THE EFFECT OF OBESITY IN THE RAT
ON THE KIDNEY AN-D URINE

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNTVERSITY
JENNY TAYLOR JOHNSON
1972

 
 
 

This is to certify that the

thesis entitled

A

THE EFFECT’ OF OBESITY IN THE RAT
ON THE KIDNEY AND URINE

presented by

JENNY TAY LOR JOHNSON

has been accepted towards fulfillment
of the requirements for

FOOD SCIENGEV fr HUMAN ‘
NUTRITION

PH'D’ #degree in?

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ABSTRACT
THE EFFECT OF OBESITY IN THE RAT

ON THE KIDNEY AND URINE

BY

Jenny Taylor Johnson

Obesity, a major problem in the human p0pulation today
is associated with an increased incidence of many degenera-
tive and disabling abnormalities including those of the
renal-cardiovascular systems. Further investigation was
considered necessary to evaluate renal function in the
obese state. The purpose of this study was to determine
the effect of dietary obesity on renal function and
histology in the laboratory rat.

Obesity was produced in Osborne-Mendel male rats by
feeding a 60% fat diet with adequate protein, vitamins and
minerals.

A light microscopy histopathological examination of
kidneys from animals at weaning and at 15, 25, 35 and 45
weeks of age, fed either a control grain ration (GR) or a
high fat (HF) ration postweaning, was completed.

The animals fed HF were significantly heavier than the
GR controls after 25 weeks of age. These animals contained
considerably more fat than the GR animals, especially in

the perirenal-retroPeritonial depots.

 

  

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In the HF-fed group, the right kidney was heavier than
the left in animals at 15, 25, 35 and 45 weeks of age.
There were no differences in the weights of the two kidneys
in the GR groups at any age examined. The total kidney
weight was significantly greater in the HF groups at 25,
35 and 45 weeks of age. The kidney weight to body weight
ratio was significantly less in HF animals at all ages.

Lesions in kidneys from GR and HF animals affected
the glomeruli and tubules. Prior to 35 weeks of age, the
incidence of lesions in both groups was almost comparable,
although the lesions in the kidneys from HF animals were
more severe and covered more of the kidney parenchyma.

By 45 weeks of age and 42 weeks on the respective
diets, the kidneys from the HF animals had considerably
more damage than those from GR rats. The total number of
lesions and the severity of these was greater in the HF
group at this age.

The effect of obesity and the HF diet on the accumu-
lation of p-aminohippurate (PAH) by renal cortical slices
was determined using an in vitro slice technique. The
accmmulation of PAH was significantly depressed in animals
fed the HF diet used to produce obesity. Accumulation of
PAH decreased with increasing age, body weight and kidney
weight independent of diet. Exchanging the diets for a
few days or a few weeks affected the transport of PAH, but
not of NMN. Animals fed GR for any period of time

immediately prior to sacrifice had kidneys which

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Jenny Taylor Johnson

accumulated PAH significantly more than those fed HF just
prior to sacrifice.

The rate of PAH uptake was determined and analyzed
kinetically using a Lineweaver-Burk plot. In GR animals

the Vm and Km were less in 60 week old animals and Km

ax
values less than the respective age controls. The decrease
in apparent affinity and maximal velocity with age and diet
could indicate non-competitive inhibition. The differences
observed in PAH tran5port in the HF animals were not the
result of differences in oxygen consumption, histology or
composition of renal cortical slices. These data are
consistent with the presence of some inhibiting factor (as
another organic acid) in the serum. However, stimulation
not inhibition was demonstrated when serum from the HF or
GR animals was added to the incubation medium.

Organic base accumulation was determined in order to
demonstrate the specificity of the effect of the HF diet
and obesity on organic acid transport. Accumulation of
N-methylnicotinamide (NMN) was not different in GR and HF
animals. There was no correlation of NMN accumulation with
body weight. In HF animals NMN accumulation by renal
cortical slices decreased with increased age and kidney
weight. Age and kidney weight in the GR animals were not

related to NMN accumulation.

 

 

 

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'I.‘.

THE EFFECT OF OBESITY IN THE RAT

ON THE KIDNEY AND URINE

BY

Jenny Taylor Johnson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1972

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In memory of my grandparents,

Mr. and Mrs. E. C. Taylor
and
Mr. and Mrs. Manning Rogers

And in honor of my parents,
Mr. and Mrs. Woodrow Taylor

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ACKNOWLEDGEMENTS

This thesis and research have become a reality only
through the assistance and encouragement of a number of
persons.

Special gratitude is extended to the following:

Dr. Olaf Mickelsen for direction, encouragement, and
support and for an appreciation of past research and
a contagious interest in that of the future.

Dr. Dena Cederquist for continued support and concern and
for her emphasis on the "human" in human nutrition.

Dr. Vance Sanger for assistance with the histological
sections and, especially for his genuine concern through—
out my graduate program.

Dr. Harold Hafs, Dr. Modesto Yang, Dr. E. P. Reineke,

Dr. Hans Lillevik and Larry Besaw for helpful suggestions
and technical assistance.

Dr. W. D. Collings for my initial introduction to Dr. Homer
Smith and the marvels of the kidney and for his
thoughtful suggestions during this study.

Dr. Jerry B. Hook for technical advice and encouragement.

Dr. John Gill and Dr. C. E. Cress for advice concerning
statistical treatment of data.

Mrs. Eleanor Smith for providing the animals used in

Part II.
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thoughtfulness, expressed in so many ways.

Mrs. Grace Scribner, a very special secretary and supporter
"par excellance" of graduate students.

Dr. Rachel Schemmel for interest and for providing the
initial breeding animals for this study.

My fellow students, who have provided encouragement during
my graduate program, especially Connie Parks, Charlotte
Foy, Ivy Woolcott, Sharon Nemeth and Eleanor Schlenker.

The Agricultural Experiment Station, the National Institutes
of Health, and the Department of Food Science and Human
Nutrition, for financial support.

Pat and Al Sculthorpe for helpful technical assistance.

A special thank you to my husband, Tom, for his technical
assistance and for his continued understanding, encourage-

ment and infinite patience.

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Part

II

TABLE OF CONTENTS

Introduction. . . . . . . . . . . . . . . . .
Bibliography. . . . . . . . . . . . . . . . .

Review of Literature. . . . . . . . . . . . .

The Functions of the Kidney . . . . . . . . .
The Effect of Diet on Kidney Function, Size
and Composition
Malnutrition
Potassium.
Sodium . .
Magnesium.

Choline.
Protein.
Fat. . .
Vitamin A.
Vitamin C.
Vitamin D.
Vitamin E. . . .
B Vitamins, Calcium,
Others . . . . . . . .
Overweight and Obesity
Bibliography. . . . . . .

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Histopathologic Description of Renal Lesions
in Rats Fed a High Fat Diet Through 45 Weeks
Of Age 0 O O O O O O O O O O O O O O O I O O 0

Introduction. . . . . . . . . . . . . . . . .
Literature Review . . . . . . . . . . . . .
Changes in Renal Function and Histology

With Age . . . . . . . . . . . . . . .
Kidney Size and Factors Influencing It . .

Dietary Nitrogen. . . .

Other Dietary Factors .

Body Weight . . .

Age . . . . . . .
Miscellaneous . .
Materials and Methods .
Animals. . . . . . .
Rations. . . . . . .
Tissue Preparation .
Statistical Analyses

Page

10
10
19
23
25
28
30
37
39
40
41
41

42
43
55

76

76
76

77
87
87
90
91
93
94
95
95
96
96
97

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Obesity and
Acids and Ba

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Model of
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Sex .
Effects
System c
Slices

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Accumula
Cortical
The Effg
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Kinetic
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Slices
Renal c
Plasma

 

 

 

 

Table of Contents (Cont'd)
Part Page
Resu1ts O O O O O I O O C I I O O I I O O O O O 9 8

Body Weights . . . . . . . . . . . 98
Kidney Weights and Kidney Weight/Body.

Weight Ratios. . . . . . . . . . . . . . . . 98
Histopathological Examination. . . . . . . . 99
Discussion. . . . . . . . . . . . . . . . . . . 125
Summary . . . . . . . . . . . . . . . . . . . . 130
Bibliography. . . . . . . . . . . . . . . . . . 132

III Kidney Function in Dietary Obesity: Effects of
Obesity and Diet on Renal Transport of Organic
ACidS and Bases O O O O O O O O O O I O O O O O 144

IntIOduCtj-ono O O O O O O C C O O O O O O O O O 144
In vitro Measurement of Renal Transport. . . 145

Model of the Renal Secretory Mechanism . . . 151

Factors Affecting Renal Tubular Transport

of Organic Acids and Bases . . . . . . . . . 153
Metabolites . . . . . . . . . . . . . . . 153
Metabolic Inhibitors. . . . . . . . . . . 154
Electrolyte Composition . . . . . . . . . 155

Compounds Transported by the Same
System. . . . . . . . . . . . . . . . . . 156
Structure . . . . . . . . . . . . . . . 158
Effects of Uremic and Normal Serum. . . . 159
The Effect of Age and Developmental
Stage of the Animal . . . . . . . . . . . 160
Sex . . . . . . . . . . . . . . . . . . . 161
Effects of Manipulations of Transport
System on 02 Consumption by Renal Cortical
Slices . . . . . . . . . . . . . . . . . . . 162
Methods . . . . . . . . . . . . . . . . . . . . 168
Accumulation of Organic Ions by Renal
Cortical Slices. . . . . . . . . . . . . . . 168
The Effect of Serum on Accumulation of PAH
by Renal Cortical Slices . . . . . . . . . . 170'
Kinetic Analyses of PAH Uptake . . . . . . . 170
Oxygen Consumption of Renal Cortical
Slices . . . . . . . . . . . . . . . . . . . 171
Renal Cortical Slice Composition . . . . . . 172
Plasma Free Fatty Acids. . . . . . . . . . 172
Histological Study of Renal Cortical
Slices . . . . . . . . . . . . . . . . . . . '173
Statistical Analyses . . . . . . . . . . . . 173
Results . . . . . . . . . . . . . . . . . . 175
Accumulation of the Organic Acid, PAH, by
Renal Cortical Slices. . . . . . . . . . . . 175
Factors Affecting the Accumulation of PAH
by Renal Cortical Slices . . . . . . . . . . 175
Rate of Initial Uptake of PAH. . . . . . . . 176

vi

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Table of Contents (Cont'd)

Part

Page

Accumulation of the Organic Base, NMN, by
Renal Cortical Slices. . . . . . . . . . . 176
Factors Affecting the Accumulation of NMN
by Renal Cortical Slices . . . . . . . . . . 177
Effect of Changing Diets on Accumulation
of Organic Acid and Base . . . . . . . . . . 177
Effect of Serum on Accumulation of PAH by

Renal Cortical Slices. . . . . . . . . . . 179

Histological Study of Renal Cortical

Slices . . . . . . . . . . . . . . . . . . 180

Kinetic Analysis of PAH Uptake . . . . . 180

Oxygen Consumption by Renal Cortical

Slices . . . . . . . . . . . . . . . . . . . 181

Renal Cortical Slice Composition . . . . . . 181
Discussion. . . . . . . . . . . . . . . . . . . 220
Speculation . . . . . . . . . . . . . . . . . . 231
Summary . . . . . . . . . . . . . . . . . . . . 233
Bibliography. . . . . . . . . . . . . . . . . . 235
Appendices

Appendix A Grain Ration . . . . . . . . . . 244

Appendix B High Fat Ration. . . . . . . . . 245

Appendix C Modified Grain Ration. . . . . . 246

Appendix D Modified High Fat Ration . . . . 247

Appendix E Parameters Evaluated in

Histological Sections of
Kidneys O O O O O O O O O O O O O 248

vii

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Table

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12

13

14

LIST OF TABLES

Summary of reports of renal function in
obese subjects . . . . . . . . . . . . . . . .

Selected reports of spontaneous changes in
renal histology in rats. . . . . . . . . . . .

Body weight, kidney weight and kidney weight/
body weight ratio of Osborne-Mendel male rats
fed GR or HF for varying periods of time . . .
Analysis of variance of body weight data . . .

Analysis of variance of kidney weight data . .

Analysis of variance of kidney weight/body
weight ratio data. . . . . . . . . . . . . . .

Percentage of animals in control (GR) and
experimental (HF) groups with histopatho-
logic kidney lesions . . . . . . . . . . . . .

Lesions observed only in kidneys from animals
45 weeks old and fed the grain or high fat
ration post-weaning. . . . . . . . . . . . . .

Lesions observed only in kidneys from animals
fed the high fat ration. . . . . . . . . . . .

Effect of diet on the accumulation (S/M ratio)
of PAH by slices of rat renal cortex . . . . .

Effect of diet on the accumulation (S/M ratio)
of NMN by slices of rat renal cortex . . . . .

Accumulation of PAH (S/M ratio) by slices of
rat renal cortex in the presence of serum. . .

Kinetic analysis of PAH uptake in rat renal
cortical slices. . . . . . . . . . . . . . . .

Effect of diet on pH of 24-hour urine samples
from lO-week old male rats . . . . . . . . . .

viii

Page

50

84

118

119

120

121

122

123

124

212

213

214

215

216

 

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List of Tables (Cont'd)

Table
15

16

17

Page

Effect of diet on the pH of the medium after
incubation of renal cortical slices. . . . . . 217

Oxygen consumption of renal cortical slices
from rats fed the grain ration or high fat
ration I O O O O 0 O C O O I O O O O O O O O O 218

Approximate composition of renal cortical
slices from rats fed the grain ration or high
fat ration O I O O O O O O I O O I O O O O 0 O 219

ix

 

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Figure

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11

LIST OF FIGURES

Kidney section from a weanling rat
(078004) 0 I C O O O O I O O O O I O O O O O O

Kidney section from an animal 15 weeks old
(078017) fed the high fat ration for 12
weeks I O O O O O I O O O O O O O O O O O O O

Kidney section from an animal 25 weeks old
(078008) fed the grain ration after weaning .

Kidney section from an animal (078010) 25
weeks old fed the high fat ration for 22
weeks showing glomerular and tubular

changes . . . . . . . . . . . . . . . . . . .

Kidney section from an animal (078010) 25
weeks old fed the high fat ration for 22
weeks showing glomerular and tubular

changes . . . . . . . . . . . . . . . . . . .

Kidney section from an animal (078024) 35
weeks old fed the grain ration for 32 weeks .

Glomerular damage in kidney section from an
animal (078026) 35 weeks old fed the high fat
ration for 32 weeks . . . . . . . . . . . . .

Tubular changes occurring in the kidney from
a rat (078027) fed the high fat ration for
32 weeks 0 O O O O O O O O O O O O O O O O O 0

Additional tubular changes occurring in the
same section as seen in the previous figure .

A glomerulus from the same section as seen
in the two previous figures . . . . . . . . .

Kidney section from an animal (080119) 45
weeks old fed the grain ration for 42 weeks
post-weaning. . . . . . . . . . . . . . . . .

Page

103

103

105

107

107

109

109

111

111

113

115

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List of Figures (Cont'd)

Figure

12

13

14

15

16

17

18

19

20

21

22

23

Glomerular and tubular changes in a section
from an animal (080116) 45 weeks old fed
the high fat ration for 42 weeks. . . . . . .

High-magnification view of tubular changes
occurring in the same kidney section seen
in the previous figure. . . . . . . . . . . .

Schematic diagram of the p-aminohippurate
(PAH) slice incubation system under several
conditions 0 O O O O O O O O O O O O O O O O 0

Model of the PAH transport mechanism in the
renal cortical slice as proposed by Foulkes
and Miller C O O O O O O O O O O O O O O O O O

Accumulation of PAH (S/M ratio) in renal
cortical slices from male rats of different
ages fed the control grain ration (GR) or the
experimental high fat ration (HF) . . . . . .

Accumulation of PAH (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against age 0 O O O O O O O O O O O O O O O O

Accumulation of PAH (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against bOdy weight 0 O C I O O C O O O O O O

Accumulation of PAH (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against kidney weight . . . . . . . . . . . .

Effect of body weight on kidney weight in
GR and HF rats. . . . . . . . . . . . . . . .

Kinetic analysis of PAH uptake by renal
cortical slices from 12—week old rats fed
GR or HF using the Lineweaver—Burk plot . . .

Kinetic analysis of PAH uptake by renal
cortical slices from 60-week old rats fed
GR or HF using a Lineweaver-Burk plot . . . .

Accumulation of NMN (S/M ratio) in renal

cortical slices from male rats of different
ages fed GR or HP . . . . . . . . . . . . . .

xi

Page

115

117

165

169

183

185

187

189

191

193

195

197

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Histolo:
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List of Figures (Cont'd)

Figure

24

25

26

27

28

29

30

Accumulation of NMN (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against age . . . . . . . . . . . . . . . .

Accumulation of NMN (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against kidney weight . . . . . . . . . . .

Accumulation of NMN (S/M ratio) in renal
cortical slices from GR and HF rats plotted
against body weight . . . . . . . . . . . .

Accumulation of PAH (S/M ratio) in renal
cortical slices from male rats fed GR and
switched to HF or fed HF and switched to

GR. . O O O O O O O O O O O O O O O O O O O

Accumulation of NMN (S/M ratio) in renal
cortical slices from male rats fed GR and
switched to HF or fed HF and switched to

GR. 0 O C O O O C O I O C O O O I O O O O O

Histological sections of kidneys from HF-
fed rats 0 O C O O O O O C O C O O O O O O O

Histological sections of kidneys from GR—
fed rats 0 O O I O O O O O O O O O O O O O O

xii

Page

199

201

203

205

207

209

211

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INTRODUCTION

Regardless of the criteria used for overweight, the
age group studied, or the country, obesity is a common
finding. The metabolic and endocrine abnormalities
associated with obesity have recently been reviewed
extensively (Gordon, 1970; Sims et aZ., 1971). These
include changes in blood lipids, in plasma insulin, gluca-
gon, and adrenal corticosteroids, in adipose tissue and
others (Sims et al., 1971). Also associated with obesity
are a number of abnormalities in organ function and struc-
ture. These include diseases of the liver and cardiovascular
system, gynecologic disorders, diabetes mellitus,
respiratory disorders and gastrointestinal disturbances.

