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THE ROLE OF ANGIOTEN SIN H AND ENDOTHELIN-l IN THE HYPERTENSION
OF EXPERIMENTAL CHRONIC RENAL FAILURE

By

Gregg Steven Potter

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

'1997

ABSTRACT

THE ROLE OF ANGIOTENSIN H AND ENDOTHELIN-l IN THE HYPERTENSION
OF EXPERIMENTAL CHRONIC RENAL FAILURE

By

Gregg Steven Potter

The primary purpose of the experiments described here was to investigate the role
of humoral factors speciﬁcally, angiotensin II (AngII) and endothelin-1 (ET-1), in the
pathogenesis of hypertension in the reduced renal mass (RRM) model of chronic renal
failure (CRF). I hypothesized that the relative contribution of these two hormones to both
short-term and long-term BP regulation in CRF differs depending on the level of salt
intake. The work is highly relevant to the treatment of human CRF, which currently
entails both regulation of dietary salt and aggressive drug therapy aimed at controlling
arterial pressure and thus slowing progressive deterioration of renal function.

My experimental approach was designed to study the mechanism(s) of
hypertension associated with CRF using the RRM animal model. I examined the effects
of acute and chronic treatment in RRM rats with speciﬁc pharmacological inhibitors of
the renin angiotensin and endothelin systems.

The results of my work demonstrate that both AngII and ET-l play important
roles in the maintenance of RM hypertension and their relative contribution depends on
the level of salt intake. Under conditions of low salt intake, inhibition of AngII formation
was shown to lower blood pressure to the greatest extent, while during high salt intake

endothelin system blockade proved to be most beneficial.

ACKNOWLEDGMENTS

Attaining a Ph.D. in Pharmacology was the most challenging undertaking of my
life thus far. The experience has profoundly changed my life (hopefully for the better). I
must thank many of those who helped me along the way.

First and foremost I want to thank my soulmate Alison for all the encouragement,
advise, and love we shared throughout these years. Your strength, determination, and
conﬁdence helped me immensely during these challenging years. I know now that we can
overcome any obstacle that comes our way. Austin and Mackenzie deserve my thanks
because they taught me to become more disciplined and focused in my work. They are
truly the source of my inspiration and they give daily meaning to my efforts of drug
discovery and cure of disease. I promise to teach you both to the best of my ability and
instill in you what is really important in life as you have taught me.

Dr. Hugh Yee and Dr. William Smith were instrumental in my decision to pursue
graduate studies and my sincere thanks are extended to both. Hugh gave me the
motivation and drive to “go back” and Bill gave me my ﬁrst shot at research.

My biggest supporter and true friend is Greg Fink. Thanks for all the help along
the way. When I stumbled, you picked me up; when I bragged, you shot me down.
Working in your lab with Matt, Renee, John, Vyvi, Robin, Jim, Dawne, Sabrina, and Ron

was a roller coaster ride with the highest of highs.

iii

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................... viii
LIST OF FIGURES ........................................................................................................ ix
LIST OF ABBREVIATIONS ....................................................................................... xiii
INTRODUCTION ............................................................................................................ 1
1. Hypertension ...................................................................................................... 1

A. Prevalence ................................................................................................... l

B. Types of hypertension ........... . ...................................................................... 1

1. Primary hypertension ........................................................................... 2

2. Secondary hypertension ....... - ................................................................ 2

H. Chronic renal failure .......................................................................................... 2

A. Background ................................................................................................. 2

B. Experimental chronic renal failure ............................................................. 3

1. Physical and metabolic changes in RRM ............................................ 4

2. Mechanisms of experimental chronic renal failure ............................. 5

3. Factors affecting the progression of renal lesions ............................... 6

C. Chronic Renal Failure and Hypertension .................................................... 8

1. Background .......................................................................................... 8

2 Hypertension development in experimental CRF ............................... 9

3. Sodium status .................................................................................... 10

4 Sympathetic nervous system activity ................................................. 11

5. Hormonal factors ............................................................................... 12

III. Renin-angiotensin system ................................................................................ 15

A. Hormonal renin angiotensin system ......................................................... 15

1. Synthesis-cascade .............................................................................. 15

2. Physiological actions ......................................................................... 17

B. Tissue renin angiotensin system ............................................................... 21

C. Inhibition of the renin angiotensin system ................................................ 22

l. Angiotensin converting enzyme actions ............................................ 22

D. Renin angiotensin system and hypertension ............................................. 24

1. Background ........................................................................................ 24

2. AngII induced hypertension ............................................................... 25

3. AngII involvement in other forms of hypertension ........................... 27

iv

E. Renin angiotensin system and chronic renal failure ................................. 29

l. Renin angiotensin system activity in chronic renal failure ................ 29

2. Other antihypertensive regimens ....................................................... 29

3. Renin angiotensin system activity in RRM model ............................ 31

IV. Endothelin system ............................................................................................ 35
A. Background ............................................................................................... 35

B. Physiological actions of endothelin .......................................................... 37

1. Hemodynamic actions ....................................................................... 37

2. Heart .................................................................................................. 38

3. Central nervous system ...................................................................... 38

4. Endocrine systems ............................................................................. 39

5. Kidney ............................................................................................... 40

C. Inhibition of endothelin system ................................................................ 41

1. Receptor agonists ............................................................................... 42

2. Endothelin converting enzyme inhibitors .......................................... 42

3. Endothelin receptor antagonists ........................................................ 42

D. Endothelin and hypertension .................................................................... 43

1. Background ........................................................................................ 43

2. Endothelin and sodium intake ........................................................... 45

3. ET-l induced hypertension ................................................................ 46

4. Endothelin involvement in experimental forms of hypertension ...... 46

5. Endothelin involvement 1n human hypertension ............................... 49

E. Endothelin and chronic renal failure ......................................................... 49

l. Endothelin activity in chronic renal failure ....................................... 49

2. Endothelin activity in RRM model .................................................... 49

V. Blood pressure regulation ................................................................................ 52
VI. Speciﬁc aims .................................................................................................... 54

METHODS

1. Animals ........................................................................................................... 55
11. Surgical procedures .......................................................................................... 55
A. General ...................................................................................................... 55

B. Reduction of renal mass ............................................................................ 56

C. Arterial and venous catheterization ......................................................... 57

III. Chronic rat model ............................................................................................ 57
IV. Hemodynamic measurements .......................................................................... 58
V. Fluid and electrolyte measurements ................................................................. 58
VI. Salt protocols ................................................................................................... 59
VII. Assays ........................................................................................................... 60
A. Plasma assays ............................................................................................ 6O

1. Blood urea nitrogen ........................................................................... 6O

2. Serum creatinine ................................................................................ 60

3. Endothelin ......................................................................................... 61

B. Urine assays .............................................................................................. 61

1. Urinary protein excretion ................................................................... 62
2. Urinary creatinine concentration ....................................................... 62
3. Creatinine clearance .......................................................................... 62
VIII. Statistics ........................................................................................................... 62
EXPERIMENTAL RESULTS ....................................................................................... 63
I. Renin angiotensin system in reduced renal mass
A. Acute experiments .................................................................................... 63
l. Bolus i.v. administration of ACEI in RRM and sham
rats on high, normal, and low salt intakes ......................................... 63
B. Chronic experiments ................................................................................. 70
1. Chronic administration of ACEI in hypertensive RRM
rats on a high salt intake .................................................................... 70
2. Chronic administration of ACEI in hypertensive RRM
and sham rats on a normal salt intake ................................................ 82
3. Chronic administration of ACEI in RRM and sham rats
on a low salt intake .......................................................................... 105
H. Endothelin system and reduced renal mass .................................................... 122
A. Acute experiments .................................................................................. 122

1. Acute i.v. administration of an ETA (PD147953)

or an ETA/ET}; (PD145065) receptor antagonist

in ET-l induced hypertension .......................................................... 122
2. Acute i.v. administration of an ETA (PD147953)

or an ETA/ET}; (PD145065) receptor antagonist

in hypertensive RRM and sham rats on a high salt

intake ............................................................................................... 126
3. Comparison of acute i.v. administration of an

ETA (PD147953) or an ETA/ETB (PD145065)

receptor antagonist, in RRM and sham rats on

high, normal, and low salt intakes ................................................... 133

B. Chronic experiments ............................................................................... 141

1. Oral ETA (PD155080) receptor antagonist treatment

in the hypertension induced by continuous i.v.

ET-l infusion ................................................................................... 141
2. Oral ETA (PD155080) receptor antagonist treatment

in RRM rats on high, normal and low salt

intakes .............................................................................................. 146
3. Oral ETA (PD155080) receptor antagonist treatment

in established hypertensive RRM rats on a normal

salt intake ......................................................................................... 153

vi

DISCUSSION .................................................................................... 168

I. Comparison of responses to alterations in salt intake in RRM and sham
rats prior to administration of inhibitors of the RAS and ET ................... 169
A. Blood pressure ........................................................................................ 169
1. Reduced renal mass ......................................................................... 169
2. Sham ................................................................................................ 170
B. Blood urea nitrogen ................................................................................ 171
C. Water intake and urine output ................................................................. 172
1. Reduced renal mass ......................................................................... 172
2. Sham ................................................................................................ 173
D. Mortality ................................................................................................. 173
H. Inﬂuence of ACEI under varying levels of salt intake ................................... 174
A. Acute ....................................................................................................... 174
B. Chronic ................................................................................................... 175
1. Support for enalapril dose used in experiments .............................. 175
2. Reduced renal mass ......................................................................... 177
3. Sham ................................................................................................ 180
III. Mechanism of action of ACEI ....................................................................... 180
IV. Mechanism of action of AngII in RRM hypertension .................................... 182
A. Role of sodium excretion ........................................................................ 182
B. Increased responsiveness in RRM .......................................................... 185
V. Inﬂuence of endothelin under varying levels of salt intake ........................... 189
A. Acute role of endothelin ......................................................................... 189
B. Chronic role of endothelin ...................................................................... 190
1. Support for PD155080 dose used in experiments ........................... 190
2. Reduced renal mass ......................................................................... 190
3. Sham ................................................................................................ 191
VI. Mechanism of action of endothelin in RRM hypertension ............................ 192
VII. Therapeutic implications ................................................................................ 193
BIBLIOGRAPHY ........................................................................................................ 210

vii

Table 1:

Table 2:

Table 3:

Table 4:

LIST OF TABLES

AngI pressor responses, enalapril dosages and blood urea nitrogen
levels in RRM rats maintained on a high salt intake .............................. 81

AngI pressor responses, enalapril dosages and blood urea nitrogen
levels in sham and RRM rats maintained on a normal salt intake ......... 96

AngI pressor responses, enalapril dosages and blood urea nitrogen
levels in sham and RRM rats maintained on a low salt intake ............. 115

Effects of PD155080 on renal parameters, ET-l plasma

concentrations, and PD155080 plasma concentrations in sham
and RRM rats maintained on a normal salt intake ............................... 167

viii

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Figure 10:

Figure 1 1:

LIST OF FIGURES

Renin angiotensin synthesis cascade ..................................................... l6

Acute i.v. enalaprilat administration in RRM and sham rats
on high, normal, and low salt intakes... ................................................. 67

AngI pressor responses in normal rats administered i.v. dextrose
or enalaprilat at 1 mg/kg ......................................................................... 69

Mean arterial pressure responses to chronic enalapril administration
in RRM rats on high salt intakes ............................................................ 76

Water intakes, urine outputs and water balances in response to
chronic enalapril administration in RRM rats on high salt intakes ........ 78

Urinary sodium excretions and sodium balances in response to
chronic enalapril administration in RRM rats on high salt intakes ........ 80

Mean arterial pressure responses to chronic enalapril administration
with and without replacement AngII in RRM rats on normal salt
intakes ..................................................................................................... 90

Mean arterial pressure responses to chronic enalapril administration
with and without replacement AngII in sham rats on normal salt
intakes ..................................................................................................... 92

Mean arterial pressure responses to chronic enalapril administration
with and without replacement AngII in sham rats on normal
salt intakes. All groups in one graph .................................................... 94

Water intakes, urine outputs and water balances in response
to chronic enalapril administration with and without replacement
AngII in RRM rats on normal salt intakes .............................................. 98

Water intakes, urine outputs and water balances in response

to chronic enalapril administration with and without replacement
AngII in sham rats on normal salt intakes ............................................ 100

ix

Figure 12:

Figure 13:

Figure 14:

Figure 15:

Figure 16:

Figure 17:

Figure 18:

Figure 19:

Figure 20:

Figure 21:

Figure 22:

Urinary sodium excretions in response to chronic enalapril
administration with and without replacement AngII In RRM
rats on normal salt intakes .................................................................... 102

Urinary sodium excretions in response to chronic enalapril
administration with and without replacement AngII in sham
rats on normal salt intakes .................................................................... 104

Mean arterial pressure responses to chronic enalapril administration
with and without replacement AngII in RRM rats on low salt
intakes ................................................................................................... 1 11

Mean arterial pressure responses to chronic enalapril administration
with and without replacement AngII in sham rats on low salt
intakes ................................................................................................... 113

Water intakes, urine outputs and water balances in response to
chronic enalapril administration with and without replacement
AngII in RRM rats on low salt intakes ................................................. 117

Water intakes, urine outputs and water balances in response to
chronic enalapril administration with and without replacement
AngII in sham rats on low salt intakes ................................................. 119

Urinary sodium excretions in RRM and sham rats administered
enalapril with and without replacement AngII on low salt
intake .................................................................................................... 121

Acute mean arterial pressure responses to ETA (PD147953)
and ETA/ET]; (PD145065) receptor antagonist infusion in
ET-l induced hypertension .................................................................. 125

Acute mean arterial pressure responses to ETA (PD147953)
receptor antagonist infusions in RRM and sham rats on high salt
intakes ................................................................................................... 130

Acute mean arterial pressure responses to ETA/ETB (PD145065)
receptor antagonist infusions in RRM and sham rats on
high salt intakes .................................................................................... 132

Acute mean arterial pressure responses to ETA (PD147953)
and ETA/ETB (PD145065) receptor antagonist infusions in
RRM and sham rats on high salt intakes .............................................. 136

Figure 23:

Figure 24:

Figure 25:

Figure 26:

Figure 27:

Figure 28:

Figure 29:

Figure 30:

Figure 31:

Figure 32:

Figure 33:

Acute mean arterial pressure responses to ETA (PD147953)
and ETA/ET]; (PD145065) receptor antagonist infusions in
RRM and sham rats on normal salt intakes .......................................... 138

Acute mean arterial pressure responses to ETA (PD147953)
and ETA/ET}; (PD145065) receptor antagonist infusions in
RRM and sham rats on low salt intakes ............................................... 140

Chronic mean arterial pressure responses to ETA (PD155080,
25 mg/kg/b.i.d.) receptor antagonist administration in ET-l
induced hypertension ............................................................................ 145

Chronic mean arterial pressure responses to ETA (PD155080,
100 mg/kg/b.i.d.) receptor antagonist administration in RRM rats
on high and normal salt intakes ............................................................ 150

Chronic mean arterial pressure responses to ETA (PD155080,
25 mg/kg/b.i.d.) receptor antagonist administration in RRM rats
on low salt intakes ................................................................................ 152

Chronic mean arterial pressure responses to ETA (PD155080,
25 mg/kg/b.i.d.) receptor antagonist administration in RRM
and sham rats on normal salt intakes .................................................... 159

Acute pressor and depressor responses to bolus i.v. ET-l

(0.5 nmol/kg) injections in sham and RRM rats on normal salt

intakes during control, treatment and recovery experimental

periods .................................................................................................. 161

Water intakes, urine outputs and water balances in response to
chronic ETA (PD155080, 25 mg/kg/b.i.d.) receptor antagonist
administration in sham and RRM rats on normal salt intakes .............. 163

Urinary sodium excretions in response to chronic ETA (PD155080,
25 mg/kg/b.i.d.) receptor antagonist administration in sham
and RM rats on normal salt intakes ................................................... 165

Mean arterial pressure measured in RRM rats on high, normal
and low salt intakes .............................................................................. 197

Relative inﬂuence on blood pressure of the fast pressor effect
of AngII in normal and RRM rats under varying salt intakes .............. 199

xi

Figure 34:

Figure 35:

Figure 36:

Figure 37:

Figure 38:

Mean arterial pressure responses to chronic low-dose enalapril
administration in sham and RRM rats on normal salt intakes .............. 201

Mean arterial pressure responses to losartan i.v. bolus
during chronic enalapril administration in normal rats
on normal salt intakes ........................................................................... 203

Mean arterial pressure responses to chronic enalapril
administration in sham and RRM rats on normal salt intakes .............. 205

Relative inﬂuence on blood pressure of the slow
pressor effect of AngII in normal and RRM rats under
varying salt intakes ............................................................................... 207

Relative inﬂuence on blood pressure of endothelin in
normal and RRM rats under varying salt intakes ................................. 209

xii

LIST OF ABBREVIATIONS

ACE .......................... angiotensin converting enzyme

ACEI ........................ angiotensin converting enzyme inhibitor
AngI ........................... angiotensin I

AngH ........................ angiotensin H

ANOVA .................... analysis of variance

ANP ......................... atrial natriuretic peptide

ATIRA ..................... angiotensin H type 1 receptor antagonist
AVP ......................... arginine vasopressin

BP ............................ blood pressure

BUN ........................ blood urea nitrogen

Ccr ............................. creatinine clearance

CNS ........................... central nervous system

CO ........................... cardiac output

CRF .......................... chronic renal failure

CVD .......................... cardiovascular disease

DOCA ...................... deoxycorticosterone acetate

ESRD ....................... end-stage renal disease

ET .............................. endothelin isoform 1

ET-l ......................... endothelin isoform 1

ETRA ...................... endothelin receptor antagonist

ETARA ..................... endothelin subtype A receptor antagonist
ETA/ETBRA ............. endothelin subtype A and B receptor antagonist
GFR ......................... glomerular ﬁltration rate

HR ........................... heart rate

i.m .............................. intramuscular

i.p. .............................. intraperitoneal

i.v ............................... intravenous

MAP ........................ mean arterial pressure

Na+lK+-ATPase ....... sodium potassium adenosine triphosphate enzyme
NO ............................. nitric oxide

OLF ......................... ouabain-like factor

1K1C ......................... one-kidney, one clip

PRA ......................... plasma renin activity

RAS ......................... renin angiotensin system

RRM ........................ reduced renal mass

RVR ........................... renal vascular resistance

xiii

SBP .......................... systolic blood pressure

s.c ............................... subcutaneous

Scr .............................. serum creatinine

SEM ........................... standard error of the mean

SHR ......................... spontaneously hypertensive rat
SNA ......................... sympathetic nervous system activity
SNGFR .................... single nephron glomerular ﬁltration rate
SNS ......................... sympathetic nervous system

SPE ............................ slow pressor effect of AngH

TPR ......................... total peripheral resistance

2KlC ......................... two-kidney, one clip

UNaV ........................ urinary sodium excretion

UO urme output

Upro ........................... urinary protein excretion

VSMC ..................... vascular smooth muscle cell

WB ............................ water balance

WI ............................ water intake

xiv

INTRODUCTION

1. Hypertension

A. Prevalence

In Westemized countries, cardiovascular disease (CVD) is the leading cause of
death. In the United States, close to 1 million people (43% of all deaths) die from CVD
each year (Whelton et al., 1995). These cardiovascular diseases include coronary artery
disease, congestive heart failure, hypertension, stroke, etc. Hypertension, or high blood
pressure, is the major modiﬁable risk factor for CVD (Burt et al., 1995). Nearly one-
quarter of Americans have hypertension and the prevalence is especially high (> 54%) in
people 60 years and older (Burt et al., 1995). Hypertension is currently considered to be a
sustained diastolic blood pressure greater than 90 mmHg, or a sustained systolic pressure
above 140 mmHg. Hypertension is the most critical risk factor for stroke and is
considered to play a major role in the pathogenesis of many other diseases. Current
pharmacological treatment of hypertension has been shown to decrease the risk of CVD.
Still, the epidemic of blood pressure related CVD compels the research community to
search for a better understanding of the mechanisms involved in hypertension.

B. Types of hypertension

The exact mechanism of most forms of hypertension is unknown. This is
complicated by the probability that hypertension is a multifactorial disease involving
many physiological disturbances. Hypertensive individuals are generally classiﬁed as

1

2

having either primary (essential) or secondary (non-essential) hypertension, depending on
whether or not a speciﬁc cause or mechanism can be determined.
1. Primary hypertension

The majority of hypertensive patients (80-90%) are classiﬁed as suffering from
primary hypertension. There is no known single cause of this disease but a variety of
pathologic factors have been implicated: increased sympathetic nervous system or renin
angiotensin system activity, abnormal insulin sensitivity, decreased renal function,
alterations in vascular structure, abnormal lipid metabolism, genetic predisposition, etc.
(Hunt and Williams, 1994; Julius, 1994). '

2. Secondary hypertension

The prevalence of secondary hypertension is thought to vary between 10—20%, and
it is classiﬁed as such when a single cause can be identiﬁed. Some major causes of
secondary hypertension that have been identiﬁed include: mineralocorticoid excess
(Gordon et al., 1994), renal artery stenosis (Aristozabal and Frohlich, 1993) and chronic
renal failure (Weidmann and Beretta-Piccoli, 1983).
H. Chronic Renal Failure

A. Background

An accelerated, progressive decline in renal function over time is termed chronic
renal failure (CRF). CRF is not a disease entity in itself, but rather a clinical condition
resulting from a number of pathologic processes that can lead to derangement and
insufﬁciency of renal excretory and regulatory function. After diabetes (33%),
hypertension (28%) is the second leading cause of CRF (Whelton et al., 1992). End-stage

renal disease (ESRD) results from decades of CRF. The prevalence, morbidity, and

3
mortality of ESRD is increasing in the last 30 years, especially due to the aging

population. In 1991 , the Federal ESRD program, which accounted for 93% of all renal
disease patients, spent $6.6 billion on approximately 165,000 enrollees. The enrollment
is predicted to reach 250,000 patients by the year 2000 (Eggers et al., 1989). The average
annual cost of one year's therapy for each patient was $29,000. Dialysis and/or renal
transplantation are usually the therapeutic endpoints for the continual loss in renal
function.

It is generally observed that once a signiﬁcant decline is renal function has been
initiated, regardless of the original insult to the kidney, there results a progressive
deterioration in function that ultimately leads to total kidney failure. A major concern is
that there is a long “silent period” from the initiation of kidney damage until the
appearance of clinical, biochemical, or laboratory markers of the disease.

B. Experimental chronic renal failure

A variety of experimental animal models have been utilized to simulate the
deterioration of renal function observed in CRF. The fawn-hooded rat is a genetic model
of spontaneous glomerulosclerosis. In these rats, systemic hypertension develops along
with glomerular hypertension leading to a decline in renal function (Simons et al., 1991).
Experimental models of diabetic nephropathy are of great interest because approximately
30% of patients with insulin-dependent diabetes mellitus develop renal failure and require
dialysis, kidney transplantation or ultimately die. Experimental diabetic nephropathy is
commonly induced by subtotal reductions in kidney mass (25%) and administration of

streptozotocin (Chen et al., 1992).

4
The most prominent model of CRF, the reduced renal mass (RRM) model, has

been mainly employed in rats and dogs. It has been demonstrated experimentally and is
also observed clinically that reductions in total kidney mass need to exceed 50% to
induce renal insufﬁciency. The RRM model of CRF has been accomplished
experimentally by two methods that are not equivalent. The excision method of RRM
involves the partial surgical excision of both poles of one kidney and the removal of the
contralateral kidney. This procedure effectively results in 66-83% ablation of total renal
mass. The remnant renal tissue undergoes functional and morphological adaptation so as
to maintain excretory function. Typically these RRM rats remain healthy for months
except when the animals are placed on a high sodium intake, where hypertension rapidly
develops and renal deterioration ensues (Ylitalo, 1976). The ligation method of RRM
involves ligating 2 of the 3 renal arteries followed by contralateral nephrectomy. The
ligation method is an inappropriate model of most clinical CRF because hypertension
quickly develops (days to weeks). It is thought that the ligation method can induce
pockets of ischemia leading to increased renin release; these abnormalities do not reﬂect
the clinical course generally observed in human CRF (Meyer and Rennke, 1988).
1. Physical and metabolic changes in RRM

Early on the rats appear healthy and normal upon gross examination. Polydypsia
and polyuria are early indicators of loss of renal concentrating ability. With continued
deterioration of kidney function, uremic toxins accumulate in the blood, and the whole
animal shifts into a catabolic state. In the ﬁnal stages of RRM, there is weight loss due
to muscle wasting, accompanied by edema and blood volume expansion due to decreased

ﬂuid excretory capacity and loss of plasma proteins.

5

Renal and cardiovascular abnormalities induced in the RRM model mimic the
changes observed with deterioration in renal function over time in CRF. These include,
but are not limited to: progressive rises in serum creatinine (Scr); blood urea nitrogen
(BUN); and urinary protein excretion (Upro). Also observed are a decline in glomerular
ﬁltration rate (GFR), creatinine clearance (Ccr), and hematocrit. These laboratory indices
are commonly monitored to assess renal function in both humans and animals.

2. Mechanisms of experimental chronic renal failure
a. Hemodynamic factors

CRF and RRM result in the permanent loss of nephrons. The decreased excretory
function results in declining GFR and leads to body ﬂuid volume excess. Structural and
functional adaptations in the surviving nephrons produce hyperﬁltration as partial
compensation (Hostetter et al., 1981). This adaptive increase in single nephron
glomerular ﬁltration rate (SNGFR) maintains total kidney GFR in the short-term. These
same adaptations also, however, contribute to the development of further glomerular
injury. Most investigators in the ﬁeld agree with the experimental evidence of Brenner
and colleagues indicating that increased intraglomerular pressure is mainly responsible
for the progressive injury to the remaining nephrons (Brenner et al., 1985). Dietary
protein restriction has been reported to preserve renal morphology and slow the
deterioration of renal function in various models of experimental renal disease (e.g.
Hostetter et al., 1986; Diamond at al., 1987) and in human CRF (Klahr et al., 1994). The
beneﬁcial effects of protein restriction are thought to be associated with a reduction in
glomerular hydrostatic pressure and a blunting of compensatory renal growth (Nath et al.,

1986). On the other hand, urinary excretion of protein increases as renal function

6

declines, presumably as a result of functional and structural damage to the glomerular
filter.
b. Hypertrophic factors

The degree of glomerular hyperplasia and hypertrophy following RRM depends
on the amount of renal mass removed and the age of the animal, but recent studies show
that hypertrophy predominates after subtotal nephrectomy (Heeg et al., 1989).
Hypertrophic stimuli (i.e. increased sodium and protein intake, glucocorticoids, growth
factors) have been found to accelerate glomerulosclerosis and overall renal deterioration
(Norman et al., 1987). Dramatic hypertrophy of all nephron segments in the renal stump
occurs due to the structural and metabolic adaptations needed to maintain excretory
capacity. Some investigators have hypothesized that stretching of the glomerular
basement membrane due to glomerular hypertrophy and enhanced glomerular capillary
pressure is the cause of the deleterious consequences to the glomeruli of RRM
(Kleinknechtet al., 1995).

