be extended to the pathophysiology of salt-induced RRM
hypertension. Increased extracellular fluid volume may increase the
responsiveness to the chronic pressor effect of ANG 11 so that normal or even
suppressed plasma concentrations of ANG 11 would be adequate to induce
hypertension. The ability of angiotensin converting enzyme inhibitors and
angiotensin antagonists to prevent hypertension development in RRM rats

could be due then to suppression of circulating ANG 11 concentrations and

143

interruption of the activity of this brainstem system. Furthermore, this
sensitivity increases with increased volume-expansion so that acute ANG II
antagonism should become more effective at reversing RRM-salt hypertension

with increased exposure to high salt, as seen in the acute losartan challenge.

 

144
V. Summary

Following is a restatement of the main hypotheses tested in this
dissertation and a brief summary of the results pertaining to each.
Hypothesis 1: Aldosterone released by elevated plasma levels of angiotensin
II contributes to angiotensin II induced hypertension. Aldosterone infusion

elevates plasma levels of aldosterone (PAC) and MAP in a dose-dependent

 

manner so that a 3-fold elevation in PAC caused a significant elevation in
blood pressure during high salt intake. Infusion of ANG II acutely elevates
plasma levels of aldosterone to hypertensive levels and elevates MAP in a
dose-dependent relationship. However, infusion of ANG II for 2 to 28 days
at the rate of 10 ng/min does not cause a sustained elevation in PAC in rats
on either high sodium intake or normal sodium intake. Therefore, the
elevated MAP during the ANG II infirsion combined with high sodium intake
was not dependent on elevated PAC. Furthermore, infusion of ANG II at
either sodium intake does not alter sodium or water balance or cause changes
in plasma electrolyte concentrations such as would be expected during PAC

CXCCSS.

Hypothesis 2: Elevations in plasma aldosterone concentration during high

145

salt intake contributes to reduced renal mass-salt hypertension. When RRM
rats with an average BUN > 30 mg % (5/6 reduction in total renal mass) are
switched from normal salt intake to high salt intake they develop a significant
elevation in MAP within 24 hours. This increase in MAP is accompanied by
a reduction in PAC, identical to the reduction seen in intact rats during the
same alteration in sodium intake. Additionally, the alteration in sodium
intake did not produce different alterations in sodium balance, water balance,
plasma electrolytes or plasma osmolality between intact and RM rats. BUN
in the RRM rats increased slightly during high salt intake and was

consistently higher than in intact rats.

Hypothesis 3: The initiation of salt-induced hypertension in reduced renal
mass rats is dependent on circulating angiotensin II. RRM rats switched
from normal sodium to high sodium intake developed the expected increase
in MAP while RRM rats treated with losartan throughout the high sodium
period did not have a significant increase in MAP. Elevation of sodium
intake did not increase MAP in intact control rats or in intact rats treated with
losartan. Furthermore, neither losartan treatment nor renal reduction affected
the time required to achieve sodium and water balance following the change

from normal to high sodium intake. Losartan did prevent a sodium-induced

146

increase in BUN in the losartan treated RRM rats. Acute losartan treatment
in hypertensive RRM rats on high sodium intake did not lower MAP after 5
days on high salt but did cause a small decrease after 10 days of high sodium
treatment. The decrease was not significant until 2 hours after losartan
challenge. The acute response to ANG II infusion at 10, 30 and 60 ng/min
was not different between RRM and intact rats and was not affected by
sodium intake. Losartan completely abolished the response to ANG II

infusion.

Hypothesis 4: Reduced renal mass rats are more sensitive to the
hypertensive effects of circulating ANG 11 than intact rats. Chronic infusion
of 10 ng/min ANG 11 into normotensive RRM rats on normal sodium intake
caused a significant and sustained increase in MAP compared to either intact
rats receiving the same dose ANG II or with control RRM rats (no ANG II).
Infusion of ANG 11 into intact rats on normal sodium intake caused an initial
increase in MAP but MAP returned to control levels during the last 6 days of
the 10 day infusion while MAP remained elevated throughout the infusion in
RRM rats. The hypertension in the RRM rats was not accompanied by
changes in water or sodium balance and MAP returned to control levels

during the recovery period.