There have been some suggestions that renal function
is impaired in obesity. Most reports, however, are
preliminary or limited in scope. Therefore, on the basis
of the high incidence of obesity and associated renal-
cardiovascular abnormalities in the obese, further investi—
gation was considered appropriate and necessary to evaluate
renal function in the obese state. The problems associated
with using humans as experimental subjects in an evaluation

of the effects of obesity on various physical parameters

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and physiological functions, however, are numerous. There-
fore, a number of animal models have been developed.

A variety of chemical compounds when injected into
experimental animals can precipitate obesity. These
include goldthioglucose (Brecher and Waxler, 1949), mono—
sodium glutamate (Olney, 1969), cortisone (Hausberger and
Ramsay, 1953), insulin (MacKay et aZ., 1940) and
bipiperidyl mustard (Rutman et al., 1966). There is no
unanimity as to the mechanism(s) whereby the obesity is
produced. Despite their limitations, induction of obesity
by chemical compounds appears to have less problems
associated with it than when obesity is produced with
electrolytic lesions.

Obesity resulting from bilaterally lesioning the
hypothalamus in rats without hypophyseal involvement was
first reported by Hetherington and Ranson (1939). Electro-
lytic destruction or electrocauterization by radio
frequency current of the ventromedial nuclei of the
hypothalamus in a number of species may produce obesity.
Several disadvantages exist when hypothalamic obesity is
used as an experimental model. These may include changes
in organ function, metabolism, and growth. Hypothalamic
obesity is a useful model in investigating the neural
factors that may regulate food intake of the organism.

Parasitic obesity has been produced in mice, rats,
deer mice and hamsters. Increased weight gains and growth

occurred in mice, for example, after the injection of

 

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spargana of Spirometra mansonoidee (Mueller, 1963). The
mechanism involved in this parasitic-induced weight gain
has not been determined.

A number of experimental models of genetic obesity
exist. These have recently been extensively reviewed by
Bray and York (1971). They include the “yellow" obese
mouse, the adipose mouse, the ob/ob or A0 mouse, the N20
mouse and the "fatty" rat. These models can be useful in
an attempt to understand the metabolic and biochemical
varieties of human obesity.

Researchers have developed a number of other proce-
dures which do not subject animals to operative procedures
or injections of chemicals and are perhaps most comparable
to the obesity in humans. These include force feeding,
feeding high caloric rations, restricting activity or a
combination of these (Fenton et al., 1951; Ingle, 1949;
Cohn, 1961, 1963; Mickelsen et aZ., 1955). The Osborne-
Mendel rat has a propensity for obesity when fed a 60% fat
diet. This model had previously been used extensively in
the laboratory in which my research was conducted
(Schemmel et al., 1969).

On the basis of the high incidence of obesity and
associated renal-cardiovascular abnormalities in obese
humans and of previous laboratory studies of kidney function
in the obese animal, further investigation was appropriate
and necessary to evaluate renal function in the dietary

obese rat. The kidney function tests were selected on the

basis of their sensitivity, their applicability to this
study, their usefulness in evaluating various parameters
of kidney function and the resources available. The experi-
ments were designed to test the hypothesis that dietary

obesity adversely affects kidney function in the laboratory

rat.

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BIBLIOGRAPHY

Bray, G. A. and D. A. York. 1971. Genetically transmitted
obesity in rodents. Physiol. Rev. 51:598-646.

Brecher, G. and S. H. Waxler. 1949. Obesity in albino
mice due to single injections of goldthioglucose.
Proc. Soc. Exp. Biol. Med. 70:498-501.

Cohn, C. 1961. Meal-eating, nibbling and body metabolism.
J. Amer. Diet. Assoc. 38:433-436.

Cohn, C. 1963. Feeding frequency and body composition.
Ann. N. Y. Acad. Sci. 110:395-409.

Fenton, P. F. and H. B. Chase. 1951. Effect of diet on
obesity of yellow mice in inbred lines. Proc. Soc.
Exp. Biol. Med. 77:420-422.

Gordon, B. S. 1970. Metabolic aspects of obesity. Adv.
Metab. Disorders 4:229-296.

Hausberger, F. X. and A. J. Ramsay. 1953. Steroid
diabetes in guinea pigs. Effects of cortisone
administration on blood and urinary glucose, nitrogen
excretion, fat deposition, and the islets of Langer—
hans. Endocrinology 53:423-435.

Hetherington, A. W. and S. W. Ranson. 1939. Experimental
hypothalamico-hypophyseal obesity in the rat. Proc.
Soc. Exp. Biol. Med. 41:465-466.

Ingle, D. J. 1949. A simple means of producing obesity
in the rat. Proc. Soc. Exp. Biol. Med. 72:604-605.

MacKay, E. M., J. W. Callaway and R. H. Barnes. 1940.
Hyperalimentation in normal animals produced by
protamine insulin. J. Nutr. 20:59-66.

Mickelsen, 0., S. Takahashi and C. Craig. 1955. Experi-
mental obesity. I. Production of obesity in rats by
feeding high-fat diets. J. Nutr. 57:541-554.

Mueller, J. F. 1963. Parasite-induced weight gain in
mice. Ann. N. Y. Acad. Sci. 113:217-233.

‘I
'P"'.O‘ ..
‘7'), 1:. r1;

fiflfl
lflw-b-V.
.
.
F'flc
Ja- vd .

u
.4
.

.: «Id

3 ‘ ;‘

Olney, J. W. 1969. Brain lesions, obesity and other
disturbances in mice treated with monosodium glutamate.
Science 164:719-721.

Ruhman, R. J., F. S. Lewis and W. D. Bloomer. 1966.
Bipiperidyl mustard, a new obesifying agent in the
mouse. Science 153:1000-1002.

Schemmel, R., O. Mickelsen and Z. Tolgay. 1969. Dietary
obesity in rats: Influence of diet, weight, age and
sex on body composition. Amer. J. Physiol. 216:373-
379.

Sims, E. A. H., E. S. Horton and L. B. Salans. 1971.
Inducible metabolic abnormalities during development
of obesity. Ann. Rev. Med. 22:235-250.

312' 1. Review ’

I IIII’IE'PIIP.
. . “
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5. u.
I

PART I . Review of literature.

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The Functions of the Kidney

The physiological and biochemical functions of the

kidney must be understood prior to selecting tests of
kidney function and assessing their results. The kidneys

hold the key position in controlling the homeostasis of

the internal environment. They accomplish this (1) by
removing waste materials and substances that are useless
to the body, regardless of their concentration in the
blood and whether they are end products of metabolism (as

urea, creatinine, etc.) or are exogenous substances (drug
metabolites, phenol red, heavy metals, etc.); (2) by
Participating in the regulation of the acid-base balance
of body fluids; (3) by regulating electrolyte composition
and osmotic balance; (4) through the production and
release of renin and aldosterone; (5) through the produc—
tion and release of erythropoietin; and (6) by the
conservation and reabsorption of substances valuable to
the 13051)! economy, such as glucose, protein, amino acids,
etc- When their concentration is commensurate with bodily
Garlands and their quantity does not exceed the maximal
ea~P<Wity of the renal tubular mass. The kidney is second
thy to the liver in synthetic activities and in other
h2.31‘3‘311‘3mica1 functions of the body. The kidney may also
Serve as a storage organ. Okuda (1962) has shown that

t
he rat kidney stores vitamin B12 that has been absorbed

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in excess of the body requirement and releases it when

the need arises. The kidney is the site of the conversion

of vitamin D2 or vitamin D3 to the active metabolite,
1,2S-dihydroxycholecalciferol (Gray et aZ., 1971) .
The kidney is an active site of glucose formation.

In addition it can synthesize amino acids, protein,

mucoproteins and fats.
The role of the kidney in protein metabolism has been

recently reviewed by Cahill and Owen (1970) . Since the

kidney is the primary excretory organ for nitrogen, the
kidney's role in overall nitrogen homeostasis is an

important one .

Urea excretion is primarily a function of the blood
Urea concentration, so the kidney's role is passive.
Ammonia excretion, in contrast, is actively regulated by
the kidney. Nash and Benedict (1921) demonstrated that
renal tubular cells form ammonia from precursors in
arterial blood and secrete it in high concentration into
tubular urine. Glutamine is the primary precursor; a-
amino nitrogen of other amino acids may also be used in
armaonia formation.

The kidney may also serve as a proteolytic organ,
b‘Jt PIObably only to small protein or peptide molecules
Capable of passing through the normal glomerulus. Under
abnormal conditions (proteinuria, nephrotic syndrome,
th.) erlich led to an excessive accumulation of protein

molecules of small molecular weight, or where there is

 

 

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increased glomerular permeability to large molecules, the
kidney may be a major site for protein catabolism (Cahill
and Owen, 1970) .

In contrast to other organs such as the liver, heart
and muscle where many endogenous substrates are utilized,
the kidney utilizes relatively few substrates for its
energy requirements in vivo (Cohen, 1964). The major
endogenous renal substrates are found in the plasma and
include nonesterified fatty acids (especially palmitate) ,
lactate, glutamine and a-ketoglutarate.

The nephron is the functional unit of the kidney.
The processes it uses to accomplish its job are filtration,
reabsorption and secretion.

The kidneys receive approximately one-fourth of the
cardiac output. From this volume, the kidneys remove by
filtration a portion of the blood with dissolved products,
excesses of electrolytes, and foreign materials.

About 99% of the material filtered at the glomerulus
is returned to the blood through the reabsorption process

as the filtrate passes through the nephron. Substances
reabsorbed in the proximal and distal tubules and in the
loop 0f Henle include water, sodium, glucose, amino acids,

b icarbonate and vitamins .

Se<=J:etion is the process by which proximal and distal
Embular cells transport materials from peritubular fluid
“to the tubular urine. Substances secreted include ions

8
“Ch as NH4+ and K"' and foreign substances such as penicillin,

phen01 red, and bromcresol green.

.N.

 

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10

The Effect of Diet on Kidney Function,

Size and Composition

According to Kark (1968), disturbances of kidney

functions and structures in animals have been produced

with some difficulty by dietary manipulation. However,

nutrition as a factor influencing kidney structure and

function in humans and animals has often been underesti-

mated. Kidney composition, size and function may be
affected by dietary deficiencies, excesses or imbalances.

The following is a review of several dietary treatments

which may affect the kidney in humans and animals.

Malnutr 1 tion

Hlamans:
The effect of malnutrition (various states of starva-

tion or protein-calorie malnutrition) on kidney function

111 humans depends in part upon the age of the individual

when the dietary restriction is imposed. Klahr et al.

( 1957) States that "careful and complete studies of renal

Ennction during severe malnutrition are scarce." An
eXamination of the literature substantiated his claim.

The question of whether malnutrition affects kidney
function in the adult has not been sufficiently determined.
8 tUdieS have been limited primarily to observations of
persons in prison or detention camps during or after wars.

E ' '
arlier reports of the effect of starvation on kidney

,4“.

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were IEVL

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.
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::.:a:.:raticn an:
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The most cor
22132;:‘shed {at

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11

function were reviewed by Keys at al. (1950) and McCance

Only a few of the starvation studies after World

(1951)-
Furthermore,

War II contain data on kidney function.

these data are incomplete, with emphasis on tests of

concentration and dilution. Despite the scarcity of

recent data, however, several conclusions can be made.

The most commonly described renal disturbances in

malnourished patients are polyuria and nocturia

(Mollison, 1946; Keys et aZ., 1950; McCance, 1951; Klahr

8t al. , 1967) . These symptoms were described in detail

in World War I victims by Schittenhelm and Schlecht in

1918 according to Klahr et al. (1967) .

Semistarved individuals have an increased urine

Volume (Keys et al., 1950; McCance, 1951; Alleyne, 1966;

Klahr et al., 1967). For example, the volume of urine

from semistarved subjects in the Minnesota study was 3-4

liters in 24 hours (Keys et al., 1950). Mollison (1946)

reported no abnormalities in examination of urine from
S tarvation cases at the Belsen detention camp. However,

determinations of glomerular filtration rate (GFR) and

renal Plasma flow (RPF) were made on only 4 subjects and

refill-ts were inconclusive. McCance (1951) reported that

undernourished men in Wuppertal responded to a water

(illution test normally, but were unable to compensate

w
hen water was withheld. Edema, not abnormal kidney

unCtJ-On, was responSible for this, according to McCance

( 1951) - Subjects in the Minnesota starvation study had

I
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12

polyuria (Keys et aZ., 1950); however, no specific deter-

minations of renal function in these men were actually

made.
Renal function was depressed in obese persons starved

for weight reduction as evidenced by 50% decreases in GFR

and CPAH (Edgren and Wester, 1970) .

In a carefully controlled experiment of protein mal—

nutrition ‘in 11 adults, Klahr et a2. (1967) reported that

a defect in renal concentrating ability was present.

Urine osmolality following 14 hours of fluid deprivation

never exceeded 600 mOsm per kg H20 in the malnourished

State; whereas, in normal subjects under similar conditions

this value approached 800 mOsm per kg H20 (Klahr et al. ,

1967) . This concentrating defect was reversible after

36-141 days of protein repletion.

The plasma potassium concentration was normal in
8“meets in the malnourished state; therefore, Klahr et al.
( 1957) reasoned that the impairment of the urinary con—

centrating mechanism was not a result of a concomitant

potassium depletion. Decreased urea concentration in the

renal medulla was suggested as the most likely cause of
the concentrating defect in malnourished patients (Klahr

‘53 t al- . 1967) .

Srikantia (1968) reported that renal clearances of

:Lnulin and diodrone in children suffering from

waShlorkor and in adults with nutritional edema were not

 

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13

abnormally low. He suggested that antidiuretic hormone

(ADH) plays a causative role in the genesis of the edema
of protein-calorie malnutrition (Srikantia, 1968) .

Some altered kidney functions and histologic
degeneration occurred when young humans were malnourished.
Glomerular hyalinization and pericapsular fibrosis were

seen in kidneys from infants and young children dying

from kwashiorkor (Davies, 1948). Stirling (1962) reported

that 22 of 31 Jamaican children suffering from malnutri-

tion had renal lesions. Furthermore, he suggested that

there may be residual renal damage even if the infants

recover from the malnutrition (Stirling, 1962) .

In contrast to Stirling (1962), Alleyne (1966) found
that kidney function in children improved with recovery.
Prior to recovery, malnourished children had depressed

glomerular filtration rates (GFR) and renal plasma flow

( RPF) Values. Furthermore, such indicators of impaired

tubular function as amino aciduria, renal phosphaturia,
lmpaired urinary concentrating ability and an inability

to excrete an acid load in these children improved on

refeeding (Alleyne, 1966).

Malnutrition in a number of forms appears to influence
\r -
arious kidney functions in children and adults. Further
Q . .
arefully controlled studies investigating a number of

a‘11‘5f‘31'fi‘P-nt kidney functions are required.

 

;;3‘.51

3:13:81 05
r; :.;::ion are
,.
:33, Dicker e:
33,3: casein d;
.r::e::. and caLc:
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are; the maxi."
:iie control ;;
25::ictio.. (Di:
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:fsaae of the ;
iilzcurished an

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14

Animals:
A number of animal studies suggest that renal size
and function are affected by various states of malnutri-

tion. Dicker et al. (1946) placed rats on turnip, carrot

and low casein diets that were markedly deficient in

protein and calories. After 36 days on the diet, the

rats did not respond normally to water concentration

and water dilution tests. Concentration tests showed a

large loss of the ability to produce a concentrated

urine; the maximum specific gravity dropped from 1.070

in the control period to 1.041 after 36 days of dietary

restriction (Dicker et al., 1946). However, this

Observation could be related to the possible retention
of some of the administered water as edema fluid in the

malnourished animals and may not reflect dietary damage

to the kidney per se.
Furthermore, glomerular filtration rate increased

in the semistarved state as the urine flow rate increased

(Dicker et al., 1946) . This suggests that the semistarved

rat PrOduces a diuresis by increasing the volume of

glomerular filtrate rather than by decreasing tubular

reabsorption as does the normal rat. Renal plasma flow

also increased in the rats on the deficient diets in

proPOrt-ion to the rate of urine flow. The total tubular

Qxcretory mass (Tm) of diodone in the malnourished

animals was about 30% of the control value (Dicker et al.,

~-)-_ ‘

 

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15

1946). In addition, Krebs et a2. (1963) reported that

renal gluconeogenesis increased in rats starved for only

48 hours.

Much work has recently been centered on the effect
of maternal malnutrition on the kidneys of the offspring.
A reduction in relative kidney size (as a percent of body
weight) was reported for newborn rats delivered by dams
fed a protein restricted ration (Zeman, 1967). This is
important since several workers have proposed that organ
size is related to the degree of maturation (Potter and
Thierstein, 1943). Furthermore, total RNA and DNA were
decreased in these kidneys at 20 days of gestation (Zeman
and Stanbrough, 1969) . Animals nursed by dams subjected
to protein deficiency during gestation also had lower
kidney weights than controls (Zeman, 1970). Furthermore

these kidneys had less RNA, DNA and total protein (Zeman,
1970).

Alkaline phOSphatase, nonSpecific esterase, leucine—
aminopeptidase, ATPase, and acid phOSphatase were studied
histochemically in kidneys from newborn rats (Zeman,
1968). Alkaline phosphatase has long been considered a

sensitive indicator of the functional capacity of the

kidney (Brain and Kay, 1927). It is suggested that this

 

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16

enzyme, which is located in the brush border of the
proximal convoluted tubules in the rat kidney (Wachstein,
1955) presumably functions in tubular absorption
phenomena (Wilmer, 1944). The acid phOSphatase is used
as an indicator of the presence of lysosomes (Straus,
1954; Miller and Palade, 1964). Zeman (1968) found no
change in ATPase, nonspecific esterase or leucineamino—
peptidase activity in kidneys from control and
maternally protein restricted rats. However, levels of
alkaline and acid phosphatase were decreased (Zeman,
1968), indicative of retarded development since enzymo-
genesis is considered to be an aspect of differentiation
according to several workers (Moog, 1952; Verne and
Hebert, 1964).

Renal immaturity was further suggested in the protein-
deficient young as indicated by increased quantities of
mesenchymal-like connective tissue, fewer identifiable
glomeruli, a larger proportion of immature glomeruli and
decreased proximal tubules or shorter tubules (Zeman,
196 3) ,

In a follow-up study Hall and Zeman (1968) measured
khflney function in newborn, 2-, 4-, and 6-day old rats
using water diuresis, osmotic diuresis, and inulin
clearance. The clearance of inulin in control animals

was four times that of the protein-deprived rats. Urine

A

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excretion was decreased in the experimental animals in
water and osmotic diuresis tests (Hall and Zeman, 1968).