3. Factors affecting the progression of renal lesions
a. Protein intake

Increasing dietary protein in rats with RRM has been shown to enhance the
progression of renal lesions leading to decreased survival in the absence of hypertension
(Kleinknecht et al., 1995). Low protein diets (7%) have been demonstrated to protect the
remnant kidney but these regimens are at the expense of undemutrition and growth
defects (Salusky et al., 1981). Increases in protein intake are thought to activate the renin
angiotensin system (RAS) thereby contributing to hypertension and renal deterioration

(Puller et al., 1986; Rosenberg et al., 1990). Other investigators have forwarded the

7

hypothesis that calorie restriction, irrespective of whether or not protein is restricted, can
retard growth and prevent the development of end-stage pathology in the RRM model
(T app et al., 1989). Protein and calorie restriction are still being investigated in RRM
models, but the application to human CRF seems of limited benefit.
b. Sodium intake

Several authors have shown that sodium restriction has protective effects on renal
function in the RRM model (Hout et al., 1983; Koletsky et al., 1959; Lax et al., 1992)
even when no changes in BP were observed (Daniels et al., 1990).

c. Coagulation abnormalities

Early studies in RRM focused on the possibility that inhibition of blood
coagulation slows the progression of hypertension and renal deterioration (Purkerson et
al., 1976; Zoja et al., 1989). Intraglomerular thrombosis and platelet aggregation have
been suggested to play a role in glomerular dysfunction in RRM. Warfarin and heparin
administration have been shown to retard the progressive increase in hypertension and
renal deterioration following RRM (Purkerson et al., 1982; Olson, 1984). Additional
reports have shown that inhibition of platelet aggregation by the thromboxane synthesis
inhibitor, OKY 1581, prevented cardiac hypertrophy and hypertension in RRM rats
(Purkerson et al., 1984). These data support a role of platelet aggregation and glomerular
thrombosis in the pathogenesis of RRM and suggests that inhibition of blood coagulation
prevents the development of hypertension and the progression of renal failure. The
inﬂuence of coagulant system activation on hypertension and renal deterioration in RRM
is still being investigated, although the emphasis of most recent research has focused on

hemodynamic factors.

8

C. Chronic renal failure and hypertension
1. Background

There exists a close relationship between CRF and systemic hypertension. High
BP is often an initiator of renal insufﬁciency leading to ESRD. High BP also acts as a
promoter of renal damage in patients with established kidney disease, e.g. diabetic
nephropathy. All levels of untreated hypertension are associated with declining renal
function (Shulman et al., 1989), but most BP related renal disease can be attributed to
mild hypertension or even high normal BP (Whelton et al., 1992). There is a beneﬁcial
effect on renal function in CRF with BP reduction acutely and chronically (Gansevoort et
al., 1994; Lebovitz et al., 1994). For examme, Upro is usually increased in CRF, but is
stabilized or even decreased by antihypertensive drug therapy.

Conversely, impairment of renal function almost always causes some elevation in
BP, but the etiology of hypertension in CRF is complex and probably multifactorial.
Some of the potential causes of hypertension in CRF are: body ﬂuid volume excess;
increased sympathetic nervous system activity (SNA); alterations in humoral factors;
structural cardiovascular changes; or some combination of these. The most common
explanation is that a loss of functioning nephrons causes a decrease in sodium and water
excretion, which leads to increased body ﬂuid volume and elevated BP. According to
the "pressure-natriuresis" theory, elevated BP restores renal ﬂuid excretion back to

normal levels (Cowley et al., 1992).

9
2. Hypertension development in experimental CRF

a. Ligation method

BP increases immediately following renal artery ligation due to the exaggerated
secretion of renin. Within weeks there is observed a severe hypertension (SBP > 180
mmHg) that is associated with volume expansion. Production of the RRM model by the
ligation method does not produce elevations in BP that are inﬂuenced by salt intake
(Kleinknecht et al., 1995). Mortality usually ensues within 6 months after the partial
ligation due to cardiovascular disease resulting from progressive increases in BP and
uremia.

b. Excision method

Three stages of hypertension can be recognized in the excision RRM model
(Gretz et al., 1993). The ﬁrst stage is characterized by a short period (i.e. days to weeks)
of acute renal failure accompanied by sodium retention and volume expansion due to
reduction in renal excretory function. Compensatory mechanisms, such as cellular
hypertrophy and glomerular hyperﬁltration, result in a steady improvement in renal
function occurring during this phase such that increases in BP or proteinuria are not
commonly observed (Gretz et al., 1993).

This is followed by a long stable phase (weeks to months) with gradual
progression of BP, proteinuria, and other signs of renal deterioration. This gradual
increase in BP can be exacerbated by increasing NaCl intake (Langston et al., 1963).
The overall rate of increase in BP and decrease in renal function in this phase is

dependent on sodium intake, dietary protein and on the original amount of renal mass

lO

removed. Therapeutic interventions effective in slowing the onset of terminal renal
failure are usually implemented during this phase.

The ﬁnal terminal phase is characterized by gross edema and uremia, very high
elevations in BP, and a generalized somatic wasting that ultimately ends in renal failure
and death (Koletsky and Goodsitt, 1960). Effective treatment of hypertension during this
malignant phase may be totally different than in previous phases and little can be done to
slow the rapid deterioration in renal function.

3. Sodium status

The degree of hypertension in advanced renal failure is frequently related to
excessive sodium chloride ingestion. Koomans reported that BP increased during
elevated salt intake in a variety of patients with different degrees of renal insufﬁciency
(Koomans et al., 1982). The BF increase tended to be larger in the patients with a greater
loss of kidney function. In fact, this "salt-sensitivity" of BP rose exponentially with the
decline in function. It is known that salt retention with extracellular ﬂuid volume
expansion can result in a raised cardiac output (CO) and elevated total peripheral
resistance (TPR), both of which are usually increased in ESRD (Textor et al., 1981). Salt
restriction lessens the accumulation of sodium and water in CRF patients, thereby
decreasing plasma volume overload, and decreasing BP (Bakris and Gavras, 1993).
Dietary salt restriction is commonly initiated in human CRF to alleviate "volume-
dependent" hypertension. Low-salt diets have played an integral role in the treatment of

hypertension in CRF patients for over 30 years.

ll

4. Sympathetic nervous system activity

An increased sympathetic nervous system activity (SNA) may contribute to
hypertension and progressive renal deterioration in patients with CRF. The kidneys are
innervated with two main types of sensory receptors: the renal baroreceptors which
increase ﬁring in response to changes in renal perfusion pressure; and the renal
chemoreceptors which may be stimulated by ischerrric metabolites or uremic toxins
(Dibona, 1982). These receptors are linked to the sympathetic centers in the central
nervous system (CNS) through renal afferent pathways (Faber and Brody, 1985).
Converse et al.,( 1992) reported that in patients with CRF, there exists a elevated
sympathetic activity, which may be due to an afferent signal from the failing kidney.
Accordingly patients with bilateral nephrectomy had lower rates of sympathetic discharge
than CRF patients with native kidneys, and this was accompanied by lower BP’s. Many
studies have implicated functional abnormalities in the sympathetic nervous system in the
hypertension observed in RRM. Elevated plasma levels of norepinephrine have been
observed in RRM rats on a high sodium intake, and epinephrine synthesis blockade with
SK&F 64139 resulted in a fall in BP (Dipette et al., 1982). Campese and colleagues
reported preventing the progression of renal disease and hypertension by renal afferent
denervation in RRM rats (Campese et al., 1995a: Campese et al., 1995b). These studies
provide evidence that neurogenic factors may play an important role in renal deterioration

and hypertension in RRM rats.

12

5. Hormonal factors
a. Aldosterone

A variety of hormonal factors also have been implicated in the hypertension
associated with CRF. Plasma aldosterone levels are increased in humans with CRF
(Mitch and Wilcox, 1982) as well as in RRM rats drinking saline compared to sham rats
under the same conditions (Chi et al., 1986). It has been shown that elevated plasma
aldosterone levels can cause hypertension in dogs particularly under conditions of high
salt (Pan and Young, 1982). But previous work in our lab demonstrated that hypertension
observed in RRM rats (excision model) is not dependent on an elevated plasma
aldosterone concentration (Kanagy, 1991).

b. Na,K-adenosine triphosphate inhibition

Some investigators have suggested that endogenous Na,K-adenosine triphosphate
(Na,K-ATPase) inhibitors may play a role in hypertensive CRF. The Na,K-ATPase
enzyme is found ubiquitously throughout the cells of the body but is most abundant in the
kidney tubules, where it is provides the energy for active sodium reabsorption from the
glomerular ﬁltrate (Lingrel et al., 1994). Hout et al in 1983 found that hypertensive
RRM rats had decreased Na,K-ATPase pump activity in vascular smooth muscle and
cardiac cells. The cardiac glycosides (i.e. digoxin) are known to inhibit Na,K-ATPase
pump activity, produce vasoconstriction and increase cardiac contractility (Vatner et al.,
1971). It is thought that an endogenous sodium pump inhibitor exists, preliminarily
described as ouabain, and is released in response to volume expansion, which contributes
to increases in BP (deWardener, 1990). Yamada et al., in 1994 reported that an

antibody to ouabain lowered BP in hypertensive RRM rats on a high sodium intake.

'13

Hanrlyn and colleagues have reported that long term ouabain administration produces
greater degrees of BP increase in RRM rats, varying proportionately with the amount of
kidney mass removed (Yuan et al., 1993). The sustained elevation in BP observed during
ouabain administration supports the possibility that endogenous inhibitors of the Na,K-
ATPase pump have a pathogenic role in RRM hypertension.
c. Arginine vasopressin

The role of arginine vasopressin (AVP) in hypertension of CRF is uncertain.
Vasopressin secretion is directly related to'plasma osmolality. AVP’s primary action is to
increase water permeability of the principal cells in the collecting ducts of the kidney.
High plasma AVP concentrations cause both renal vasoconstriction and glomerular
mesangial cell proliferation. These abnormalities can eventually result in renal
deterioration and increased systemic BP. Yet in 1993, Yamada and co-workers reported
that even with relatively high plasma concentrations in CRF patients, AVP does not
participate in hypertension. Oral administration of an AVP-V1 receptor antagonist, OPC-
21268, did not result in any BP changes‘in 7 hypertensive CRF patients. Likewise in
hypertensive RRM rats, treatment with this AVP antagonist produced only a minimal
decrease in established hypertension (Gavras, 1982). In contrast, in two different
experimental models of renal failure, deoxycorticosterone-salt (DOCA-salt) and
adriamycin-induced nephropathy, Okada reported that combined therapy of OPC-21268
and the AVP-V2 selective receptor antagonist, CFC-31260, prevented hypertension
development and the progression of renal injury (Okada et al., 1994). To date, the
relative importance of increased AVP plasma levels in renal pathophysiology and

hypertension in the RRM model remains to be clariﬁed.

14
d. Natriuretic peptides

CRF is associated with expansion of extracellular ﬂuid volume, and this volume
overload is thought to elicit increases in circulating concentrations of natriuretic peptides.
Elevated atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) plasma
concentrations have been reported in patients with CRF (Rascher et al., 1985; Totsune et
al., 1994). ANP secretion is increased in response to the distention of the atria which
occurs during plasma volume expansion in CRF. ANP increases urinary sodium
excretion by increasing GFR, inhibiting sodium reabsorption by the medullary collecting
duct, and indirectly by inhibiting renin and AngH-induced aldosterone secretion (Vander,
1991). These actions of ANP serve to regulate total body sodium and ﬂuid homeostasis.
ANP has also been reported to play a role in the adaptive hemodynamic and excretory
responses observed in the RRM model under normal and high sodium conditions (Zhang
et al., 1994; Brandt et al., 1989). It has been shown that rats subjected to RRM excrete
elevated amounts of sodium and water per remnant nephron as a compensatory response
to overall reduced excretory capacity (Zhang et al., 1994). Enhanced ANP secretion may
promote the compensatory increase in sodium and water excretion per individual
nephron. Increases in plasma ANP levels in RRM have been shown to follow rather than
accompany the development of hypertension, and the increased plasma concentrations
reported are not sufﬁcient to effect BP (Brandt et al., 1989). It seems unlikely then that
ANP plays a major role in the maintenance of RRM hypertension. Currently, the focus
of most research on the hormonal basis of hypertension in RRM is on angiotensin II

(AngH) and endothelin-1 (ET-1).

15

TH. Renin-angiotensin system
A. Hormonal renin angiotensin system
1 . Synthesis-cascade

The RAS is of major importance in sodium and water homeostasis, but also
inﬂuences a plethora of other physiological functions. The RAS is usually described in
terms of its synthesis cascade (Figure 1). Angiotensinogen is synthesized in the liver and
released into the bloodstream. Granular cells of the juxtaglomerular apparatus in the
kidney secrete the peptidase, renin, into the bloodstream in response to decreased renal
perfusion pressure or altered sodium chloride delivery. Renin, the rate-limiting enzyme
of the RAS, cleaves circulating angiotensinogen to the decapeptide angiotensin I (AngI).
AngI is then further cleaved by angiotensin converting enzyme (ACE) to form the
octapeptide AngH. ACE is a non-speciﬁc protease, found primarily in vascular
endothelial cells of the lungs, which degrades other peptides in addition to cleaving
AngI. AngH is the principally active component of the RAS. AngH acts upon two main
types of angiotensin receptors designated as angiotensin H type 1 (AT1), and angiotensin
H type 2 (AT2) (Wong et al., 1990). These receptors are located throughout the body in a
wide variety of tissues. It is believed that AT; receptors mediate most of the
physiological effects of AngH. A wide variety of proteases such as neutral endopeptidase
terminate the actions of AngH (Poulsen and Jacobsen, 1993). A few of the peptide
metabolites of AngH, i.e. angiotensin (1-7), angiotensin HI, angiotensin (38), may also
exert physiological effects, but their relative importance is questionable and needs to be

determined.

16

   
    
 

 

 
 
  
 
       
     
    
 

Angrgf‘rgrogen Kininogens
Prorenin __.> Renin renin inhibitor kallikrein
(kidney) - A74273
Angiotensin I bradykinin
. . ACEI
éngrotensrn _ captopril 32 -receptor
onvertrng , .
- enalapn] antagonist
Enzyme - lisino ril - HOE 140
(ACE) P
T peptide
fragments
Angiotensin II
Bradykinin 32
Neutral receptor
Endopeptidase
ATI rec§=pt0r 24.11 -VSMC,kidney,
antagonrst lung,brain,etc.
- Losartan AT2 receptor
-Valsartan antagonist

- PD123177

inactive metabolites ?
AT, receptor AT2 receptor

. -Angiotensin 1-7
-VSMC,krdney, -kidney,heart -Angiotensin IH
liver,lung,brain,etc. brain,etc. -Angiotensin (3-3)

~Angiotensin IV

Figure 1: Renin angiotensin synthesis cascade

l7
2. Physiological actions

a. Hemodynamic actions
Most of AngH physiologic actions serve to increase BP and/or the renal retention
of sodium and water. Thus it is not surprising that salt deﬁcit and hypotension are the
two most potent stimuli for RAS activation. AngH induces contraction of blood vessels,
causing an increase in TPR which results in an elevated BP. AngH also is a growth factor,
and may be involved in vascular hypertrophy or remodeling, which can cause long-term
increases in TPR and result in hypertension (Grifﬁn et al., 1991).
b. Heart
AngH tends to slightly increase the force of heart contractions and heart rate by its
facilitory actions on sympathetic outﬂow (Garrison and Peach, 1990). As blood volume
increases due to the renal actions on AngH, increases in left ventricular preload result and
therefore augments cardiac output. It must be kept in mind that in intact animals,
increased circulating concentrations of AngH increase systemic BP and baroreﬂex
discharge but these actions may initiate reﬂex vagal activity sufﬁcient to slow the heart.
c. Central nervous system
The brain contains all components of the RAS and AngH may serve as a
neurotransmitter or neuromodulator at many sites within the CNS. In addition,
circulating AngH can gain access to the brain through the circumventricular organs and
elicit cardiovascular responses. The central and peripheral actions of AngH on the
nervous system include: stimulation of drinking behavior and AVP release, increasing

SNA, and enhancing norepinephrine release from sympathetic nerve terminals (Vander,

1991).

18

Hypovolemia is known to stimulate the RAS. This stimulation is associated with
a compensatory increase in water intake and sodium appetite to which renin from both the
kidney and brain may contribute. AngH is the most potent dipsogenic substance yet
discovered and it stimulates drinking activity by a direct action on the CNS (Epstein et
al., 1970). The effect of AngH on stimulating thirst has also been observed clinically. In
patients with severe renal disease exhibiting high plasma renin concentrations intractable
thirst was relieved following bilateral nephrectomy (Brown et al., 1969). AngH has also
been implicated in a centrally mediated increase in sodium appetite. Circulating AngH is
not thought to be a powerful stimulus to sodium appetite, but i.c.v. AngH has been shown
to induce increased sodium chloride intake in rats when maintained under sodium replete
conditions (Fitzsimons, 1993).

d. Endocrine systems
i. Aldosterone

AngH stimulates the synthesis and release of aldosterone from the adrenal cortex
which then acts on the collecting duct in the kidney to cause retention of sodium. The
synthesis and release of aldosterone is enhanced under conditions of hyponatremia and
suppressed during sodium replete conditions following the inverse relationship between
RAS activity and sodium intake.

ii. Arginine vasopressin

The role of AngH in the regulation of AVP secretion was first described by
Bonjour and Melvin who demonstrated that i.v. infusions of AngH increase plasma AVP
concentrations (Bonjour and Malvin, 1979). It is currently believed that high levels of

exogenous AngH that border on the supraphysiological are required to cause increases in

l9
AVP release (Brooks and Malvin, 1993). The receptors that mediate AngH effects on

AVP secretion are located in the brain and AngH is thought to utilize the
circumventricular organs to gain access to these regions. AVP and the RAS are linked by
a negative feedback mechanism. In opposition to the stimulatory action of AngH on
vasopressin secretion, it is well established that AVP inhibits renin release (Brooks and
Malvin, 1993).
iii. Atrial natriuretic peptide

ANP appears to be a physiological counterpart to activation of the RAS because
ANP opposes the majority of actions elicited by AngH and aldosterone. These include:
inhibition of AngH-mediated modulation of glomerular ﬁltration, antagonism of AngH-
induced proximal tubule sodium reabsorption, attenuation of the vasoconstrictor effects
of AngH, suppression of aldosterone secretion and antagonism of aldosterone-mediated
sodium reabsorption from distal nephrons (Richards and Nicholls, 1993). Changes in
sodium and water status that induce activation of the RAS elicit reciprocal decreases in
ANP activity. For example, plasma ANP concentrations rise in proportion to increasing
salt intakes whereas the RAS is suppressed.

e. Kidney

As described above, renin released from the granular cells of the juxtaglomerular
apparatus in the kidney is the rate-limiting step in the RAS which results in the
production of AngH. The control of renin secretion is quite complex. A variety of
hormonal and nervous system imputs inﬂuence renin secretion: (1) intrarenal
baroreceptors, (2) tubular sodium or chloride delivery to the macula densa, (3) renal

sympathetic nerves, and (4) AngH (negative feedback) (Vander, 1991). Stimulation of

20

renin release and activation of the RAS ultimately leads to increases in AngH and
aldosterone release which both cause significant effects within the kidney.
i. Renal hemodynamics
The overall renal effect of AngH is to increase renal vascular resistance (RVR)
and consequently decrease renal blood ﬂow (RBF) due to direct vasoconstriction of the
renal vasculature, which is quite sensitive to the peptide. These responses are observed at
plasma levels that have little effects on systemic arterial pressure. The renal vascular
responses to AngH depend partly on total body sodium status and are affected by the
inverse relationship between RAS activity and sodium intake (Hollenberg et al., 1974).
ii. Glomerular function
Increases in circulating AngH cause decreases in GFR due to both renal
hemodynamic effects and direct contraction of mesangial cells. Mesangial cell
contraction results in a decrease in glomerular capillary ﬁltration coefﬁcient (Ky)
(Dickinson et al., 1963). The decreased Kf decreases the glomerular ﬁltration surface and
this leads to reductions in GFR. Changes in afferent and efferent arteriole resistance
greatly inﬂuence GFR. Micropuncture studies have shown that AngH disproportionately
increases efferent resistance resulting in increased glomerular capillary hydraulic pressure
(Dickinson et al., 1963). Some investigators have implicated an increased glomerular
pressure as the cause of renal deterioration in CRF (Meyer et al., 1987).
iii. Tubular function
One of the important renal actions of AngH is to promote sodium reabsorption
from the proximal tubule, but the single most important controller of sodium reabsorption

is aldosterone (Vander, 1991). Synthesis and release of aldosterone from the adrenal

21
cortex is stimulated by AngH (Vander, 1991). The principal cell in the cortical collecting

duct is acted upon by aldosterone to stimulate sodium reabsorption. Reﬂex pathways
exist to keep sodium balance within a very tight range. Baroreceptors in the kidney and
the carotid sinus, sensitive to changes in extracellular sodium and plasma volume,
regulate GFR and sodium reabsorption. The tubuloglomerular feedback loop involves
detection of increased sodium concentrations by the macula densa which generates a
signal to ultimately decrease GFR, thereby decreasing sodium retention. Activation of
ﬂuid-retaining mechanisms resulting in water and sodium volume expansion play a part
in the long-term actions of AngH on BP regulation. It should be noted that when
circulating AngH levels are high enough to raise systemic pressure, an AngH mediated
pressure-natriuresis counteracts the sodium retaining actions of the peptide in the
proximal tubules and collecting ducts. When the pressure-natriuresis relationship is
impaired, AngH leads to excessive sodium retention, volume expansion and hypertension.

B. Tissue renin angiotensin system

Recent evidence suggests that in addition to the classical endocrine RAS, there
exists a “local RAS” that is thought to act in an autocrine or paracrine fashion and is
differentially regulated from the circulating RAS (Campbell, 1987). These angiotensin
generating systems have been described in many organs in the body including: brain,
heart, kidney, adrenal, and blood vessels (Phillips, 1993). Tissues in these organs have
been shown to contain mRNA for all the various components of the RAS:
angiotensinogen, renin, ACE, etc. Plasma derived renin of kidney origin, however, is the
major source of vascular renin and is considered to be the main regulator of vascular

AngH production (Dzau and Re, 1994: Kato et al., 1993). Changes in activity of these

22

local systems are not thought to inﬂuence plasma AngH concentrations. Campbell and
others have promoted the viewpoint that the circulating RAS provides homeostatic
responses to acute changes in BP and ﬂuid and electrolyte status (Campbell, 1987). In
contrast the local RAS may affect BP regulation by exerting more of a tonic inﬂuence in
the tissues where they exist (i.e. regulation of vascular tone). Even though the existence
of local RAS were described over a quarter of a century ago, their physiological and
pathophysiological relevance remains to be deﬁned.

C. Inhibition of the renin angiotensin system

The RAS can be inhibited at several different points in the synthesis cascade.
Generally speaking the RAS is suppressed under conditions of high salt intake (H8) or
increasing cumulative sodium balance. This may also be true but probably to a lesser
extent under conditions where water intake is inappropriately elevated. The RAS also can
be inhibited by pharmacological intervention. For example, beta-adrenoceptor blockers
decrease renin secretion (Vander, 1991). Direct inhibitors of renin have also been
developed (Kleinert et al., 1992). But the drugs used most commonly to impair RAS
activity are the ACE inhibitors, e.g. captopril, lisinopril and enalapril. Competitive,
reversible angiotensin H receptor antagonists are available for both the AT; (AT IRA) (i.e.
losartan, and EXP3174), and AT; (ATzRA) (i.e. PD123177) receptors.

1. Angiotensin converting enzyme actions

As mentioned above, ACE is a non-speciﬁc protease that can act on a variety of
substrates besides AngH such as: bradykinin (BK), enkephalin, and neurotensin (Skidel
and Erdos, 1993). ACE is responsible for inactivation of the vasodilator BK (Figure 1),

therefore some of the hypotensive effects of ACEI have been proposed to be due to the

23

accumulation of endogenous BK (Williams and Hollenberg, 1977). BK causes relaxation
of VSMC but its role under basal conditions is considered to be minor (Carretero and
Scicli, 1993). Under pathological conditions, low sodium intake, or when degradation is
inhibited, BK either directly or via various intermediates may cause: diuresis, natriuresis,
antiproliferative and antihypertrophic actions, antithrombotic and ﬁbrinolytic effects
(Carretero and Scicli, 1993). There are many published reports on the potential
signiﬁcance of BK in cardiovascular control. Unger and colleagues reported that chronic
administration of a bradykinin Bz-receptor antagonist, HOE 140, partially attenuated the
antihypertensive effect due to the ACEI ranripril in two-kidney, one-clip hypertensive rats
(Bao et al., 1992). Chen demonstrated similar ﬁndings in a dissimilar model of
hypertension (DOCA-salt). They showed that the reduction in BP due to the ACEI
captopril was abolished acutely by HOE 140 administration (Chen et al., 1996). There is
much experimental evidence to refute the role of BK in mediating the hypotensive
response of ACEI. Plasma kinins are reportedly unchanged or only moderately increased
after ACEI administration (Carretero and Scicli, 1988), yet it has been shown that kinin
concentrations need to be elevated approximately 20X normal values to cause acute
decreases in BP in some forms of experimental hypertension (Salgado et al., 1986).
Kohzuki reported in SHR that co-administration of cilazepril (ACEI) and HOE 140
induced no changes in BP other than those associated with cilazepril alone (Kohzuki et
al., 1995). A powerful argument that many investigators cite is the lack of an increased
hypotensive response to enalapril when compared to AngH receptor antagonist
administration (Siegl et al., 1995; Okada et al., 1995). These studies suggest that the

hypotensive effects due to ACEI derive from inhibition of AngH formation only. There

24

still exists a good deal of controversy over the mechanisms responsible for the BP
lowering effects of ACEI, however, and further investigation is warranted.

D. Renin angiotensin system and hypertension

1. Background

The notion that the RAS is involved in hypertension evolved from work done by
Goldblatt in the 1930’s. Goldblatt theorized that essential hypertension was caused by
the release of a renal pressor substance in response to renal artery constriction. This
pressor substance was later characterized as renin and there is now unequivocal evidence
that the RAS participates in the pathogenesis of hypertension.

It is generally agreed that essential hypertension is multifactorial, therefore
abnormalities of the RAS may only play a partial role in BP elevation. Hypertensive
patients are sometimes classiﬁed into two groups based on their plasma renin
concentrations. Patients with low-renin hypertension are considered to have plasma
renins that are low for their level of salt intake (Swales, 1993). Generally low-renin
hypertensives exhibit volume expansion and do not respond well to antihypertensive
therapy directed at inhibiting or blocking the RAS. High-renin hypertensives have
plasma renin levels above the normal range expected at their level of salt intake. These
patients often have increased SNA and cardiac outputs (Esler et al., 1978).
Antihypertensive therapy aimed at inhibiting the RAS in these patients usually has
beneﬁcial effects when compared to low-renin hypertensives on the same therapy. These
classiﬁcations are based on historical perspectives, and may not provide an accurate

description of patients in light of recent studies involving the development of more

25

selective antagonists, better biochemical measurements and the discovery of local tissue
renin angiotensin systems.
2. AngH induced hypertension

Chronic i.v. administration of low doses of AngH causes the development of a
sustained hypertension in normal rats (Kanagy et al., 1990). The degree of increase in BP
depends mainly on the infusion rate of AngH. In this model of hypertension, elevation of
BP is completely reversed upon discontinuation of exogenous AngH infusion. It has been
demonstrated that AngH-induced hypertension is initially dependent on direct
vasoconstriction due to the fast pressor effect of AngH, but chronic increases in BP are
due to the slow pressor effect (Brown et al., 1981).

a. Fast pressor effect of AngH

Large infusion rates of AngH (i.e. > 30 ng/kg/min) administered parenterally in
experimental animals and humans elicit large, rapid increases in BP referred to as the fast
pressor effect of AngH. BP is increased within seconds to minutes and this effect lasts
only as long as the peptide is administered (Brown et al., 1981). Upon discontinuation of
AngH infusion, BP returns to normal values within minutes. Tachyphylaxis occurs and
BP gradually falls if these high doses of AngH are continuously infused for several days
(Dickinson and Lawrence, 1963). It is now well established that the mechanism of the
fast pressor effect of AngH is direct contraction of vascular smooth muscle cells via AT.
receptors resulting in vasoconstriction (Brown et al., 1981).

b. Slow pressor effect of AngH
The slow pressor effect (SPE) of AngH occurs when low amounts of the peptide

(i.e. < 10 ng/kg/min) are infused which do not invoke a fast pressor response but cause

26
BP to gradually rise over several days (Brown et al., 1981). Tachyphylaxis is not

observed to the SPE and upon discontinuation of AngH infusion, BP returns to pre-
infusion levels only after a prolonged period (i.e. hours to days). Several theories have
been proposed, but the exact mechanism(s) of the SPE are still being investigated.