 

147

V1. Conclusions

The results from these experiments help to answer two different
questions. The first is the main one addressed by this dissertation. Namely,
what are the mechanisms which initiate and maintain the salt-induced
hypertension in RRM rats? The second is the more general question of the
mechanism of ANG II-induced hypertension.

Work by other investigators has established that hypertension in RRM
rats is dependent on elevated TPR and not on increased CO since changes in
MAP parallel the changes TPR but not CO (Lombard et al. 1989). The
observation in our study that exaggerated sodium retention does not
accompany the initiation of RRM-salt hypertension confirms that RRM
hypertension is not associated with volume expansion.

Secondly, earlier investigations have also found evidence to suggest
that the elevated TPR in RRM appears to be dependent on either increased
activity of the SNS as evidenced by the elevated plasma levels of
catecholamines or on increased response of VSMC to sympathetic
stimulation. The partial depolarization of vascular smooth muscle membranes
recorded in hypertensive RRM rats and the elevated constriction of VSMC to
both NE and electrical stimulation in the presence of plasma from

hypertensive RRM rats further suggests that a humoral factor caused the

 

148
depolarization of the VSMC. While our studies do not address the role of the

SNS in RRM hypertension, activation of the SNS during hypertension in
RRM is consistent with the results in our studies

From our studies, it is concluded that the initiation of RRM-salt
hypertension is dependent on the activation of ANG II AT, receptors,
confirming the earlier findings with CEI. The location of the receptors
responsible for this action is not known. Our results suggest that vascular
AT, receptors are not involved as evidenced by the time course of the acute
antihypertensive effect of losartan in RRM-salt rats and by the acute response
to an ANG II bolus.

Furthermore, we established that normotensive RRM rats on normal
sodium intake have an increased MAP response to circulating ANG II
chronically but not acutely. This suggests that the slow pressor response to
ANG II is heightened in RRM rats. Together with the evidence that
increased sodium intake is associated with an apparent increased response of
the SNS, we hypothesize that ANG 11 may act to augment SNA.

Finally, we can conclude that the elevated PAC observed by other
investigators in RRM rats appears to be a result of electrolyte imbalances
induced by loss of renal mass, and does not cause the hypertension.

On the more general question of ANG II-dependent hypertension, we

 

149

conclude from the first experiment that elevated PAC does not play a role in
the hypertension caused by chronic infusion of ANG II. The increased
response in RRM rats to circulating ANG 11 may also indicate the mechanism
elevating MAP. First, it has been demonstrated that sodium-loading, which
predisposes to ANG II hypertension in intact rats, expands sodium space F

(Ando et 01.1990, Krieger and Cowley 1990) and stimulates cardiopulmonary :,

 

baroreceptors (Andresen et al. 1989). RRM should also stimulate

‘MJ! - '_.

cardiopulmonary baroreceptors by expanding body fluid volume

Secondly, desensitization of the cardiopulmonary baroreceptor reflex
has been implicated in the slow presser effect of ANG II (Matsukawa and
Ried 1990) through the action of ANG II on area postrema (AP) neurons.
The AP is necessary for the development of chronic ANG II hypertension
(Fink er al. 1987), and lesion of the AP prevents ANG II modulation of the
baroreflex control of heart rate (Matsukawa and Ried 1990).
Electrophysiological connections from the AP to the parabrachial nucleus and
the NTS, two regions which appear to control baroreflex responses (Cechetto
and Calaresu 1983, Hamilton and Ellenberger 1981) have been established
(Shapiro and Miselis 1985, Papas and Ferguson 1990). The AP also receives
direct inputs from both the carotid sinus and aortic depressor nerves (Ciriello

et al. 1981, Davies and Kalia 1981, Wallach and Loewy 1980). Finally,

150

microinjection of ANG 11 into the AP selectively stimulates neurons that also
respond to changes in blood pressure (Papas et al. 1990). These studies
suggest that ANG II actions at the AP may be able to alter the baroreflex
response to changes in body fluid volume produced by high salt intake. This
inhibition of the cardiopulmonary baroreflex may be the common mechanism
underlying the increased sensitivity to ANG II in salt loading and RRM

hypertension.

 

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