Chow and co-workers investigated kidney function in
offspring from dams fed, during gestation and lactation,
a normal ration restricted to 50% of that consumed ad
Zibitum by controls (Lee and Chow, 1965, 1968; Roeder and
Chow, 1969). Animals from these underfed dams, although
fed ad Zibitum post weaning, excreted greater amounts of
nitrogen. Furthermore, there was an abnormal distribution
among the nitrOgen-containing components of the urine
(Lee and Chow, 1965). Animals from restricted mothers
had a lower mean nitrogen retention at 6, 10, 14 and 18
months. Urine from rats born to dams fed restricted
amounts of the ration during pregnancy and lactation had
a significantly larger proportion of total amino acids
and a significantly smaller prOportion of urea, creatinine,
and ammonia (Lee and Chow, 1965). This increased urinary
eXCretion of amino acids could result from high blood
levels of these, or impaired renal tubular absorption.

Urine from experimental animals at 3 months of age
had a larger percent of ammonia than that from control
animals. This suggested to these workers that in the
animals from restricted dams there was an impaired acid-
base balance or a higher level of circulating amino acids
as urinary ammonia comes from hydrolysis of blood

glutamine in the kidneys and oxidative deamination of

 

 

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18

blood amino acids. In later work Lee and Chow (1968)
showed that progeny from restricted animals excreted more
free basic amino acids, such as arginine and histidine.
Recently, Roeder and Chow (1969) looked at the con-
centrations of RNA, DNA and activities of glutaminase I
and cathepsin in the kidneys from progeny of restricted
animals. At 13 months of age there was no significant
differences in these parameters. At 19 months, glutaminase
I activity per unit wet weight was the same in progeny
from dams restricted during gestation and lactation and
control groups although both groups were fed ad Zibitum
post weaning. When this activity was expressed per unit
of DNA to estimate enzymatic activity per cell, the values
were lower in the animals from restricted dams. Cathepsin
activity per unit wet weight or per unit of DNA was higher
in progeny from restricted dams than in control groups.
It increased significantly in the restricted animals bet—
‘Wken 13 and 19 months of age. These data suggested the
early'appearance of senescent changes in the kidneys from
PrOgeny of dams restricted in feed consumption during
Pregnancy and lactation (Roeder and Chow, 1969) .
Widdowson and McCance (1956) could detect no differ-
ences in loss of kidney weight as a proportion of body
weight in growing and adult rats subjected to complete
Starvation and to undernutrition. Stewart (1919) reported

that kidney weights were increased in rats subjected to

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19

repeated periods of fasting from birth until age 11-22
days. In contrast, Brown and Guthrie (1968) found the
absolute weight of kidneys in rats deprived during and
after weaning was less than that in control animals.
Furthermore, upon refeeding these animals a nutritionally
adequate diet for up to 10 weeks, kidney weights were
less than 80% of those of control rats (Brown and
Guthrie, 1968).

As reviewed above, there is considerable evidence

that malnutrition affects renal function in animals.

Potassium

Humans:

Renal abnormalities are produced in humans by a
potassium deficiency. According to Conn and Johnson
(1956), kaliopenic nephropathy should be recognized as
an established clinical and pathologic entity.

Conn and Johnson (1956) reviewed the early reports
0f renal tubular lesions accompanying cases of chronic
difsentery, bacillary dysentery and other chronic intesti—
na1 diseases. Perkins et al. (1950) were the first to
aSsociate these renal tubular changes with a potassium

deficiency. Since then several reports have related the

tubular lesions ("clear cell nephrosis" or "vacuolar
nephropathy") to the state of potassium deficiency
associated with chronic intestinal disease (Keye, 1952;

Schwartz and Relman, 1953; Achor and Smith, 1955).

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20

A hypokalemic state resulting from excessive use of
laxatives produced impairment of renal function in two
humans studied by Schwartz and Relman (1953). These sub-
jects had a depressed clearance of p—aminohippurate and
an inability to concentrate urine maximally (Schwartz and
Relman, 1953).

Humans with primary aldosteronism show the same
tubular lesion morphologically that have been observed in
autopsy cases with histories of potassium depletion
resulting from some chronic intestinal disease (Conn and
Johnson, 1956). Furthermore, these clinical cases of
primary aldosteronism show histoloqic lesions similar to
those in animals on a potassium-deficient diet (Schrader
et al., 1937; Follis at al., 1942; Follis, 1943; Newberne,
1964; Segar and Schulz, 1965) or fed an adequate diet
but given large doses of desoxycorticosterone (Darrow
and.Miller, 1942). Muehrcke and Rosen (1964), however,
S'llg'gested that the site of alterations in the kidney may
TKTt be the same in potassium-deficient humans, as in
IKItassium-deficient animals. Whether potassium deficiency
Produces changes in urinary diluting capacity is unclear
CLevitin at aZ., 1960).

Renal lesions resulting from an induced depletion of
Potassium in adult humans appear to be reversible (Blahd
and Bassett, 1953; Fourman, 1954; Evans et aZ., 1954;

Womersley and Darragh, 1955; Clarke et aZ.,l955).

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Animals:

As a result of many studies in animals, it is evident
that renal cells, eSpecially tubular cells, need adequate
potassium to preserve their integrity. In fact,
Hollander at al. (1958) suggested that permanent renal
damage may result from an acute episode of potassium
depletion. They observed that rats after 3 days of
potassium deficiency followed by adequate dietary
potassium were unable to concentrate urine maximally.

In contrast, Holliday and Egan (1962) found that
rats on a potassium deficient diet for 30 days had GFR

and CH 0 values which were less than those in controls.
2

When these animals were refed potassium for 5-13 days,
values for GFR and CH20 returned to normal (Holliday and
Egan, 1962). Segar and Schulz (1965), exposed rats to
repeated periods of potassium deficiency interSpersed
wi'tlhperiods of adequate dietary potassium. They
reported that although kidneys from these rats showed
tulDular dilation, the rats could concentrate urine
maximally.

Microsc0pic tubular lesions in rats, mice, dOgS,
Cats and monkeys with potassium deficiency have been

reported (Schrader at al., 1937; Follis et al., 1942;

Follis, 1943; Newberne, 1964; Muehrcke and Rosen, 1964;

Segar and Schulz, 1965).

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22

Concentrations of the enzymes, glutaminase and
carbonic anhydrase are increased in kidneys from potassium-
deficient rats (Iacobellis et aZ., 1954). Polydipsia and
polyuria (Brokaw, 1953), decreased creatinine clearance
(Muntwyler and Griffin, 1953), renal hypertension
(Grollman and White, 1958), increased protein excretion
(Morrison and Gardner, 1963), and renal hypertrOphy
(Durlacher at al., 1942; Follis et al., 1942; Fuhrman and
Brokaw, 1951; Brokaw, 1953; Muntwyler and Griffin, 1953;
Perey et al., 1967a, 1967b) also occurred in potassium-
deficient animals.

Inclusion of rubidium and, to a lesser extent, cesium
in potassium-deficient diets protected rats from the
renal lesions normally seen with potassium deficiency
(Follis, 1943). The distribution, biological properties
and excretion of rubidium are very similar to those of
Potassium. Rubidium was actively transported into the
rabbit lens in competition with potassium (Becker, 1962) .
Furthermore rubidium replaced potassium in the activation
as Na-K ATPase in this system (Bonting et al., 1963).
Whether rubidium acts similarly in the kidney has not
beendetermined.

Perey and co-workers (1967a, 1967b) induced multiple
Cysts in the kidneys of fetal rabbits. A single injection
Of a long-acting adrenal corticosteroid (as 9-fluoro-
prednisolone) into the rabbits at birth produced cystic

changes in the kidneys. These changes could be prevented

 

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23

by repeated injections of potassium chloride (Perey at aZ.,
1967a, 1967b). Therefore, the hypokalemia or a general
systemic effect produced by the corticosteroids could be
the causative factor (Perey at al., 1967a, 1967b). This
finding could have great implications considering the
problem of congenital polycystic renal disease in man.
Additional evidence for the kidney's requirement for
potassium comes from tissue culture studies. Crocker and
vernier (1970) found that the fetal mouse kidney required

a high concentration of potassium for normal development.

Sodium
Humans:

Information concerning various states of sodium
status and renal histology and function in humans is
Sparse.

Black et al. (1950) reported altered tubular function
in humans fed a salt-poor diet for only a few days. The
reabsorption of sodium by the renal tubules was increased
10-2 hours after hypertonic saline was given intravenously
to these subjects.

Hypertensive patients on a low salt diet had
depressed GFR, RBF and TmPAH values (Chasis et al., 1950).
Sodium metabolism in pregnancy relative to kidney

function has been inadequately investigated (Robinson,
1958; Lindheimer and Katz, 1970). These authors concluded

that further research is needed in this area since now

 

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24

some doctors consider the pregnant patient to be an

"insidious salt retainer" while others consider pregnant

subjects to be "insidious sodium wasters."

Animals :

High sodium chloride and low sodium chloride diets
produce a number of changes in the kidneys.

A common finding associated with high dietary
intakes of sodium chloride was renal hypertrophy (Krakower
and Heino, 1947; Sapirstein et al., 1950; Auerbach at al.,
1953; Fregly, 1960; Dahl and Schackow, 1964; Hall and
Hall, 1966) . A number of pathological changes were
GVident in kidneys from animals on a high sodium intake.
Glcunerular changes in the rat included hypertrophy, lipid
deposition and degeneration (Meneely et al., 1953b) .
ASSociated with these glomerular changes were edema and

p0lyuria (Meneely at al., 1953a) . Krakower and Heino

(1947) reported glomerular hypertrOphy and glomerular and
tubular degenerative changes in kidneys from chickens on
El Iligh sodium chloride intake. Tubular dilation and
tieaths from uremia occurred in rats on a chronic excess
Se-:Lt ingestion (Dahl and Schackow, 1964) . Among the

szt extensive renal lesions observed with a high salt
diet were reported by Auerbach et a2. (1953) in rats fed
a diet of 7.0 to 9.8% NaCl. Abnormalities included

a2Otemia, enlarged glomeruli with swelling and

 

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25

vacuolation, and dilated tubules. These were present in

15% of the animals studied (Auerbach et aZ., 1953).

Hypertension was a consistent finding in animals

eXposed to a high salt diet (Meneely at aZ., 1953a, 1953b;

Auerbach at aZ., 1953; Dahl and Schackow, 1964). The

relationship of the hypertension to the renal changes

observed, especially the glomerular changes, has not

been differentiated.
A number of pathological lesions were reported in

rats fed simulated Japanese diets (Hilker et aZ., 1965) .
Whether the sodium content or some other factor in the

diert.was reSponsible for the lesions was not determined

(Hilker et aZ., 1965).
Sodium deprivation also produced considerable renal
1948; Marx and Deane, 1963;

dainage (Cuttino et aZ.,
Ganguli et aZ., 1969a, 1969b).

WaI‘l‘dlaw and Pike, 1963;
ReI‘lal hemorrhage and dilated tubules (Cuttino et aZ.,

1948) , decreased proximal tubular lumens (Marx and

[Deeéine, 1963), and juxtaglomerular alterations (Ganguli

1963; Wardlaw and

e t aZ., 1969a, 1969b; Marx and Deane,

P:the, 1963) occurred in rats on low sodium diets.

 

Magnesium deficiency in man was recently reported
( Flink, 1956; Vallee et aZ., 1960); however, renal changes

asSociated with this deficiency in man were not described.

'If. I'WJ

 

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26

Magnesium deficiency in infants with kwashiorkor has also
been reported (Montgomery, 1960), but no effect of a
magnesium deficiency per as on kidney function was investi-

gated.

Animals:

Rats fed a magnesium deficient diet developed
calcification of soft tissues, particularly renal tissues
(Tufts and Greenberg, 1936; Watchorn and McCance, 1937;
Greenberg et aZ., 1938; Sullivan and Evans, 1944; Hess
et aZ., 1959; McAleese and Forbes, 1961; Welt, 1964;
Schneeberger and Morrison, 1965; Jacob and Forbes, 1969;
Farnell and Whitehair, 1971). On the contrary, however,
Schrader at al. (1937) did not observe calcification in
rats fed a magnesium-deficient diet for 34 days.

KK mice (unlike ICR, C57BL and CFI mice) showed renal
calcification when fed a low magnesium, high phosphorus
diet (Hamuro et aZ., 1970). Therefore, Hamuro et al.
(1970) suggested that there was a strain difference in
susceptibility to the renal calcification produced by the
magnesium-deficient diet. Magnesium deficiency and
calcification of renal tissue has also been produced in
the dog (Featherston et aZ., 1963), guinea pig (Pyke at aZ.,
1967) and cotton rat (Constant and Phillips, 1952).

A number of other degenerative changes affecting
tubules and glomeruli were reported in the early studies

(Df magnesium deficiency (Watchorn and McCance, 1937;

 

 

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27

cramer, 1932; Sullivan and Evans, 1944). The diets used
in some of these studies, however, were inadequate or
imbalanced in respect to some nutrients other than
Inagnesium. Also, the changes observed were described
primarily as histological changes but no estimation of
the functional capacity was made.

Other changes seen in magnesium deficient animals
inraluded nephrosis (Barron et aZ., 1949); renal fibrosis
(Barron et aZ., 1949); increased proteinuria (Cramer,
.19132; Greenberg et aZ., 1938); glomerular degenerative
Changes (Cramer, 1932; Sullivan and Evans, 1944); and
tllkrular degenerative changes other than calcification
(Czlramer, 1932; Schrader et aZ., 1937; Greenberg at aZ.,

1938; Sullivan and Evans,l944; Hess at aZ., 1959).

Whether a magnesium deficiency produces a polyuria

(Cramer, 1932; Greenberg et aZ., 1938; Smith et aZ.,

1962) or no change in urine volume (Manitius and Epstein,
1963; Schneeberger and Morrison, 1965) has not been
easitablished. Manitius and Epstein (1963) found that a
potassium depletion can be induced in rats by a magnesium
Cieef'icient diet with potassium levels that would normally
k3€e considered adequate. This result makes it more
clifficult to interpret much of the earlier work concerning
1:11e effect of a magnesium deficiency on renal function
andhistology.

Renal tubules in rats fed a magnesium—deficient diet

with adequate potassium showed significant functional

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28

impairment which closely resembled that of potassium
depletion (Smith at aZ., 1962) . Rats showed a 33%
depression of concentrating capacity and a decreased
reSponse to an acid load when compared with rats on a
control diet (Smith et aZ., 1962).

The production of calcification of soft tissues,
especially renal tissue by a magnesium deficient diet
has provided a convenient tool for investigating factors
that influence soft tissue calcification (Jacob and Forbes,
1969). L-thyroxine (Jacob and Forbes, 1969); Vitamin D
depletion (Jacob and Forbes, 1970) and thyrocalcitonin
(Parnell and Whitehair, 1971) prevented or lessened the
Severity of renal calcification normally seen in rats on

a magnesium deficient diet.

Choline

\__
fitnmans:

Choline may also play a role in maintenance of normal
kidney structure in humans. Arends and Nieweg (1954)
JIVE—sported two cases of choline-like deficiency with renal
Eirld hepatic lesions closely resembling those produced
GeJ-cperimentally in animals. Enlarged kidneys, tubular

lesions, hemorrhage and casts were observed (Arends and

Nieweg, 1954) .

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29

Animals:
Acute choline deficiency in weanling rats is associ-
ated with hemorrhagic nephropathy accompanied by an
increase in kidney weight, in part due to cellular proli-
1939; Hartroft, 1948;

1969) .

feration (Griffth and Wade,

Levenson at aZ., 1968; Parks and Smith, 1968,

Monserrat at al. (1968) suggested that, in weanling rats,

lysosomal alterations are the outstanding pathological
change in choline deficiency renal necrosis.
Renal hypertension developed in rats and dogs sub—

jected to a choline deficiency (Hartroft and Best, 1949;

Grollman and White, 1958) . Nagler et a2. (1968, 1969)

rePorted that feeding diets low in choline to weanling

rats produced an imbalance in vasoactive mediators due

to a fall in tissue acetylcholine. They pr0posed that

tl‘lis led to vasospasm, ischemia, vascular rupture and the
tubular necrosis seen in the nephropathy of acute choline

deficiency (Nagler at aZ., 1968, 1969).

Renal tubular degeneration and non-calcified, mucoid

structures in the urinary bladder were seen in kidneys

fJi‘om rats subjected to diets marginal in choline and

methionine (Newberne and Young, 1966) . The "hemorrhagic

kidney syndrome" of weanling rats was used as a bioassay
to test the nutritional adequacy of various diets in

supporting methylneogenesis for the synthesis of choline

(Woodard, 1970) .

Ettanin B, L

b

..;‘r ‘Ett'v' ac)“;
"Ab" - ‘

 

waned the y\
a: fatty live:

I
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30

Vitamin 812 (Hawk and Elvehjem, 1953) and medium
chain fatty acids in coconut oil (Zaki et aZ., 1966)
protected the young rat from hemorrhagic renal necrosis
and fatty liver when fed a choline deficient diet.
Short and long chain fatty acids aggravated the

deficiency manifestations (Zaki et aZ., 1966) .

Protein
Humans:

Kidney function may be altered in kwashiorkor (Davies,
1956) and in other states of protein deficiency as
Previously discussed under the topic of malnutrition.

High protein levels had no adverse effect on renal
function of humans according to Strouse and Kelman (1923) .
These workers fed 150 g of protein daily. Similarly,
tWO Artic explorers who consumed an exclusive meat diet
for 1 year had no albumin, casts or blood in urine
SE>ecimens (Lieb, 1929). Furthermore, the urea clearance
and phenol red excretion were normal. One subject
followed by Newburgh et a1. (1930) consumed 333 g protein
Ciatily. Albuminuria occurred after 6 weeks and hyaline
and granular casts were evident after 7 weeks on this
regimen. The urine was normal after the subject was fed
a high carbohydrate diet for 10 days (Newburgh et aZ.,

l 9 30) .
The effect of the protein level on the urea clearance

(Curea) in man was extensively reviewed by Schmidt-Nielsen

a

I
s

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url”

L

7‘

 

.

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r” "v

 

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3.
..,..