One mechanism proposed for the development of the SPE is vascular remodeling
and/or hypertrophy. These structural changes can occur throughout the vasculature and
may contribute to the increase in total peripheral resistance that is commonly observed in
hypertension (Heagerty et al., 1993). Yet it is generally thought that hypertrophy and
remodeling of vascular tissue requires weeks to months to develop, which does not
correlate well with the SPE that is apparent within days (Lundgren, 1974). Accordingly,
vascular remodeling may play a role in BP regulation over much longer periods of time,
but its inﬂuence on initiation of the SPE is not supported by the current experimental
evidence.

AngH is known to elicit physiological responses from interactions with receptors
within the central nervous system (CNS); therefore, these cardiovascular centers may be
an important site of action for the SPE. Circulating AngH can interact with the CNS via
receptors in circumventricular organs outside the blood brain barrier to augment
sympathetic tone (Lappe and Brody, 1984). Luft and coworkers in 1989 demonstrated
that SNA was increased in rats receiving chronic low dose AngH infusions. Blockade of
the sympathetic nervous system (Yu and Dickinson, 1971) and ablation of the area
postrema (Fink et al., 1987), a circumventricular organ, have been reported to prevent the
development of the SPE in rats. On the contrary, other investigators have demonstrated

that an enhanced slow pressor response to AngH in spontaneously hypertensive rats

27
(SI-IR) was still intact after sympathectomy (Li and Jackson, 1989). The relative role of

the CNS on the SPE is still a matter of investigation and controversy.

The SPE has been proposed to result from AngH-induced increases in total body
sodium and ﬂuid volume (DeClue et al., 1978). Salt loading was shown to increase the
magnitude of the SPE of AngH, whereas salt restriction diminished this effect (Cowley
and DeClue, 1976). Our laboratory has found that subpressor rates of AngH infusion do
not affect sodium balance or increase plasma aldosterone concentrations, thereby arguing
against sodium retention being responsible for the SPE.

Although no one of the aforementioned possible mechanisms discussed appears to
be totally responsible for the SPE, it is quite probable that each mechanism plays a role
and that their additive effects are needed for the expression of the SPE.

3. AngH involvement in other forms of hypertension

Since in the majority of hypertensive patients there is no deﬁnable cause, a great
deal of research has focused on developing experimental models of essential
hypertension.

a. Spontaneously hypertensive rat

The SHR was derived from selective breeding by Aoki in 1963 (Okamoto and
Aoki, 1963). This genetically hypertensive strain is the most widely used experimental
model of hypertension. Increased BP develops very early and is accompanied by left
ventricular hypertrophy, increased SNA and nephrosclerosis (Kurtz et al., 1995). The
involvement of the RAS in SHR has been investigated extensively in recent decades. The

majority of these studies demonstrate that inhibition of the RAS with ACEI and ATIRA

28

prevents and reverses the progression of hypertension and renal damage in SHR (Kohara
et al., 1993; Cachofeiro et al., 1995).
b. Goldblatt renal hypertension

A well studied model of renal hypertension referred to as Goldblatt hypertension
is produced by constriction of one or both 'of the renal arteries with adjustable silver clips
(Goldblatt et al., 1934). The procedure that most closely resembles human renovascular
hypertension is the two-kidney, one-clip (2K1C) model where both kidneys are present,
but one renal artery is clipped and partially occluded. This procedure causes a dramatic
increase in renin secretion from the affected kidney (Martinez-Maldonada, 1991) and
renin inhibitors, ACEI and ATIRA are effective antihypertensive agents (McMahon et
al., 1995; Wallace and Morton, 1984; Thurston, 1994). Thus, this model of hypertension
is referred to as “renin-dependent”.

c. Transgenic models of hypertension

The causes of essential hypertension are still poorly deﬁned, but genetic factors
are known to play an important role. Techniques have recently emerged that allow for the
overexpression or deletion of speciﬁc genes in experimental animals. Transgenic animals
have new genetic material incorporated into their genome through microinjection into
germ cells. Ganten and coworkers were the ﬁrst to overexpress the renin gene in rats
(Mullins et al., 1990). Fulminant hypertension resulted and heterozygous animals
developed systolic BP of up to 250 mmHg at 10 weeks of age. Other genes inﬂuencing
BP are currently being identiﬁed so that manipulations of the genomes will permit
investigators to examine the effect of these alterations in vivo. The generation of

laboratory animals expressing candidate genes involved in cardiovascular disease may

29

provide for further investigation of regulatory mechanisms of the gene products and

possible pathophysiological consequences of their abnormal expression.

E. Renin angiotensin system and chronic renal failure
1. Renin angiotensin system activity in chronic renal failure
The contribution of the RAS to hypertension associated with human CRF has
been studied for many years. Many investigators have demonstrated the efﬁcacy of ACEI
treatment in arresting the progression of hypertension and the deterioration of renal
function in human CRF. The beneﬁcial effects of ACEI treatment have been reported in
many types of renal failure, i.e.; hypertensive non-insulin dependent diabetes mellitus
(Lebovitz et al., 1994), diabetic nephropathy (Mulec et al., 1994), and non-diabetic CRF
(Becker et al., 1994). Additionally, Gansevoort et al., in 1994 reported a lowering of BP
and a decrease in urinary protein excretion with the ATIRA, losartan, in hypertensive
patients with renal disease. Thus the RAS, speciﬁcally AngH, is involved in both the
hypertension and the renal deterioration observed in human CRF and experimental RRM.
2. Other antihypertensive regimens

A variety of antihypertensive agents have been shown to have some protective
actions against CVD and renal deterioration in CRF patients. What has been more
difﬁcult to demonstrate with these medications is a slowing of renal deterioration
independent of BP lowering effects. ACEI, on the other hand, have been shown to exert
beneﬁcial effects on the kidney independent of BP lowering effects (Kasiske et al., 1993;
Liou et al., 1995; Mann et al., 1990). When compared to ACEI, most other
antihypertensive regimens elicit undesirable effects that may be detrimental. For

example, thiazide diuretics act in a beneﬁcial way to cause sodium excretion and reduce

30

peripheral vascular resistance but they tend to induce lipid abnormalities and their
efﬁcacy is greatly reduced in patients with renal impairment (National High Blood
Pressure Education Program, 1991).

Beta-adrenoceptor blockers are capable of lowering BP effectively and to the
same extent as ACEI in CRF. But many studies have concluded that ACEI slow the
progression towards end stage renal disease and prolong kidney survival better than beta-
blockers, probably through mechanisms in addition to antihypertensive effects
(Hannedouche et al., 1994). The beta-blockers also are associated with some detrimental
side effects in CRF patients. For example this class of antihypertensives may induce
carbohydrate intolerance and exacerbate diabetes mellitus. Propranolol has been
reported to cause a 10-20% reduction in GFR (Vulpis et al., 1991).

The calcium channel blockers (CCB) cause moderate reductions in systemic BP
in CRF patients. But in experimental models of renal failure this BP decrease is
accompanied by renal afferent arteriole dilation, thereby permitting an increased
glomerular capillary pressure to persist (T olins and Raij, 1990). This lack of effect on
glomerular hypertension is less effective in preventing hemodynamically-mediated
progressive glomerular injury. It has recently been hypothesized that non-hemodynamic
effects of CCB’s may play a role in slowing renal deterioration. CCB’s have been shown
to inhibit mesangial cell proliferation and the generation of inﬂammatory mediators by
endothelial cells (Shultz and Raij, 1989; Tolins et al., 1989). Zucchelli and colleagues
studied the progressive rate of renal insufﬁciency in 142 hypertensive patients over four
years and found that both CCB and ACEI possess a renoprotective effect that is no greater

with either treatment (Zucchelli et al., 1992).

_31

3. Renin angiotensin system activity in RRM model
a. Ligation vs. excision method of RRM

The majority of studies investigating the role of the RAS in RRM have utilized
the ligation method. As previously mentioned, the ligation method is not the best model
for the study of hypertension in CRF, because the resultant pockets of ischemia that are
produced cause exaggerated release of renin and a rapid increase in BP. The hypertension
is associated with high intrarenal renin concentrations and is not affected by changes in
sodium intake (Kleinknecht et al., 1995), unlike human CRF.

With the excision method there is little change in BP during the ﬁrst weeks and
BP rises progressively only as renal deterioration develops. The hypertension is
associated with very low renin concentrations and is directly proportional to the level of
salt intake (Kleinknecht et al., 1995). Because of these differences, extrapolations of data
from one model to the other are hazardous and a closer examination of the experimental
evidence directed towards the proper model of RRM hypertension is warranted here.

b. RAS in ligation method of RRM

The evidence for the involvement'of the RAS in the ligation method of RRM is
extensive. There is an increased tissue renin content, renin mRNA and renin synthesis in
the ligated kidney (Correa-Rotter et al., 1992). In the systemic vasculature it has been
shown that there is increased tissue RAS activity (Kuczera et al., 1990). ACEI have been
widely reported to slow progression of hypertension and renal deterioration in the ligation
method (e.g. Anderson et al., 1985; Brunner et al., 1989). This is in spite of studies
demonstrating that activity of the circulating RAS is not elevated (Smith et al., 1992).

Since inhibition of AngH production is the main action of ACEI, it is likely that a

32
reduction in AngH activity is responsible for the beneﬁcial effects of ACEI ( e.g.

Lafayette et al., 1992; Pelayo et al., 1990). This suggests that in RRM either local tissue
formation of AngH is enhanced or there is an increased responsiveness to circulating
AngH.

ACEI have also been reported to reverse established hypertension in this model
(Meyer er al., 1987; Brunner et al., 1989). Reversal of established hypertension more
closely resembles the clinical setting of CRF in human patients where diagnosis and
treatment usually occur well after development of signiﬁcant renal disease. Both CCB
(Dworkin et al., 1993) and ACEI (Katsumata et al., 1990) have been shown to attenuate
glomerular hypertrophy and thereby reduce glomerulosclerosis in RRM but many
investigators have argued that ACEI posses unique beneﬁcial effects on ameliorating
functional and structural damage to the glomerulus (Jackson et al., 1988; Brunner et al.,
1989; Tolins et al., 1990). It seems clear from the literature that ACEI may have
therapeutic advantages in RRM attributable to mechanisms independent of systemic
blood pressure reduction.

Pharmacological studies using ATlRA’s have shown that systemic blockade of
AngH receptors lowers BP and limits glomerular injury in RRM (Lafayette et al., 1992;
Pollock et al., 1993). These newly developed antagonists have recently been compared to
ACEI in RRM. In a study by Lafayette, losartan lowered BP and protected the kidney
from further deterioration in RRM but not to any greater extent than that observed with
enalapril treatment (Lafayette et al., 1992). When the investigators combined losartan
with enalapril they observed no additional beneﬁt over single administration of either

drug. These studies support the idea that reducing AngH activity exerts beneﬁcial

33
antihypertensive and renoprotective effects in RRM whether it is accomplished by AngH

receptor blockade or by inhibition of AngH formation.
c. RAS in excision method of RRM

Evidence supporting the involvement of the RAS in hypertension and renal failure
in the excision method of RRM is less convincing. It must be kept in mind that RAS
activity in the excision method of RRM is determined in part by the level of salt intake.
The inverse relationship between sodium intake and the activity level of the RAS has
been reported by Ylitalo and coworkers (Ylitalo et al., 1976). They proposed that excess
extracellular levels of sodium observed in RRM exert a negative feedback on the
production of angiotensinogen and renin. In RRM rats sodium restriction stimulated the
RAS, and excess sodium suppressed it. They observed a decrease in plasma AngH
concentration and kidney renin concentration in RRM rats placed on an elevated daily
sodium intake of 10-15 mEq. Yet, it has been demonstrated that the development of RRM
hypertension in rats on a increased salt intake progresses at a faster rate than in rats on
normal or low salt intakes (Douglas et al., 1964). The literature is sparse on the effects of
blocking the RAS in RRM under conditions of elevated salt intake. Terzi and coworkers
reported that inhibition of AngH formation by enalapril did not affect BP in RRM rats fed
a high salt diet (T erzi et al., 1992). Yet Kanagy et al in 1993 showed that the AT] RA,
losartan completely prevented hypertension development in RRM rats on high salt intake
(HS). Since CRF patients typically consume elevated amounts of salt, at least until the
disease is diagnosed, it seems necessary to evaluate further the role of the RAS in the

maintenance of elevated BP in RRM during HS intakes. I speciﬁcally intend to

34
investigate the role of the RAS in reversal of established RRM hypertension when rats are

kept on HS.

In RRM rats (excision model) on a normal salt intake (NS), recent studies have
shown that prophylactic administration of ACEI prevents the development of
hypertension and inhibits the decline in renal function (Ashab et al., 1995; Amann et al.,
1993). Reports investigating the reversal of these parameters in established RRM rats
maintained on NS are non-existent. Once again, this leads to a gap in our knowledge of
the involvement of the RAS in CRF and needs to be investigated further.

Very few reports detail the effectson BP and the RAS of lowering salt intake in
RRM. An early report by Ylitalo and colleagues showed that when RRM rats were
maintained on a low salt intake (LS), elevations in BP were prevented (Ylitalo et al.,
1976). They reported that plasma AngH and kidney renin concentrations were
considerably elevated from RRM rats maintained on NS. Others have conﬁrmed
Ylitalo’s results and recently Terzi tested ACEI in RRM rats under conditions of
moderate sodium restriction (Terzi et al., 1992). These rats exhibited lesser degrees of
renal damage than groups on HS and did not become as hypertensive. Enalapril treatment
decreased BP below normotensive levels in these moderately sodium restricted rats. The
protective effects of salt restriction do not appear to be unique to the excision method nor
to this model of hypertension. Salt restriction has been shown to prevent glomerular
injury in RRM rats prepared using the ligation method and displaying established renal
disease (Dworkin et al., 1996; Lax et al., 1992). In these studies, BP still increased when
on LS but not to the extent that was observed in RRM rats on NS. In other models of

hypertension such as SHR, sodium restriction lowered BP by 15% (Ely et al., 1990). Salt

35

restriction alone appears to be an excellent therapeutic tool for patients with CRF but care
should be taken when extrapolating studies in experimental animals to humans. There
are risks associated with dietary sodium restriction and some abnormalities have been
observed in experimental animals. These include: an abnormal sensitivity to blood loss,
attenuated responses to stress situations, cardiac structural compensations, compensatory
increases in SNA as well as enhanced activity of the RAS (Ely et al., 1990).

These studies suggest the involvement of the RAS in the development of RRM
hypertension, yet it seems likely that the contribution of the RAS to control of arterial
pressure in RRM rats varies with salt intake and the method of RRM. Much of the
previous work has looked at inhibiting or lowering the activity of the RAS early on in the
progression of CRF. Most of my experimental approach is directed towards determining
the contribution of AngH to RRM hypertension under varying conditions of salt intake
when the disease is well established.

IV. Endothelin System

A. Background

The endothelins are a family of 21~ amino acid peptides consisting of 3 isoforms
called endothelin-1, endothelin-2, and endothelin-3. These isoforms are produced in a
variety of cell types and are found throughout the body in many tissues. There are
speciﬁc patterns of isoform expression in individual tissues. Most of the studies have
focused on endothelial cell ET-l because it appears to be the most widely distributed
is oform and is most often implicated in cardiovascular control. The lung and the kidney
have been shown to be the predominant sites of ET-1 production (Rubanyi and Polokoff,

1994). The production of ET-1 is clearly linked to regulation of transcription of ET

36

mRNA. A variety of stimuli have been shown to increase message levels for ET
including growth factors and cytokines such as thrombin (Emori et al., 1992), TGFﬁ
(Kurihara et al., 1989), and insulin (Hu et al., 1993). Vasoactive substances i.e. AngH
(Dohi et al., 1992), AVP (Irnai et al., 1992), and bradykinin (Marsden et al., 1991) have
also been reported to increase mRNA expression in endothelial cells. ET synthesis
begins as prepropeptides which are cleaved by a protease into inactive intermediates
called big ET-1,-2, and -3. Big ET is activated via cleavage by a speciﬁc endopeptidase
called endothelin-converting enzyme (ECE), forming the biologically active ET-1,-2, and
-3 (Opgenorth et al., 1992). In terms of biological activity, ET-l has been demonstrated
to be a 140 fold more potent vasoconstrictor than big ET-1 and the prepropeptide is
devoid of vasoconstrictor action (Code et al., 1990). Membrane metalloendopeptidase I
(a.k.a. neutral endopeptidase and enkephalinase) has been shown to efﬁciently cleave
mature ET-l, rendering the peptide biologically inactive (Sokloovsky et al., 1990). The
plasma half-life of ET-1 injected i.v. in the rat is about 60 seconds with the lungs
removing approximately 90% of the bolus (Rubanyi and Polokoff, 1994). ET is subject
to a high degree of plasma protein binding (> 98%) and serum albumin may act as a
“pseudo-receptor” to bind ET and ET receptor antagonists (W u-Wong et al., 1996)

Of the 3 isoforms, ET-1 is considered to mediate most of the physiologically
important cardiovascular effects. Vascular endothelial cells produce only ET-l , and they
appear to be the most abundant source of ET-1 in vivo (Yanigasawa et al., 1994). Yet,
ETs are ubiquitous peptides and their receptors are distributed in almost all tissues
(Koseki et al., 1989). These receptors all have 7 transmembrane domains and are coupled

to G-proteins. Their locations are tissue speciﬁc and their physiologic actions are quite

37

diverse. ET acts on two pharmacologically and molecularly distinct receptor subtypes
identiﬁed as ETA and ETB receptors. The afﬁnity of the 3 isoforms of ET differs for the
ET receptor subtypes. At the ETA receptor subtype the afﬁnity is as follows: ET-l > ET-
2 > ET—3. At the ETB receptor subtype each isoform has an equal binding afﬁnity (ET-1 =
ET-2 = ET-3). The general consensus, until recently, was that ETA receptors mediate
direct vasoconstrictor actions and ETB receptors produce vasodilator effects via the
release of nitric oxide and cyclooxygenase products. But Seo et al., in 1994 reported that
both ETA and ETB receptors are involved in vasoconstriction in human blood vessels.
Vasoconstrictive ETB receptors were located on vascular smooth muscle cells. Many
others have conﬁrmed the existence of two ETB receptor subtypes in various animal
species. The current classiﬁcation of ETB receptor subtypes is in the process of
modiﬁcation where endothelial cell receptors producing vasodilation will putatively be
designated ETBI and vascular smooth muscle cell receptors producing vasoconstriction
will be designated ETBZ. Cardiovascular responses to exogenously administered ET-l
may be difﬁcult to interpret because the relative contribution of the ETA and ETB
subtypes varies depending on the vascular (bed and species studied.

B. Physiological actions of endothelin

1. Hemodynamic actions

ET administered intravenously produces a rapid and transient vasodilation,
followed by a profound and long-lasting vasoconstriction (Yanagasawa et al., 1988). It is
thought that the long-lasting pressor response to exogenous ET-l administration is not
due to the peptide’s prolonged presence in the plasma, but rather to slow dissociation

from the receptors. ET-1 is the most potent vasoconstrictor of isolated blood vessels

38

identiﬁed to date (Rubayani and Polokoff, 1994). This vasoconstriction leads to increases
in systemic BP which are due to increased total peripheral resistance (Rubayani and
Polokoff, 1994). In addition to ET’s direct vasoconstrictor effects, the peptide can
potentiate the contractile responses to other vasoconstrictors such as norepinephrine and
serotonin (Tabuchi et al., 1989). Other potential ways in which ET may act as a regulator
of vessel reactivity and vascular tone have not yet been clearly deﬁned.
2. Heart

ET-l has direct actions on the heart that include positive inotropic and
chronotropic effects in addition to prolongation of action potential duration (Rubayani
and Polokoff, 1994). The coronary circulation is particularly sensitive to the
vasoconstrictor effects of ET (Kurihara et al., 1989). The involvement of ET in the
physiological and pathophysiological mechanisms of heart failure is an ongoing area of
much research.

3. Central nervous system

ET’s are considered to be neuropeptides because they are: localized in the brain,
bind speciﬁcally in some brain tissues, and i.c.v. injections of ET have been shown to
signiﬁcantly change cardiovascular, respiratory, and neuroendocrine system function
(Rubayani and Polokoff, 1994). These i.c.v. injections produce profound vasoconstrictor
and pressor responses (Siren and Feurerstein, 1989). Additionally, centrally administered
ET-l activates SNA and AVP release (Matsumura et al., 1991). Even though activation
of the baroreﬂex stimulates ET-l release into the plasma, i.v. ET-l does not affect

baroreceptor reﬂex control of heart rate (Knuepfer et al., 1989).

39

4. Endocrine systems .

ET can interact with a variety of hormones at both the level of biosynthesis and

the site of biological action.
a. Renin angiotensin system

Much evidence exists for the existence of an interaction between the renin
angiotensin and ET systems. ET stimulates aldosterone secretion through direct actions
on the zona glomerulosa of the adrenal gland (Rubayani and Polokoff, 1994). Some
controversy exists as to whether ET stimulates or suppresses renin secretion. Most
studies in vitro have found that ET suppresses renin secretion from the kidney (reviewed
by Rubanyi and Polokoff, 1994). Generally, in vivo studies are in agreement with in vitro
work and demonstrate that ET administered i.v. or intrarenally suppresses or does not
change PRA in dogs, rats or humans. ET has been shown to stimulate AngH production
in vitro and act synergistically with many of the biological actions of AngH in viva. ET
stimulates ACE activity and dose-dependently increases the conversion of Aug] to AngH
in cultured pulmonary artery endothelial cells (Kawaguchi et al., 1990). Previous work in
our lab has demonstrated that ET-1 induced hypertension produced by continuously
administered i.v. ET-l (5.0 pmol/kg/min) could be prevented by co-infusion of captopril
(Mortensen and Fink, 1992). Yet captopril administration after ET-l induced
hypertension was established did not produce an antihypertensive result. The
mechanisms of the interactions between the renin angiotensin and ET systems are still

being elucidated and will require additional investigation.

40

b. Arginine vasopressin

It is generally considered that ET and AVP act synergistically as vasoconstrictors
and ET-l potentiates the vasoconstrictor action of AVP (Rubayani and Polokoff, 1994).
ET given i.v. and i.c.v. has been shown to stimulate AVP release from the
neurohypophysis, increase circulating AVP plasma levels, and elevate BP (Nakamoto et
al., 1991; Yamamoto et al., 1991).

c. Atrial natriuretic peptide

In general there exists a functional antagonism between ET and ANP in most
biological systems. ANP is a vasodilator that causes natriuresis thereby reducing plasma
volume and osmolarity. ET is a potent vasoconstrictor that decreases sodium excretion.
ET stimulates the release of ANP from atrial myocytes which results in elevations in
circulating plasma levels (Rubayani and Polokoff, 1994). Additionally, ANP has been
demonstrated to reduce ET production in cell culture (Hu et al., 1992). In a
comprehensive study in human patients, ET-l effectively antagonized the cardiovascular,
renal and endocrine actions of ANP (Ota et al., 1992).

5. Kidney

Many cell types within the kidney are known to produce ET and they can act in
both a paracrine and an autocrine fashion on different ET receptors. High afﬁnity binding
sites for ET are found throughout the kidney although the renal medulla has been reported
to contain the greatest density of receptors (Kohan et al., 1996). The vasculature of the

kidney seems particularly sensitive to the physiologic effects of endogenous ET.

41

a. Renal hemodynamics
Systemic and intrarenal infusion of ET increases RVR and decreases RBF
resulting from constriction of both afferent and efferent arterioles (Badr et al., 1989).
This vasoconstriction is often preceded by a transient renal vasodilation like that observed
in the systemic effect of ET, but vasodilatory prostaglandins in addition to NO have been
implicated in attenuation of the vasoconstrictor effect of ET in the kidney (Chou et al.,
1990).
b. Glomerular function
ET reduces GFR and causes contraction of mesangial cells leading to reductions
in K; and ﬁltration surface area (Badr et al., 1989; Ferrario et al., 1989). ET is also a
potent mitogen and activates many cellular signaling pathways in mesangial cells
(Simonson et al., 1989). These properties of ET lead to diminished excretory capacity of
the kidney.
c. Tubular function
Intravenous infusions of ET decrease sodium excretion partly by decreasing
ﬁltered load and partly by increasing aldosterone secretion (Goetz et al., 1988). The
decreased diuresis observed in response to ET is thought to be due indirectly to a
reduction in RBF and GFR.
C. Inhibition of endothelin system
The development of inhibitors of ET synthesis, ET receptor agonists and ET
receptor antagonists (ETRA) has provided pharmacological tools for the identification of

multiple receptor subtypes and the physiological responses following receptor activation.

42

1. Receptor agonists
ET-1 is considered a non-selective agonist for all ET receptor subtypes, but this
isoform has greater afﬁnity than the other isoforms at the ETA receptor. There are no
known selective ETA agonists, but there are several ETB agonists such as: sarafotoxin 6c
(STX 6c), sarafotoxin 6b (STX 6b), and IRL 1620. Most of these agonists have been
shown to elicit NO or prostacyclin release from endothelial cells, but may also cause
VSMC contraction in some instances.
2. Endothelin converting enzyme inhibitors
Endothelin converting enzyme (ECE) inhibitors have only recently been
investigated in disease states where endothelin is thought to play a role. The ﬁrst ECE
inhibitor was phosphoramidon which has been shown to be an effective inhibitor of ET-1
production (Sawamura et al., 1990). In 'many vascular preparations, inhibition of ET
mediated responses by blocking the enzymatic activity of ECE have been demonstrated
with phosphoranridon (Fukuroda et al., 1990). Phosphoramidon and similar drugs may
not be ideal therapeutic tools because decreased ET production would be expected to
result in both decreased ETA and ETB receptor activation. The discovery of selective,
potent peptide and non-peptide receptor antagonists, however, has facilitated the search
for ET involvement in normal functions and disease states.
3. Endothelin receptor antagonists
Selective antagonists exhibiting high afﬁnities (K, = nM-pM) for the two ET
receptor subtypes are currently available. Two of the most commonly used ETA receptor
antagonists (ETA RA) are BQ-123 and FR139317. These antagonists have approximately

1000 fold selectivity for ETA vs ETB receptors in rat preparations and are thought to bind

43
to the ETA receptor subtype very tightly (Doherty et al., 1993). Recently developed non-

peptide orally active ETA RA (i.e. PD155080, PD156707, Ro 46-2005) are now being
investigated in animals models of disease. .

Endothelin subtype B receptor antagonists (ETBRA) such as BQ-788 and RES
701-1 have been used in some experiments recently, but the results are difﬁcult to
interpret because these blockers bind to both endothelial ETBI receptors and VSMC ETBZ
receptors. The only reported endothelial ETB. receptor antagonist is RES701-1, but
recent comparisons of this compound in various animal species suggest that RES701-1 is
much less selective for ETm receptors in rats than in other species ( Tanaka et al., 1995).