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31

(1958). In general, the Curea varies with the protein

content of the diet. When the protein level is low,

particularly over a prolonged period, the Curea drops

considerably (COpe, 1933; Goldring et aZ., 1934; Nielsen

and Bang, 1948, 1949; Pullman at aZ., 1949). Increasing

the protein level of the diet will increase the Curea

(Addis and Drury, 1923; COpe, 1933; Farr, 1936; Longley

and Miller, 1942; Nielsen and Bang, 1948, 1949). Gold-

ring et al. (1934) did not report an increased Curea

The control protein

when

the protein intake was increased.
level, however, was 100 gm/day which would be considered

by some researchers as a high level.

The maximum difference in Curea between normal and

low protein intakes was found at low urine flow rates
(Schmidt-Nielsen, 1958) . Whether the change in Curea is

due primarily to a change in glomerular or tubular function
has not been established (Nielsen and Bang, 1948; Schmidt-
Nielsen, 1958).

Bolourchi and co-workers (1968) presented data which

S"~lggest that the Curea may be affected by the type, as

Well as by the dietary level, of protein.

The Specific gravity of urine (Addis and Shevky,
1922) and the GFR (Nielsen and Bang, 1948; Pullman et aZ.,
l 949) decreased when subjects were fed a low protein diet.
The effective renal plasma flow (ERPF) was depressed 2%

in females on a low protein diet (Nielsen and Bang, 1948) -

‘ ' V‘ ’-
.-,"ye:361\‘ ' tilt
w" ‘

 

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32

Conversely, the ERPF was increased 18% when the protein
content of the diet was increased to 200 g/day (White and

Rolf, 1948) . An increase in ERPF was also reported by

(1949) when subjects were given 2.3 to
The GFR was

Pullman et a1.
3.0 g protein per kg body weight per day.
also increased in persons on a high protein diet (White

and Rolf, 1948; Pullman et aZ., 1949).

Animals :
In animals, especially the dog, variations in protein

intake have a greater effect on renal hemodynamics than

in man (Schmidt-Nielsen, 1958).

Urea Clearance and Excretion: Factors affecting the

urea excretion in dogs, seals, ruminants, rodents and other
Inanimals was extensively reviewed by Schmidt-Nielsen (1958) .
Urea clearance in the dog in the postabsorptive
S"Ilate (i.e., 18 hours postprandially) was increased on
meat diets when compared with a low protein (cracker meal)
or mixed diet (Jolliffe and Smith, 1931a, 1931b). Similar
reports relating urea clearance to the protein level of

1ll‘ie diet were published by numerous researchers (Rhoads

et aZ., 1934; VanSlyke et aZ., 1934; Pitts, 1935).
Herrin et al. (1937) reported that the Curea was

increased when dogs were given casein, butter, glycine,

alanine, glutamic acid, deaminated glycine, lactic acid,

pYruvic acid, acetic acid and prOpionic acid. Gluconic

aCid did not elevate the Curea in these dogs.

w.

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33

Recently, workers demonstrated that the site of the
accumulation of urea in the renal medulla was different
in dogs on low and high protein diets (Truniger and
Schmidt-Nielsen, 1964; Schmidt-Nielsen and Robinson, 1970).
Furthermore, the excretion of urea is apparently depend-
ent on the quality of protein as well as the amount of
protein. When rats were fed casein or gluten so that
nitrogen intakes and digestibility were equal, the urea
excretion was increased in the gluten-fed group
(Kiriyama and Ashida, 1964).

Glomerular Filtration Rate and Effective Renal Plasma

Flow:

 

According to Smith (1951), the most complete study
of the effects of protein on renal function in the dog
was made by Moustgaard (1948) who showed that after a high
protein meal the GFR and ERPF of dogs were increased. No
alterations in the reabsorptive capacity for glucose in
these animals was reported (Moustgaard, 1948). Smith
(1951) reviewed in detail other studies completed by
Moustgaard.

The GFR increased in dogs (Pitts, 1944; Ayer et aZ.,
1947) and in rats (Dicker, 1949) when the protein content
of the diet was increased. Glycine when fed or infused
produced increases in the GFR and ERPF in the dog

(Friedman, 1946). Rats similarly treated showed no changes

in the GFR or ERPF.

rats were fed
azierate till-1L;
iii Curtis, 1
grziuced by t
3:133 (Newbu:

MEdlar 5
Pittein diet;
3.6 PrOXimal
these rats.

 

 

34

No changes of an inflammatory or degenerative nature
were observed microscopically in the kidneys of rats fed
a 75% protein diet (Osborne et aZ., 1923). Some minute
tubular and glomerular changes occurred, but these were
insignificant (Osborne et aZ., 1927).

In contrast, rats fed less than a year a diet con-
taining 40% or more liver had "granular" kidneys with
degeneration of tubular epithelium and fibrosis (Newburgh
and Curtis, 1928; Newburgh and Johnston, 1931). When
rats were fed casein at a 75% level, however, only
moderate tubular injury was evident at 16 months (Newburgh
and Curtis, 1928). A partial explanation for the lesions
produced by the liver may be its high content of nucleic
acids (Newburgh and Johnston, 1931).

Medlar and Blatherwick (1937) reported that high
protein diets produced chronic degenerative nephritis in
rats. Although there were glomerular and tubular changes,
the proximal convoluted tubules were normal in kidneys of
these rats.

Polvogt et al. (1923) fed rats various proteins at
a level of 31 to 41%. The kidneys of the original
animals plus 5 of their subsequent generations (ages 5
months to 485 days)were examined histologically. Renal
changes observed included hyalinization of glomeruli,
congestion, degeneration of the tubular epithelium,

glomerular adhesions, and hyaline casts. These researchers

 

1
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a

new ‘._

 

a n
215‘ la! wan
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rats fed an 17:

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35

ascribed the renal damage to the excessive amounts of the
end-products of protein metabolism (Polvogt et aL., 1923).
Osborne at al. (1927) observed glomerular and
tubular changes in rats fed high protein diets. Renal
tubules were dilated throughout the kidneys in rats fed
diets containing 70% or more protein. Moise and Smith
(1927) examined the remaining kidney of uninephrectomized
rats fed an 18% or 85% protein ration. No anatomic
evidence of significant renal damage in the 18% protein
group was reported. The high protein group, on the
other hand, showed significant glomerular and tubular
changes. The lesions were conspicuous and relatively wide-
spread, becoming progressively more marked with increased
time on the diet. Glomerular changes included prolifera-
tion of the epithelium of Bowman's capsule with and
without glomerular adhesions, fibrous thickening of
Bowman's capsule, partial fibrosis of the glomerular tuft
and some infiltration of round cells in fibrotic areas
(Moise and Smith, 1927). Concomitantly, tubular changes
reported were desquamation of the epithelial lining,
some dilation and amorphous material within tubular lumina.
Moise and Smith (1927) suggested that these changes were
late manifestations of injury or irritation to which the
kidney was subjected. In contrast, Addis et a1. (1926)
reported no difference in the kidneys of control and high-
protein or high-cystine fed rats upon microscopical

examination.

 

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36

Hogs fed a 42% crude protein diet had considerable
renal damage when compared to controls fed 13.6% protein
(Terrillet aZ., 1952). Tubular changes in the high-
protein fed group included dilation and amorphous
deposits.

A deficiency of protein may produce alterations in
kidney functions in animals. Dicker (1950) reported
that the GFR and clearance of water in protein deficient
rats was depressed when compared to controls. The concen-
tration and total amount of sodium and chloride in the
urine was decreased (Dicker, 1950). Dicker et al. (1946)
reported that when rats were fed low protein diets
(carrot or turnip diet), some histological changes in
the tubules occurred. No glomerular abnormalities were
observed. Tubular changes consisted of necrosis and
calcification of the broad limbs of Henle. Protein-
deficient rats responded less well to urinary concentration
and dilution tests than the same animals on a standard

diet. The total tubular excretory mass (Tm ) of

Diodrast
rats on the turnip diet was below control values. In
contrast to normal rats, the GFR of the protein-deficient
rats was significantly correlated with the rate of urine
flow. Guggenheim (1956) reported that protein-deficient
rats had normal GFR values, but abnormal responses to a

water load.

Xylose Clearance: The glomerular clearance of xylose

 

was increased in dogs fed meat or casein (Shannon et al.,

1932; Pitts, 1935).

 

q'prfl'ZI

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».4

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37

fiypertrophy: High protein levels produced renal

 

hypertrOphy in a number of species. These results are
discussed in the section on factors affecting renal size.
In conclusion, the quantity and the quality of

protein fed may affect renal function and structure.

§a_t

When rats were fed diets virtually free of fat,
kidneys were mottled with surface indentations (McAmis
et aZ., 1929). When Burr and Burr (1929) excluded fat
from the diet of rats, kidneys were "mottled, spotted,
abnormal." Furthermore, concretions in the bladder and
hematuria were present. The kidneys from these rats
were further examined by Borland and Jackson (1931).
Grossly these were pale and larger than controls. No
glomerular changes were observed. Lesions included
tubular calcification, cortical tubular epithelium
degeneration, increased intracellular fat in proximal and
distal convoluted tubules, round cell infiltration and
large quantities of "fatty or albuminous material" in
the medullary area. Addition of lard at a dietary level
of 2-20% prevented or cured these renal disorders. In
the control animals, changes reported were round cell
infiltration, some tubular degeneration and an increase
in intracellular fat (Borland and Jackson, 1931). Later
Rice and Jackson (1934a, 1934b) determined that the fat

accumulation in the kidney was due to the high carbohydrate

1L7:- ‘ T'“. '0“. ‘TL_4 v

z... (1929) . 3'

Tie C510 1 1

23333515 was 1

2: corn 0‘1 :
"is level
.1 .u V! I

reguirenents .

 

 

38

content of the diet and the general state of undernourish-
ment and not to the "fatless" quality of Burr and Burr's
diet (1929). With the advent of more chemically defined
diets, the effects of fatty acid deficiency should be
examined anew.

The choline requirement necessary to prevent renal
necrosis was increased when rats were fed cocoa butter
or corn oil at a 40% level (O'Neal et aZ., 1961). Butter
at this level, however, did not increase the lipotrOpic
requirements.

Numerous calculi occurred in kidneys of rats fed
methyl esters of fatty acids at a 10% level (Spining
et aZ., 1964).

Rats fed cyclized fish oil for 19 weeks had signifi-
cantly lower urine specific gravities than animals fed a
control diet. After 18 hours of water deprivation, the
specific gravity of the urine of animals fed the fish oil
was significantly less than that of the urine of control
animals (Gottenbos and Thomasson, 1965). No histological

changes in the kidney function were evident, however.

Thomasson et a2. (1966) found no disturbances of urinary
concentrating capacity in rats fed hydrogenated fats
(soya bean, linseed, olive or butterfat). Furthermore,
the urine aspartate transaminase activity, which is
increased in renal pathological conditions, was lower

in the animals given the hardened oils than in controls

fed unhardened soya-bean oil.

 

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£15.12 cement C :

 

39

Vitamin A

 

Humans:

Herrin and Nicholes (1940) reported that the Curea
was increased in humans given vitamin A supplements for
2-3 weeks with a maximum response at 8 weeks. Further-
Inore, these researchers suggested that individuals with
excessive subcutaneous fat are most likely to show an
enhancement of the Curea with vitamin A administration.

Similar results were reported by Taylor et al.
(1943). Human patients given 100,000 to 400,000 I.U. of
vitamin A for 5-90 days had increased values for GFR and
renal plasma flow. These workers suggested that large

doses of vitamin A could be used therapeutically in patients

with degenerative renal diseases.

Animals:

Herrin and Nicholes (1939) observed that diets
containing 150 g of butter or cod liver oil produced urea
clearances in dogs that were 147 and 130% of control values.
When vitamin A was given at a level of 50,000 I.U. daily

the Curea increased from 41 to 94% in a week. The maximum

increase occurred after vitamin A supplementation for 96

days. In contrast to these findings, Bing (1943) found

of dogs

no change in the GFR, ERPF, Tm AH' or Tm

P Diodrast

given daily 5,000 to 50,000 I.U. vitamin A orally. When
200,000 I.U. were added to the diet daily, a significant

increase in TmD or TmPAH occurred. The GFR and ERPF were

r11

 

‘ &
:wacts con.
‘- J'! ..

H" H.»
.evEO inese

fliction wher.

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40

moderately increased with the filtration fraction remaining
unchanged (Bing, 1943).
Rats when given high doses of vitamin A had increased

values for GFR, RPF and Tm (Dicker and Heller,

Diodrast
1946). Corcoran and Page (1947) reported that when crude
extracts containing 2000 I.U. of vitamin A were injected
to rats daily intramuscularly, the TmPAH was increased
100%. These investigators found no enhancement of renal
function when crystalline vitamin A was given and con-
cluded that the changes previously reported to be due to
vitamin A were due to some other factor in the commercial
concentrates. Corcoran and Page (1947) did not explain
what factor in the butter and cod liver oil fed by Herrin
and Nicholes (1939) was reSponsible for the increased

C Herrin and Nicholes (1939) found that vitamin D

urea’

was not reSponsible.

Vitamin C

 

Renal function was examined in 14 patients with florid
scurvy by Eales (1956). The GFR was slightly reduced. A
significant decrease in the ERPF was reported. The most
striking change was a decrease in the effective renal
blood flow (ERBF). The ERBF was related to a lower
hematocrit primarily, but also to a diminution in the
ERPF. The total excretory mass as measured by TmPAH was
normal in 2 subjects and slightly decreased in a third

(Eales, 1956).

3 :9! ‘ ’
”ArcaLC‘Jr 5 ~

p
“1"
a.

72:: can lead

 

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ii not inf 1;
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iegressed 423

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Accordir

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tn: 3‘]

 

 

41

Vitamin D

 

An excess of vitamin D can produce hypercalcemia,
hypercalciuria, and metastatic calcification in the kidney.
This can lead to fatal or reversible renal failure.

Dietary imbalances, such as a high-calcium, low—phOSphorus
diet, can also lead to renal disorders (Kark, 1968).

Vitamin D when administered overseveral weeks to dogs

did not influence the C (Herrin and Nicholes, 1939).

urea

When high levels of vitamin D were fed, the Curea was

depressed 42%.

Vitamin E

 

According to Herrin and Nicholes (1939) high levels
of vitamin E were ineffective in altering the Curea in
dogs.

Kidneys from vitamin E deficient rats underwent
rapid postmortem autolysis (Moore et aZ., 1958; Emmel and
LaCelle, 1961; Gyorgy at aZ., 1966). Vitamin E, choline,

methionine and vitamin B were active in preventing this

12
tendency to rapid autolysis (Gyorgy et aZ., 1966). Emmel
and LaCelle (1961) found that the increased rate of post-
mortem autolysis in the kidney was always preceded by a
decrease in the renal toc0pherol content.
The incidence and morphology of centrioles in the

proximal convoluted tubules of rat kidney were altered by
the dietary content of vitamin E (Hess and Menzel, 1968).

Whether this would affect proximal tubular functional

changes was not determined.

S
"'s \‘
.4
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o ' v-
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42

B Vitamins, Calcium, PhosPhorus and Others

 

Hamar (1940) reported that the tubular reabsorption
of glucose was decreased in dogs fed a vitamin B1
deficient diet.

Rats suffering from a lack of pantothenic acid,
pyridoxine and riboflavin had depressed values for GFR
and a water dilution test (Guggenheim, 1956). These
returned to normal when the deficient animals were given
ACTH or cortisone. Thiamine deficient rats also exhibited
a delayed diuretic response to a combined inulin-water
load, however, these were not restored when ACTH or
cortisone was given (Guggenheim, 1956).

Pyridoxine deficiency in the rat was associated with
a number of changes in the kidneys. These included
hypertrOphy (Agnew, 1951; Seronde, 1960), calcium or
oxalate deposits (Agnew, 1951; Gershoff and Andrus, 1961)
and casts (Agnew, 1951). Other workers, however, reported
that when the pathologic manifestations due exclusively to
pyridoxine deficiency were separated from the effects due
solely to undernutrition in rats of similar sex, age, and
weight, pyridoxine deficient animals had no renal hyper-
trOphy or lesions (Wirtschafter and Walsh, 1964).

The effect of folic acid on producing renal hyper-
trophy is discussed in the section on factors affecting

renal size.

._-1I

 

,
;s a nip-:32”.

'-'r-'= fed 6. 16:1

"'5

1: rats fed

\—

.Lcaroonate}

 

C"
(D
m
D
P‘
1
7

—

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43

Calcium metabolism and its relationships to the
kidneys of humans were reviewed by Kushner (1956). A
number of diseases associated with hypercalcemia and
metastic calcification produce renal damage. Calcium
is a component of most primary urinary calculi.

Calcification of the kidneys was produced when rats
were fed diets high in calcium, phOSphorus and phOSphoric
acid for 3 weeks (Ham, 1940). High levels of phosPhate
also produced damage in kidneys of rats (MacKay and
Oliver, 1935; McFarlane, 1941).

Addis and co-workers (1926) found a number of changes
in rats fed acid (calcium chloride) or alkali (sodium
bicarbonate) diets. The protein excretion of rats on the
acid diets was one half that of controls. Significant
hematuria and hydronephrosis occurred in rats on the

alkali diet (Addis et aZ., 1926).

Overweight and Obesity
Humans:

The incidence of cardiovascular—renal deaths in over—
weight persons is significantly higher than in those of
normal weight (Mayer, 1968). The existence of renal and
cardiovascular abnormalities among the obese appears to
be an important factor responsible for the shortened life
expectancy of this group according to Ross (1960).

The effects of overweight on kidney function have

been investigated less than most other areas. As early

 

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ofifl
I. J}:
1: '4b4,

35:53:85 th a t
r

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44

as 1925, Dublin et al. reported that albuminuria was more
frequent in overweight persons than those of normal weight.
This was emphasized by Armstrong et al. (1951) who
reported that statistical studies of periodic health
examination data showed that the proportion of persons
with albuminuria and glycosuria of significant degree was
higher among overweight persons than among average and
underweight persons. The occurrence of granular and
hyaline casts was "noticeably greater" among overweights
(Dublin et aZ., 1925). Chronic and acute nephritis were
also increased in the overweight (Dublin et aZ., 1925:
Britten, 1933).

The hazardous effects of perirenal fat deposition in
the obese has been emphasized by Weil (1955). The pro-
posed that in excessive overweight "large deposits of fat
cover the viscera and fat infiltrates the parenchyma of
vital organs." These morphological changes then could be
reflected in a wide variety of physiological aberrations
according to Weil (1955). On this basis, perirenal fat
deposition in excessive amounts could lead to abnormal
kidney function.