Additionally, non-selective peptide (i.e. PD145065) and non-peptide (i.e.
bosentan, SB209670, BM8182874) endothelin receptor antagonists (ETA/ETBRA) have
been developed. Some investigators have theorized that blockade of both receptor
subtypes might result in additional beneﬁts over blockade of ETA receptors alone, and
comparisons of efﬁcacy of these selective and non-selective antagonists are currently
being evaluated.

All of these antagonists are classiﬁed as being competitive and reversible, but
their tight binding and long receptor occupation simulates the appearance of a non-
competitive irreversible binding situation.

D. Endothelin and hypertension
1. Background

Since its discovery in 1988 by Yanigasawa et al., ET-l’s potent vasoconstrictor

effects and long duration of action have led many investigators to study its role in

hypertension. ET has been postulated to be involved in the pathogenesis of hypertension

44

because many of its biological actions lead to elevations in peripheral vascular resistance.
ET may inﬂuence the short-term regulation of BP by direct vasoconstriction, which has
been reported in rats (McMurdo et al., 1993) and humans (Sorensen et al., 1994). ET
may also act through vascular remodeling as a result of smooth muscle proliferation to
inﬂuence the long-term regulation of BP (Ohlstein et al., 1992).

ET involvement in mild to moderate hypertension is controversial, but its role in
malignant hypertension is supported by convincing evidence. Plasma ET levels are not
elevated in most forms of experimental hypertension (Rubayani and Polokoff, 1994). In
contrast, when severe or malignant hypertension is accompanied by end-organ damage
(i.e. arteriosclerosis, renal failure), circulating ET concentrations are consistently elevated
(Yokokawa et al., 1991; Luscher et al., 1990).

There may be an increased responsiveness of VSMC to the vasoconstrictor actions
of ET in hypertension (Miyauchi et al., 1989; Dohi and Luscher, 1991), but this
phenomenon has not been demonstrated unequivocally (W inquist et al., 1989; Dohi and
Luscher, 1991). Therefore, this concept requires more investigation. In contrast to the
inconclusive results on increased responsiveness to ET, subpressor concentrations of ET
have been shown to potentiate the vasoconstriction induced by other agonists in many
hypertension models (Tabuchi et al., 1989; Dohi and Luscher, 1991).

ET may activate speciﬁc areas of the CNS that can result in increases in
sympathetic tone or enhanced release of vasoconstrictor hormones such as AVP or
norepinephrine (Vanhoutte, 1993). Both acute and chronic i.c.v. administrations of ET
have been reported to elevate arterial pressure in a dose-dependent manner (Ouchi et al.,

1989; Nishimura et al., 1991). Additionally, ET injected into the dorsolateral

45
periaqueductal gray area (PAG) increased BP (D’Amico et al., 1995). These

investigators also demonstrated that stimulation of the pressor neurons in the PAG by ET
produced an increase in sympathetic tone. Work utilizing ETRA in the CNS has just
begun, and much more effort will be needed to characterize the central role of ET in
cardiovascular regulation.

As previously mentioned, ET has profound renal effects (i.e. decreased RBF and
GFR) at concentrations that do not alter systemic hemodynamics. These effects may play
a crucial role in pressure-volume regulation and may be extremely important in the
development of hypertension. Tomobe and co-workers have shown an increased
reactivity to ET in renal arteries from SHR (Tomobe et al., 1988). Slight alterations in
pressor responsiveness in the renal vasculature can have profound effects on long term
regulation of systemic BP.

2. Endothelin and sodium intake

The inﬂuence of sodium intake on the physiologic and pathophysiologic functions
of ET are uncertain. Clozel in 1993 demonstrated an increased antihypertensive effect
using the ETA/ETBRA, Ro 46-2005, in normotensive sodium-depleted squirrel monkeys
compared to sodium-replete conditions. In contrast to these results, some investigators
have shown that ETRA are more effective in lowering BP in animal models of salt-
dependent hypertension than in other types of experimental hypertension (Schiffrin et al.,
1995: Doucet et al., 1996). There have been no reports comparing the effects of ETRA
treatment on the progression of hypertension and renal deterioration in RRM animals

under varying sodium intakes. Since sodium balance plays an integral part in the

46

development and progression of renal failure in RRM, my experiments were designed to
characterize the relationship between ET and sodium intake in this model.
3. ET-l induced hypertension

Yanagisawa’s original work with ET showed that bolus injections of the peptide
had long-lasting and potent vasoconstrictor effects (Yanagisawa et al., 1988). Since then
it has repeatedly been demonstrated that chronic i.v infusions of ET cause large and
sustained increases in BP (Mortensen and Fink, 1991; Yasujima et al., 1991, Wilkins et
al., 1993). The pressor effect has been shown to increase in a dose-dependent manner
and cease within hours upon cessation of the infusion.

4. Endothelin involvement in experimental forms of hypertension
a. Spontaneously hypertensive rat

ETRA’s have only recently been administered in experimental hypertension.
Some groups have demonstrated an antihypertensive effect in SHR due to systemic ETA
receptor blockade both acutely over minutes to hours (Douglas et al., 1994; Ohlstein et
al., 1993) and chronically over several days (Bird et al., 1995). But studies utilizing
blockade of ET formation or blockade of both ETA and ETB receptor subtypes have
produced conﬂicting results. Early work by McMahon and colleagues reported that the
ECE inhibitor, phosphoramidon, lowered BP in SHR to a greater extent then ETA
receptor blockade with BQ-123 (McMahon et al., 1993). Blocking ET formation would
be expected to cause decreased binding of ET to ETA and ETB receptor subtypes thereby
theoretically lessening ET’s inﬂuence on VSMC vasoconstrictor actions and on
endothelium induced vasorelaxation. Much work by Schiffrin and co-workers with

bosentan (ETA/ETBRA) has suggested that ET does not play a role in the maintenance of

47
hypertension or in the vascular hypertrophy in SHR (Li and Schiffrin, 1995). They

treated hypertensive SHR for 4 weeks with bosentan and did not observe any changes in
BP. The variable antihypertensive efﬁcacy of ET blockade in these studies may be due to
differences in the degree of ETA vs. ETB subtype blockade. The inﬂuence of each
receptor subtype on systemic hemodynamics is still being deﬁned and the involvement of
ET in SHR is still controversial and will require more investigation.

b. Goldblatt hypertension

In the 2K1C model of Goldblatt hypertension, chronic endothelin receptor
antagonism has not been demonstrated to exert antihypertensive effects. Both selective
ETA (Bazil et al., 1992) and non-selective ETA/ET]; receptor blockade (Li et al., 1996;
Schricker et al., 1995) were not associated with hypotensive responses in 2K1C, so ET
does not appear to play a major role in this so-called renin-dependent model of
hypertension.

Another type of Goldblatt hypertension is the one-kidney, one-clip (1K1C) model
which involves clipping of one renal artery and contralateral nephrectomy. The
contralateral nephrectomy drastically alters the development of hypertension in 1K1C
relative to that of 2K1C. 1K1C hypertension is generally more severe than in 2K1C, and
is associated with suppressed RAS activity. This variant of Goldblatt hypertension is
thought to resemble the RRM model in that there exists a volume expansion along with
expansion of exchangeable body sodium (McAreavey et al., 1984). Schiffrin and
colleagues have reported an increased vascular and cardiac ET gene expression in 1K1C
rats 24 weeks after application of the clip (Sventek et al., 1996). When these

investigators administered bosentan to 1K1C rats during this time interval, no

48
antihypertensive effect was observed ( Li et al., 1996). They concluded that even in the

presence of increases in ET-l gene expression, an ET component of BP elevation is not
evident in renovascular hypertension in rats.
c. Mineralocorticoid

Excess secretion of mineralocorticoids (e.g. aldosterone, deoxycorticosterone)
causes an antinatriuresis and kaliuresis which leads to increases in BP (Kenyon and
Morton, 1994). In experimental mineralocorticoid-induced hypertension,
deoxycorticosterone—acetate (DOCA) is usually administered subcutaneously and
accompanied by unilateral nephrectomy and a high-sodium diet. This model of
hypertension is associated with suppression of renin activity due to sodium retention
(Kenyon and Morton, 1994). Much evidence now exists for the involvement of ET in the
DOCA-salt model of hypertension. Suzuki et al., in 1990 reported that vascular ET
reactivity was increased in DOCA-salt hypertensive rats. More evidence comes from
Schiffrin and colleagues who demonstrated that ET gene expression is enhanced in blood
vessels from DOCA-salt rats (Schiffrin et al., 1996). In this experiment, vascular
expression of ET was not enhanced in rats treated with DOCA or salt alone, even when
BP rose to hypertensive levels. Acute blockade of endothelin receptors with the ETARA,
BQ-123 and FR139317, decrease BP in DOCA-salt rats over a period of minutes to hours
(Okada et al., 1994; Fujita et al.,1995). Chronic blockade of endothelin receptors with
bosentan (ETA/ETBRA) has been shown to partially attenuate the progressive rise in BP
observed in DOCA-salt rats (Li et al., 1994). The conclusion from these experiments was
that ET activity is increased in DOCA-salt and ET played a role in the maintenance of BP

in this experimental form of hypertension.

49

5. Endothelin involvement in human hypertension
ET was the cause of hypertension associated with an endothelin secreting
malignant hemangioendothelioma in humans (Yokokawa et al., 1991). In two reported
cases, changes in plasma ET concentrations correlated directly with changes in BP. Other
preliminary investigations have reported that single doses of bosentan administered to
hypertensive patients produce signiﬁcant reductions in diastolic BP that are maintained
for 24 hours (Warner et al., 1996).
E. Endothelin and chronic renal failure
1. Endothelin activity in chronic renal failure
Conﬂicting evidence is found in the literature for the involvement of ET in renal
disease in humans. It must be kept in mind that the causes of kidney injury are unknown
in most CRF cases because the disease is usually not detected until signiﬁcant renal
damage has occurred. In humans with CRF, urinary excretion of ET-1 has been reported
to be both elevated (Ohta et al., 1991; Roccatello et al., 1994) and decreased (Saito et al.,
1991). Additionally, plasma ET has been reported to be both elevated (Saito et al., 1991;
Koyama et al., 1989) and unchanged (Brooks et al., 1991: Totsune et al., 1989). Since
pharmacological inhibition or blockade of ET effects have just recently become available,
there is a scarcity of information characterizing the role of ET in human cases of CRF and
we must currently rely on animals models to advance our understanding of this disease.
2. Endothelin activity in RRM model
In the RRM model in rats, many investigators have found an increase in plasma
ET levels (Orisio et al., 1993). Yet other investigators have demonstrated plasma ET

levels in RRM rats are the same or even numerically lower than in sham rats (Benigni et

50

al., 1991). The current consensus is that plasma ET concentrations in RRM rats are
usually measured to be within the range found in normal rats unless malignant
hypertension or end-organ disease is present. Systemic plasma concentrations of ET may
correlate poorly with ET-induced pressor effects, however, because: ET secreted from
endothelial cells acts mainly on closely apposed vascular smooth muscle cells; circulating
ET is rapidly and efﬁciently cleared by the lungs (t”2 = 1 min); and ETB receptor
stimulation often leads to formation of physiological antagonists such as nitric oxide and
prostacyclin (Yanagisawa et al., 1994). Also, most experiments reporting ET levels
represent the family of ET's, and not exclusively the biologically active ET-l.

Since there is an abundance of ET synthesized in the kidney and both receptor
subtypes are present, work investigating the pathophysiological role of ET in the kidney
is of great interest. As mentioned before, ET is a potent mitogen and it promotes growth
of mesangial cells. This proliferation can lead to decreases in: ﬁltration coefﬁcient, GFR
and functioning of the kidney. Some investigators have proposed that inﬂammatory
diseases of the glomerulus, regardless of the original insult, are associated with activation
of ET synthesis (Luscher and Wenzel, 1995).

a. Endothelin activity in the ligation method

RRM rats prepared by the ligation method exhibit an increased renal ET gene
expression. This correlates with renal synthesis of ET, measured as increased ET
excretion in urine. Expression becomes greater as renal failure progresses (Brooks et al.,
1991; Orisio et al., 1993). Urinary excretion of ET appears to be a good marker of renal

deterioration in contrast to plasma ET levels, which do not change over the course of the

disease (Benigni et al., 1991). Since the development of ETRA's in the last few years,

51
reports deﬁning ET's involvement in models of RM hypertension have begun to appear

in the literature. Benigni et al., in 1994 reported that FR139317 (ETARA) reduced
proteinuria, attenuated the progression of hypertension, and slowed renal deterioration in
RRM rats. They administered FR139317 once daily by i.p. injection at a dose of 32
mg/kg for 53 days and recorded BP by tail plethysmography. Of particular interest was
that Benigni did not demonstrate reversal of hypertension or renal deterioration with the
ETARA in this study. In 1996, Benigni and coworkers reported that administering
bosentan to RRM rats also slows renal deterioration, attenuates progression of
hypertension, and reduces proteinuria. They administered bosentan once daily by gavage
at a dose of 100 mg/kg for 120 days and recorded BP by tail plethysmography. As in the
previous study utilizing FR139317, bosentan did not reverse the hypertension or renal
deterioration in these ligated RRM rats but only attenuated their progression. Benigni’s
report of beneﬁcial results using bosentan in RRM are a little surprising because one
would expect that blocking endothelial ETB receptor mediated release of NO should
oppose blockade of ETA receptor mediated vasoconstriction. These investigators did ﬁnd
that there is up-regulation of the ETB receptor gene and increased mRNA levels in RRM
as the disease progresses. Whether this is a counterregulatory mechanism in response to
vasoconstriction or involved in the maintenance of that vasoconstriction is currently being
investigated. Not all reports have demonstrated beneﬁcial effects when administering
ETRA’s in RRM. One group of investigators gave A-127722 to RRM rats and found that
prophylactic administration of this ETARA immediately following completion of the
partial nephrectomy by the ligation method did not prevent hypertension development nor

retard renal deterioration (Polakowski and Pollock, 1996 ).

52

b. ET activity in the excision method

To date there has been no studies investigating the role of ET in the RRM model
utilizing the excision method. Since the excision method of RRM is the appropriate
model of CRF, investigations utilizing ETRA’s in this model could provide information
that is clinical relevant.

From these studies it seems clear that further investigation is needed to understand
the roles that each ET receptor subtype plays in renal disease and to determine if
optimization of drug therapy can lead to the ultimate goal of reversal of hypertension and
renal deterioration in RRM. The second major part of my experimental approach was
directed towards determining the contribution of ET to RRM hypertension and renal
deterioration, particularly in the excision method and when the disease is well
established.

V. Blood Pressure Regulation

The major purpose of my research is to study the mechanism(s) of hypertension
associated with CRF (using the RRM excision model). It is important therefore to review
current understanding of how BP levels are established. The mechanisms by which BP is
regulated in the short-term are not identical to the mechanisms involved in long-term BP
control. Rapid alterations in arterial pressure are generally achieved through adjustment
of vascular resistance by local tissue mechanisms (myogenic and humoral), and reﬂexly
regulated neural and hormonal systems. These vasoconstrictor effects increase BP over
relatively short periods of time, i.e. seconds to minutes, in response to a variety of
external and internal stimuli. Long-term control mechanisms serve to establish a

relatively stable "setpoint" for BP over time periods of weeks to months. The major

53

means by which this type of regulation is achieved are believed to be: adjustment of body
ﬂuid volumes by the kidney; and the development of vascular wall thickening due to
smooth muscle proliferation or remodeling. Neural and hormonal signals also might
contribute to long-term BP regulation, either through modulating renal function or
vascular structure, or by directly affecting cardiac or vascular function.

Hypertension may result from a disorder of short-term or long-term BP control
mechanisms. The implication for my work is that a complete evaluation of the possible
causes of hypertension in CRF requires investigation of physiological systems involved in
both short-term and long-term BP regulation. This will be achieved by examining the
effects of acute and chronic therapy with speciﬁc pharmacological inhibitors of the renin-

angiotensin and endothelin systems.

54
VI. Speciﬁc aims

The overall purpose of my thesis work was to understand the role of hormonal
factors in the hypertension that is commonly associated with CRF. My general
hypothesis was that hormonal factors, speciﬁcally, AngH and ET-l, contribute to the
maintenance of hypertension in RRM rats, and that their relative importance differs
depending on the dietary intake of salt. I tested the following speciﬁc hypotheses in
conscious, chronically instrumented rats:

(1) If the RAS exerts short-term control of BP in hypertensive RRM rats, acute ACEI
treatment will lower blood pressure over minutes to hours.
(2) If the RAS exerts long-term control of BP in hypertensive RRM rats, chronic

ACEI treatment will lower BP over days to weeks.

(3) ACEI lower BP in hypertensive RRM rats only by decreasing the concentration
of AngH in the blood.

(4) The level of salt intake inﬂuences the antihypertensive actions of ACEI in RRM.

(5) If ET-l exerts short-term control of BP in hypertensive RRM rats, acute ETRA
treatment will lower BP over minutes to hours.

(6) If ET-l exerts long-term control of BP in hypertensive RRM rats, chronic ETRA
treatment will lower BP over days to weeks.

(7) The level of salt intake inﬂuences the antihypertensive actions of ETRA

in RRM hypertension.

METHODS

I. Animals

Male Sprague-Dawley rats (Sasco-King Animal Laboratories, Madison, WI)
weighing between 200-250g were used in all experiments. The rats were housed in clear
plastic boxes with woodchip bedding in a climate controlled room with a 12 hour light-
dark cycle. Rats were housed two to a box and allowed unlimited access to standard
rodent chow (Rodent Laboratory Chow #5001, Purina, St. Louis, MO) and tap water
while awaiting surgery.
H. Surgical procedures

A. General

Surgical anesthesia was performed with pentobarbital sodium 45-50 mg/kg i.p.
(NembutalQ, Abbott Laboratories, Chicago, IL). Atropine sulfate 0.2mg/kg i.p. (Sigma
Chemical Co., St Louis, MO) was also administered prior to all surgical procedures.
Methohexital sodium 5-10 mg/kg i.v. (Brevital®, Eli Lilly, Indianapolis, IN) was used
for supplemental anesthesia during catheterization. Surgical instruments were sterilized
by autoclaving at 200 0C for 40 minutes. Aseptic procedure was followed throughout all
surgical procedures and loss of body temperature was prevented by operating while the
rats were on a heating pad. Post-operatively, a 0.5mg/kg subcutaneous dose of
butorphanol tartrate (Stadol®, Bristol Laboratories, Princeton, NJ) was given as an
analgesic.

55

56

B. Reduction of renal mass

A 5/6 reduction of renal mass was accomplished by a two-stage subtotal
nephrectomy. The ﬁrst stage of the procedure involved the surgical excision of
approximately 2/3 of the left kidney mass. Under anesthesia, the left ﬂank was shaved
and washed with an iodine antiseptic cleanser (Betadine®, Purdue Frederick Co.,
Norwalk, CT). A lateral incision was made, the kidney was exteriorized, and the renal
artery and vein were isolated. The vessels were brieﬂy occluded with a bulldog clamp.
While the vessels were occluded, the two poles of the left kidney are surgically excised
with a scalpel. The exposed surfaces of the remnant kidney were cauterized and lightly
covered with sterile thrombin for topical use (Thrombostat®, Parke-Davis Co., Ann
Arbor, MD to prevent excess bleeding. Total ischemia lasted less than two minutes. After
bleeding had completely stopped, the kidney was replaced into the abdominal cavity.
Muscle and skin were individually sutured and a prophylactic dose of ticarcillin sodium
40mg/kg s.c. (T icar®, Smith Kline-Beecham, Pittsburgh, PA) was given. The rats were
allowed 7 days for surgical recovery and nephron adaptation before the second stage of
the nephrectomy was started. Using the same anesthetic method, the right ﬂank was
shaved as described previously and the right kidney was exteriorized. This time, the renal
artery and vein were ligated and the right kidney was removed. Antibiotics and analgesia
were given as previously described. Kidney weights from both surgeries were recorded to
calculate the reduction in renal mass. The sum of the two stages of surgical nephrectomy
resulted in approximately a 5/6 reduction of kidney mass with a functional remnant
kidney left intact. The rats were housed in clear plastic boxes as described previously

while awaiting catheterization.

57

C. Arterial and venous catheterization

Catheters were constructed of polyvinyl chloride (Tygon®, Microbore) with
silicone rubber tubing (Silastic®, Dow Corning, Midland, MI) attached to the
intravascular end. Catheters were advanced through the internal iliac artery and vein to
the abdominal aorta and vena cava. The catheters were tunneled s.c. along the back and
exteriorized at the head. The catheters were fed through a stainless steel spring which
was then anchored to the skull using jeweler's screws and dental acrylic. The arterial
catheter was ﬁlled with a heparinized sucrose solution and occluded when not in use.
The venous catheter was attached through a hydraulic pivoting swivel to a 5 ml syringe.

This syringe was ﬁlled with a NaCl solution which was infused continuously at a rate of

5ml/24hrs (2 mEq Na+/24hrs) with a Harvard infusion pump. Post surgery, animals were
placed in metabolism cages while awaiting experimentation. During the next three
recovery days, 40mg/kg ticarcillin sodium i.v. was administered to each rat. This
antibiotic regimen was also given as needed during the experimental protocol.
IH. Chronic rat model

After catheterization, rats were individually housed in metabolism cages to allow
daily monitoring of water intake, urine output and urinary electrolytes. A hydraulic
swivel mounted above the cage allowed the animals freedom of movement and unlimited
access to sodium-deﬁcient rat chow (Teklad, Madison, WI) and drinking water. In rats
receiving continuous i.v. drug treatment, pharmacological agents were added to the

intravenous NaCl infusion on the appropriate experimental days.

58

IV. Hemodynamic measurements

BP was recorded daily for 15-20 minutes between 8:00 am and 12:00 pm from
the arterial catheter using a pressure transducer (Model P50, Statham). The pressure
signal was run through a digitizer (Stiemke, Madison, WI) and recorded on a polygraph
(Model 7, Grass Instrument Corp). HR was determined directly from the trace of the
polygraph. To test the blockade of ACE when administering ACEI, a 50ng bolus of AngI
was given. The magnitude of ACE inhibition by ACEI‘s was estimated from the pressor
response to the AngI bolus. Any changes in BP were recorded for up to 30 seconds after
the injection. Blockade of endothelin receptors by endothelin receptor antagonists was
assessed by inhibition of the pressor and depressor responses to exogenously
administered i.v. bolus of ET-1 at 0.5 nmol/kg. Inhibition of depressor responses due to
ETB receptor blockade were recorded over a period of 0-15 seconds. In contrast, changes
in pressor response to ET-l due to ETA blockade were monitored for 15-20 minutes after
the ET-l bolus and the peak pressor response over that time frame was reported.
V. Fluid and electrolyte measurements

Voluntary water intake (WI) was measured from calibrated cylinders and total WI
was calculated by adding the water drank in 24 hours and the volume of the intravenous
saline infusion. Urinary output (UO) was collected over 24 hours in a calibrated cup.

Water balance (WB) was calculated by subtracting UO from total WI. Urinary sodium

(UN,+) was determined by sample analysis with a ﬂame photometer (Model 1L943,

Instrumentation Lab.) Total urinary sodium excretion (UNaV) was calculated by

multiplying UN; times the 24 hour UO.

59
VI. Salt protocols

Experiments involving ACEI and ETRA in RRM rats were designed to compare
their effects on BP under differing NaCl intakes. The ACEI and ETRA were tested during
3 distinct periods that represented the extremes of salt intake. High salt (HS) rats drank
isotonic saline (0.9% NaCl) starting one day following completion of the reduction in
kidney mass or sham operation. Rats were allowed a two week period of time to develop
hypertension and renal failure. They were then chronically instrumented with arterial
and venous catheters and placed in metabolism cages. After arterial and venous
catheterization, all rats were fed sodium deﬁcient rat chow (0.002 mEq Na+ lgm) and
received an additional Na” intake of 2mEq/24 hours through the venous catheter. These
HS rats remained on the oral and venous saline solutions for the remainder of the
experiment, and this resulted in a 5-6 fold increase in daily salt intake (normal:
approximately 2 mEq/24hr on standard rat chow). Normal salt (NS) rats were kept on
normal rat chow and distilled drinking water for 30 days after partial renal ablation or
sham operation, while awaiting catheterization. After catheterization and throughout the
rest of the experiment, the rats ate sodium deﬁcient chow and received 2 mEq/24 hr NaCl
through the venous catheter. These procedures resulted in a normal salt intake of 2
mEq/24 hr in the rat. The low salt (LS) group of rats were kept on a sodium deﬁcient
chow and distilled water for 60 days following completion of the 5/6 nephrectomy or
sham-operations while awaiting catheterization. After catheterization, all rats received a

dextrose solution through the venous catheter replacing the normal NaCl solution given.

60
VH. Assays

A. Plasma assays

After the daily hemodynamic parameters were recorded, blood samples were
drawn directly from the exteriorized arterial catheter into a syringe containing heparin (5
USP units/ml [32 ug/ml]). Samples were immediately spun in a refrigerated
microcentrifuge to separate the plasma, which was then stored frozen at -70°C until
assayed. All blood samples were analyzed in the same assay to control for interassay
variability.

1. Blood urea nitrogen

Blood samples of 0.7m] were collected in a 1.0ml syringe containing 0.05m] of
heparin sodium. Blood urea nitrogen (BUN) was determined by a colorimetric assay
using a prepared assay kit, (# 640, Sigma Chemical Co., St. Louis, MO.), involving
ammonia production. A plasma sample of 10ul was assayed against an individual
standard curve for each assay. Normal rat values for BUN vary from 15-22 mg/dl (Biven
et al., 1979).

2. Serum creatinine

Blood samples of 0.7ml were collected in a 1.0ml syringe containing 0.05m]
heparin sodium. Serum creatinine (Scr) was determined by a colorimetric assay using a
prepared kit (#555, from Sigma) involving the alkaline picrate method. A serum sample
of 300ul was assayed against an individual standard curve for each assay. Mean normal
rat serum creatinine ranges from 0.4 to 1.0 mg/dl depending on the analytical method

used (Biven et al., 1979).

6 l
3. Endothelin

Two m1 of blood collected into an inhibitor cocktail containing EDTA (2mg/ml)
and heparin (5 USP units/ml) was microcentrifuged to separate 1.0 ml of plasma on days
differing from other plasma sampling. Blood cells were resuspended in normal saline and
injected back i.v. into the rat. Plasma ET-l [ET] p concentrations were determined using
a solid phase ELISA kit (Parameter, R & D Systems, Minneapolis, MN, USA). ET-1 was
extracted from 1 ml of plasma with 1.5 ml of extraction solvent composed of acetonele
HCszater (40:1:5). The mixture was centrifuged for 20 min at 3000 rpm at 4° C. The
supernatant was dried down with a centrifugal evaporator, the pellet reconstituted in
sample diluent and assayed. Optical density readings of unknown samples were plotted
against a standard curve of synthetic ET-l spiked rat plasma samples over a range of 1-
113 pg/ml. The recovery from the extraction procedure was 36 i 3%. The interassay
variation was 8.24% and the intra-assay variation was 10.15%. Cross-reactivity with
other isoforms was demonstrated to be less than 1%. Assays were performed by Edie
Quemby-Brown at Parke-Davis Pharmaceuticals Research Division.

B. Urine assays

Urine samples were taken from 24 hour urine collections into calibrated cylinders.
All samples were spun in a refrigerated microcentrifuge to separate any particulate matter
and then stored at - 700 C until assayed. All urine samples were analyzed in the same
assay to control for interassay variability. The urinary values reported were normalized

per 100 grams body weight of each rat.

62

1. Urinary protein excretion
Urine samples of 100ul were collected and protein concentration was determined
by a colorimetric assay using a prepared kit (#541, Sigma) involving a modiﬁed version
of the Lowry method. Urinary protein excretion (Upro) was calculated by multiplying the
daily urine output times the protein concentration determined from this assay. Normal rat
values for total protein excretion are reported to be < 30 mg/dl (Biven et al., 1979).
2. Urinary creatinine concentration
A urinary sample of 300ul was collected and assayed to determine urinary
creatinine concentration (Ucr) using the same assay as used for serum creatinine from
Sigma.
3. Creatinine clearance
Creatinine clearance (Ccr) was calculated by the formula UO x Ucr/ Scr.
VIH. Statistics
Data were analyzed using a mixed-design ANOVA. Post-hoc tests included: least
signiﬁcant difference, simple main effects, Dunnetts, t-test, Bonferroni’s, and analysis of
contrasts. Criterion for statistical signiﬁcance was a "p-value" less than 0.05. Computer

software (Crunch® Version 4) was used for statistical analysis.

EXPERIMENTAL RESULTS

1. Renin angiotensin system in reduced renal mass
A. Acute experiments
1. Bolus i.v. administration of ACEI in RRM and sham rats on high,

normal, and low salt intakes.
a. Rationale
The purpose of this experiment was to determine the contribution of the RAS to
short-term BP regulation in RRM rats under the three different salt extremes. Since the
primary short-term mechanism of BP regulation by the RAS is through direct
vasoconstriction, a process which can be initiated or terminated within seconds to a few
minutes, acute BP responses were measured to i.v. bolus injections of the ACEI,
enalaprilat, in RM and sham rats. The hypothesis was, if the RAS exerts short-term
control of BP in RRM rats during any of these 3 salt intakes, acute ACEI treatment
should lower BP within minutes to hours.
b. Protocol
Daily control measurements were taken prior to administration of an i.v. bolus of
enalaprilat at 5mg/kg in RM and sham rats maintained on the 3 levels of salt intake. BP

was recorded from 5 minutes up to 24 hours after drug administration.

63

c. Results

Figure 2 presents BP data from acute treatment with enalaprilat in RRM and
sham rats. The top graph illustrates the stratiﬁcation of BP observed in RRM rats during
the different levels of NaCl intake prior to enalaprilat administration. HS rats started off
with the highest BP, followed by the NS group of rats. The BP of LS rats was only
mildly elevated and these values are not considered to be in the hypertensive range. All
the RRM rats maintained on HS and NS had higher resting BP’s than the corresponding
sham groups during the control measurements. In all 3 groups of RRM rats, BP was not
acutely lowered over minutes to hours following enalaprilat administration. NS rats did
show a delayed antihypertensive effect at 6 and 24 hours after enalaprilat.

The bottom graph illustrates the hypotensive effect of enalaprilat in sham rats
under varying salt intakes. BP was decreased at every time point in each treatment group,
but only reached statistical signiﬁcance. in sham rats on a low salt intake, under
conditions when the RAS is known to be stimulated. In normal rats, a lower dose (1
mg/kg) of enalaprilat (Figure 3) was shown to signiﬁcantly inhibit the AngI pressor
response for up to 5 hours.

(1. Interpretations

Other investigators have demonstrated the salt sensitivity of BP in RRM rats and
have observed similar differences in BP (Langston et al., 1963). It was concluded from
these results that AngH plays an important role in the maintenance of short-term BP
regulation only in normal rats on a low salt intake. It would be expected that the RAS
exerts its greatest role under conditions of salt deprivation. What was unexpected was

that when RRM rats were kept in a salt depleted state, ACEI treatment did not lower BP

65
acutely. A possible explanation for the differing hypotensive effect in RRM vs. sham rats

at all salt intakes is that RRM rats have lower circulating levels of AngH than the
corresponding sham rats. AngH levels were not measured in these experiments, but it is
generally agreed that the RM model is a low renin model of hypertension. In fact, most
studies have shown that plasma AngH levels in RRM rats are normal or decreased relative
to that of sham rats. (Ylitalo, 1976). Another possible explanation for the lack of an
antihypertensive effect in RRM is that the doses used were ineffective in inhibiting AngH
formation. Data presented in Figure 3 demonstrate that even at lower doses of enalaprilat
(1mg/kg), pressor responses to AngI are inhibited acutely. AngI has little pressor activity
in itself and the pressor response to AngI in untreated animals is mainly due to its
conversion by ACE to the vasoconstrictor AngH. These data show that the doses used
were effective in inhibiting ACE over the course of my experiment, and that the negative
results reported are not due to inadequate inhibition of the enzyme.

These data indicate that AngH does not exert a direct vasoconstrictor fast pressor
effect in RRM rats to maintain BP. On the contrary, the maintenance of BP in normal rats
maintained on a low salt intake appears .to be partly dependent on the vasoconstrictor

properties of AngH.

66

Figure 2: Acute i.v. enalaprilat administration in RRM and sham rats on high,
normal, and low salt intakes. *Asterisks indicate reductions in MAP from the zero

control value.

MAP (mmHg)

MAP (mmHg)

180

160

140

120

100

80

120

100

80

67

H High salt (5)
H Normal salt (8)
H Low salt (7)

 

 

 

 

 

 

SHAM

 

I-I High salt (3)
H Normal salt (5)
H Low salt (5) -

 

 

01 5' 15'

Enalaprilat
(5 mg/kg)

30' 60'

Time (min)

Figure 2

II
120' 360' 24HR

68

Figure 3: AngI pressor responses in normal rats administered i.v. dextrose or
enalaprilat at 1mg/kg. *Asterisks indicate reductions in pressor response from average of

control values.

69

 

 

 

 

 

75

   

Dextrose

Egg
gig/Z

    

 

 

5
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70

B. Chronic experiments
1. Chronic administration of ACEI in hypertensive RRM rats on a
high salt intake.
a. Rationale

A clinically relevant model of CRF in humans is RRM rats placed on an elevated
NaCl intake. The increased salt intake is generally considered to be detrimental to renal
function, and increased salt intake is known to suppress the RAS. This experiment was
designed to determine the involvement of the RAS in maintaining chronic hypertension
when RRM rats are on a high salt intake. I hypothesized that, if the RAS exerts long
term control of BP in RRM rats on a high salt intake, then chronic ACEI treatment
should lower BP over days to weeks.

b. Protocol

All rats were subjected to RRM and drank isotonic saline starting one day
following completion of the surgery. As previously described in the methods section,
these HS rats were allowed a two week period of time to develop hypertension while on
increased salt intake. The rats were divided into two experimental groups after
catheterization. One-half the rats received enalapril at 250mg/L in the oral isotonic
saline solution. The other rats drank only the isotonic saline solution, and they served as
the control group. Three days of control measurements were followed by 14 days of
enalapril treatment or vehicle during the HS period. Then, distilled water was substituted
for the oral saline solution for the last 7 days resulting in a normal salt intake (2mEq/24

hours). Rats in the treatment group still received enalapril during NS intake. BUN

71

samples and AngI challenges were carried out during control, high, and normal salt
experimental periods in both groups.
c. Results

Figure 4 shows BP throughout the 24 days of this experiment. During the 3
control days, BP was slightly elevated and similar in both groups of RRM rats. A
progressive increase in BP was seen in both the enalapril and vehicle treated groups
during HS. The magnitude of pressure change was not different between groups.
Likewise, a similar fall in BP was seen in the two groups after initiation of NS.
Moreover, during these last 7 days of the experiment while the rats were on NS no
difference in BP between groups was recorded.

Since some investigators believe that a primary mechanism by which AngH acts
on long-term BP regulation in CRF is through stimulating tubular retention of water and
sodium (Bakris, and Gavras, 1993), water and sodium balance were recorded throughout
the experiment. Data on WI, U0, and WB from the vehicle and enalapril treated RRM
rats during conditions of HS and NS are shown in Figure 5. Because of the inﬂuence of
an increased salt appetite in RRM rats, both groups had an increased WI and U0
throughout the 14 days of HS administration. These measurements returned to a more
normal level immediately after discontinuation of the salt addition in the drinking water.

Figure 6 presents urinary sodium excretion (UNaV) and sodium balance (NaB) in
the two groups over the course of the experiment. Both groups of RRM rats showed a
signiﬁcant natriuresis during the high salt administration days when compared to the
normal salt days. The addition of NaCl to the drinking water resulted in an increased

sodium excretion. Inhibition of AngH formation by enalapril did not affect UN3V or N aB

72

during HS or NS experimental periods. There were no sustained differences in UNaV or
NaB between the vehicle and enalapril treated groups throughout the experiment.

Table 1 shows the pressor response to a 50ng i.v. bolus of Angl during control,
high, and normal salt days in both the vehicle and enalapril treated RRM rats. The
average daily dose of enalapril in this eXperiment was calculated to be 13.5 mg. A
signiﬁcant decrease in the Angl pressor response was found during enalapril treatment as
compared to both the within group control value and the between group non-treated
values. This indicated persistent, successful blockade of ACE in enalapril treated rats.

Table 1 also presents BUN values in vehicle and enalapril treated RRM rats
during control, high, and normal salt intake periods. Both of the RRM groups had mildly
elevated BUN levels throughout the protocol compared to normal values reported in our
lab and by others (Kanagy, 1991; Pollock et al., 1993). During NS, elevated BUN values
from controls were measured in both groups.

(1. Interpretations

From these blood pressure data, it was concluded that when RRM hypertension
was allowed to develop in rats on a high salt intake, the RAS played no role in the long-
term regulation of BP, even when the rats were subsequently allowed to ingest a more
"normal" level of salt. The lack of an antihypertensive effect in enalapril treated rats was
not due to inadequate blockade of ACE because pressor responses to Angl during
enalapril administration were successfully blocked. These data also suggest that ACEI
do not reduce BP by mechanisms other than decreasing AngH formation.

Mildly elevated BUN levels reﬂect a decreased GFR in both groups of RRM rats

and suggests some decrease in renal function occurred. Any progression of renal

73

deterioration that might have occurred was not measurable during the high salt
administration. BUN values have been shown in some studies to progressively increase
over time in RRM rats but a two week interval may not be sufﬁcient to clearly
demonstrate such a change (Kaufman er al., 1974). Conversely, during NS, clearly
elevated BUN values were measured in vehicle and enalapril treated rats. The increased
BUN values during the NS in both groups may be explained due to a sudden drop in BP
in addition to a slowly progressive deterioration of renal function. Sudden falls in
pressure can result in decreases in renal perfusion pressure with resultant decreases in
excretion of urea nitrogen. One explanation for the observation that the BUN values in
the enalapril treated rats were elevated when compared to those of vehicle treated rats
during normal salt intake might be that a larger decrease in glomerular hydrostatic
ﬁltration pressure due to vasodilation of the efferent arteriole by enalapril may have
caused a larger decline in GFR in the treated rats.

In this experiment, inhibition of AngH formation by enalapril administration did
not affect WI, UO or WB. Yet, AngH is known to stimulate drinking behavior under
normal conditions. This may be explained due to the low RAS activity normally
observed in HS situations. Under conditions of increased sodium intake, the
physiological actions of AngH on stimulating drinking behavior would be expected to be
minimal. These data suggest that the inﬂuence of AngH on WB under conditions of high
salt is negligible.

The NaB data agree with the WB data described above in that the inﬂuence of

AngH on NaB in RRM rats under high salt conditions was negligible. It was concluded

74

that the RAS does not contribute to chronic water and sodium balance in RRM rats under
these conditions.

Chronic experiments involving enalapril in sham rats maintained on HS were not
performed because of the lack of an antihypertensive effect in RRM rats under these

conditions.

75

Figure 4: Mean arterial pressure responses to chronic enalapril administration in

RRM rats on high salt intakes.

 

 

MAP (mmHg)

160

140

120

100

80

76

 

 

 

 

 

 

 

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77

Figure 5: Water intakes, urine outputs and water balances in response to chronic

enalapril administration on high salt intakes.

 

 

Water intake (mL/24hr)

Urine output (mL/24hr)

Water balance (mL/24hr)

100

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79

Figure 6: Urinary sodium excretions and sodium balances in response to chronic
enalapril administration in RRM rats on high salt intakes. All rats drank an isotonic saline
solution and were administered 2 mEq/day NaCL i.v. *Asterisk indicates reduction in

sodium balance from the control values.

Na+ Excretion (mEq/24hr)

Na+ Balance (mEq/24hr)

80

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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' 82
2. Chronic administration of ACEI in hypertensive RRM and sham

rats on a normal salt intake.
a. Rationale
Part of the work described in this thesis was aimed at characterizing how salt

intake inﬂuences ACEI in RRM hypertension. Given the inverse relationship between
dietary salt intake and RAS activity, it is reasonable to assume that the relative
importance of the RAS in the pathophysiology of renal failure is larger under conditions
of lower dietary salt intake. This issue is particularly important when considering the
therapy of CRF, since dietary salt restriction is an established part of the treatment regime
(Brown et al., 1971). Thus, the next series of experiments were designed to determine
the contribution of the RAS to long-term BP regulation in RRM rats on a lower, more
normal salt intake. Based on previously published reports it was expected that ACEI
treatment would lower BP in RRM rats on NS. But the mechanism by which this occurs
is not fully understood. As mentioned in the introduction of this thesis, factors such as
inhibition of local tissue RAS and/or kinin potentiation have been proposed to mediate
most or all of ACEI effects on BP reduction. A corollary question addressed in this
experiment was: do the ACEI produce antihypertensive effects in RRM rats only through
the inhibition of AngH formation?

b. Protocol

The rats were maintained on the normal salt protocol as described in the methods

section. After arterial and venous catheterization, RRM and sham rats were divided into
four treatment groups: vehicle group, oral enalapril at 250mg/L, and in two groups of rats

drinking enalale at 250mg/L, constant i.v. infusions of 2 and 4 ng/min of AngH were

83

administered to restore circulating AngH concentrations. The experiment lasted 18 days
total: 3 control days, followed by 1 day of i.v. ACEI treatment, followed by 7 days of oral
ACEI treatment and ending with 7 recovery days with no treatment. IV ACEI treatment
consisted of a 5mg/kg bolus of enalaprilat which was administered to all groups that
subsequently received the oral enalapril (250mg/L). The vehicle group of rats served as
control, receiving no i.v. or oral ACEI. BUN samples and Angl challenges were carried
out during control, treatment and recovery periods in all groups.
c. Results

Figure 7A presents data from chronic BP recordings in RRM rats on NS. Enalapril
treatment at 250mg/L in hypertensive RRM rats on NS prevented the continuing rise in
BP observed in vehicle treated animals. The chronic treatment data from this experiment
suggest that the RAS plays a signiﬁcant role in long-term BP regulation in RRM
hypertension. The known ability of these drugs to inﬂuence BP through mechanisms
other than inhibition of AngH formation led me to test directly the mechanism of action
of enalapril. It was hypothesized that it is only the blockade of AngH formation by
enalapril that prevents the progressive rise in BP observed in the vehicle treated rats. To
test this hypothesis, enalapril treated rats received continuous infusions of AngH at rates
designed to restore normal circulating concentrations of the peptide. If enalapril was
preventing hypertension development in RRM rats only by blocking AngH formation, this
treatment should fully restore hypertension.

BP data from the groups of RRM rats drinking enalapril at 250mg/L and receiving
continuous i.v. replacement AngH at 2 and 4 ng/min is shown in Figures 7B and 7C.

Replacement of circulating AngH at 2 ng/min during enalapril treatment restored the

84

progressive rise in BP observed in the vehicle group during the treatment. Daggers
indicate a signiﬁcant increase in BP from the vehicle group in rats administered
replacement AngH at 4 ng/min (Figure 7C). All groups are shown for comparison with
no error bars, asterisks, or daggers in Figure 7D.

Figure 8 shows data from chronic BP recordings in sham rats on NS. Enalapril
treatment at 250mg/L in normotensive sham rats decreased BP from the 3 control days
every day during the treatment (Figure 8A). These data suggest that AngH is a necessary
component of long term BP regulation in normal rats on a normal salt intake. It was tested
in sham rats if the effects on BP during enalapril treatment could be reversed by
continuous infusions of AngH at rates designed to restore normal circulating
concentrations of the peptide. An additional group of sham rats given enalapril and
administered lng/min AngH infusion was incorporated into the study to further
characterize the level of replacement AngH needed to restore BP to levels measured in
untreated sham rats. Data from 3 different AngH infusion rates in enalapril treated sham
rats on NS are shown in Figures 8B-D. Rats administered enalapril and 1 ng/min i.v.
AngH still had signiﬁcantly reduced BP from control levels (Figure SB). Replacement of
circulating AngH at a rate of 2 ng/min i.v. during oral enalapril treatment prevented the
reduction in BP recorded in the enalapril only group (Figure 8C). Additionally, this rate
restored the normal BP observed in the vehicle group. When the replacement rate of
AngH was increased to 4ng/min, BP was again restored to the normal BP observed in the
vehicle group while not being elevated into the hypertensive range (Figure 8D). Figure 9

shows all sham groups for comparison of BP with no error bars or asterisks.

85
The pressor responses to 50ng bolus’s of Angl were signiﬁcantly inhibited in all

RRM and sham rats drinking enalapril during the treatment period (Table 2). The
signiﬁcant decrease in Angl pressor responses found during treatment indicates the
successful blockade of ACE and the suppression of endogenous AngH formation in all
RRM and sham groups administered enalapril.

Table 2 also contains BUN values in sham and RRM rats during control,
treatment, and recovery periods. All of the RRM groups had elevated BUN levels
compared to sham values. Enalapril treatment alone or with replacement AngH did not
alter BUN levels in sham or RRM rats during the treatment period.

There were no measurable differences in WI, UO, or WB in any of the RRM
(Figure 10) or sham (Figure 11) rats throughout the experiment when groups were
individually compared to their control days. Enalapril treatment alone or with the
addition of low infusion rates of AngH, did not affect overall WB in any group of RRM
or sham rats. There was an increased WI and compensatory UO in RRM rats when
compared to the sham rats in each treatment group.

Figure 12 presents UNaV in all groups of RRM rats over the course of the
experiment. The UMV were generally in the 1-2 mEq/24hr range which correlated well
with the i.v. saline infusion administered at a rate calibrated to deliver 2 mEq Na+l24hr.
There were no measurable differences in UNaV in any of the RM rats when compared to
the within group control days, except for the last 3 recovery days in the enalapril 250
mg/L + 4ng AngH group. Enalapril treatment alone or with the addition of low infusion

rates of AngH did not affect natriuresis or overall NaB in any group of RRM rats.

86

Figure 13 shows UMV in ﬁve groups of sham rats on NS. Enalapril treatment
alone in sham rats was associated with a significant natriuresis that developed only after
several days on enalapril administration. The natriuretic effect of enalapril was reversed
by two days after enalapril discontinuation. In fact, the majority of the recovery days
were associated with a signiﬁcant sodium retention. There was no change in UNaV in the
vehicle group or any of the groups drinking enalapril and receiving replacement AngH.

d. Interpretations

The data from this experiment support a role of the RAS in long-term BP
regulation in RRM rats on NS. The progressive rise in BP observed in non-treated RRM
rats was prevented by ACEI treatment; this effect was totally reversed by continuous i.v
infusion of AngH at a rate of 2 ng/min. This low replacement rate of AngH was expected
to produce systemic peptide concentrations within the physiologic range as has been
demonstrated by other investigators (Hall and Brands, 1993). If enalapril was working
through mechanisms other than the inhibition of AngH formation, then replacing AngH
systemically should have not been able to reverse ACEI full effect on BP. Data from this
experiment support the hypothesis that ACEI prevents the progression of hypertension in
RRM by blockade of endogenous AngH formation and not by other putative mechanisms.

The data from this experiment also supports the role of the RAS in long-term BP
regulation in sham rats on NS. Exogenous AngH replacement at a continuous rate of 1
ng/min partially attenuated the hypotensive effect of enalapril observed in untreated sham
rats and 2 ng/min restored the basal level of BP. These data suggest that AngH is a

necessary component of BP regulation in normal rats under NS conditions. Data from

87
this experiment support the hypothesis that ACEI lower BP in normal rats only by

blockade of endogenous AngH formation.

One of the experimental strategies was to inhibit the endogenous production of
AngH with ACEI. The signiﬁcant decreases in Angl pressor responses found during the
treatment period indicate successful blockade of ACE and the suppression of endogenous
AngH formation in all RRM and sham groups administered enalapril. Suppression of
endogenous AngH formation facilitated an accurate assessment of the involvement of the
RAS through the use of intravenously administered AngH.

The elevated BUN levels in the RRM groups reﬂect a decreased GFR which
suggests RRM groups had a signiﬁcantly reduced renal function. None of the sham
groups exhibited elevations in BUN from normal measurements. There was only a slight
progression in BUN levels that did not reach signiﬁcance over the course of this
experiment in RRM receiving no treatment (37.3 i 3.5 to 43.0 i 3.5 mg/dl). Enalapril
treatment alone or with replacement AngH did not alter BUN levels in any group of RRM
rats during the treatment period, which suggests a lack of a renoprotective effect of ACEI
under these conditions of salt intake and length of investigation.

Enalapril dosages were similar in each of the sham groups and generally less than
in any of the RRM groups (Table 2). The increased enalapril consumption in RRM
groups was due to an elevated WI. As shown from data above, all of these dosages
inhibit AngH formation to a similar extent yet some evidence suggests that ACEI in
dosages in excess of those required to lower BP may impart additional beneﬁt to

glomerular structure and function (Ikoma et al, 1991). This seemed not to be the case in

88

this experiment because enalapril administration at 250 mg/L did not reverse or change
the progression of renal deterioration in RRM rats.

Even though when compared to sham, RRM rats had increased WI and U0, these
measures offset each other so as to prevent increases in WB. One of the main results of
activation of the RAS is an increased Na+ and H20 reabsorption from the proximal tubule
(Bakris and Gavras, 1993) therefore inhibition of AngH production would be expected to
result in a natriuresis and diuresis. This was not observed during treatment in RRM rats.
It must be kept in mind that BP was lower in enalale treated than untreated RRM
groups. The pressure-natriuresis theory dictates that lower BP’s cause decreases in
sodium and water excretion. This phenomenon may have canceled out any loss of AngH
stimulated tubule reabsorption during ACEI administration. Another consideration is that
the activity of the RAS is low to normal in RM and the reduction of an AngH mediated
effect due to inhibition of already low plasma AngH concentrations may not exert an
effect readily observable using our methods.

The natriuretic response observed in enalapril treated sham rats during the
treatment period was most likely due to decreased AngH stimulated tubular reabsorption.
This natriuretic effect may even have been greater if it were not for the BP lowering
inﬂuence on natriuresis. The substantial hypotensive effect of enalapril suggests that
activity of the RAS is signiﬁcant in sham rats on NS. This explains why the natriuretic
response to ACE inhibition was measurable in sham rats. The decrease in sodium
excretion after withdrawal of enalapril supports the role of AngH in mediating sodium

reabsorption.

89

Figure 7: Mean arterial pressure responses to chronic enalapril administration with
and without replacement AngH in RRM rats on normal salt intakes. Panel A depicts
vehicle and enalapril only treated groups. Panel B depicts vehicle and group administered
enalapril + AngH replacement at 2ng/min. Panel C depicts vehicle and group
administered enalapril + AngH replacement at 4ng/min. Panel D depicts all groups with
no error bars or signiﬁcance denotations for overall comparisons. *Asterisks indicate
reductions in MAP from the vehicle group. ’r Daggers denote increases in MAP from the

vehicle group.

 

MAP (mmHg)

0
MAP (mmHg)

MAP (mmHg)

U

MAP (mmHg)

180

160

140

120

100
200

180
160
140
120
100

200
180
160
140
120
100

 

 

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Control

Figure 7

Treatment
Protocol Day

Recovery

91

Figure 8: Mean arterial pressure responses to chronic enalapril administration with
and without replacement AngII in sham rats on normal salt intakes. Panel A depicts
vehicle and enalapril only treated groups. Panel B depicts vehicle and group administered
enalapril + AngII replacement at lug/min. Panel C depicts vehicle and group
administered enalapril + Angl] replacement at 2ng/min. Panel D depicts vehicle and
group administered enalapril + AngH replacement at 4ng/min. *Asterisks indicate

reductions in MAP from the vehicle group.

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Figure 8

Treatment
Protocol Day

Recovery

93

Figure 9: Mean arterial pressure responses to chronic enalapril administration with
and without replacement AngII in sham rats on normal salt intakes. This graph depicts

all groups for overall comparisons with no error bars or significance denotations.

 

 

MAP (mmHg)

94

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Figure 10: Water intakes, urine outputs and water balances in response to chronic
enalapril administration with and without replacement AngII in RRM rats on normal salt

intakes.

Urine Output (mL/24hr) Water Intake (mL/24hr)

Water Balance (mL/24hr)

98

H Vehicle (8)

RRM H Enalapril 250mg/L (8)

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Treatment Recovery
Protocol Day

99

Figure 11: Water intakes, urine outputs and water balances in response to chronic
enalapril administration with and without replacement AngH in sham rats on normal salt
intakes. *Asterisks indicates reduction in WB from within group control values.

1 Daggers denote increases in WB from within group control values.

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Water Balance (mL/24hr)

100

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101

Figure 12: Urinary sodium excretions in response to chronic enalade administration
with and without replacement AngH in RRM rats on normal salt intakes. All rats were
maintained on a ﬁxed sodium intake of 2 mEq/day administered i.v. *Asterisks indicate

increases in sodium excretion from within group control values.

Na+ Excretion (mEq/day)

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102

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103

Figure 13: Urinary sodium excretions in response to chronic enalapril administration
with and without replacement AngH in sham rats on normal salt intakes. All rats were
maintained on a ﬁxed sodium intake of 2 mEq/day administered i.v. *Asterisks indicate
reductions in sodium excretion from within group control values. 1‘ Daggers denote

increases in sodium excretion from within group control values.

Na" Excretion (mEq/day)

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104

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105

3. Chronic administration of ACEI in RRM and sham rats on a low
salt intake.
a. Rationale
Since the inverse relationship between dietary salt intake and RAS activity

appears to hold true in renal failure (Ylitalo et al.,l976), it was hypothesized that the
relative importance of the RAS in the pathophysiology of renal failure is largest under
conditions of a dietary intake devoid of sodium. As mentioned earlier, this is a critical
point given that dietary salt restriction is an important part of the therapy of CRF in
humans. The purpose of this experiment was to conﬁrm the importance of the RAS in
long-term BP regulation in RRM rats on a low intake of NaCl.

b. Protocol

In this experimental protocol the same enalapril treatment that was used in the NS

experiments was used except now rats were maintained on LS. As described in the
methods section, this LS protocol involves sham and RRM rats fed a sodium deﬁcient
chow for 8 weeks prior to experimentation. After arterial and venous catheterization,
RRM and sham rats were divided into four treatment groups: vehicle group, oral enalapril
at 250mg/L, and in two groups of rats drinking enalapril at 250mg/L, constant i.v.
infusions of 4 and 10 ng/min of AngH were administered to restore circulating AngH
concentrations. The experiment lasted 18 days total: 3 control days, followed by 1 day of
i.v. ACEI treatment, followed by 7 days of oral ACEI treatment and ending with 7
recovery days with no treatment. IV ACEI treatment consisted of a 5mg/kg bolus of
enalaprilat which was administered to all groups that subsequently received the oral

enalapril (250mg/L). The vehicle group of rats served as control, receiving no i.v. or oral

106

ACEI. BUN samples and Angl challenges were carried out during control, treatment and
recovery periods in all groups.
c. Results

Figure 14 outlines BP data from 4 groups of RRM rats on a LS. In Figure 14A,
enalapril alone signiﬁcantly reduced BP from the 3 control days during the treatment
period and 2-3 days after enalapril was replaced by distilled water in the recovery period.
This hypotensive effect in the recovery period after discontinuation of enalapril treatment
in RRM rats was not recorded in sham rats on the same experimental protocol (Figure
15A). Figures 14B and 14C demonstrate that replacement of endogenous AngII at rates of
4 and lOng/min does not restore the basal level of BP observed in the vehicle group of
RRIVI rats. Figure 14D depicts BP from all groups without error bars or asterisks for
comparison.