Table 1 is a summary of reports of renal function in
obese patients. The purpose of most of the investigations
cited was not related to determining kidney function;
therefore, some of the data are incomplete. It is

apparent from a number of studies that weight loss improves

 

i.

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.

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r“*,“f‘ed ‘1‘-

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45

renal function in obese subjects (Weil, 1955; Lillington
et aZ., 1957a, 1957b).

The glomerular filtration rate (GFR), renal blood
flow (RBF) and filtration fraction (FF) were significantly
depressed in obese subjects (Bansi and Olsen, 1959;

Olsen et aZ., 1961). Olsen and his co-workers (1959;
1961), as well as others (Bittnerova et aZ., 1968), have
reported that obese persons have disturbances in water
metabolism. A recent review (Gordon, 1970) points out
that the obese are unable to excrete both salt and water
normally. Bansi and Olsen (1959) suggested that in obese
patients inadequate renal blood flow might be an essential
factor in these disturbances. Triiodothyronine (T3)
restored the GFR, RBF, and FF to normal in obese patients
(Bansi and Olsen, 1959; Olsen et aZ., 1961). The mode of
action of T3 is not apparent since the extracellular fluid
volume was normal in these patients both before and after
T3 treatment. Some evidence suggests that antidiuretic
hormone (ADH) and aldosterone may be related to these
disturbances. This suggestion is based on the increased
blood levels of ADH (Bansi and Olsen, 1959; Olsen et aZ.,
1961) and increased aldosterone levels (Gordon, 1970) in
obese subjects.

German workers recently reported somewhat different
results in obese women. Bittnerova et a2. (1968) found
that the GFR was normal in their obese patients. The RFB

and renal plasma flow (RPF), however, were depressed.

 

 

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46

Furthermore, upon water loading 53% of the obese subjects
had a retarded excretion. The absolute values of the
body water spaces were increased in the obese. These
workers did not find differences in ADH plasma activity
between obese and control subjects, however (Bittnerova
at aZ., 1968).

In conclusion, there is evidence to suggest that some
renal functions are altered in obese humans. However,
the degree of alteration and the mechanisms responsible
for these alterations are not agreed upon by all investi-

gators.

Animals:

The association of kidney damage with increased body
weight in animals has been noted by several workers.
Benedict et al. (1932) observed "congested and large"
kidneys in an Osborne-Mendel rat that had reached a
maximum weight of 822 g. Kidney lesions were also re-
ported in the first hypothalamic obese rats (Brobeck et
aZ., 1943). The obese rats when killed 6 to 9 months
after hypothalamic lesioning had advanced kidney disease
and excessive proteinuria. These workers observed
urinary casts, hematuria and histological glomerular and
tubular changes. They called this condition "chronic
glomerulonephritis." Kidney damage in hypothalamically
obese rats has been confirmed by a number of workers

(Brooks and Lambert, 1946; Stevenson, 1949; Kennedy, 1951,

1957; Long, 1957; Hausberger et aZ., 1964).

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47

Kennedy (1951, 1957) concluded that the lesions
observed in the hypothalamic obese rats were identical
to those occurring spontaneously in old rats.

Considerable renal damage was observed in obese
animals between 12-15 months of age, while no renal abnor-
malities were seen in control animals until 21 months.
Later, Kennedy (1960) stated that if the protein in the
diet had been increased from 15% casein to 20-25% casein,
the lesions in the obese animals would have developed
more rapidly. Goldblatt (1947) also observed that the
changes in renal tissues were the same in obese younger
animals as those in older normal weight animals.

The clearance of creatinine was depressed in hypo-
thalamic obese rats (Stevenson, 1949). Other changes
included a retardation in plasma flow and inability to
respond to a water load (Stevenson, 1949). Similarly
Long (1957) reported that albuminuria, hematuria and
renal hypertrophy occurred in hypothalamic obese rats.
He observed histological damage to the kidneys including
hyalinization and fibrosis of the glomerulus, dilated
tubules with thin epithelium and filled with amorphous
material.

In rats made obese by purely dietary means, kidney
abnormalities have been seen by several workers.
Barboriak et a1. (1958) observed tubular lesions in rats
fed high fat diets. Preliminary studies at NIH

(Yamamoto et aZ., 1959) indicated an increase in

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48

proteinuria in obese rats, with males showing a greater
increase than females. These workers attributed the
increased proteinuria to an increase in the excretion of
proteins with the same mobility as albumin and y-
globulin of serum. Craig (1952), using rats from the NIH
colony, observed a statistically significant difference
between the absolute kidney weights of obese and nonobese
animals, but reported an absence of structural and
functional alterations upon gross observation although
large deposits of fat surrounded the kidneys. Naimi et
al. (1965) found no abnormalities in kidney histology
when rats became obese on a high-fat (butter) diet.

These workers, however, looked at only 6 males at 6 months
of age.

Rats with unrestricted feed intakes may also have an
increased incidence of renal lesions. Berg and co-workers
(Berg and Harmison, 1957; Berg, 1960; Berg and Simms,
1960) found chronic nephrosis practically nonexistent in
old growth-retarded rats, but found a high incidence and
increased severity of glomerular lesions in animals with
unrestricted feed intakes. Bras and Ross (1964) found
that in normal rats the shortest life expectancy was
associated with ad Zibitum food intake and concomitantly
the highest incidence of progressive glomerulonephrosis.

Genetically obese mice and rats also show considerable
renal damage. The hyperlipemic "fatty" rat is frequently

nephrotic (Zucker and Zucker, 1962; Zucker, 1965, 1967).

 

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The kidneys were hypertrophied and frequently contained
kidney "sand" or stones (Zucker and Zucker, 1962).
MicroscoPically, kidney lesions included thickened
glomerular basement membranes, extensive tubular plugging
and dilation and progressive replacement of nephrons by
connective tissue (Zucker, 1965). Proteinuria and hypo-
albuminemia frequently occurred especially in the males
(Zucker, 1965, 1967). These renal lesions contributed to
the shortened life span of the "fatty."

The ob/ob hereditary obese mice also exhibited renal
lesions including lipohyalin deposits in the glomerulus
(Hellman, 1965).

When obesity was produced in mice by injections of
goldthioglucose (GTG) a number of changes in renal size
and function were reported according to several workers
(Waxler and Enger, 1954; Larsson, 1957). The kidneys
were normal histologically in mice examined by Brecher and
Waxler (1949) and Drachman and Tepperman (1954). The kid-
neys examined by Brecher and Waxler (1949) however, were
from animals killed 14 weeks after GTG-injections. The long
term effects of GTG-obesity on the kidney are not known.

In conclusion, obesity produced in animals by a
number of different procedures is associated with
abnormalities of renal structure and function. In fact,
some workers have suggested that the renal damage contri-
butes in large part to the premature death of obese

animals.

 

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BIBLIOGRAPHY

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Addis, T. and D. R. Drury. 1923. The rate of urea
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Addis, T. and M. C. Shevky. 1922. A test of the capacity
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Agnew, L. R. C. 1951. Renal lesions in pyridoxin-
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55

It

 

+u
.'
‘

 

si
lStOlC

v

P

a

n, G.

L
T.“

ieve
lint: .

II".
Che
'IC

vol
..;,3.W.

'. .
H”

 

. .h.‘
.n n
a
:3
re
5..
ode

56

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Becker, B. 1962. Accumulation of rubidium-86 by the
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Berlyne, G. M. 1958. The cardiorespiratory syndrome of
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.. l
31:23.13- *3;

in man.

 

 

Siourchi. E
flour,
in adul
843.

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b In

Mr? O m U’

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2
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57

Blahd, W. H. and S. H. Bassett. 1953. Potassium deficiency
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Bonting, S. L., L. L. Caravaggio and N. M. Hawkins. 1963.
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58

Burr, G. 0. and M. M. Burr. 1929. A new deficiency disease
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Carroll, D. 1956. A peculiar type of cardiopulmonary
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Chasis, H., W. Goldring, E. S. Breed, G. E. Schreiner and
A. A. Bolomey. 1950. Salt and protein restriction.
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Clarke, E., B. M. Evans, I. MacIntyre and M. D. Milne.

1955. Acidosis in experimental electrolyte depletion.
Clin. Sci. 14:421-440.

Cohen, J. J. 1964. Specificity of substrate utilization
by the dog kidney in vivo. In: Renal Metabolism and
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Conn; J. W. and R. D. Johnson. 1956. Kaliopenic nephropathy.
Amer. J. Clin. Nutr. 4:523-528.

Constant, M. A. and P. H. Phillips. 1952. The occurrence
of a calcinosis syndrome in the cotton rat. I. The
effect of diet on the ash content of the heart. J.
Nutr. 47:317-326.

Cope, C. L. 1933. Studies of urea excretion. VIII. The
effects on the urea clearance of changes in protein
and salt contents of the diet. J. Clin. Invest. 12:
567-572.

Corcoran, A. C. and I. H. Page. 1947. Effect of renal
function of rats of substances containing vitamin A.
Fed. Proc. 6:91 (Abstract).

Counihan, T. B. 1956. Heart failure due to extreme obesity.
Brit. Heart J. 18:425-426.

Craig, C. E. 1952. Production of obesity in Sprague-Dawley
rats by tube feeding a high caloric diet. Washington,
D. C.: Georgetown University, M. S. thesis.

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124 (Abstract).

 

 

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71

Rapoport, A., G. L. A. From and H. Husdan. 1965.
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72

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73

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74

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239-243.

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171.

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INTRODUCTION

A number of manipulations, including dietary ones,
produce changes in kidney structure, size and function
in the rat. In addition, the histological appearance of
the kidney is affected by age. Lesions have been
described in kidneys from rats made obese by hypothalamic
lesions, genetic selection, and dietary procedures. The
histological changes occurring in the kidneys of rats
made obese by feeding a high fat diet have not been
described. The objectives of this investigation were to
describe the histological changes in kidneys of male rats
as they became obese and to compare these changes with

those normally occurring in rats with aging.
LITERATURE REVIEW

According to Snell (1967), literature dealing with
the influence of diet on renal lesions is "somewhat

contradictory and difficult to assess." These contra-

dictory results could reflect differences in the inherited

characteristics of the strain of rats, differences in
basal dietary components and differences in criteria for

evaluation of the sections.'

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The rat is remarkably resistant to the develOpment
of spontaneous renal tumors. These rarely occur in rats
fed control rations (Snell, 1965, 1967). Curtis et al.
(1931) reported that of 24512 rats of 7 strains investi-
gated, only eleven females and 1 male had renal tumors.
In another study, the incidence of spontaneous renal
tumors was only 5 in 468 animals (Ratcliffe, 1940).
Kidney neoplasms occurred in 4 of 1342 rats; however, the
ages of the 3 males and 1 female with these tumors
exceeded 2 years (Gilbert and Gillman, 1958). When rats
were irradiated at 230 or 320 rads, renal tumors
developed in 41% and 43% of the animals respectively,
while none occurred in the control animals (Rosen et al.,
1962). A single pleomorphic renal tumor (Babcock and
Southam, 1961) and a renal adenosarcoma (Lillie and

Engle, 1935) also have been observed in rats.
Changes in Renal Function and Histology with Age

Renal function and histology are related to the age
of an animal or human.
Humans:

The newborn infant has impaired renal function
(Edelmann and Spitzer, 1969). At the other end of the
life span, that of old age, the kidney is also less capable
of functioning maximally. In subjects from 20 to 90 years
of age, the glomerular filtration rate (GFR), the effec-

tive renal plasma flow (ERPF) and the excretory capacity

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78

or tubular maximum for Diodrast (Tm ) decreased 46,

Diodrast
53 and 44%, respectively (Davies and Shock, 1950; Shock,
1952, 1956). There was a gradual diminution of the urea
clearance and a progressively increased BUN with advancing
age (Shock, 1952). In addition, the excretory capacity of
the renal tubules for p-aminohippurate (PAH) (TmPAH) and

for glucose (Tm ) decreased as age increased (Shock,

Glucose
1952). The decreased ERPF cannot be explained fully on

a basis of structural changes in renal blood vessels as
renal arterioles in the aged kidney were capable of
dilating when subjected to a standardized pyrogen test
(McDonald et al., 1951). The ability of the distal

tubule of the older individual to perform osmotic work
when provided with a standardized amount of antidiuretic
hormone (ADH) was also impaired (Miller and Shock, 1953).
On the basis of the observed changes in a number of kidney
functions and the constant ratio between GFR and Tm over

7 decades, Shock (1952) suggested that the nephron loses
its function as a unit.

In contrast to the other functions described above,
normal acid-base balance was maintained in spite of
depressed kidney function (Shock and Yiengst, 1950).
Acid-base balance, however, is not regulated totally by

the kidney.

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Animals:

The kidneys from newborns of a number of species are
less capable of transporting organic acids and bases than
those from mature animals (Williamson and Hiatt, 1947;

New et al., 1959; Rennick et al., 1961; Hirsch, 1970).

A number of other renal functions are immature in the
newborn (McCance & Widdowson, 1954, 1955, 1957). The kidney
of the newborn rat contains a nephrogenic zone with
malpighian corpuscles at various stages of formation and
incompletely formed proximal tubules (Bogomolova, 1966).
Baxter and Yoffey (1948) suggested that only a proportion
of renal tubules are fully functional in the rat at birth.
Other workers (Enesco and LeBlond, 1962) found that the
number and size of renal cells increased in the rat kidney
from 7 to 95 days of age. Kunkel (1930) reported that the
number, size and capillary surface of glomeruli were more
closely related to body surface than any other measure-
:ment. There is a progressive decrease in the number of
nephrons in the rat kidney after middle age (Arataki,
1926).

Kidney cortical slices from old rats were less able
tn) transport PAH and a-aminoisobutyric acid (AIB) than
yrnanger adults (Adams and Barrows, 1963; Beauchene et al.,
1965) . Old rats were less able to concentrate their urine
than young adults (Dicker and Nunn, 1958) . An intracarotid
ijyjection of hypertonic NaCl produced a smaller anti-

djnxretic effect in old rats. Dicker and Nunn (1958)

 

 

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suggested that this defect was of renal rather than of
neurohypoPhyseal origin in contrast to earlier reports by
Friedman and Friedman (1957).

Histological changes in kidneys from old rats included
disintegration of mitochondria in the epithelium of
degenerating nephrons, hyaline droplets within tubular
cells and hyaline casts within tubular lumens (Bogomolova,
1966). Kennedy (1957) has suggested that although in old
age the kidneys differ histologically from the normal
kidney of youth, it is not always a simple task to distin-
guish the end results of senile atrophy from those of
pathological injury. A review of the literature on the
effects of various diets on renal histology substantiates
Kennedy's suggestion.

Saxton and Kimball (1941) reported renal lesions in
male Osborne-Mendel rats from 259-620 days old fed
regimens including high protein and low protein levels.
Forty-four percent of the rats dying natural deaths
showed lesions indicative of "chronic nephrosis." The
histological observations included hyaline casts in
tubules,dilation of glomerular capsules, and hyaline
thickening of the basement membranes. Not all tubules
and.glomeru1i, however, were affected even in the most
seveme cases. Saxton and Kimball (1941) suggested that
‘these lesions constituted a spontaneous disease of rats
and were not specifically produced by diet. The lesions,

however, were modified by diet as they were more severe in

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81

animals fed high protein diets than in those fed low
protein diets, in rats fed casein rather than liver, and
in rats allowed food on an unrestricted basis when com-
pared to those on a restricted regimen (Saxton and
Kimball, 1941).

Similar lesions in kidneys of rats were described
by numerous workers (Moise and Smith, 1927; Blatherwick
and Medlar, 1937; Wilens and Sproul, 1938; Simms and Berg,
1957; Andrew and Pruett, 1957; Kennedy, 1957; Gray, 1963;
Kennedy and Parker, 1963b; Foley et al., 1964; Bras and
Ross, 1964; Gray and Purmalis, 1965; Bras, 1969), and
classified under a variety of names including nephrosis,
nephritis, pyelonephritis and glomerulosclerosis. Table 2

is a compilation of selected reports of spontaneous
changes in renal histology observed in rats. It is
apparent from this table that a variety of terms have
been used to classify similar changes.

Kidneys from rats made obese by a number of procedures
show alterations in structure microscopically. Kennedy
(1951) described the changes in kidneys from hypothalamic
obese rats as follows:

"The damage to the kidney appears to

fall primarily on the tubules. The
epithelium becomes atrophic and the lumen
is full of hyaline debris. There is
relatively little glomerular or vascular
damage until a late stage, and although
the animals are sometimes hypertensive,
this is not constant. Histologically

it has the appearance of pyelonephritis,
which Goldblatt has shown to be common

 

 

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in some strains of rats. But none of our

controls have shown kidney lesions, and

we have found no sign of infection at any

stage in the fat animals. Although it

might be tempting to regard it as due to

a relative deficiency of lipotrophic

substances, the slight degree of liver

involvement is against this."
He suggested that the obesity could be a non-specific
stress and that the kidney damage was secondary to over-
activity of the adrenals (Kennedy, 1951). The adrenals
in these animals were hypertrophied. Brobeck, Tepperman
and Long (1943) observed these lesions in hypothalamically
obese rats and called the condition "chronic glomerulo-
nephritis." Kennedy, after confirming these lesions
(1951, 1957, 1960), suggested that they were identical
with those that occur spontaneously in senility.

Bras and Ross (1964) reported that restriction of
intake, whether of protein alone, of carbohydrate alone
or of both had a beneficial effect upon the incidence of
spontaneous renal disease. The incidence was greatest in
rats fed a commercial diet ad libitum. Obesity was common
in these animals.
The type of fat used to produce dietary obesity could

also influence the occurrence of renal lesions. Gydrgy
et al. (1966) reported that the renal changes in rats fed
lard and cod liver oil were more severe than in those fed
Crisco. Kaunitz et al. (1970) suggested that the minor

dif ferences in various fats including their methods of

processing could influence the incidence of renal lesions.

 

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The histological lesions observed by Kaunitz and co-workers
(1970) included renal tubular calcification, atrophy or
dilation of tubules by hyaline casts. Glomerular changes
were mild and included thickening and calcification of
Bowman's membrane. All of these lesions are the same as
seen in secondary hyperparathyroidism (Kaunitz et al.,
1970).