Figure 15 presents BP data from 4 groups of sham rats on LS. In Figure 15A,
enalapril alone signiﬁcantly reduced BP from the control days during the treatment
period. Figures 15B and 15C show that replacement of exogenous AngII at rates of 4 and
IOng/min does not restore the basal level of BP observed in the vehicle group. Figure
15D depicts BP from all groups without error bars or asterisks for comparison.

The pressor responses to Angl were signiﬁcantly inhibited in RRM and sham
groups drinking enalapril during the treatment period (Table 3). The signiﬁcant decrease
in Angl pressor responses found during enalapril treatment indicates successful blockade
of ACE and the suppression of endogenous Angl] formation in enalapril treated rats.

Data from Table 3 also shows BUN values in sham and RM rats during control,

treatment, and recovery periods. All of the RRM groups had elevated BUN levels

107
compared to sham values. These elevated BUN levels reflect a decreased GFR which

suggests the RRM groups did have decreased renal function. Enalapril treatment alone or
with replacement AnglI did not alter BUN levels in sham or RRM rats during the
treatment period.

Figure 16 shows WI, U0, and WB in RRM rats on LS. In general WI and U0
were slightly increased in RRM rats as compared to the sham rats (Figure 17) in each
group. WB was not consistently changed from control levels in the groups receiving
enalapril alone or with the addition of replacement AngII. Figure 17 presents WI, U0 and
WB in sham rats on LS. WB was not changed from control levels in the groups receiving
enalapril alone or with the addition of any infusion rate of AngII.

Figure 18 shows UNaV in both RRM and sham rats maintained on LS. Urinary
sodium excretion was not affected by enalapril administration alone or in combination
with AngII replacement in either RRM or sham rats under these sodium deplete
conditions.

(1. Interpretation

Urinary Na“ excretion and BUN data conﬁrm that experimental manipulations
were successful in achieving the desired effects on salt intake and renal function. The
Na” excretory rates in both sham and RRM rats (zero to 0.1 mEq/24h) were barely
detectable and suggest that interventions aimed at restricting salt intake were successful.
Additionally, BUN levels in RRM rats were all elevated after 2 months, even in the
absence of hypertension, suggesting that the desired reduction in renal function by partial

kidney ablation was accomplished in this experiment.

108

Since blockade of endogenous AngII formation by enalapril lowered BP in the
normotensive rat on a sodium-restricted diet, these results suggest that Angl] is required
for the maintenance of basal BP under these conditions. Additionally, this experiment
supports the role of the RAS in long-term BP regulation in RRM rats on LS. It is
interesting that BP in RRM and sham rats was not different prior to treatment. Each
group of rats was maintained on a low NaCl diet for two months following subtotal renal
ablation. The original expectations were that B? would be elevated in the hypertensive
range in RRM rats. A thorough literature search revealed that some investigators have
shown an increased BP in RRM rats maintained on low salt intakes (Purkerson et al.,
1976; Lax et al., 1992) but most have not (Dworkin et al., 1996; Ylitalo et al., 1976;
Dipette et al., 1983). Also unexpected was that the hypotensive effect of enalapril
treatment in sham and RRM rats was similar in magnitude in both groups during the
treatment period. I anticipated that enalapril would lower BP to a greater extent in sham
than RRM rats based on my NS conclusions. Nonetheless, there is some reason to
believe that the mechanism of the hypotensive effect of enalapril was different in sham
and RRM rats. First, the fall in BP in sham rats was rapid (Figure 15A ), but required
many days to reach a maximum in RRM rats (Figure 14A). Second, during the recovery
period RRM and sham groups exhibited a disparate pattern in restoration of basal BP: the
RRM rats had a prolonged hypotensive effect after discontinuation of enalade as
compared to the sham rats. The delayed restoration of basal BP could be due to a
lingering inhibition of serum ACE by enalapril. Yet Angl pressor responses were back to
control levels during days 2—3 of the recovery period, suggesting that ACE activity was

intact during this recovery (Table 3). Another difference between sham and RRM was the

109

responses to replacement of Angl] at 10 ng/min during the enalapril treatment period.
Sham rats exhibited less of an hypotensive effect than RRM rats in the ﬁrst two days of
treatment. This difference suggests that higher doses of AngII may be required in RRM
rats when compared to sham rats to fully reverse enalapril’s hypotensive effects. These
results are consistent with the possibility that in sham rats on a very low sodium intake
Angl] affected basal BP via the fast pressor effect, but that in RRM rats under similar
conditions it was the slow pressor effect of AngII that contributed to basal BP regulation.
The failure to restore normal BP in sodium-depleted, ACEI treated rats with even a high
infusion rate of AngII (10 ng/min) was not unexpected, because both the fast and slow
pressor effects of Angl] are signiﬁcantly impaired by sodium restriction (Cowley and

McCaa, 1976).

110

Figure 14: Mean arterial pressure responses to chronic enalapril administration with
and without replacement AngH in RRM rats on low salt intakes. Panel A depicts vehicle
and enalapril only treated groups. Panel B depicts vehicle and group administered
enalapril + AngH replacement at 4ng/min. Panel C depicts vehicle and group
administered enalapril + AngH replacement at IOng/min. Panel D depicts all groups with
no error bars or signiﬁcance denotations for overall comparisons. *Asterisks indicate

reductions in MAP from within group control measurements.

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125

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111

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112

Figure 15: Mean arterial pressure responses to chronic enalapril administration with
and without replacement AngII in sham rats on low salt intakes. Panel A depicts vehicle
and enalapril only treated groups. Panel B depicts vehicle and group administered
enalapril + AngII replacement at 4ng/min. Panel C depicts vehicle and group
administered enalapril + AngH replacement at 10ng/min. Panel D depicts all groups with
no error bars or signiﬁcance denotations for overall comparisons. *Asterisks indicate

reductions in MAP from within group control measurements.

113

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 16: Water intakes, urine outputs and water balances in response to chronic
enalapril administration with and without replacement Angl] in RRM rats on low salt
intakes. *Asterisks indicate increases in water balance from within group control

measurements.

 

Urine Output (mL/24hr) Water Intake (mL/24hr)

Water Balance (mL/24hr)

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Figure 16

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117

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118

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119

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120

Figure 18: Urinary sodium excretions in RRM and sham rats administered enalapril
with and without replacement AngII on low salt intakes. All rats were maintained on a

sodium deﬁcient diet and dextrose i.v. infusion.

 

N 3+ Excretion (mEq/24hr)

N a+ Excretion (mEq/24hr)

121

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122

II. Endothelin system and reduced renal mass
A. Acute Experiments
1. Acute i.v. administration of an ETA (PD147953) or an ETA/ET};
(PD145065) receptor antagonist in ET-l induced hypertension.
a. Rationale
In this study, the efﬁcacy of PD147953 and PD145065 in reversing the chronic
hypertensive response in rats caused by systemic infusion of ET-1 was tested. PD147953
has been characterized as a selective ETARA from binding studies in the rat and rabbit
(Doherty et al., 1993). PD147953 exhibits 1000 times more selective binding for ETA
verses ETB receptors in the rat VSMC. PD145065 has been characterized as a non-
selective ETA/ETBRA from binding and functional studies (Doherty et al., 1993).
Previous work in our lab has shown that continuous i.v. administration of ET-1 at 5
pmol/kg/min produces a sustained hypertension in normal rats (Mortensen and Pink,
1992b). The speciﬁc aim of this study was to determine if acute i.v. administration of
either peptide receptor antagonist would lower BP in this model. The current view is that
the endothelial cell ETBI receptor initiates release of nitric oxide and/or prostacyclin
upon ET-l binding, which results in vasodilation. It was expected that a mixed
ETA/ETBRA would be less efﬁcacious in lowering BP because of blockade of the release
of vasodilators.
b. Protocol
Two groups (n=5) of male Sprague-Dawley rats weighing 350-400gm were
catheterized for hemodynamic measurements and the continuous i.v. infusion of ET-1 at

5 pmong/min in a saline solution calibrated to deliver a sodium intake of 6.0 mEq/24

123
hours. The experiment lasted a total of 15 days; 3 control days followed by 7 days of

continuously administered ET-1, and ending with 5 recovery days. One-half hour
infusions of each ETRA (0.1 mg/kg/min) were administered on four representative days
covering each experimental period. BP was recorded at 5‘,15‘,30‘,60‘, and 120‘ after the
start of each ETRA infusion on these 4 days.
c. Results

Continuous infusion of ET-1 at a rate of 5 pmol/kg/min in normal rats produced a
sustained increase in BP. Data are shown in Figure 19, from experimental days when the
antagonists were administered. Thirty minute i.v. administration of PD147953
signiﬁcantly reduced BP from daily control values between 30-120‘ on both ET-l
infusion days. This effect was not observed during non-ET-l infused days (control and
recovery). PD145065 significantly reduced BP on protocol day E3 from 15-120‘ during
ET-l infusion but on day E7 the BP lowering effect did not reach statistical significance.
The antihypertensive effect was not observed during non-ET—l infused days (control and
recovery).

d. Interpretations

These results demonstrated that PD147953 and to a less consistent extent
PD145065 were able to reversibly inhibit the chronic hypertensive response to exogenous
ET-l. The latency of the antihypertensive effect is thought to be due to the difﬁculty of
displacing endogenous ET from its binding sites. ET-l increases BP mainly by
stimulating ETA receptors. The dose used here was the starting point for additional
experiments designed to evaluate the role of endogenous ET-1 in short-term BP

regulation in RRM rats under varying salt intakes.

124

Figure 19: Acute mean arterial pressure responses to ETA (PD147953) and ETA/ETB
(PD145065) receptor antagonist infusion in ET-l induced hypertension. Each antagonist
was infused at a rate of 0.1 mg/kg/min for 30 minutes in normal rats (n=5). Infusion
lengths are indicated by representative arrows during each protocol day. *Asterisks

indicate decreases in pressure from daily control measurement.

 

125

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126
2. Acute i.v. administration of an ETA (PD147953) or an ETA/ETB

(PD145065) receptor antagonist in hypertensive RM and sham

rats on a high salt intake.

a. Rationale

ET-l has the potential to act both acutely and chronically to promote hypertension

through a combination of short-term systemic vasoconstriction, and long-term effects on
vascular structure, renal function or neural BP control mechanisms. The purpose of this
experiment was to deﬁne the contribution of ET to short-term BP regulation in RRM rats
on a high salt diet. The hypothesis was: if ET exerts short-term control of BP in RRM
rats on a high salt diet by direct vascular actions, acute ETRA treatment should lower
BP over seconds to minutes, since theses drugs are known to rapidly block the vascular
responses to ET (Gardiner et al., 1994). Since the acute antihypertensive effect of the
ETRA’s in the ET-l infusion study did not result in the complete normalization of BP, it
was decided to add two larger doses of these ETRA in this experiment involving RRM
and sham rats.

b. Protocol

Rats were subjected to either RRM or sham operation. All rats drank isotonic

saline starting one day following completion of the surgery according to the HS protocol.
Control BP measurements were taken prior to the start of twenty minute infusions of each
antagonist. PD147953 and PD145065 were infused i.v. at 3 different rates: 0.1, 0.3, and
1.0 mg/kg/min in RRM and sham rats on separate days. BP was recorded from 5 minutes
to 6 hours after the start of the antagonist infusions and was taken for the last time 24

hours later. Each rat received both antagonists separated by at least 2 days.

127

c. Results

Figure 20 presents BP data from acute ETARA treatment in RRM and sham rats
on HS. The RRM rats all had an elevated BP compared to the sham rats prior to the start
of each antagonist infusion. In RRM rats there was a delayed, dose-dependent decrease
in BP from 30 minutes to 2 hours after the administration of PD147953. Some initial
lowering of BP was observed at each infusion rate but the most pronounced and
prolonged effect was observed with the highest infusion rate (1.0 mg/kg/min). In sham
rats after 20 minute infusions of the ETARA, there were no changes in BP from 5
minutes up to 24 hours at any dose.

BP data are contained in Figure 21 from acute ETA/ETBRA (PD145065) treatment
in the same RM and sham rats on HS. In RRM rats, there was a delayed, dose-
dependent decrease in BP from 30 minutes to 2 hours after administration of each of the
higher antagonist infusion rates (0.3 - 1.0 mg/kg/min). In fact each of these infusion rates
produced the same fall in BP in RRM rats. In the sham rats after 20 minute infusions of
PD145065, there were no changes in BP from 5 minutes up to 24 hours at any dose.

(1. Interpretations

These data show that each ETRA, at the infusion rates used, does not lower BP in
sham normotensive rats on HS. These results suggest that ET-1 is not involved in short-
terrn BP regulation in normotensive rats when on HS. On the other hand, the magnitude
and the duration of the fall in BP was similar after ETARA and ETA/ETBRA treatment in
RRM rats especially at the 1.0 mg/kg/min rate. These data suggest that ET, acting
, primarily on the ETA receptor subtype, contributes to the short-term regulation of BP in

RRM rats on HS. The ETB receptor subtype does not appear important in short-term

128
regulation of BP in RRM rats. Subsequent studies in RRM used the highest infusion

rate from this study to compare the antihypertensive effects of selective versus non-

selective endothelin receptor blockade, in rats on lower salt intakes.

129

Figure 20: Acute mean arterial pressure responses to ETA (PD147953) receptor
antagonist infusions in RRM and sham rats on high salt intakes. The antagonist was

infused at three different rates for 20 minutes. *Asterisks indicate decreases in pressure

from ZCI'O COl‘ltl‘Ol measurement.

180

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131

Figure 21: Acute mean arterial pressure responses to ETA/ET}; (PD145065) receptor
antagonist infusions in RRM and sham rats on high salt intakes. The antagonist was
infused at three different rates for 20 minutes. *Asterisks indicate decreases in pressure

from zero control measurement.

 

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132

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133
3. Comparison of acute i.v. administration of an ETA (PD147953) or

an ETA/ETB (PD145065) receptor antagonist, in RRM and sham
rats on high, normal, and low salt intakes.
a. Rationale
Currently, the inﬂuence of salt intake on the physiological and pathophysiological
functions of ET are uncertain. Previous work in our lab demonstrated the salt-
dependency of hypertension produced by chronic i.v. infusion of ET-1 in normal rats
(Mortensen and Fink, 1992b). Also, an antihypertensive action of ETRA's has been
demonstrated in RRM rats on HS. Since sodium balance plays an integral role in the
development of hypertension in RRM, the purpose of this study was to characterize the
relationship between ET, BP, and sodium intake in this model. In this experiment, the
hypothesis was investigated that the contribution of ET to short-term BP regulation in
RRM rats was altered by varying salt intake.
b. Protocol
The three levels of NaCl intake described in the methods section were utilized for
the acute administration of ETARA and ETA/ETBRA in RRM and sham rats. Control BP
recordings were taken prior to 20 minute i.v. infusions of PD147953 and PD145065 at
1.0 mg/kg/min on separate days. BP recordings were taken acutely from 5 minutes to 24
hours after the start of the infusion.
c. Results
Figure 22 presents BP data following acute PD147953 and PD145065 treatment
in RRM and sham rats on a high salt intake. These are the same data shown in the last

experiment but now I have only reported the 1.0 mg/kg/min infusion results. BP was

134

severely elevated in RRM rats as compared to sham prior to antagonist administration.
Each antagonist caused a delayed decrease in BP in RRM but not in sham rats. The
magnitude and the duration of the decrease in BP was similar after ETARA and
ETA/ETBRA treatment. I

Figure 23 presents BP data following acute PD147953 and PD145065 treatment
in RM and sham rats on a normal salt intake. BP was modestly elevated in RRM rats
as compared to sham prior to antagonist administration. Twenty minute infusions of
either antagonist did not cause a decrease in BP in RRM or sham rats on NS.

Figure 24 shows BP data following acute PD147953 and PD145065 treatment in
RRM and sham rats on a low salt intake. As in previous experiments, hypertension was
not observed in RRM rats maintained on LS. Therefore at the start of the experiment, BP
in RM and sham groups was not different and within the normotensive range. Neither
antagonist caused a decrease in BP in RRM or sham rats on LS. Once again, the lack of a
hypotensive effect in RM and sham rats on LS after ETARA and ETA/ETBRA
administration suggests that ET-1 does not appear important in short-term arterial
pressure regulation under low salt conditions.

(1. Interpretations

It was concluded from these data that the lack of an antihypertensive effect in
RRM or a hypotensive effect in sham rats on NS or LS after ETARA and ETA/ETBRA
treatment suggests that ET—1 does not appear important in short-term arterial pressure
regulation under these conditions. The overall conclusion from this set of experiments
was that ET contributes to short-term arterial pressure regulation through actions at the

ETA receptor subtype in hypertensive RRM rats only during high salt intake.

135

Figure 22: Acute mean arterial pressure responses to ETA (PD147953) and ETA/ET};
(PD145065) receptor antagonist infusions in RRM and sham rats on high salt intakes.
Each antagonist was infused at 1.0 mg/kg/min for 20 minutes. *Asterisks indicate

decreases in pressure from zero control measurement.

MAP (mmHg)

MAP (mmHg)

200

180

160

140

120

100

160

140

120

100

 

 

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137

Figure 23: Acute mean arterial pressure responses to ETA (PD147953) and ETA/ET};
(PD145065) receptor antagonist infusions in RRM and sham rats on normal salt intakes.

Each antagonist was infused at 1.0 mg/kg/min for 20 minutes.

 

160

80

138

Normal salt

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139

Figure 24: Acute mean arterial pressure responses to ETA (PD147953) and ETA/ETB
(PD145065) receptor antagonist infusions in RRM and sham rats on low salt intakes.

Each antagonist was infused at 1.0 mg/kg/min for 20 minutes.

 

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MAP (mmHg)

160

140

120

100

80

160

140

120

100

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140

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141

B. Chronic experiments
1. Oral ETA (PD155080) receptor antagonist treatment in the
hypertension induced by continuous i.v. ET-l infusion.
a. Rationale
One of the main goals of this research was to uncover evidence for a chronic
inﬂuence of ET-1 on arterial pressure regulation in RRM using ETRA. Therefore, initial
experiments were performed to demonstrate the efﬁcacy of ET-1 receptor blockade in
reversing long-term cardiovascular effects of ET-1. To this end rats were made
hypertensive by continuous i.v. infusion of ET-1 for several days, then treated with the
ETARA, PD155080 to establish the antihypertensive speciﬁcity of this drug and to
determine a dose to use in the RRM model. PD155080 is a potent competitive inhibitor
of both ETA and ETB receptors having ICso values of 7.4 and 4500 nM respectively for
each receptor subtype (Doherty et al., 1995). This orally active compound has a
bioavailability of 87% and a half-life of Shours in the rat so it was a good candidate for
chronic administration in these studies (Doherty et al., 1995).
b. Protocol
Normal Sprague-Dawley rats weighing 350—400gm were instrumented with
arterial and venous catheters for hemodynamic measurements and continuous i.v.
infusions of ET-1. The rats were divided into 4 groups: the vehicle group (vehicle, n=5)
was administered an i.v. saline solution calibrated to deliver 6.0 mEq Na+l24 hours. In
another group of rats (ET-1 2.5, n=5) 2.5 pmol/kg/min ET—1 was continuously
administered in the i.v. saline solution. PD155080 was given orally to the last two groups

of rats that received ET-l i.v. at 2.5 pmol/kg/min (ET-1 2.5 + PD155080, n=5) or 5

142
pmol/kg/min (ET-1 5.0 + PD155080, n=5). The experiment lasted a total of 12 days; 2

control days were followed by 10 days of continuously administered saline alone or
saline plus ET-l. During ET-l administration, 3 days of infusion were allowed to
establish an increased BP, at which time ETRA treated rats were given PD155080 (25
mg/kg bid.) for 5 days. PD155080 was given orally in powder form mixed with sodium
deﬁcient chow in powder form. All rats in the study were given chow twice a day; 5
grams in the morning (9-10 am) and 7.5 grams in the evening (5-8 pm). The experiment
ended with two days of recordings after PD 155080 had been discontinued. The 3
groups receiving ET-l infusions were kept on the infusions these last two days while the
vehicle group just received the saline solution.
0. Results

Continuous infusion of ET-1 at a rate of 2.5 pmol/kg/min in normal rats produced
a slowly developing, sustained hypertension (Figure 25A). Oral administration of
PD155080 for 5 days resulted in a complete normalization of BP. After discontinuation of
PD155080, BP returned to hypertensive levels within one day. Continuous infusion of
ET-1 at a rate of 5 pmol/kg/min in normal rats produced a rapidly developing, sustained
hypertension (Figure 25B). The magnitude of the BP increase was greater in these rats
than in rats given 2.5 pmol/kg/min. Oral administration of PD155080 for 5 days resulted
in a sustained antihypertensive effect that did not quite reach normotensive levels. After
discontinuation of PD155080, BP returned to hypertensive levels within one day. Figure
25B also shows PD155080 administration in rats that received saline only i.v. Saline

infusion alone in normal rats did not increase blood pressure. PD155080 resulted in a

143

slight hypotensive effect during the ﬁrst 2 days of administration. Upon discontinuation
of PD155080, BP rose to mildly hypertensive levels during 2 recovery days.
d. Interpretations
These results show that PD155080 at 25mg/kg bid was able to fully and
reversibly inhibit the chronic hypertensive response to exogenous ET-l at 2.5 and 5.0
pmol/kg/min. Thus, this antagonist dose was utilized in subsequent experiments designed

to evaluate the effect of endogenous ET-l on BP regulation in RRM rats.

144

Figure 25: Chronic mean arterial pressure responses to ETA (PD155080, 25 mg/kg
b.i.d.) receptor antagonist administration in ET-l induced hypertension. All rats were
administered a saline infusion calibrated to deliver a sodium intake of 6.0 mEq/day
through the i.v. catheter. Panel A depicts MAP responses to PD155080 treatment in
normal rats continuously infused ET-l (2.5 pmol/kg/min). Panel B depicts MAP
responses to PD155080 treatment in normal rats continuously infused ET-l (5.0
pmol/kg/min) or saline infusion alone. *Asterisks indicate increases in blood pressure
from within group control measurements. 1‘ Daggers denote decreases in blood pressure

from within group control measurements.

MAP (mmHg)

MAP (mmHg)

180

160

140

120

100

80

180

160

140

120

100

80

 

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145

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Figure 25

146
2. Oral ETA (PD155080) receptor antagonist treatment in RRM rats

on high, normal and low salt intakes.
a. Rationale
Pilot experiments were performed to determine the optimal antihypertensive dose
of PD155080 in RRM rats. In addition, the relative importance of ET-1 was tested in the
maintenance of BP under varying salt intakes. My previous results with acute
administration of peptide ETRA’s suggested that RRM rats on higher salt intakes would
respond best to chronic endothelin receptor blockade.
b. Protocol
i. High salt, normal salt
In this pilot study, I tested the antihypertensive effect of PD155080 at 100 mg/kg
b.i.d. in RRM rats maintained on high and normal salt intakes. Two rats from each group
were catheterized and given PD155080 as previously described for 2 days following 2
days of control measurement. BP was recorded for 2 recovery days after PD155080
discontinuation to monitor for reversal of any drug effect.
ii. Low salt
Two rats maintained on L8 were catheterized and given PD155080 for 5 days
following 3 days of control measurement. They were given PD155080 at the lower dose
of 25 mg/kg b.i.d. BP was recorded for 3 recovery days after PD155080 discontinuation
to monitor for reversal of any hypotensive effect.
c. Results
Figure 26 shows BP in RRM rats maintained on HS or NS. During the 2 control

days, both groups had severe and sustained hypertension. PD155080 administration

147
substantially lowered BP in both groups with NS RRM rats exhibiting the largest fall in

BP. The antihypertensive effects of PD155080 were totally reversed at 24 hours after the
discontinuation of ETRA treatment in both groups.

PD155080 lowered BP throughout all 5 treatment days in RRM rats maintained
on LS and this hypotensive effect was reversed during the recovery days (Figure 27). But
similar to my previous experiments, RRM rats in this experiment did not reach
hypertensive levels when kept on LS.

d. Interpretations

The results from these pilot studies suggest that ET is involved in BP regulation in
RRM over a period of days. The extraordinary antihypertensive effect of PD155080 in
RRM rats maintained on HS and NS actually gave cause for concern. This work has
focused on antihypertensive therapies in experimental renal failure, but drastic falls in
blood pressure over such a short period of time can actually initiate acute renal failure.
Therefore, lower doses of PD155080 were tested in RRM rats to look for the lowest dose
necessary to achieve a signiﬁcant reduction in BP. It was determined from these pilot
studies that 25 mg/kg given twice daily was an effective antihypertensive dose in RRM
rats. These preliminary experiments established a dosing regimen for further
investigation over longer periods of ETRA administration in RRM. From these results it
was decided to focus on the involvement of ET in RRM only under NS conditions for a
variety of reasons. RRM rats maintained on NS most closely resemble the clinical setting
of CRF and results obtained under these conditions would have the most therapeutic
relevance. The other conditions represent extremes of salt intake; therefore, experimental

pitfalls occurred. An unacceptable degree of mortality (50% at 1 month) was observed in

148
RRM rats given saline to drink in the HS protocols. Under LS conditions, RRM rats did

not become hypertensive over the time frame of investigation (3 months). The other
important factor in these investigations was the availability of the newly developed
ETRA. PD155080 is not commercially available and was a generous gift from Parke-

Davis Pharmaceutical Research, so supplies were limited.

149

Figure 26: Chronic mean arterial pressure responses to ETA (PD155080, 100
mg/kg/b.i.d.) receptor antagonist administration in RRM rats maintained on high and

normal salt intakes.

150

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o—o Normal Salt (2)

 

 

 

 

 

 

 

 

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Figure 26

 

15]

Figure 27: Chronic mean arterial pressure responses to ETA (PD155080, 25

mg/kg/b.i.d.) receptor antagonist administration in RRM rats on low salt intakes.

152

Low Salt (2)

 

 

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Control

PD155080

Protocol Day

Figure 27

Recovery

 

153
3. Oral ETA (PD155080) receptor antagonist treatment in

established hypertensive RRM rats on a normal salt intake.
a. Rationale
The mechanisms by which ET may play a role in long-term BP regulation are
currently speculative. As mentioned before, a clinically relevant scenario would be
reversal of hypertension by ETRA administration without worsening renal function. The
next experiment therefore was designed to determine the contribution of ET-1 to long
term BP regulation in RRM rats one month after renal ablation. The hypothesis was that
chronic administration of the oral ETARA, PD155080, would lower BP in RRM rats with
established hypertension on N S.
b. Protocol
PD155080 (25mg/kg b.i.d.) was administered in powder form mixed into sodium
deﬁcient chow in powder form as described previously. The experiment lasted a total of
15 days; 3 control days followed by 7 treatment days and ending with 5 recovery days.
Arterial blood was sampled for ET-l plasma levels and PD155080 plasma concentrations
during each of these experimental periods. To assess the degree of endothelin receptor
blockade, i.v. bolus’s of ET-1 were administered and the level of blockade was estimated
from the inhibition of the pressor (mediated by ETA receptors) and depressor responses
(mediated by ETB receptors). Changes in renal function were monitored by measuring
BUN, serum creatinine, urinary protein excretion, and creatinine clearance.
0. Results '
Figure 28 presents the effect of PD155080 on BP in RRM and sham rats in NS.

Untreated RRM rats demonstrated a sustained, gradually progressive hypertension

154
throughout the experiment. Hypertensive RRM rats receiving PD155080 exhibited a

signiﬁcant and well-maintained decline in BP throughout the treatment period. This
antihypertensive effect was reversed by 24 hours after discontinuation of PD155080. In
fact, during the recovery period, BP was signiﬁcantly higher than during the pretreatment
control period. Norrnotensive sham rats given PD155080 showed a slight, inconsistent
hypotensive effect during the treatment period when compared to the BP during the 3
control days.

Figure 29 shows pressor and depressor responses to exogenously administered
ET-l (0.5 nmol/kg) in sham and RRM rats during each experimental period. Depressor
responses were unchanged in individual groups from the control period values except for
the sham rats given PD155080 (-19.8 mmHg control vs. -13.6 mmHg treatment). Pressor
responses during all experimental periods in RRM rats were generally lower than those
observed in the sham groups, but PD155080 treatment did not signiﬁcantly impair acute
pressor responses to ET-1 in either group of rats

There were no measurable differences in WB (Figure 30) or UNaV (Figure 31)
throughout the experiment in untreated or PD155080 treated sham rats. In RRM rats, the
ﬁrst day of PD155080 administration resulted in a signiﬁcant decrease in UNaV when
compared control days (treatment: 1.22 i 0.1 vs. control: 1.68 i 0.2 mEq Na+/24 hours).
A concomitant increase in WB was observed on the ﬁrst day of PD155080 treatment
(treatment: 20.2 i 3.6 vs. control: 11.0 i 2.1 ml/24 hours) but this did not reach
statistical signiﬁcance. Upon discontinuation of PD155080 in RRM rats during the ﬁrst
recovery day, a signiﬁcant increase in UNaV was observed when compared to control days

(recovery: 2.20 :t 0.3 vs. control: 1.68 p i 0.2 mEq Na+l24 hours). This signiﬁcant

155

increase in UNaV was coupled with a decrease in WB during the ﬁrst recovery day, which
did not reach statistical signiﬁcance (recovery: 8.2 i 2.1 vs control: 11.0 i 2.1 ml/24
hours

Indices of glomerular function during PD155080 treatment in sham and RRM rats
are shown in Table 4. As expected, BUN, Scr and Upro were all elevated and Ccr
decreased in RRM rats compared to sham rats. In sham rats, 7 day treatment with
PD155080 caused no signiﬁcant changes in glomerular function. Likewise PD155080
administration in RRM rats did not cause signiﬁcant changes in glomerular function,
despite a strong antihypertensive effect observed during the treatment period.

(1. Interpretations

This study showed in RRM rats studied 4 weeks after reduction in renal mass that
BP was signiﬁcantly higher than that of sham rats. But plasma ET-l concentrations were
similar in the two groups (Table 4), conﬁrming an earlier published report (Benigni et al.,
1991). ET—l plasma levels were not signiﬁcantly different between any groups during the
experiment except for a slight elevation in non-treated RRM rats during the recovery
period (control: 1.0 :t 0.1 vs. recovery: 1.7 i 0.] pg/ml). Nonetheless, one-week
treatment with PD155080 caused a signiﬁcant and sustained decrease in BP in RRM rats,
while producing only a modest hypotensive effect in normotensive sham-operated
animals. It is noteworthy that PD155080 administration in both sham and RM groups
did elevate plasma ET-l concentrations from the control levels, but these changes did not
reach statistical signiﬁcance. The difference in BP response was not due to impaired
elimination of PD155080 in rats with remnant kidneys, since plasma levels of the drug at

the time of BP measurements were similar in both sham and RRM rats (sham: 7.50 i 1.9

156
vs. RRM: 6.26 i 4.1 ug/ml). It is not possible from these data to determine if the

synthesis and release of ET-1 from vascular endothelial cells (or other tissues) is
increased in RRM rats. Schiffrin and colleagues (Schiffrin et al., 1995; Sventek et al.,
1996) reported increased vascular ET-l gene expression in several models of
hypertension in rats, but this has not been investigated in the RRM model. Failure to
observe elevated plasma levels of ET-1 in the RRM rats in this study does not rule out
increased ET—l release from endothelial cells, because most of this secretion probably
occurs abluminally.

It has been established that release of ET-1 from endothelial cells in the systemic
vasculature causes vasoconstriction and smooth muscle cell growth by stimulating ETA
receptors (Rubanyi and Polokoff, 1994). The time course of the antihypertensive response
to PD155080 in this experiment was too short for reversal of vascular structural changes.
Therefore, inhibition of ET-1 induced vasoconstriction probably accounted for the BP
lowering effect of ETA receptor blockade in the RRM rats. Some of the data though seem
inconsistent with this conclusion. For example, measurement of acute BP changes to
bolus injections of ET-1 revealed that neither pressor (presumably mediated via vascular
ETA receptors) nor depressor (presumably mediated via endothelial ETBI receptors)
responses were signiﬁcantly inhibited in rats receiving PD155080; yet resting BP in RRM
rats was markedly decreased. Since only a single, high dose of ET-1 was used to assess
receptor blockade acutely, these results may simply reﬂect the expected greater difﬁculty
in showing receptor antagonism against higher doses of agonist. An alternative
explanation is that endogenous ET-l raises BP in RRM rats by an action on receptors

distinct from those affected by acute i.v. boluses of the peptide, perhaps in the brain,

157

adrenal gland or other organs involved in BP regulation.

Increased ET-l gene expression in the kidney and elevated urinary excretion of
ET-1 occur in RRM rats, indicating enhanced intrarenal synthesis of the peptide (Orisio et
al., 1993). Renal ET-l causes sodium retention so blockade of renal ET receptors by
PD155080 could cause a fall in BP by promoting ﬂuid excretion via the kidney. Recent
work suggests, however, that most actions of ET-1 in the rat kidney are mediated through
ETB receptors, making this explanation unlikely (Pollock et al., 1993; Wellings et al.,
1994: Qiu et al., 1995). These results also do not support such an explanation in that
PD155080 administration in RRM rats was associated with sodium and water retention
rather than diuresis and natriuresis.

These ﬁndings indicate that ETA receptor blockade may be an effective therapy
for the hypertension associated with CRF. It is obviously important, however, that any
such therapy not further impair renal glomerular function. It is therefore noteworthy that
the antihypertensive response to PD155080 in RRM rats was not accompanied by any
measurable decrease in creatinine clearance, or increase in plasma creatinine, urea

nitrogen or urinary protein excretion over the short time-course of this experiment.

158

Figure 28: Chronic mean arterial pressure responses to ETA (PD155080, 25
mg/kg/b.i.d.) receptor antagonist administration in RRM and sham rats on normal salt
intakes. *Asterisks indicate decreases in pressure from within group control

measurements. 1‘ Daggers denote increases in pressure from within group control values.

 

MAP (mmHg)

MAP (mmHg)

180

160

140

120

100

80

140

120

100

80

 

 

159

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Protocol Day

Figure 28

160

Figure 29: Acute pressor and depressor responses to bolus i.v. ET-l (0.5 nmol/kg)
injections in sham and RRM rats on normal salt intakes during control, treatment and
recovery experimental periods. *Asterisks indicate decreases in responses from within
group control measurements. 1 Daggers denote decreases in pressor responses between

RRM and sham groups within each individual experimental period.

 

ET-l Depressor (mmHg) ET-l Pressor (mmHg)

50

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-20

-30

-40

-50

161

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162

Figure 30: Water intakes, urine outputs and water balances in response to chronic
ETA (PD155080, 25 mg/kg/b.i.d.) receptor antagonist administration in sham and RRM

rats on normal salt intakes.

 

 

 

163

H sham (5)
H sham + PD155080 (5)
0—0 RM (5)
H RRM + PD155080 (5)

 

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80-

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Protocol Day
Figure 30

 

164

Figure 31: Urinary sodium excretions during chronic ETA (PD155080, 25
mg/kg/b.i.d.) receptor antagonist administration in sham and RRM rats on normal salt
intakes. All rats were maintained on a ﬁxed sodium intake of 2 mEq/day administered in
the i.v. saline solution. *Asterisk indicates decreases in sodium excretion from within
group control values. 1‘ Dagger denotes increases in sodium excretions from within group

control values.

 

Na” Excretion (mEq/day)

3.0

2.5

2.0

1.5

1.0

0.5

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165

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DISCUSSION

A close relationship exists between hypertension and CRF. Hypertension, the
second leading cause of renal disease, is commonly observed in renal failure patients that
have progressed to ESRD. Long-term multicenter studies of renal disease patients have
concluded that elevations of BP are a strong independent risk factor for ESRD (Klag et
al., 1996). Many recent studies have shown that control of BP is associated with a
slower rate of decline in renal function (Brazy et al., 1989; Pettinger et al., 1989) and
reduced risk of hypertension-related diseases.

The primary purpose of the experiments described here was to investigate the role
of humoral factors, speciﬁcally, Angl] and ET, in the pathogenesis of hypertension in the
RRM model of CRF. I hypothesized that the relative contribution of these two hormones
to both short-term and long-term BP regulation in CRF differs depending on the level of
salt intake. The work is highly relevant to the treatment of human CRF, which currently
entails both regulation of dietary salt, and aggressive drug therapy aimed at controlling
BP.

My experimental approach was designed to study the mechanism(s) of
hypertension associated with CRF using the RRM animal model. The mechanisms by
which BP is regulated in the short-term are not identical to those involved in long-term

BP control and hypertension may result from a disorder of any of these. I decided that a

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complete evaluation of the possible causes of hypertension in CRF required investigation
of physiological systems contributing to both short—tenn and long-term BP regulation.
This was achieved by examining the effects of acute and chronic therapy with speciﬁc
pharmacological inhibitors of the renin-angiotensin and endothelin systems.

Previous investigation led me to hypothesize that the relative importance of each
of these systems in BP control differs depending on the dietary intake of salt. It is known
decreasing salt intake causes activation of the RAS, whereas changes in salt intake do not
appear to affect ET production (Schiffrin et al., 1996). Since salt intake is so relevant to
the pathophysiology of human CRF, I decided to fully investigate the inﬂuence of each on
HP under different levels of salt intake.

1. Comparison of responses to alterations in salt intake in RRM and sham rats prior
to administration of inhibitors of the RAS and ET.

A. Blood pressure

1. Reduced renal mass: HS>NS>LS

The development of hypertension during the 3 levels of salt intake was very
diverse and directly related to the amount of salt consumed (Figure 32). Even though the
HS groups were studied after the least amount of time following partial ablation (2
weeks), they exhibited the highest BP. On the other end of the salt intake spectrum, the
LS groups never became hypertensive even 12 weeks following RRM.

Some investigators have proposed that all hypertension is due primarily to a renal
defect restricting the excretion of sodium (de Wardener, 1990b). During increases in salt
intake in individuals where renal function is compromised, the kidneys experience an

inability to excrete the excess salt leading to sodium and water retention. This volume

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expanded state leads to a temporary elevation of cardiac output (CO). Later in the disease
progression sodium and water balance are re-established through pressure
diuresis/natriuresis and the hypertension maintained by a chronically elevated peripheral
vascular resistance (PVR)(Lombard et al., 1989). An elevated PVR is the basic
hemodynamic abnormality underlying the maintenance of most forms of hypertension. In
RRM hypertension this elevated PVR has been associated with an increased SNA,
elevated levels of natriuretic hormones, and an altered activity of the RAS.

During sodium restriction RRM rats have diminished serum sodium
concentrations, reduced intravascular volume, and an activated RAS, but BP remains in
the normal range (Ylitalo et al., 1976). The progression of BP is attenuated when
excision method RRM rats are maintained on sodium restricted diets (Ylitalo et al., 1976;
Terzi et al., 1992) and exacerbated by sodium loading (Koletsky, 1959; Ylitalo et al.,
1976; Douglas et al., 1964). The general consensus is that BP elevation observed in the
ligation method of RM is not salt sensitive.

2. Sham: HS=NS=LS

Wide variations in salt intake had no effect on BP when renal function was
unaltered, as exhibited in sham rats in these experiments. All groups of sham rats
remained with normal BP ranges (100-110 mmHg). Intrarenal and hormonal
compensations apparently were sufﬁcient to maintain sodium and water balance without
the recruitment of alterations in systemic BP. My results conﬁrm earlier reports by
Ylitalo and colleagues who demonstrated that alterations in salt intake ranging from highs
of 10-15 mEq/day to lows of O mEq/day did not alter BP over a period of 4-6 weeks from

levels observed in rats maintained on NS (Ylitalo et al., 1976). Elevations in BP

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associated with drinking 1.0% saline have been reported in normal rats but these
increases were not observed in all rats (66%) and only after 6-12 months on the increased
salt intake (Koletsky, 1959).

B. Blood urea nitrogen: NS=LS>>HS

Changes in BUN levels were utilized throughout my thesis as an estimate of a
decline in GFR. A declining GFR is the hallmark of diagnosis in patients with CRF. For
the most part, BUN levels increased as the time following renal ablation increased. In the
HS rats, slightly elevated to high normal BUN levels were measured, suggesting that little
overall renal deterioration had yet occurred.

This ﬁnding is surprising given the approximately 50% mortality in the HS rats.
It is likely that HS rats did not die due to renal dysfunction per se but may have
experienced some other lethal cardiovascular event (i.e. myocardial infarction or stroke)
due to the increased pressure and excessive salt intake. High salt itself is probably
responsible in part for the low BUN values observed in these rats. Guyton and co-
workers measured a signiﬁcant increase in GFR when isotonic saline was substituted for
tap water in partially nephrectomized dogs (Langston et al., 1963). During the switch to
isotonic saline BUN values that had more than doubled following partial nephrectomy
were reduced almost back to normal levels (tap water: 53.1 -_1-_ 2.1 vs. saline: 27.0 i 1.0
mg/dl). Similar ﬁndings were reported by Koletsky who showed that renal excretory
function was less compromised in RRM rats drinking 1% saline than in RRM rats
drinking tap water (Koletsky, 1959).

The BUN levels measured in the NS and LS experiments were similar in

magnitude even though BUN in LS rats was measured twice as long after nephrectomy.

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This may be due to a protective effect of LS on glomerular injury. BP was in the
normotensive range in LS, therefore renal deterioration due to an elevated BP exerted
little or no role in the progression of renal disease under these conditions. Also Dworkin
and colleagues have reported in RRM that salt restriction lessens renal deterioration in the
presence and absence of BP reduction (Lax et al., 1992; Dworkin et al., 1996). The
renoprotective effect of salt restriction in the absence of reductions in BP has been
observed in other models of renal disease such as the uninephrectomized SHR (Benstein
et al., 1990). It has been suggested that the beneficial effects of salt restriction are related
to inhibition of compensatory renal growth. Sodium restriction has been reported to slow
the growth of the kidney as well as other organs and inhibit tubular cell hyperplasia
(Solomon et al., 1972; Gallaher et al., 1990; Ostlund et al., 1991). These studies support
my experiments in RRM on LS in that salt restriction was sufﬁcient to slow the
progression of renal deterioration.

C. Water intake and urine output

1. Reduced renal mass: HS>NS>LS

Increased W1 is commonly observed in CRF and RRM due to water loss caused
by osmotic diuresis and impaired renal concentrating ability. Thirst can be stimulated by
the retention of osmotically active substances or by high AngH levels under certain
conditions (Mitch and Wilcox, 1982). A diminished ability to concentrate the urine in
RRM leads to increases in U0 and WI.

All groups of RRM rats in my experiments had elevated WI and U0 when
compared to sham animals maintained on the same level of salt intake. Increases in UO

paralleled WI so that WB was not increased in RRM. This was to be expected because

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ﬂuid intake and output must be precisely balanced under steady-state conditions or
continuous expansion would lead to circulatory collapse within days.

WI and U0 were greatest in RRM rats on HS and least under LS conditions.
Similar results have been reported in rats undergoing excision of renal mass and varying
salt intake (Ylitalo et al., 1976; Ylitalo and Gross, 1979). A centrally mediated increase
in circulating AVP has been implicated in the increased ﬂuid intake during HS. Adding
salt to the drinking water increases the osmolarity of the blood which is detected by
osmoreceptors in the hypothalamus. Activation of hypothalamic osmoreceptors initiates
increases in AVP release and mechanisms that involve somatic responses leading to the
consumption of water. Increased plasma levels of AVP have been found in RRM rats in
addition to elevated WI (Gavras, 1982).

2. Sham: HS>NS=LS

The addition of salt to the drinking water elicited an increase in WI in sham rats
when compared to rats maintained on NS or LS. No differences in WI were observed
between sham rats kept on NS or LS. Others have reported increased WI in normal rats
on elevated salt intakes (Ylitalo and Gross, 1979; Ylitalo et al., 1976). The mechanisms
are linked to activation of osmoreceptors in the hypothalamus and a sodium appetite in
the rat.

D. Mortality

One interpretation from these data is that the best therapy for CRF is a dietary
intake very low in NaCl. I did not speciﬁcally monitor pathological factors associated
with low NaCl intake, but upon general observation, the groups of rats maintained on this

regimen looked the healthiest (i.e. gained weight) and survived the longest as compared

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to any other level of salt intake. Mortality estimates were proportional to the level of salt
intake and inversely proportional to the length of time following partial renal ablation.
Roughly 50% of RRM rats maintained on HS, 20% on NS and < 10% on LS died before
completing the study. The degree of hypertension associated with each of these levels of
salt intake surely played an important role in these mortality estimates. Others have
shown that partial nephrectomy plus salt reduces life span by almost 25% over rates
observed with partial nephrectomy alone (Koletsky, 1959). The LS experimental data
strongly suggest that patients with renal disease be kept on a restricted salt intake.
H. Inﬂuence of ACEI under varying levels of salt intake

A. Acute

The acute experiments described above were designed to evaluate the role of the
RAS short-term BP regulation under varying salt intakes. Acute results following ACE
inhibition in RRM demonstrated that BP was not altered at any level of salt intake (Figure
2). On the contrary in sham rats, BP was lowered during all levels of salt intake while
only reaching signiﬁcance during LS. Bolus administration of enalaprilat was designed to
acutely inhibit the formation of AngH over a period of minutes to hours. From this type
of approach I could evaluate the contribution of AngII’s fast pressor effects on the
maintenance of BP. High AngII levels are required to cause direct contraction of the
vasculature through the fast pressor effect. When circulating levels of Angl] are high, it
would be expected that administration of ACEI would reduce AngII production thereby
lowering BP. It is evident from these acute results that direct vasoconstriction by AngII

operating through the fast pressor mechanism plays little role in short-term BP regulation

175
BP control under LS conditions when the RAS is known to be highly activated. Figure

33 summarizes the relative theoretical inﬂuence on BP due to the fast pressor effect in
normal and RRM rats under varying salt intakes.

B. Chronic

1. Support for enalapril dose used in experiments

Other investigators have shown hemodynamic effects with ACEI in RRM rats
with lower doses of enalapril than were used throughout these studies. Enalapril
administration of 25 mg/L (Lafayette et al., 1992) and 50 mg/L (Anderson et al., 1985) in
the drinking water has been reported to slow the rise in BP in RRM rats NS. On the
contrary, pilot studies in our lab showed that enalapril in the drinking water at 50 mg/L
did not signiﬁcantly attenuate the rise in BP observed in vehicle treated RRM rats over 7
days (Figure 34). The differences in efﬁcacy of enalapril may due to the different
experimental approaches used. First of all, Lafayette and Anderson measured BP by tail
plethysmography, which is not as reliable a measurement of BP obtained from an arterial
catheter. Secondly, they achieved the reduction in renal mass by the ligation method,
whereas I utilized the excision method. Lastly, enalapril treatment in their studies began
immediately following the completion of the ablation. In my experiment, enalapril
treatment was initiated 4 weeks after ablation. This period of time allows for
compensatory renal changes to occur, and hypertension to develop. This approach is
better suited for the evaluation of the inhibition of the RAS in established RRM
hypertension. The inability of enalapril administration at 50 mg/L to signiﬁcantly lower

BP in my experiment when RRM rats were maintained on NS may also be attributed to a

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shorter treatment time (1 week) compared to the other studies (3-5 weeks). This duration
of administration may not permit enalapril's full antihypertensive effects to be realized.

The difference in antihypertensive effectiveness in my normal salt studies
between the 50 mg/L and 250 mg/L dosages can not be attributed to an inadequate
inhibition of ACE in the 50 mg/L group because both groups had similar inhibition of
Angl pressor responses during treatment days (enalapril 50 mg/L control: 34.5 i' 3.7 vs.
treatment: 3.4 i 1.2 mmHg; enalapril 250 mg/L control: 32.7 i 4.6 vs. treatment: 5.2 i
1.1 mmHg). Similar ACE inhibition was achieved in spite of large differences in the
average daily dose of enalapril between 50 mg/L (2.6 mg/day) and 250 mg/L (13.5
mg/day) administration. As mentioned above, both Anderson and Lafayette have shown
dosages of 25-50 mg/L to signiﬁcantly block Angl pressor responses and decrease BP in
hypertensive RRM rats albeit in the ligation method. These factors suggest that enalapril
did reach the systemic circulation in sufﬁcient quantities to inhibit ACE in each of my
treatment groups.

Another justiﬁcation for the dose of enalapril used throughout my thesis work was
the report of additional renal beneﬁt with doses higher of enalapril than those needed to
control systemic BP (Ikoma et al., 1991). These investigators suggest that ACEI in
dosages in excess of those required for antihypertensive effects have the potential to
preserve glomeruli not yet exhibiting sclerotic lesions and to reverse early glomerular
lesions. Most of my studies were designed to initiate ACEI therapy well after partial
nephrectomy which would be similar to human CRF. The potential to reverse glomerular

lesions with high dose enalapril seemed especially attractive.

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2. Reduced renal mass

This work was aimed at characterizing how salt intake effects the actions of ACEI
in RRM hypertension. The hypothesis was that the relative importance of the RAS in the
elevated BP associated with renal failure was largest under conditions of a dietary intake
devoid of sodium. As mentioned earlier, this is a critical point given that dietary salt
restriction is an important part of the therapy of CRF in humans.

My experimental data support the original hypothesis that the activity of the RAS
is inversely proportional to the level of salt intake in RRM. Under conditions of HS in
RRM rats, enalapril did not change the progressive increase in BP observed in non-
treated rats (Figure 4). My results are in agreement with reports from Terzi who showed
that RRM rats on a 0.50% sodium diet (normal-high) developed progressive hypertension
over a 12 week period (Terzi et al., 1992). Enalapril by gavage at 3 mg/kg/day starting
one week after ablation failed to cause any signiﬁcant change in the progression of
hypertension.

Elevations in BP during HS are thought to result from intravascular ﬂuid volume
expansion. This is secondary to excessive retention of sodium and water, and is
associated with suppression of the RAS. Since the role of the RAS is minimal under
these conditions, it was not surprising that enalapril was unable to lower BP in my
experiment. Other mechanisms involved in the progression of hypertension in RRM
during high salt intakes have been proposed. Vasoconstrictors other than AngII may play
more of a role in BP regulation under high salt conditions. Much of my experimental
results have focused on the role of ET in RRM on HS and this will be discussed in detail

later in this paper. Dipette and co-workers have suggested a neurogenic mechanism for

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salt-induced hypertension in RRM and have measured increased plasma norepinephrine
concentrations (Dipette et al., 1982). Salt is known to stimulate growth of many cell
types (i.e. mesangial cells) and these actions may effect renal function and/or vascular
hypertrophy.

When i.v saline administration was fixed at levels known to approximate salt
intakes observed in normal rats, enalapril treatment attenuated the progressive rise in BP
that was recorded in non-treated RRM rats (Figure 7). Other investigators have reported
similar beneﬁcial results using ACEI in RRM rats maintained of NS over longer periods
of time. All of these studies that utilized the excision method of RRM report prevention
or slowing of renal deterioration and hypertension with prophylactic administration of the
ACEI. Enalapril administration for 8 weeks (Amann et al., 1993) or captopril for 12
weeks (Ashab et al., 1995) immediately following partial renal ablation slowed the
development of hypertension and renal deterioration observed in non-treated RRM rats.
Results from my experiment using RRM rats maintained on NS for 4 weeks prior to
enalapril treatment also show a prevention‘of the progression of hypertension over 1 week
administration of the ACEI. I hypothesized further that 1 week of enalapril
administration would reverse established RRM hypertension when rats were kept on NS.
This hypothesis was incorrect. Perhaps longer administration of enalapril might have
lowered established hypertension in my RRM rats but that is only speculation at this time
and is not supported by the literature. My results from this experiment suggest that AngH
plays a role in long-term BP regulation in RRM under NS conditions. These data support
the work of others who have demonstrated that AngH is a necessary component of the

progressive elevations in BP observed in RRM rats.

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Experimental data from LS studies also support the original hypothesis that the

activity of the RAS is inversely proportional to the level of salt intake in RRM.
Norrnotensive RRM rats maintained on LS exhibited the largest decrease in BP in
response to enalapril administration. Enalapril’s hypotensive effect in RRM on L8 was
quite remarkable considering BP fell 25-30 mmHg below what is considered the normal
range for rats.

Others have investigated the role of the RAS in RRM (excision method) during
salt restriction. Work by Brenner has shown that enalapril was more efﬁcacious in
reducing proteinuria and preventing the progression of hypertension when RRM rats were
maintained on a ﬁve fold reduction from normal salt intake (Brenner et al., 1989).
Additional results from Terzi in RRM rats demonstrate that moderate sodium restriction
retards the progression of hypertension and causes an antihypertensive effect of enalapril
that was not observed when RRM rats were maintained on a moderately elevated salt
intake (Terzi et al., 1992). Once again these studies incorporated prophylactic
administration of enalapril to prevent progressive renal deterioration and hypertension.

These experiments were designed to demonstrate reversal of established
hypertension in RRM rats. The lack of even a moderately elevated BP in rats maintained
on LS for 8 weeks was unexpected. This might have been due to the severity of salt
restriction and/or the lack of sufﬁcient renal mass reduction. The dramatic fall in BP
following enalapril administration in RRM rats on LS suggests that the RAS plays an
important role in long-term BP regulation under these conditions and that activation of

the RAS is enhanced by salt restriction. Overall my experiments show that the relative

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inﬂuence of the RAS in long-term BP regulation in RRM is largest under conditions of
lower dietary salt intakes.
3. Sham

Long-term enalapril administration in sham rats maintained on HS was not
addressed in my thesis work because of the lack of an effect on BP with enalapril in RRM
rats under these conditions. Enalapril lowered resting BP in sham rats maintained on NS
and LS with the largest reduction recorded in the latter group. It is not surprising that
ACEI in LS sham rats (where the activation of the RAS is highest) lowered BP. It was
unexpected that enalapril would lower BP to such an extent in sham rats on NS. Not all
reports in the literature (Amann et al., 1993) conﬁrm my results in NS rats but others in
our lab have published similar results (Melaragno and Pink, 1995). These data suggest
that AngII is an important contributor to basal levels of BP in normal rats maintained on
NS and LS. They also support the role of the RAS in long-term regulation of BP under
these conditions.
111. Mechanism of action of ACEI

One of my main objectives in this thesis was to determine the mechanism of
action of the BP lowering effect of ACEI in RRM rats. The mechanism of action of ACEI
has been debated for years. ACEI were rationally designed to inhibit the formation of
circulating AngII. Most evidence supports the hypothesis that ACEI exert their
antihypertensive effect through inhibition of AngII formation. The experimental data
supporting this hypothesis is extensive although agreement is not universal. Some of the
most convincing evidence comes from studies comparing the effects of ACEI to AT,

receptor antagonists in RRM. Losartan alone produced the same magnitude of

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antihypertensive effect as enalapril, and combination of these two agents produced no
additional benefit on BP (Lafayette et al., 1992). Some investigators have suggested that
the antihypertensive effectiveness of these inhibitors involves a variety of other pathways
such as: decreased degradation of BK, increased production of vasodilatory
prostaglandin’s, inhibition of local tissue RAS, and increased NO production. Studies
designed to investigate these other pathways have not produced convincing evidence that
ACEI lower BP by mechanisms other than by a reduction in AngH production.