Renal lesions can result from a variety of other
dietary manipulations including a deficiency of choline
(Hartroft, 1948), potassium (Schrader et al., 1937;
Follis et al., 1942; Follis, 1943; Newberne, 1964),
protein (Dicker et al., 1946) and magnesium (Watchorn and

McCance, 1937; Cramer, 1932; Sullivan and Evans, 1944).

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87

Kidney Size and Factors InfluencingIt

 

A number of factors may influence the size of the
kidneys. Increased kidney weight may result from an
increase in cell number (hyperplasia), cell size (hyper-
trOphy), intracellular material or a combination of these.

Renal growth may result from subjecting an animal
to: partial nephrectomy, nephrectomy serum, ureteral
ligation, intraperitoneal injection of a protein, a high
sodium diet, a low potassium diet, NH4C1, folic acid,
thyroxine, testosterone, cold, growth hormone and
mineralocorticoids (G033 and Dittmer, 1969). A number of

these will be discussed in detail.

DietarygNitrogen

 

The amount and nature of the nitrOgen in the diet
may affect kidney weight. A.diet with 75% protein of
vegetable or animal origin produced kidneys which were
2 times the average weight of those from control rats
(Osborne et al., 1923, 1927). No microsc0pic or degenera-
tive changes were observed in kidneys from rats fed these
diets although the kidney weight/body weight (KW/BW)
ratios were twice those of control rats (Osborne et al.,
1923, 1927). Other workers have reported increased kidney
weights in hogs (Terrill et al., 1952), piglets (Filer
et al., 1960), and dogs (Allen and COpe, 1942) with an
increased dietary protein intake. Filer et al. (1960)

reported increased kidney weights with increased protein

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88

content in the diet whether it was a high fat or low fat
diet. A number of other workers have reported increased
kidney weights with a high protein diet (MacKay et al.,
1926b, 1926c; MacKay et al., 1928a, 1928b; MacKay and
MacKay, 1931a, 1931b; Medlar and Blatherwick, 1937; Chanutin
and Ludewig, 1939a, 1939b; Walter and Addis, 1939; McCay

et al., 1941).

Reid (1963) found that differences in casein levels
from 20-70% produced no significant changes in kidney
weights of guinea pigs. The kidneys of guinea pigs fed
soy protein at a level of 30 to 70% protein were signifi-
cantly heavier than those of guinea pigs fed soy protein
at a level of 20 or 25% protein (Reid, 1963). Reid (1963),
however, emphasized that comparisons between the rat and
guinea pig as to effect of protein level of kidney size
were not justified. She pointed out that the guinea pig
studies dealt only with the early stages of growth (6
weeks) whereas most of the work with rats was with older
animals (Reid, 1963).

The type of protein may affect the degree of hyper-
trOphy. When rats were fed gelatin their kidney weights
were significantly heavier than those of rats fed caseinogen
or liver (Wilson, 1933). The mechanism for the gelatin
effect on kidney weight has not been determined
(Halliburton, 1969). The degree of compensatory renal
hypertroPhy after nephrectomy was directly proportional to

the dietary protein content (Smith and Moise, 1927).

89

MacKay et al. (1926a, 1931) reported increased kidney
weights in rats fed urea or urea plus protein. The hyper-
trOphy was less than that produced by the same nitrogen
consumption obtained by protein alone. Thus, the absolute
nitrogen consumption did not explain the hypertrOphy
observed with an increased dietary protein intake. In
contrast, glycine, glutamic acid and gluten produced
increased kidney weights in preportion to the nitrogen
consumed (Wilson, 1933). The type of protein or nitrogen
source differed in these studies and may partially explain
the apparent discrepancy in these results when compared to
those of MacKay's group.

The hypertroPhy observed in animals fed a high protein
diet was apparently not associated with an increased urine
volume. The increased urine volume is a concomitant of
the high protein intake (Stier and Hayman, 1938).

The blood urea nitrogen (BUN) of rats increased with
an increased protein intake. When the (BUN) 2/3 was

plotted against the renal weight per 100 cm2

of body sur-
face the relationship was linear (MacKay et al., 1928a).

A protein deficiency in the dam may affect the weights
of the kidneys of rats in utero. Zeman (1967) reported
that kidneys in newborn rats from dams fed an 18% casein
diet during pregnancy were 1.014% of the body weight.

When dams were fed a 6% casein diet during pregnancy,

their offSpring at birth had kidneys which were only 0.788%

of the body weight (Zeman, 1967). Other workers have

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reported that kidney weights of older rats on diets devoid
of protein are almost directly prOportional to their
endogenous protein metabolism (MacKay and Cockrill,

1931b).

Other Dietary Factors

 

Kidney weights were increased in potassium-deficient
rats although body weights were less than those of controls
(Follis et al., 1942; Fuhrman and Brokaw, 1951; Muntwyler
and Griffin, 1953; Brokaw, 1953). Deficiences of
Vitamin 86 (Seronde, 1960) and choline (Griffth and Wade,
1939; Parks and Smith, 1969) also produced kidney hyper-
trOphy. Animals on a high sodium chloride intake of
dietary origin (food or water) had significantly larger
kidneys than control animals (Krakower and Heino, 1947;
Sapirstein et al., 1950; Auerbach et al., 1953; Fregly,
1960; Dahl and Schackow, 1964; Hall and Hall, 1966).
Whether the increased kidney weight in animals on a high
sodium chloride intake is related to the concomitant
hypertension in these animals has not been determined.

A single injection of folic acid also produces renal
hypertrophy (Threlfall, 1969; Hirsch and Hook, 1969).

When young rats were fed rations with increased
levels of phosphate, kidney weights of these animals were
increased (MacKay et al., 1926a). Rats on a fat-free
diet also had hypertrophied kidneys at ages 2.5 to 19

months (Borland and Jackson, 1931). There were no

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91

statistical differences in the weights of kidneys when

adult female rats were fed a 68% sucrose, 18% casein,

10% vegetable oil diet or a 48% cornstarch, 30% egg white,

14% cottonseed oil diet (Peters and Krijnen, 1966).

Wierda (1950) reported changes in kidney weights when

animals were fed diets that were unbalanced in various

nutrients. He reported kidney weights of animals fed a

25% casein, 69% lard diet were greater than those from

animals fed a 14% casein, 83% cornstarch ratio.
Underfeeding or short periods of fasting also affected

the kidney weights (Stewart, 1919; McCay et al., 1939a,

 

1939b; Widdowson and McCance, 1956; Peters and Boyd, 1965;
Brown and Guthrie, 1968). Stewart (1919) reported that
underfeeding rats until age 11-22 days by repeated periods
of fasting from the day of birth produced a 90% increase
in the absolute kidney weight in males and females. The
kidney weights were decreased in rats retarded by limiting
caloric intake (McCay et al., 1939a). Other workers
reported that after two weeks of reduced food intake the
changes in kidney weights decreased approximately in
pr0portion to changes in body weight (Peters and Boyd,

1965, 1966).

Body Weight

 

Absolute kidney weights increased linearly as body
weight increased in rats after a body weight of 50 gms

(Hatai, 1913). Similar results were reported in rats by

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92

Webster et al. (1947) and Widdowson and McCance (1960).
In contrast, Forster (1947) found no close relationship of
kidney weights and body weights in rabbits.

The relative kidney weight or kidney weight/body
weight ratio (KW/BW) ranged from 1.16 to 0.74 and decreased
with increasing weight from 50 to 200 g (Webster et al.,
1947). Similarly, Widdowson and McCance (1960) reported
that the kidneys of rats grew faster than the body as a
whole initially and the KW/BW ratio exceeded 1. With
increasing age the KW/BW ratio decreased.

An increase in the absolute renal weights occurred in
obese mice whether the obesity was genetically transmitted
(ob/ob), GTG-induced or produced by hypothalamic lesions
(Marshall et al., 1957). The KW/BW ratio decreased in
these obese animals. Marshall et al. (1957) suggested that
the renal enlargement was due to a nonSpecific effect of
the hyperphagia and obesity.

The kidney weight on a dry or defatted basis was not
increased in proportion to increases in body weight as
mice injected with GTG became obese (Waxler and Enger,
1954). When these GTG-obese mice were reduced to body
weights equal to that of controls, the absolute weights
of the kidneys were less than those of control animals

(Waxler and Enger, 1954).

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Age
MacKay and MacKay (1927) concluded that in the albino

rat the kidney weight had practically the same relation to
body surface at all ages. Later these workers reported
that the kidney weight to body surface ratio fell slightly
with increasing age (MacKay and MacKay, 1934). They
suggested that this was due to a decreased protein intake
in relation to body surface in the older animals. Other
workers reported that kidney weights were related more to
body weight than to age (Widdowson and McCance, 1960).

In humans, kidneys at birth represented a higher pro-
portion of the body weight than they did at any other age
(Widdowson and Dickerson, 1960).

The effect of dietary protein on renal growth may be
modified by age.

MacKay et al. (1928b) and MacKay and MacKay (1931a)
reported that age up to 346 days had no influence on the
linear relationship between the renal weight and protein
intake. The absolute and relative increase in renal weight
which followed a given percentage increase in protein
intake, however, decreased as age increased (MacKay et al.,
1928b). From 346-400 days of age increased protein in the
diet had little effect on kidney size in males (MacKay
et al., 1926b). In the female of all ages there was a
linear relationship between the renal weight and protein

intake (MacKay and MacKay, 1931b).

 

L
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94

Miscellaneous

 

Kidney weights of rats were influenced by strain and
sex (Freudenberger, 1932). Eaton (1938) reported that
sex and genetics influenced kidney weights of guinea pigs.
Exposure to cold caused hypertrOphy of kidneys in rats of
both sexes (Emery et al., 1940). Exercise affected
kidney size in rats according to Bloor et al. (1968).

When animals were given thyroxin (Walter and Addis, 1939)
or thyroid (MacKay and MacKay, 193lc) renal weight was
increased above that of control animals.

Chronic acidosis in rats also resulted in renal
enlargement (Janicki, 1970). Constantinides (1951)
reported that stresses (formalin injections, cold, forced
exercise) for 20-48 hours duration caused an immediate
enlargement of the kidneys. Dinitrophenol produced
hypertrOphy of the kidney in albino rats (Murphy, 1938).

Kidney weight may be influenced by a number of
dietary and nondietary factors (G033 and Dittman, 1969;
Halliburton, 1969). The mechanisms reSponsible for the
renal hypertrOphy produced by these has not been elucidated.

The objectives of this experiment were (1) to describe
the lesions occurring in kidneys from animals made obese by
dietary means, (2) to compare these lesions with those
occurring spontaneously in rats and (3) to determine the

influence of dietary obesity on renal weights.

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MATERIALS AND METHODS

Animals

Osborne-Mendel rats bred in the Department of Foods
and Nutrition were chosen for this study for two reasons:
(1) They are relatively resistant to upper respiratory
infection and are thus suitable for long term studies
(Mickelsen et al., 1955); (2) They attain a large body
size and become obese, as measured by total ether-
extractable body fat, when fed a 60% hydrogenated fat
semi-synthetic diet (Mickelsen et al., 1955; Schemmel
et al., 1969).

Within 48 hours after birth, litters were cut to
eight pups by keeping all of the males and making the
litter to eight with females. If less than eight animals
were born, all were saved. Each litter was weaned at an
average age of twenty-three days. Grain ration and water
were available in the breeding cage at all times.

After weaning, males were paired but housed in
separate wire-bottom cages in a temperature and light
controlled room. One male of each pair was fed a grain
ration (GR) and the other a semi-synthetic high fat

ration (HF). Females were not used in this experiment.

95

96

Rations

The two rations fed in this study were develOped by
Smith (1969). They are modifications of a grain ration
(Campbell et al., 1966) and a semi-synthetic ration
containing 60% fat (Mickelsen et al., 1955). Composition
of the modified rations are shown in Appendices C and D.
The diets were altered by Smith (1969) so that animals
on the grain ration or high fat ration would consume the
same quantities of calcium, magnesium, and phoSphorus
per week. The calcium to phOSphorus ration in these diets
is similar to that recommended for the growing rat by the
National Research Council (Committee on Animal Nutrition,
1962). The two rations and distilled water were avail-

able ad libitum.

Tissue Preparation

 

A total of 73 animals were used in this experiment.
At predetermined ages (weaning, nine, fifteen, twenty-five,
thirty-five and forty-five weeks), the animals, after
being weighed, were killed instantly' by using an
overdose of ether in a closed chamber. Kidneys were
removed from capsules, blotted, and weighed separately.
Kidneys were cut transversely in 1.0 mm sections. Separate
pieces were fixed in 4% acetate-buffered formaldehyde,
modified Zenker's fixative, or Carnoy's fixative (Lillie,
1965; Armed Forces Institute of Pathology, 1968). After

12-24 hours, Zenker-fixed tissues were washed in running

 

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97

tap water 8 hours, and then stored in 80% ethanol. The
sections in Carnoy's solution were fixed for 6-12 hours
and then placed in absolute alcohol. All samples were
stored at room temperature. The entire cross sectional
kidney slices were dehydrated, embedded in paraffin and
cut at 5 microns. Hematoxylin-eosin stain was used
routinely (Lillie, 1965). Special stains were applied
where needed. These included Oil Red 0, PAS, Alcian Blue,
and Congo Red.

Parameters evaluated in histological sections of

kidneys are shown in Appendix E.

Statistical Analyses

 

The paired Students "t" test was used to assess the
significance of differences between group means. This
test was also used to assess the differences in the
weights of the right and left kidneys within the same age
and dietary group.

An analysis of variance (Steel and Torrie, 1960) was
used to delineate factors affecting kidney weight, body

weight and the kidney weight/body weight ratio.

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RESULTS

Body Weights:

 

Animals fed either ration increased in weight as they
grew older, but animals fed the HF ration increased their
weights at a faster rate than those fed the control diet
(Table 3). By ages 25, 35, and 45 weeks, animals fed the
HF diet weighed significantly more than animals fed the
GR diet (25 and 35 weeks-P<0.001; 45 weeks-P<0.05).

From gross observation, the HF animals contained consider-
ably more carcass fat than the GR animals, particularly
perirenal fat. The effect of age and diet upon body
weight were each statistically significant (P<0.01) for
all rats (Table 5). Furthermore, the interaction of diet

and age was statistically significant (P<0.01).

Kidney Weights and Kidney Weight/Body Weight Ratios

 

In the HF fed group, the right kidney was heavier
(15, 35 and 45 weeks-P<0.02; 25 weeks-P<0.01) than the
left kidney at all ages (Table 3). There was no differ-
ence (P>0.05) in the weights of the two kidneys in the
GR groups at any age (Table 3).

The total kidney weight was significantly greater in
HF animals at 25 (P<0.02), 35 (P<0.01) and 45 (P<0.01)
weeks than in GR animals (Table 3). The effect of diet

was statistically significant (P<0.01) for all rats

98

99

(Table 5). Furthermore, the interaction of diet and age
was statistically significant (P<0.05).

The kidney weight/body weight ratio (KW/BW) (Table 3)
was significantly less in HF animals at all ages (15 weeks-
P<0.05; 25 and 45 weeks-P<0.001; 35 weeks-P<0.0l). Diet
and age had a statistically significant effect on the
KW/BW ratio at the 1% and 5% level of significance,
respectively (Table 6). The interaction of diet and age

was statistically significant (P<0.05).

Histopathological Examination

The incidence of lesions observed histologically in
kidneys from GR and HF animals is shown in Table 7.

Kidneys of weanling animals (Figure 1) had small
densely cellular glomeruli. The outer cortex had a
nephrogenic zone with undifferentiated glomeruli and
tubular cells. No inflammation or degenerative changes
were observed in these kidneys.

At 15 weeks changes were observed in kidneys from
GR and HF animals (Table 7). Kidneys from HF animals
(Figure 2) had considerable glomerular damage. Tubules
contained some cellular debris. Although there were
degenerative glomerular changes in this kidney, there were
areas with normal appearing glomeruli.

The incidence of different types of lesions was
greater in the 15 week GR group than the HF group
(Table 7). The lesions observed in the HF group were more

wideSpread within the kidney.

 

I'I'I"

100

Kidneys from GR animals at 25 weeks (Figure 3) had
large, well-defined glomeruli. Tubules were relatively
free of debris or secretion. In the section pictured in
Figure 3 no inflammation, edema, fibrosis or hemorrhage
was evident. Some kidneys from 25 week old control
animals had occasional glomeruli undergoing atrOphic
degenerative changes.

Glomerular and tubular changes were observed in
kidney sections from 25 week old animals fed HF (Figure 4,
Figure 5). The tubules in the section in Figure 4 had
swollen nuclei and epithelial cells. Tubular lumens
contained proteinaceous debris. Tubular cells were
swollen. Glomerular changes observed in this section
were swelling and increased cellularity. Extensive
glomerular changes in this group as seen in Figure 5 were
infrequent.

Kidneys from GR animals at 35 weeks (Figure 6) had
for the most part large glomeruli with open Bowman‘s
Spaces. There were some atrOphic glomeruli and cystic
tubules in these kidneys.

Glomerular changes of considerable magnitude were
observed in kidneys of 35 week old animals fed the HF
ration (Figure 7, Figure 10). These changes included
hypertrophy of the capsular epithelium, swelling of
some capillary loops and atrOphy of others.

Tubular changes in the kidneys of 35 week old obese

animals were extensive (Figure 8, Figure 9). These changes

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included amorphous debris and proteinaceous material in

the lumens, swelling of tubular epithelial cells and cystic
tubules. These sections were negative when stained with
Congo Red and Alcian Blue.

Glomerular crescents (Figure 10) were observed in
kidneys from two experimental animals. These develOped
in response to some injurious stimuli.

A number of lesions were seen in the 45 week groups
of animals fed GR and HF (Table 8) which were not observed
in other groups. Kidneys from control animals at 45 weeks
(Figure 11) had some degenerative changes in glomerular
and tubular structure. For the most part, however,
glomerular tufts were distinct with no hyperplasia or
swelling. Bowman's space was narrow and Open.

The 45 week old animals fed HF postweaning had kidneys
with extensive damage (Table 9). This group showed
considerably more degenerative changes and of greater
magnitude than any other group (Table 7). An example of
the extensive glomerular and tubular damage observed in
these animals is illustrated in Figure 12. In this section
tubules were dilated and contained proteinaceous secretion.
Glomerular changes were also visible. A higher magnifica-
tion of the tubular changes observed in most of the
45-week HF group is shown in Figure 12.