I hypothesized that ACEI affect BP in RRM by inhibiting the production of
physiological amounts of AngII. Infusion at physiological rates of AngII into RRM rats
given enalapril during NS conditions restored the progressive rise in BP observed in
untreated rats. A necessary prerequisite for this type of experimental approach was to
establish that ACEI completely inhibit endogenously produced AngII. Work done in our
lab has shown that when an AT; receptor antagonist (losartan) was given to rats
chronically receiving ACEI (enalapril, 250 mg/L), BP did not further decrease over the
next 24 hours (Figure 35). In untreated normal rats this dose of losartan caused a
signiﬁcant decrease in BP and blocked pressor responses to exogenous AngII (Sacerdote
et al., 1995). This additional evidence supports the assertion that enalapril treatment at
250 mg/L successfully blocks ACE, and suggests that enalapril reduces endogenous
AngH formation to a functionally insigniﬁcant level.

In the normal salt scenario, exogenous AngII replacement at a rate of 2 ng/min
restored the progressive rise in BP normally observed in RRM rats. Likewise in sham
rats on a normal salt intake, AngII at 2 ng/min restored the basal level of BP. This low

replacement rate of AngII is considered to produce systemic Angll concentrations within

182
the physiologic range. This work supports the hypothesis that enalapril prevented rises in

BP only through inhibition of Angl] formation. If enalapril was working through
mechanisms other than the inhibition of AngII formation, then I should have not been
able to reverse the ACEI’s full effect on B? by replacing AngH systemically.

IV. Mechanism of action of AngII in RRM hypertension

A. Role of sodium excretion

AngII exerts multiple actions to control body ﬂuid volume, sodium excretion and
blood pressure. Extrarenal (i.e. stimulation of thirst, activation of the sympathetic
nervous system, secretion of aldosterone and AVP) and intrarenal (i.e. efferent arteriole
constriction, proximal tubule sodium reabsorption) effects make Angl] one of the body’s
most powerful controllers of sodium and ﬂuid homeostasis and long-term BP regulation.
Most evidence suggests that direct intrarenal actions of AngII play the major role in the
excretion of sodium under physiological conditions (Hall and Brands, 1993). Sodium
retention is achieved mainly through AngH mediated increases in proximal tubular
reabsorption. Blockade of AngII formation (i.e. ACEI) reduces sodium reabsorption
whereas high AngII levels (i.e. AngII infusions) elevate sodium reabsorption.

High circulating AngII concentrations cause increases in BP, and the resulting
increase in renal perfusion pressures initiates a transition from sodium retention to
sodium excretion via pressure-natriuresis. Thus, the natriuresis associated with high rates
of AngH infusion is not caused by decreases in proximal tubular reabsorption (Hall and
Brands, 1993). The net effect of AngII on sodium excretion depends on the balance of
direct antinatriuretic actions of AngII and the natriuresis resulting from increasing renal

perfusion pressure.

183
In my first experiments in RRM rats maintained on HS, the addition of NaCl to

the drinking water resulted in increased sodium excretions (Figure 6). Inhibition of Angl]
formation by enalapril did not affect UNaV, sodium balance, or BP during HS or NS
experimental periods. These results were not unexpected because of the anticipated lack
of RAS involvement during HS conditions. Therefore it is doubtful that and AngII
mediated antinatriuretic effect played a role in RRM hypertension under these HS
conditions.

In RRM rats maintained on NS, no changes in UNaV were measured even though
signiﬁcant changes in BP occurred during some treatments (Figure 12). These results do
not support increases in sodium reabsorption as the cause of progressive elevations in BP
observed in RRM rats on NS. Enalapril administration alone, which successfully
inhibited AngH formation, was expected to elicit an increase in sodium excretion. But BP
was lower in enalapril treated than untreated RRM groups. The decreased BP, which
lessens sodium excretion may have opposed any inhibition of sodium tubule reabsorption
during ACEI administration. Another consideration is that the activity of the RAS is low
to normal in RRM and the reduction of an Angl] mediated effect due to inhibition of
already low plasma AngII concentrations may not exert an effect readily observable using
our methods. It was expected that the highest rate of AngII replacement (4 ng/min)
would increased sodium excretion due to the elevation in BP overwhelming directly
mediated AngII proximal tubule reabsorption. This was not observed. My data suggest
that the hemodynamic effects of AngII on natriuresis counteract the intrarenal effects on

the proximal tubule resulting in no net change in sodium excretion.

184

In sham rats on NS, enalapril treatment alone was associated with a signiﬁcant
natriuresis that developed only after several days on enalapril and was reversed by two
days after enalapril discontinuation. In fact, the majority of the recovery days were
associated with a signiﬁcant sodium retention. The natriuretic response observed in
enalapril treated sham rats during the treatment period was most likely due to decreased
Angl] stimulated tubular reabsorption. This natriuretic effect may even have been greater
if it were not for the BP lowering inﬂuence limiting natriuresis. The substantial
hypotensive effect of enalapril suggests that activity of the RAS is greater in sham than in
RRM rats on NS. This explains why the natriuretic response to ACE inhibition was only
measurable in sham rats. The decrease in sodium excretion after withdrawal of enalapril
supports the role of AngII in mediating sodium reabsorption.

The natriuresis observed after enalapril did not contribute to the hypotensive
effect of ACEI. My data show than very low rates of AngH infusion (1 ng/min) were
enough to reverse the natriuretic effect of ACEI (Figure 13). This reversal occurred
without a full restoration in BP. In fact the hypotensive effect due to enalapril alone or
enalapril plus 1 ng/min AngII was not different. My data suggest that in sham rats the
actions of AngII in the proximal tubule on sodium reabsorption are more sensitive than
the actions of AngII on the systemic vasculature. Work by Hall and colleagues has shown
that proximal tubule transport is approximately 1000 fold more sensitive to AngII than
contraction of aortic vascular smooth muscle (Hall and Brands, 1993). Upon infusion of
higher rates of AngII, no additional changes in sodium excretion or BP were observed.
This is the case because the normalization of BP elicited an increase in sodium excretion

that offset the AngII stimulated increase in tubule reabsorption of sodium. I concluded

185

from this experiment that Angl] mediated effects on sodium reabsorption do not play a
signiﬁcant role in the maintenance of BP in normal rats on NS.

Urinary sodium excretion was not affected by enalapril administration alone or in
combination with AngII replacement in either RRM or sham rats under sodium deplete
conditions (Figure 18). The substantial BP lowering effect of ACEI predicts that a
decrease in sodium excretion would occur, whereas loss of intrarenal Angl] would be
expected to increase sodium excretion. These effects appeared to cancel one another, but
subtle changes in sodium excretion may not have been detected because the sodium
excretory rates in both sham and RRM rats were barely measurable (zero to 0.1
mEq/24h). Infusion of exogenous Angl] during enalapril treatment also did not
measurably affect sodium excretion, probably because proximal reabsorption of sodium
was already maximal under LS conditions, even in the absence of AngII. Because BP fell
in the absence of any preceding alterations in sodium excretion, I concluded that changes
in sodium balance due to inhibition of AngII formation were unlikely to exert an effect on
BP under these LS conditions.

B. Increased responsiveness in RRM

I hypothesized that an increased sensitivity of AngH AT] receptors in the
vasculature could be involved in the hypertension observed in RRM. Activation of ATI
receptors in the vasculature results in contraction of VSMC leading to vasoconstriction an
elevated BP. If RRM or salt increases the sensitivity of these vascular receptors to Angl]
then bolus challenges of Angl should elicit greater pressor responses in RRM rats. This
did not occur in these experiments. This indicates that reduction of renal mass did not

cause an increased vascular response to Angl]. Thus, there was no change in the fast

186

pressor response to AngII in RRM. Previous studies support my observations that acute
pressor responses to AngII were not different between RM and normal rats on both HS
and NS (Kanagy et.al., 1993).

Another potential mechanism that was addressed in my experiments was that
RRM rats exhibited an increased chronic responsiveness to circulating AngII. Days after
discontinuation of enalapril treatment, BP in RRM rats maintained on NS did not return
to the rate of increase that was recorded in the vehicle group of RRM rats (Figure 7A).
This effect persisted throughout the recovery period and was not observed in sham rats on
the same dose of enalapril (Figure 8A). A possible explanation for this sustained
antihypertensive effect was persistent inhibition of plasma ACE. Yet Angl pressor
responses were back to control levels 2-3 days into the recovery period. Another
possibility is that in RRM there is a slowly developing pressor effect of AngII that was
inhibited during enalapril treatment. The “slow pressor effect” as described in the
introduction is the phenomenon observed when low doses of AngII i.v. infusion produce
increases in BP over hours to days. This is in contrast to the fast pressor effect of AngH,
which is caused by higher doses of AngH via direct vasoconstriction. If Angll was
working to raise BP in RRM through fast pressor effects, then restoration of circulating
AngII concentrations after discontinuation of enalapril should have increased BP within
minutes to hours. This did not occur. Furthermore, if the fast pressor effect of AngII was
operative in RRM rats on NS, acute enalaprilat treatment should have lowered BP, an
outcome also not supported by the data (Figure 2).

I hypothesized instead that RRM rats exhibit increased responsiveness to the slow

pressor effects of AngH, and that this accounted for the ability of “normal” levels of

187

circulating AngII to restore hypertension development. To test this idea, I examined the
difference in pressor responsiveness between RRM and sham rats drinking enalapril and
receiving replacement AngII at a rate of 4ng/min (Figure 36). Sham rats drinking only
enalapril had a BP drop of 10-20 mmHg during the treatment period from the 3 control
days of the experiment. This hypotensive effect was reversed by infusion of AngII at
4ng/min and BP levels returned to normal. Thus, the peak BP change in sham rats while
on enalapril and administered AngII at 4ng/min was 20-25 mmHg. RRM rats drinking
enalapril had a BP drop of approximately 5-10 mmHg during the treatment period from
their respective 3 control days. Yet, in the RRM rats also administered 4ng/min AngII,
the peak change in BP was 40-45 mmHg. These results suggest an enhanced
responsiveness to the SPE in RRM rats on NS. This enhancement could explain why BP
is elevated in RRM rats despite their having plasma AngII concentrations in the normal
physiologic range.

Additional support for the hypothesis of increased responsiveness of the SPE in
RRM rats comes from previous work in our lab by Dr. Kanagy, who demonstrated that a
10 ng/min continuous i.v. infusion of AngII in untreated RRM rats on NS elevates BP by
30-35 mmHg over a period of 7-10 days (Kanagy, 1991). This was in contrast to only
mild elevations of BP (10—15 mmHg) recorded in sham rats receiving the same infusion
rate of AngH.

The role of the SPE in RRM was also evaluated in RRM rats during LS
administration. In comparing enalapril administration between sham and RRM rats, it is
clear that there was a slower developing hypotensive effect in RRM rats. Likewise, days

after discontinuation of enalapril treatment, BP in RRM rats maintained on LS did not

188

exhibit the rate of increase back to pre-treatment levels that was recorded in the sham rats
given the same treatment (Figure 14A vs. Figure 15A). This lack of BP restoration in
RRM rats persisted throughout most of the recovery period and was not observed in sham
rats. Here again, Angl pressor responses were back to control levels 2-3 days into the
recovery period. These results suggest the slow pressor effect of AngII plays a major role
in the maintenance of BP in RRM rats on LS.

On the contrary it is evident that the mechanism by which ACEI chronically
decrease BP under LS conditions in sham rats is by inhibiting the fast pressor effect of
AngH. ACEI lowered BP faster in sham rats than in RRM rats on LS (Figure 14 vs.
Figure 15) because plasma AngH concentrations are greater in sham than in RRM rats
when both are on a low salt diet (Ylitalo et al., 1976).

One question still remains. Why didn’t replacement of Angl] restore BP to pre-
treatment levels in rats on LS even with higher doses of the peptide? I did not observe a
complete reversal of enalapril’s hypotensive effect in RRM or sham rats. Administration
of AngII i.v. at rates of 10 ng/min partially blunted enalapril’s hypotensive effect in sham
and did not restore BP levels measured prior to enalapril treatment in RRM. In support of
my results, Cowley and coworkers in 1976 reported that sodium-depleted dogs did not
show an increase in BP when given Angl] at an i.v. rate of 23 ng/kg/min for 9 days
(Cowley and DeClue, 1976). It is likely that slightly higher rates of infusion would have
been sufﬁcient to restore normal BP. These date are consistent with existing evidence
that the SPE of Angl] is inhibited by LS (Cowley and McCaa, 1976). Figure 37
summarizes the relative theoretical inﬂuence on BP due to the SPE in normal and RM

rats under varying salt intakes.

189

V. Inﬂuence of endothelin under varying levels of salt intake

A. Acute role of endothelin

The direction of my early studies investigating the role of ET in RRM evolved
from previous work done in our lab demonstrating the salt dependency of ET-l-induced
hypertension (Mortensen and Fink, 1991). At the time I only had access to peptide
ETRA’s that were restricted to parenteral administration. I hypothesized that ET may be
involved in RRM hypertension when rats were maintained on an elevated salt intake. By
this time my experiments using ACEI in RRM on HS had demonstrated that the RAS is
not involved in BP regulation under HS conditions. I decided to examine the
hemodynamic response to both selective ETA (PD147953) and non-selective ETA/ETB
(PD145065) receptor antagonism in RRM rats on HS. Since these antagonists were
relatively new and little was published about their speciﬁcity and potency, I conducted a
series of experiments to validate their speciﬁcity in ET-l-induced hypertension and
determine potential dosing regimens (Figure 19). After I was satisﬁed with these
experiments I tested the speciﬁc hypothesis that ET exerts short-term control of BP in
RRM rats by immediate and direct contraction of the vasculature. A corollary question
that I examined was: does the level of salt intake inﬂuence the hemodynamic effects of
ET receptor blockade? The results from acute administration of each antagonist in RRM
on the 3 levels of salt intake showed that an antihypertensive effect was only observed in
RRM rats on HS. The results from these experiments suggest that ET contributes to
short-term BP regulation in RRM mainly through activation of ETA receptors, and
preferentially during high salt intakes. The mechanism of this effect is uncertain, since

ET formation is not generally responsive to differences in salt intake.

190

B. Chronic role of endothelin
1. Support for PD155080 dose used in experiments

Since PD155080 was a newly developed non-peptide ETARA, dosing regimens
were not in place when my experiments started. I developed the dosing regimen of 25
mg/kg b.i.d. from a series of pilot studies involving RRM and sham rats. Since my
original observations other evidence has been generated supporting the appropriateness of
this dose. In vivo evaluations suggest that plasma PD155080 concentrations ranging
from 5 to 60 jig/ml exert selective antagonism of ETA receptors in the rat. Plasma
concentrations of PD155080 in RRM and sham rats given PD155080 chronically were
within that range (Table 4). The most convincing evidence supporting this dose however,
comes from its proven efficacy at reversal of ET-1 induced hypertension (Figure 25).

2. Reduced renal mass

The lack of information regarding the involvement of ET in the excision method
of RRM provided an opportunity to be the ﬁrst to characterize the contribution of ET in
this model. I was speciﬁcally interested in what role ET plays in long-term BP regulation
in RRM and how salt intake may inﬂuence its actions. My acute studies suggested that
ET’s involvement in RRM may be greater under HS conditions. Pilot experiments using
PD155080 demonstrated an antihypertensive effect at each level of salt intake, with the
largest fall in BP observed under NS conditions (Figure 26). Because of these
encouraging results and a variety of other reasons already mentioned, I decided to
concentrate on the involvement of ET in long-term BP regulation in RRM rats on NS.

One week treatment with PD155080 caused a signiﬁcant and sustained decrease

in BP to normotensive levels in RRM on NS (Figure 28). This antihypertensive effect

191
was produced in RRM rats four week following partial nephrectomy and exhibiting a

sustained hypertension. The effect of PD155080 on BP was observed within 1 day after
administration and reversed by 1 day following discontinuation. These novel ﬁndings
suggest that ET, acting through ETA receptors, has an integral part in the maintenance of
hypertension in RRM rats on NS.

The overall implication of my chronic experiments utilizing ETRA’s in RRM was
that ET may play some role in the long-term control of BP at all salt intakes while it
exerts a predominant role under NS conditions.

3. Sham

In sham rats PD155080 was given chronically to test the hypothesis that ET was
involved in the maintenance of basal levels of BP. After getting preliminary results that
suggested ET’s involvement in RRM hypertension was greater under HS conditions, I
evaluated the inﬂuence of salt intake on ETA receptor antagonism in normal rats. In sham
rats maintained on a saline infusion calibrated to deliver 6.0 mEq Na+lday (HS),
PD155080 resulted in a slight hypotensive effect during the ﬁrst 2 days of administration
(Figure 25). In sham rats maintained on a saline infusion calibrated to deliver 2.0 mEq
Na+lday (NS), PD155080 resulted in a slight, inconsistent hypotensive effect during the
treatment period when compared control days (Figure 28). My data demonstrated that HS
conditions did not alter the BP response to ETARA administration in normal rats.
Schriffrin has reported that elevations in salt per se did not increase tissue ET-l content
nor elevate circulating levels of the peptide, suggesting that salt did not stimulate ET
production or release by itself (Schiffrin et al., 1996). Given that the hypotensive effects

due to PD155080 in my experiments were relatively modest and inconsistent, these data

192

do not support a role of ET in the maintenance of normal levels of BP. My observations
are consistent with most reports that do not support a role of ET in the maintenance of
basal vascular tone in normal rats (Ohlstein et al., 1993; Teerlink et al., 1995). Figure 38
summarizes the relative theoretical inﬂuence on BP due to ET in normal and RRM rats
under varying salt intakes.

VI. Mechanism of action of endothelin in RRM hypertension

A variety of mechanisms that are inﬂuenced by ET may be involved in RRM
hypertension. I did not attempt to identify these mechanisms or their relative
contribution. Yet through my experimental design some direction of future investigation
may be determined.

It has been established that release. of ET-1 from endothelial cells in the systemic
vasculature causes vasoconstriction and smooth muscle cell growth by stimulating ETA
receptors (Rubanyi and Polokoff, 1994). The time course of the antihypertensive response
to PD155080 in my chronic experiments was too short for reversal of vascular structural
changes. If vascular endothelial cell production of ET is increased in RRM, I might have
expected to see increased circulating levels of the peptide. ET plasma levels were not
different between sham and RRM rats on NS suggesting that increased ET production did
not occur. Because the release of ET from endothelial cells is directed towards VSMC, it
is possible that tissue levels of ET increase in the absence of increased plasma levels.
This scenario has been reported by Schiffrin and colleges to occur in DOCA-salt rats
(Schriffrin et al., 1996). I did not measure vascular tissue content of ET directly so I can

not rule out increased vascular ET production and direct vasoconstriction as a contributor

193

to RRM hypertension. Therefore, inhibition of ET-1 induced vasoconstriction could have
accounted for the BP lowering effect of ETA receptor blockade in the RRM rats.

An alternative explanation is that endogenous ET-l raises BP in RRM rats by an
action on receptors distinct from those affected by acute vasoconstrictor actions of the
peptide, perhaps in the brain, adrenal gland or other organs involved in BP regulation. In
DOCA-salt rats, i.c.v. administration of an ETARA was shown to elicit an acute
antihypertensive effect (Mortensen and Haywood, 1995). A centrally mediated
hypertensive mechanism involving ET may be involved in RRM but this was not
addressed in my study nor has it been in the literature.

As mentioned in the introduction, previous evidence suggests that alterations in
renally synthesized ET may be involved in RRM hypertension. Increases in urinary
excretion of ET occur as renal disease progresses and appear to be a good marker of renal
deterioration (Benigni et al., 1991). Whether increases in renally derived ET are a
marker, or a cause, of renal deterioration and elevated BP is not known. In my
experiments, blockade of renal ET receptors by PD155080 could have caused a fall in BP
by promoting sodium and ﬂuid excretion via the kidney. My results from RRM rats
maintained on NS do not support such an explanation in that PD155080 administration
was associated with sodium and water retention rather than diuresis and natriuresis.
PD155080 may exert a variety of beneﬁcial effects on the remnant kidney, but
investigation of these was not the focus of my experiments.

VII. Therapeutic implications
There are a variety of antihypertensive agents currently on the market. To date

only ACEI have been shown to exert protective effects on the kidney while lowering BP

194
in human CRF. Thus, ACEI are currently the drug of choice in CRF. They may be most

beneﬁcial when hypertension exists in the compliant patient where salt restriction can be
successfully maintained. ACEI inhibitors generally have a good side-effect proﬁle with
persistent cough being the most common adverse effect. These drugs have not been
shown, however, to decrease mortality or time to transplantation in patients with CRF.
Furthermore, there are instances where ACEI are not the best therapy in CRF. ACEI are
contraindicated in pregnancy because of their teratogenic potential. ACEI seem to have
limited if any beneﬁcial effect when patients do not comply with restriction of salt intake.
It also must be kept in mind that the risk of acute renal failure is increased when ACEI are
administered during low salt intakes (Hall and Brands, 1993). Low salt intakes increase
the dependence of the renal circulation on the RAS and preferential dilatation of the
efferent arterioles by ACEI can further decrease GFR. Therefore discovery of novel
therapies that lower BP and prevent renal deterioration could be of great beneﬁt
clinically.

ET receptor antagonists may be useful in reducing BP and preventing renal
deterioration in situations where ACEI are not appropriate. My work suggests that ETRA
treatment may be more effective in CRF patients on high salt intakes. Currently this class
of drugs is not known to be teratogenic and therefore may be useful in women with renal
insufﬁciency or in pregnancy-associated hypertension (preeclampsia). Preeclampsia is
associated with a generalized endothelial cell dysfunction and signiﬁcant elevations in
plasma ET levels have been reported (Rubanyi and Polokoff, 1994).

My ﬁndings indicate that ETA receptor blockade may be an effective therapy for

the hypertension associated with CRF. It was noteworthy that the antihypertensive

195

response to PD155080 in RRM rats was not accompanied by any measurable decrease in
renal function. Further studies involving longer periods of administration will be
necessary to speciﬁcally address the potential renoprotective effects of ET receptor

antagonism in RRM rats.

 

 

 

196

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198

Figure 33: Relative inﬂuence on blood pressure of the fast pressor effect of AngII in
normal and RRM rats under varying salt intakes. Dashed vertical lines represent points
on the graph used to estimate the relative inﬂuence of blood pressure under differing salt
intakes. The theoretical contribution of the fast pressor effect to blood pressure is

estimated by the product of the responsiveness of blood vessels to AngII times AngII

levels.

 

 

Theoretical Activity (Arbitrary Units)

10-

 

 

199

Fast pressor effect of AngII

H Angll levels in normal rats
0—0 AngII levels in RRM rats
A- - ‘4 Pressor responsiveness (normal = RRM)

 

 

 

 

Normal salt High salt
FPE : pressor x AngII = relative
responsiveness levels inﬂuence on BP
FPEWWII : l x 6.0 = 6
L . _
ow FPERRM . l x 4.5 — 4.5
N l FPEnom“ : 2 x 2.0 = 4
orma
FPERRM :2x l.0=2
H' h FPEmm' : 3 x 0.3 = 0.9
'3 FPERRM : 3 x 0.2 = 0.6

 

 

 

Figure 33

200

Figure 34: Mean arterial pressure responses to chronic low-dose enalapril

administration in RRM rats on normal salt intakes.

 

180

160

MAP (mmHg)
8

120

100

201

[El—E] Vehicle

(3)

H Enalapril 50mg/L (7)

 

"Ha

 

 

IV

 

L—— ACEI —q

Oral

 

,xifi‘h'

 

4‘
r

 

‘-

 

 

Control

Treatment

Protocol Day

Figure 34

Recovery

202

Figure 35: Mean arterial pressure responses to losartan i.v. bolus during chronic

enalapril administration in normal rats on normal salt intakes.

 

120

110

MAP (mmHg)
8

on
G

60

203

H Enalapril 250mg/L

n=6

 

U

 

J I

I

 

 

 

 

 

E1 E2

Days

Losartan
(lime/kg)

Figure 35

1 1
E3 E4 E5 E6+5' 15' 30' 60'120'240‘360'24H48H
Time Post Losartan

 

204

Figure 36: Mean arterial pressure responses to chronic enalapril administration in
sham and RRM rats on normal salt intakes. Panel A depicts sham rats administered
vehicle and enalapril + AngII replacement at 4ng/min. Panel B depicts RRM rats

administered vehicle and enalapril + AngII replacement at 4ng/min.

 

>

Change in MAP (mmHg)

Change in MAP (mmHg)

205

 

 

 

 

 

 

 

 

 

 

B—EI SHAM Enalapri1250mg/L (5)
H SHAM Enalapri1250mg/L + 4ng AH (5)
60
. SHAM
40 '-
20 -
0 b " a: 'i'
’ I! V
'20 - ' . :I
_40 I I I I I I I I I I I I I I
G—O RRM Enalapril 250mg/L (8)
H RRM Enalapril 250mg/L + 4 ng All (7)
60
_ RRM
40 ..
20 -
0 - W
-20 b
.40 I I I I I I I I I I I I I I
Treatment Recovery

Protocol Day

Figure 36

206

Figure 37 : Relative inﬂuence on blood pressure of the slow pressor effect of AngII in
normal and RRM rats under varying salt intakes. Dashed vertical lines represent points on
the graph used to estimate the relative inﬂuence of blood pressure under differing salt
intakes. The theoretical contribution of the slow pressor effect to blood pressure is

estimated by the product of the responsiveness of blood vessels to AngII times AngII

levels.

 

Theoretical Activity (Arbitrary Units)

 

207

Slow pressor effect of AngII

H AngII levels in normal rats

 

 

 

 

 

 

‘0 ‘ ._. AngII levels in RRM rats
9 _ D * D Pressor responsiveness in normal rats
0 ~ 0 Pressor responsiveness in RRM rats
8
7 ‘ I
6 - . > ' to
l 0 ' C1
5 ‘ I
l I O I
4 7 o
. d , a
3 ‘ I .0 I
v _ Cl '
2 ‘ .,' . . - - I
l-o'”.El _D“ i I
. » D ‘3" ~
0 I E I
Low salt Normal salt High salt
SPE : pressor x AngII = relative
responsiveness levels influence on BP
L SPEWmI : 0.1 x 6 = .6
”w SPERRM :l.5x4=6
Normal SPEW“.III : 1.5 x l = 1.5
SPERRM : 3.0 x l = 3
H' h SPEWMl : 3 x 0.3 = .9
'g SPERRM : 6 x 0.1 = .6

 

 

 

Figure 37

208

Figure 38: Relative inﬂuence on blood pressure of endothelin in normal and RRM
rats under varying salt intakes. Dashed vertical lines represent points on the graph used to
estimate the relative inﬂuence of blood pressure under differing salt intakes. The
theoretical contribution of ET to blood pressure is estimated by the product of the

responsiveness of blood vessels to ET times ET levels.

 

209

Pressor effect of ET
10 T H Pressor responsiveness to ET in RRM rats
9 .4 H Pressor responsiveness to ET in normal rats
A- - 4 ET levels
8 .—
7 _

 

 

Theoretical Activity (Arbitrary Units)

 

Low salt Normal salt High salt

pressor x ET = relative
responsiveness levels inﬂuence on BP

 

L normal:0.1x2=0.2
0“ RRM :1.0x2=2

 

Normal normal : 2.0 x 2 = 4
RRM :3.5 x 2 = 7

 

- normal :3.0x2= 6
mg” RRM :4.5x2=9

 

 

 

Figure 38

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