Special staining procedures were inconclusive.
Sections as illustrated in Figure 12 were negative when

stained with Congo Red, Alcian Blue and 011 Red 0.

 

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Figure 1.

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Figure 2.

102

Kidney section from a weanling rat (078004).
Numerous glomeruli are seen, inactive and
active ones. Tubules are open and clear or
may be collapsed as a result of inactivity.
The brush border of the proximal convoluted
tubules is incomplete due to immaturity. No
inflammation or degenerative changes were seen
in this kidney. Hematoxylin and Eosin, X160.

Kidney section from an animal 15 weeks old
(078017) fed the high fat ration for 12 weeks.
The tubules are functional, but some contain
hypertrophied cells. An occasional epithelial
cell has become detached and lies free in the
tubule lumen. The most significant change is
the glomerular degeneration as indicated by
glomeruli A and B. Glomerulus A remains only
as a dense atrophic mass while B is larger and
has blood passing through the capillaries. The
entire capillary tuft in B is smaller and is
irregular compared to others. Hematoxylin and
Eosin, X160.

 

103

 

Figure 1

 

Figure 2

 

Figure 3.

104

Kidney section from an animal 25 weeks old
(078008) fed the grain ration after weaning.
Glomeruli are large, well-defined and func-
tional. Tubules are normal, Open, and free
of secretion or debris. No inflammation,
edema, fibrosis or hemorrhage to indicate any
disease process was found in this section.
Occasional glomeruli were undergoing atrOphic
degenerative changes, probably as a result of
the normal aging process. Hematoxylin and
Eosin, X160.

105

 

Figure 3

 

Figure 4.

Figure 5.

106

Kidney section from an animal (078010) 25 weeks
old fed the high fat ration for 22 weeks showing
glomerular and tubular changes. The most
prominent change in this section is the swelling
of the nuclei and epithelial cells of the
tubules (arrow). The tubules contain
proteinaceous debris (A) and epithelial cells
are swollen. The glomerular tuft is swollen and
is more cellular than normal (B). Hematoxylin
and Eosin, X270.

Kidney section from an animal (078010) 25 weeks
old fed the high fat ration for 22 weeks showing
glomerular and tubular changes. The glomerular
tuft in this photomicrograph remains only as an
atrOphic mass floating in secretion (arrow).

The capsule is enlarged and Bowman's Space con-
tains secretion as a result of blockage below
this level. Vacuoles indicate fat and the

small dark globules are probably protein of
serum origin, although they could be remnants

of degenerating cells. The former is more
likely. In this kidney section, occasional
small lymphocytic foci are present but inflamma-
tion and general hemorrhage are not present with
the exception of a small amount of edema and
serous‘fluid collected around one arteriole.
Hematoxylin and Eosin, X270.

 

 

Figure 5

 

Figure 6.

Figure 7.

108

Kidney section from an animal (078024) 35 weeks
old fed the grain ration for 32 weeks. Glomeruli
appear normal. There is a fragment of capillary
loop lying free in the glomerular Space (arrow).
Tubules appear open and normal. Hematoxylin and
Eosin, X160.

Glomerular damage in kidney section from an
animal (078026) 35 weeks old fed the high fat
ration 32 weeks. Capillary loops are swollen

so detail is obscured. HypertrOphy of capsular
epithelium is visible in about half of the
capsule surface (arrow). This has not increased
to the point that it could be called an
epithelial crescent. Other changes seen in this
kidney included occasional cystic tubules with
secretions or hyaline casts, somewhat shrunken
glomeruli probably indicating early atrophic
changes. There are a few collecting tubules in
the medulla containing hyaline casts. These
lesions were not extensive, however. Hematoxylin
and Eosin, X270.

109

 

Figure 6

 

Figure 7

 

Figure 8

Figure 9.

110

Tubular changes occurring in the kidney from a
rat (078027) fed the high fat ration for 32 weeks.
Changes include proteinaceous material in the
lumens (arrow), swelling of tubular epithelial
cells and amorphous debris in the tubules. There
is a small focus of lymphocytes (A). Hematoxylin
and Eosin, X270.

Additional tubular changes occurring in the same
section as seen in the previous figure. A cystic
tubule (A) and tubules containing secretion which
appears to have coagulated (arrow) are evident.
Smaller tubules contain debris in lesser quantities.
This section was negative when stained using Congo
Red and Alcian Blue. Hematoxylin and Eosin, X160.

111

 

Figure 9

 

Figure 10.

112

A glomerulus from the same section as seen in
the two previous figures. Glomerular capillary
and basement membrane detail are indistinct
(arrow) although some loops are functional as
indicated by the presence of erythrocytes in
the lumens. A large epithelial crescent has
formed at one area (A). The remainder of
Bowman's capsule appears to be unaffected.
Some material, which appears to be protein,
is lying in Bowman's space. Hematoxylin and
Eosin, X270.

113

 

Figure 10

 

Figure 11.

Figure 12.

114

Kidney section from an animal (080119) 45 weeks
old fed the grain ration for 42 weeks post-
weaning. Glomerular tufts are distinct (A),
capillary 100ps are well defined and Bowman's
Space is narrow and clear (narrow arrow).
Bowman's membrane is normal (broad arrow).

The brbken capsule at the upper left surface is
an artifact. Tubules are open and clear.
Hematoxylin and Eosin, X160.

Glomerular and tubular changes in a section from
an animal (080116) 45 weeks old fed the high fat
ration for 42 weeks. Extreme degenerative changes
are indicated by the large number of dilated
tubules (A) and the obsolescence of glomeruli
(arrows) in this section. Dilated tubules were
distributed throughout this section extending
from the surface throughout the cortex and into
the medulla. Most of these tubules were widely
dilated and some contained hyaline casts and
secretion with protein. Many of the tubules were
dilated, but empty. The epithelial cells were
flattened and pressed close to the basement mem-
brane. This could have been caused by the

-retained fluid which escaped when the sections

were cut. In other areas the casts have remained
and are visible in the dilated tubules. This
section was negative when stained with Congo Red,
Alcian Blue, and Oil Red 0. Hematoxylin and
Eosin, X60. . .

115

 

 

Figure 11

 

Figure 12

 

7

Figure 13.

116

High-magnification view of tubular changes
occurring in the same kidney section seen in
the previous figure. The tubular lumens
contain protein. Strands of fibrin attach the
protein masses to the epithelial cell surfaces.
Some epithelial cells have undergone degenera-
tion and are no longer evident (arrow).
Hematoxylin and Eosin, X270.

117

 

Figure 13

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122

Table 7. Percentage of animals in control (GR) and experimental (HF) groups with

histopathologic kidney lesions.

 

Lesion or Change
+

Group *

 

q Weanling

W 15 weeks-GR

(n 15 weeks-HF

m 25 weeks-GR

H 25 weeks-HP

“+ 35 weeks-GR

fl 35 weeks-HF

a 45 weeks-GR

w 45 weeks-HF

 

Kidney Capsule
Irregular
Bowman's Capsule
Thick
Fibrosis
Hyaline
Crescent
Necrosis
Irregular
Glomerular Tuft
AtrOphic (Immature?) 88
Adhesions
Fibrosis
Degeneration
Bowman's Space
Blood
Protein
Tubular Epithelium
Hyperplasia
Degeneration
Necrosis
Cystic with Filtrate
Swelling
Cellular Casts
Fibrosis
Lymphocytes & NeutrOphils
Basement Membrane Thickened
Henle's Leaps
Cell and Protein Casts
Hyaline Casts
Hemoglobin Casts
Oorticomedullary Junction
Cystic Tubules with Filtrate
Protein Debris in Tubules
Tubular Necrosis
Collecting Tubules
Casts
Epithelium Degeneration 13
Hemoglobin and Protein in Lumen 13
Renal Papillae
Protein in Tubules
Renal Pelvis
Hemorrhage
Miscellaneous
Calcification
Cortical Hemorrhage
Nephritis
Focal Hemorrhage

20

40

60'

60
20

20

20

60
20

20

20

40
20

20
2O

80

100

20

20
20
20
20

20

20
60

20
4O

20

20

60

20
20

20

20

25
50

25
50

25

25

11

56

22

22

67
33

ll

22
44

ll
11

11
ll

11

11

33

11
100

22
ll

11

33
22

33
67
56
33

11

22

90

ll
56
11

44

ll

11
ll

Table 8.

123

Lesions observed only in kidneys from animals
45 weeks old and fed the grain or high fat
ration post-wean1ng.

Hyperplasia of Tubular Epithelium

Necrosis of Tubular Epithelium

Cellular Casts

Fibrosis

Lymphocytes and Neutrophils

Thickened Basement Membrane

Nephritis

Focal Hemorrhage

Table 9.

124

Lesions observed only in kidneys from animals
fed the high fat ration.

Casts in Collecting Tubules
Hyperplasia of Tubular Epithelium
Degeneration of Tubular Epithelium
Fibrosis of Tubular Epithelium
Thickened Basement Membrane

Hemoglobin and Protein in Collecting Tubule
Lumens

Renal Pelvis Hemorrhage
Nephritis
Focal Hemorrhage

Hyalin in Bowman's Capsule

DISCUSSION

The Osborne-Mendel male rat when fed a high fat diet
has a propensity for obesity (Mickelsen et al., 1955;
Schemmel, 1967; Schemmel et al., 1969). Animals in this
study fed the high fat diet (HF) were Significantly
heavier than controls fed a natural grain ration (GR) at
25, 35 and 45 weeks of age (Table 3). Grossly these
animals contained more carcass fat, particularly in the
perirenal depots. In most obese animals the kidneys were
completely embedded in adipose tissue. No brown fat was
observed in the renal area except in the weanling animals.
Other researchers have reported that animals made obese
by feeding high fat diets contain more carcass fat (Wierda,
1950; Mickelsen et al., 1955; Schemmel, 1967; Schemmel
et al., 1969). The body weight was significantly influenced
by age, diet and the interaction of these two factors
(Table 3).

Arataki (1926) reported that the right kidney of
female and male control rats was 2.3% and 2.1% heavier,
respectively, than the left. Similarly, Wachtel et al.
(1966) found that the ratio of the weight of the left
kidney to the right kidney was always less than 1.0. In

this study, however, there were no significant differences

125

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126

in the two kidney weights in the GR animals at the ages
studied (Table 3). If the differences in kidneys weights
are analyzed on a percentage basis, the right kidney was
at least 2% heavier than the left in the 15, 25, 35 and
45 weeks groups although these differences were not
statistically Significant (P>0.05; paired "t" test). The
right kidney was significantly heavier than the left in
HF animals at all groups postweaning. Whether this
difference is physiologically significant is not known.
One could speculate that the hemodynamics are altered in
the obese animals and that these changes could lead to
the differences observed. Blood flow measurements have
not been made in these animals however.

The absolute weight of both kidneys was Significantly
greater in the HF rats at 25, 35 and 45 weeks of age
(Table 3). These differences cannot be explained on a
basis of the dietary nitrogen intake. The percent of
protein in the HF and GR diets is approximately the same.
The GR animals would normally consume more protein per
day as their food intakes are greater on an absolute, but
not caloric, basis. Thus the endogenous protein presented -
to the kidney is greater in the control animals. Some
workers have suggested that renal enlargement in obese
animals resulted from a nonspecific effect of hyperphagia

and the obesity (Marshall et al., 1957).

127

Other workers have reported that in rats absolute
kidney weights increased linearly as body weight increased
(Hatai, 1913; Webster et al., 1947; Widdowson and McCance,
1960). Obese rats were not used in any of these studies,
however, and it is doubtful that the relation of kidney
weight to body weight is linear in excessively overweight
animals.

The relative kidney weight or kidney weight/body
weight ratio (KW/BW) ranged from 1.30 in the weanling
animals to 0.522 in obese animals (Table 3). This ratio
was less than that of controls in all HF groups. Other
researchers have reported a KW/BW ratio greater than unity
in young animals (Webster et al., 1947; Widdowson and
McCance, 1960). In these studies, this ratio decreased
as age and body weight increased.

Marshall et al. (1957) found that kidney weights of
animals made obese by several procedures (genetic, hypo-
thalamic, GTG) were heavier than controls. The KW/BW
ratio in these obese animals, however, was less than that
of controls.

Grossly, the kidneys from HF and GR animals appeared
normal. No pale, granular, pitted kidneys were observed
in any of the animals. Similarly, none of the animals,

HF or GR, had hydronephrosis, ureteral blockage or bladder

stones.

.I~.rblu..5.v 1k! III. lie"!
1.. q . i

128

Kidneys from the weanling animals can best be des-
cribed as immature. The glomeruli and tubules appeared
undifferentiated in the nephrogenic zone below the
capsule (Figure 1). These kidneys were similar to those
described in young animals by other workers (Bogomolova,
1966; Hirsch et al., 1971). No evidence of inflammation
or degenerative changes were seen in kidneys in this
group.

The absolute incidence of renal lesions in the 15 and
25 weeks groups of control animals was greater than that
of the obese animals (Table 7). The severity of the
lesions, however, was greater in the HF groups.

By 35 weeks of age the obese animals had considerably
more histological renal damage than control animals
(Table 7). Glomerular and tubular lesions similar to
those described as occurring spontaneously in rats with
aging were observed (Saxton and Kimball, 1941; Simms and
Berg, 1957; Andrew and Pruett, 1957; Kennedy, 1957; Bras
and Ross, 1964; Gray and Purmalis, 1965). Cystic tubules
with flattened epithelium (Figure 9; Figure 12) were
particularly prevalent in kidneys from the obese animals.
Glomerular damage leading to crescent formation (Figure 10)
was Similar to that reported by other researchers also
(Gray, 1963; Bras and Ross, 1964).

The results reported may be complicated by the

occurrence of an epidemic in the animal laboratory during

129

the course of this investigation. The etiologic agent
isolated was Pasteurella pneumotropica. However, all
animals except the weanlings were in the laboratory at
the time of the epidemic. In spite of this complication,
several conclusions can be made concerning the renal

lesions in obese animals, especially as the animals aged.

SUMMARY

A histopathological examination of kidneys from
animals at weaning and at 15, 25, 35 and 45 weeks of
age, fed either a grain ration (GR) or a high fat (HF)
ration postweaning, was completed.

The animals fed HF were significantly heavier than
the GR controls after 25 weeks of age. These animals
contained considerably more fat than the GR animals,
eSpecially in the perirenal-retr0peritoneal depots. Age
and diet each had a significant effect upon body weight.
In addition, the interaction of diet and age on body
weight was statistically significant.

In the HF-fed group, the right kidney was heavier
than the left in animals at 15, 25, 35 and 45 weeks of
age. There were no differences in the weights of the
two kidneys in the GR groups at any age examined. The
total kidney weight was significantly greater in the HF
groups at 25, 35 and 45 weeks of age. The effect of diet
on kidney weight was statistically significant as was the
interaction of diet and age. The kidney weight to body
weight ratio was significantly less in HF animals at
all ages. Furthermore, this ratio was Significantly
influenced by the diet, age and the interaction of diet

and age.

130

131

Lesions in kidneys from GR and HF animals affected
the glomeruli and tubules. Prior to 35 weeks of age, the
incidence of lesions in both groups was almost comparable,
although the lesions in the kidneys from HF animals were
more severe and covered more of the kidney parenchyma.

By 45 weeks of age and 42 weeks on the reSpective
diets, the kidneys from the HF animals had considerably
more damage than those from GR rats. The total number of
lesions and the severity of these was greater in the HF
group at this age.

In conclusion, dietary obesity had an adverse effect
on the kidneys. A histopathological examination of the
kidneys by light microscopy without Specific staining for
key enzymes may be a less sensitive tool for examining
this effect of obesity on the kidneys than tests of

functional capacity or examination by electron microscopy.

qr. lbfii . 5V. 2’ shilling

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PART III. Kidney function in dietary obesity: Effects
of obesity and diet on renal transport of

organic acids and bases.

INTRODUCTION

Renal tubular secretion, the transport of materials
from peritubular fluid to tubular lumen is a process by
which many substances are added to the urine. Substances
actively tranSported across the tubular epithelium
include certain organic acids and bases and hydrogen
ions (H+). Many compounds foreign to the body such as
penicillin are transported by such a system, which is
localized in and restricted to the proximal tubule
(Wesson, 1969).

Organic ion secretory systems may be studied by at
least three different approaches: (1) the intact
organism may be used to study various agents and condi-
tions that affect secretion, (2) the appearance and
approximate concentration of visible dyes in the lumina
of isolated tubules can be observed microscopically, and
(3) an in vitro slice or isolated tubule technique using
a suitable nutrient medium can be used (Wesson, 1969).
The first method is limited to circumstances which are
tolerated by the animal, and furthermore, extra-renal
factors may affect the results. The visual method
has the advantage that luminal accumulation of substrate
can be distinguished from intracellular accumulation;
however, quantitation is less precise and only colored

or fluorescent compounds can be studied. The slice

144

145

technique permits the use of a wide range of environmental
conditions (pH, temperature, ion concentration, etc.) and
permits examination and precise quantitation of a great
number of variables. Although the secretory system is
incomplete in the in vitro preparation, the advantages of
the technique are considerable.

For these reasons the in vitro slice technique
developed by Cross and Taggart (1950) was used to elucidate
the effect of obesity produced by feeding a high fat diet

on tubular secretion in the male rat.

In vitro Measurement of Renal Transport

 

The technique develOped by Cross and Taggart (1950)
has been widely used to study the secretion of organic
compounds. Very thin kidney cortical slices are incu-
bated in an oxygenated buffered salt medium and the
intracellular accumulation of compounds such as
p-aminohippurate (PAH) is used as an indication of the
activity and capacity of the secretory system. Accumula-
tion of PAH and other organic compounds is expressed as
the final ratio of concentrations in the slice and
medium, or the slice/medium ratio (S/M ratio).

The in vitro methods used to study renal tubular
tranSport have several advantages (Cross and Taggart,
1950) including the following: permit observation of
events that accurately reflect tubular tranSport; can be

0
used to Simultaneously measure transport and certain

146

metabolic activities such as oxygen consumption; permit
variation of experimental conditions (pH, ion concen-
tration, temperature, etc.) over a broad range which
would produce systemic toxicity in the intact animal;
make it Simple to rigidly control the chemical composi-
tion of the ambient fluid; and allow exclusion of
extrarenal factors (hormones, blood flow, etc.) that may
alter tubular secretion in the intact animal.

The in vitro slice preparation is a useful techni-
que even though the secretory system is incomplete. The
slice preparation does not allow for a continuous
filtration process, with the resulting disturbance of
normal concentration relationships between cells and
tubular urine. Also, the PAH, once it has accumulated
intracellularly in the in vitro technique, is able to
diffuse out of the cortical slices back into the medium,
in contrast to the unidirectional movement of PAH in the
intact kidney. Another disadvantage of this system is
the inability to distinguish between the accumulation of
substrate within the cells from that accumulated within
the tubular lumen. While these differences undoubtedly
influence certain quantitative aspects of PAH tranSport,
numerous experimental observations suggest that the same
biochemical processes Operate in both systems.

The slice technique is a reliable estimate of

organic acid and base tranSport as shown by a number of

147

reports comparing tranSport in vivo and in vitro. In the
rabbit in vivo studies have shown the maximum capacity of
the renal cortex to concentrate PAH is 4-5 umoles/g
tissue, and similar values have been obtained using kidney
slices (Foulkes and Miller, 1959a). Penicillin, PAH, or
carinamide, competitive inhibitors of the acid transport
system, have similar action, both in vivo and in vitro,
on the intracellular accumulation of phenol red (Forster
and Copenhaver, 1956). Other inhibitors such as
dibenemine and dibenzyline depress N-methylnicotinamide
(NMN) tranSport in vitro and in vivo (Ross et al., 1968a).
Mudge and Taggart (1950b) demonstrated that acetate and
lactate have stimulatory effects on PAH transport in both
preparations while succinate and fumarate uniformly
depressed transport. Cross and Taggart (1950) and Mudge
and Taggart (1950b) presented similar data supporting
their conclusion that the accumulation of PAH by kidney
slices in vitro and the tubular excretion of this compound
in the intact animal are closely related phenomena.

A schemmatic representation of the data obtained
from the slice incubation system is shown in Figure 14.
The S/M ratio develops with time and reaches a plateau
after 60-90 minutes. The system may be manipulated
employing inhibitors or stimulators so that a wide range
of S/M ratios may be obtained. A nitrOgen atmOSphere
prevents active accumulation of PAH so that the S/M

ratio approaches 1, indicating diffusion only.

148

The S/M ratio is the net result of intracellular
accumulation, binding within the cell and extrusion out
of the cell (runout). Intracellular accumulation
involves an active transport process described below.

The efflux mechanism, although incompletely characterized
at this time, is distinctly different from the process
mediating uptake (Farah et al., 1963; Ross et al., 1968b).
Efflux, or runout, is thought to involve passive
diffusion. There is also some evidence that an energy-
requiring tranSport process may be associated with

runout since inhibitors of PAH uptake such as dinitro-
phenol (DNP), octanoate, iodopyracet, and probenecid can
decrease PAH runout (Kinter and Cline, 1961; Wilbrandt
and Rosenberg, 1961).

Active tranSport refers to an energy-requiring
process whereby a substance is transported across a
biological membrane against an electro-chemical gradient.
The energy required is obtained from cellular metabolism.
Inhibition of a transport process by anoxia or by
inhibitors of selective enzymes involved in the energy
producing mechanism of the cell suggests that PAH trans-
port is active. Low concentrations of azide, cyanide,
and arsenite, exposure to cold and anaerobiosis all block
PAH uptake (Wesson, 1969).

Theoretically, any active transport process has

quantitative limits. In defining these limits it is

149

necessary to postulate the existence of some form of
carrier system. The number of reactive sites on the
carrier system is limited; that is, the system can only
be transporting a limited number of molecules at a given
moment. The active transport system for organic anions
in the kidney exhibits a non-linear relationship between
concentration and transport rate. The rate of transport
increases as substrate concentration increases until
saturation of the mechanism occurs. This fixed maximal
transport rate suggests that active transport involves
the reversible combination of the transported compound
with a carrier system, which is limited. Also consistent
with the concept of a cellular carrier of limited capacity
is the demonstration of competitive phenomena associated
with active transport systems such as penicillin com-
peting with PAH for tranSport.

Active transport can also be limited by decreasing
the energy available to the carrier system. Aerobic
metabolism provides energy for the tubular secretory
mechanisms for organic acids and bases. Tubular
secretion is blocked by anoxia, by inhibitors of the
cytochrome electron tranSport system, and of the oxidative
reactions of the tricarboxylic acid cycle (Taggart and
Forster, 1950; Shideman and Rene, 1951; Maxild and
M¢ller, 1969). The energy-rich phosphates, such as

adenosine triphosphate (ATP), generated by the oxidative

1..'~P.'.D biz";
.. _ JIM.

 

150

reactions apparently play a central role in providing
energy for tranSport systems and may explain the depend-
ence on aerobic metabolism. DNP, which uncouples
oxidation and phOSphorylation, depresses PAH transport
in vivo and in vitro without affecting tubular
reabsorption of glucose or amino acids. This strongly
suggests that phOSphate bond energy is involved in
tubular secretory transport (Taggart, 1958).

Although the carrier system for organic acids has
not been isolated and characterized, considerable evidence
suggests that proteins are involved in organic ion
transport. Ross et al. (1969) and Magour et al. (1969)
used a receptor protection technique to provide evidence
that the renal carrier of NMN is in the protein fraction.
Similar techniques have not been utilized to identify the
carrier for PAH since a Specific, irreversible inhibitor
of PAH transport is not yet available. The NMN studies
were possible because Ross et al. (1968a) demonstrated
that dibenamine Specifically and irreversibly blocked NMN
transport. That the transport system for PAH is protein
is suggested by the work of Hirsch and Hook (1970c) who
showed penicillin stimulation of PAH S/M ratios in young
animals was associated with enhanced renal cortical

protein synthesis.

151

Model of the Renal Secretory Mechanism

 

The secretory mechanism in mammalian renal cortical
slices is thought to involve two distinct processes, the
transport from peritubular fluid into the cell and the
movement from the cell into the tubular lumen (Taggart,
1958). Movement into the cell requires active transport,
whereas the step involving movement across the luminal
border is downhill and involves diffusion only (Foulkes
and Miller, 1959b, Tune et al., 1969).

The multifaceted nature of the transport process was
elucidated by two groups. Foulkes and Miller (1959b),
using renal cortical slices, showed that there are two
intracellular fractions of PAH, a fraction which rapidly
diffuses and equilibrates with extracellular PAH and a
fraction which is slowly equilibrating and is reSponsible
for the high slice to medium ratio. Foulkes (1963) and
Welch and Bush (1970) also presented evidence for at
least two intracellular pools--a bound or compartmen-
talized fraction and a freely diffusible pool.

0n the basis of these results, Foulkes and Miller
(1959b) proposed a 4 step mechanism of renal tubular
secretion of PAH (Figure 15). The proposed steps are as
follows: step one consists of diffusion of PAH from the
medium to the interstitial fluid; step two involves
facilitated diffusion at the peritubular cell membrane

from the interstitial fluid into the cell; step three

152

consists of an active accumulation of a high tissue con-
centration of PAH; and step four results in the transfer
of PAH across the luminal border of the cell into the
tubular lumen.

Since the S/M ratio is the net result of uptake into
the slice, accumulation in the cells and runout back into
the medium, uptake may involve the first three steps and
runout may involve the fourth. Ross et al. (1968b)
presented data suggesting that the uptake of organic
acids and bases occurs at the peritubular side and that
runout occurs at the luminal side of the proximal tubular
cell.

Shefikh and M¢ller (1970) reported that, in contrast
to earlier views, active transport of PAH ascribed to
the rapid component and slow component may be due to
intracellular compartmentalization. These workers used
separated renal tubules and eliminated the extracellular
space and thus the possibility that the slow component of
PAH tranSport might be due to a diffusion barrier between
the exterior and interior parts of the slices. Both of
these occur in the slice technique.

Recently, Tune et al. (1969), using isolated tubules,
proposed a model similar to that one of Foulkes and
Miller (1959b). These workers prOpose that entry into
the tubular fluid occurs down a concentration gradient

by a process consistent with simple diffusion.

153

Furthermore, active tranSport into renal tubular cells
across the peritubular membrane with subsequent diffusion
down a concentration gradient into the tubular lumen
follows. Tissue PAH levels reach a steady state when
passive efflux equals the active influx (Tune et al.,

1969).

Factors Affecting Renal Tubular TranSport

 

of Organic Acids and Bases

 

A number of factors affect the renal tubular tranSport

of organic acids and bases in vitra and in viva.

Metabolites

 

Intermediary metabolites may act to stimulate or
inhibit the organic acid transport system. For instance,
acetate enhances PAH transport in man (McDonald et al.,
1951), dog (Mudge and Taggart, 1950b), and rabbit (Cross
and Taggart, 1950). Lactate and pyruvate, precursors of
acetate, also increase PAH uptake by renal kidney slices
in vitra (Cross and Taggart, 1950) and PAH tranSport
in viva in man (McDonald et al., 1951). Low concentra-
tions of dicarboxylic acids of the citric acid cycle, as
succinate and fumarate (Cross and Taggart, 1950), fatty
acids of intermediate carbon chain length (CG-Clo) (Cross
and Taggart, 1950; Schacter et al., 1955) and the amino
acids L-alanine and L-glutamate (Schacter et al., 1955;

Cross and Taggart, 1950), markedly depress the uptake of

154

PAH by renal cortical slices. The maximal tubular
excretory capacity of PAH (TmPAH) is depressed in the
dog by succinate (Mudge and Taggart, 1950b), fumarate
(Mudge and Taggart, 1950b) and a-ketoglutarate (Knoefel
and Huang, 1959). Balagura-Baruch and Stone (1969)
demonstrated that in the dog PAH transport was depressed
by a—ketoglutarate when plasma levels of PAH were both

greater than and less than the Tm This depression

PAH'
was not overcome by the addition of acetate. These
workers concluded that a-ketoglutarate is a noncompetitive
or mixed inhibitor of the PAH tranSport system. Farah

at al. (1963) showed that acetate and lactate decreased

while succinate and a-ketoglutarate increased the rate

of runout of PAH from renal slices.

Metablic Inhibitors

 

Metabolic inhibitors also depress transport of organic
acids and bases. Taggart and co-workers (Cross and
Taggart, 1950; Mudge and Taggart, 1950a; and Taggart and
Forster, 1950) have shown in vitra and in viva that
2,4-dinitr0phenol, cyanide, and arsenite depress PAH,
phenolsulfonthalein (PSP) and Diodrast uptake. They
proposed that these compounds depress organic acid
tranSport by acting directly on some component of the
cellular transport mechanism, probably by inhibiting
aerobic phosphorylation. Maxild and M¢ller (1969)

demonstrated that inhibiting the Krebs cycle and

155

carbohydrate metabolism virtually abolishes PAH accumula-
tion. Metabolic inhibitors may also depress organic

acid S/M ratios by increasing runout (Kinter and Cline,
1961; Farah at al., 1963). As with acids, organic base
transport is inhibited by metabolic inhibitors

(Shideman and Rene, 1951; Farah et al., 1959).

Electrolyte Composition

 

The electrolyte composition of the bathing medium
and of the slices influences organic acid and base
tranSport.

The H+ concentration of the medium may affect the
transport system, and the optimum pH for maximum
accumulation is Species specific (Copenhaver and Davis,
1965; Ross at al., 1968b; Hirsch, 1970).

Potassium is necessary for the transport system to
function. Taggart at al. (1953) found kidney slices lost
the ability to accumulate PAH in a K+-free medium and
suggested K+ was necessary for maintenance of cell
integrity. Foulkes and Miller (1960) prOposed that K+
has a Specific role at the cell membrane. They found
that adding K+ to K+-deficient slices stimulated PAH
uptake prior to a significant increase in intracellular
K+. Furthermore, they suggested that K+ is required as
a part of the intracellular PAH—concentrating mechanism.

Hirsch (1970) found that renal cortical slices from

156

immature and adult rabbits required K+ in order to
accumulate PAH. Burg and Orloff (1962a) reported that
the digitalis glycoside, strOphanthidin, interferes with
PAH accumulation in slices of rabbit renal cortex, pre-
sumably by decreasing the K+ content. When K+ was
increased in the medium, PAH accumulation was not
depressed by strophanthidin. Dantzler (1969) similarly
reported that increased K+ in the medium overcame the
depressing effects of ouabain, a cardiotonic steroid, on
PAH accumulation. Ross et al. (1968b) demonstrated that
decreased K+ in the medium depressed uptake of PAH and
NMN by dog renal slices without affecting runout. Chung
at al. (1970) reported a critical role for Na+ and Ca++
in addition to a requirement for K+ for accumulation of
‘PAH and PSP by rabbit kidney slices. Dantzler (1969)
reported that snake and chicken kidney slices were unable
to accumulate PAH and urate normally in the absence of
K+. If K+ was sufficient, transport of PAH was not
affected unless Na+ was greatly reduced. Thus a number

of electrolytes have been reported to influence organic

acid and base tranSport.

Compounds Transported by the Same System

 

Other compounds tranSported by the same system may
affect the tranSport of PAH or NMN in the in vitra slice
preparation. Chlorothiazide (Beyer and Baer, 1961) pro-

benecid (Kinter and Cline, 1961; Huang and Lin, 1965),

157

DNP (Huang and Lin, 1965), free fatty acids of medium
chain length (Schacter et al., 1955), Diodrast (Cross
and Taggart, 1950), penicillin (Cross and Taggart, 1950),
carinamide (Cross and Taggart, 1950), PSP (Forster and
COpenhaver, 1956), triiodothyronine (T3) and tetra-
iodothyronine (T4 or thyroxine) (Nepomuceno and Little,
1964a; 1964b; Hirsch, 1970), have all been shown to
depress PAH accumulation.

The PAH transport system in the immature animal may
mature or develop at a faster rate if one of these com-
pounds which inhibits in vitra is given in utera or
shortly after birth. T3 given to weanling rats increased
the ability of renal cortical slices to tranSport PAH and
increased the kidney weight (Hirsch and Hook, 1969b).
Since T3 when added to slices in vitra depressed PAH
uptake these workers suggested that the compound is
tranSported by the organic acid system and specifically
stimulates the tranSport system. Folic acid when
administered to rats several days prior to sacrifice also
stimulated PAH accumulation by renal cortical slices
(Hirsch and Hook, 1969c).

Treating nursing rats or pregnant rabbits with
penicillin increased PAH accumulation when measured in
vitra (Hirsch and Hook, 1969a, 1970a, 1970b), presumably
by increasing the synthesis of Specific tranSport pro-

tein(s) (Hirsch and Hook, 1970c). NMN transport was

158

unaffected in treated animals, thus the substrates of the
organic acid tranSport system were apparently specific
stimulators of the PAH system.

Farah et al. (1959) reported that transport of NMN
by the organic base system in renal slices from dogs was
depressed by tetraethylammonium (TEA) and darstine, both
bases and both presumably tranSported by the same system.
Ross et al. (1968a) found dibenamine and dibenzyline
depressed NMN transport in viva and in vitra 80-90% with-
out affecting organic acid transport as measured by PAH
accumulation. TEA uptake was inhibited by
triiodotherprOpionic acid and triiodothyroacetic acid,
according to Nepomuceno and Little (1964b). Domer (1960)
force fed NMN to dogs at lZ-hour intervals for seven days
and determined that renal tubular transport of NMN and
priscoline (tolazoline) in viva was increased.

Numerous studies have shown that other compounds
tranSported by the same system may interfere with organic

acid and base transport.

Structure

 

Compounds transported by the organic acid transport
system may have a variety of chemical structures
(Taggart, 1958). There may be intramolecular reactive
groups possessed in common (DeSpopoulous, 1965). Weiner
at al. (1964) point out that protein binding, acidic

strength, lipid solubility, urinary pH, urinary flow, and

159

plasma level all may affect the transport of organic
acids in a particular Species. They also state that all

acid compounds tranSported are transported as anions.

Effects of Uremic and Normal Serum

 

A number of workers have demonstrated that addition
of "uremic" serum to the incubation system depressed PAH
tranSport (White, 1966; Preuss et al., 1966; Bourke et al.,
1967; Hook and Munro, 1968; and Orringer et al., 1971).
Hook and Munro (1968) found that fasting rats for 24 hours
prior to removing the kidneys for the in vitra study also
depressed PAH tranSport without affecting NMN transport.
Adding acetate to the slice increased PAH S/M ratios to
control values. Serum from nephrectomized animals also
depressed PAH uptake but did not change NMN transport.

Orringer et al. (1971) found when normal serum was
used at high concentrations, accumulation of PAH by rat
renal cortical slices was depressed while at low concen-
trations it was stimulated. Azotemic serum, at any
concentration studied, depressed transport (Orringer
at al., 1971). They suggested that, in high enough
concentration, some constituent in normal serum com—
petitively inhibited organic anion tranSport. Preuss
at al. (1966) found that dialyzing uremic serum prior to
adding it to the in vitra slice preparation prevented the

inhibition.

160

Uninephrectomy in rats increased hippurate tranSport
by the remaining kidney transiently while no change was
seen in organic base tranSport (Goldberg et al., 1970).
These workers suggested the early increase in organic
acid tranSport in hypertrophying kidneys is secondary to

substrate induction in the growing kidney.

The Effect of Age and Developmental Stage of the Animal

 

Barrows and co-workers (Adams and Barrows, 1963;
Beauchene at al., 1965) reported depressed transport of
PAH and a-aminoisobutyric acid (AIB) in kidney cortical
slices from old rats. Associated with this depressed
transport was a 10% decrease in deoxyribonucleic acid
(DNA) and depressed sodium-potassium-activated adenosine
triphOSphatase (ATPase) activity in the older animals
when compared with the younger ones.

Kidney cortical slices from the newborn of a number
of species are incapable of accumulating as much organic
acid and base as those from a mature animal (Williamson
and Hiatt, 1947; New at al., 1959; Rennick et al., 1961;
Hirsch, 1970).

The depth of the cortex from which the slices are
taken may also affect the accumulation. The inner slices
of renal cortex are more developed at birth than the
outer slices of the "nephrogenic zone" (Wachstein and
Bradshaw, 1965). In addition, the state of development

of the animal at birth influences the transport system.

161

The guinea pig, which can take solid food at birth, has
a well developed transport system at birth while the renal
tubular transport system in the newborn rabbit is less
mature (Hirsch, 1970).

The TmPAH was 2-4 times greater in premature
infants fed a high protein diet for four weeks than in
those fed a low protein diet, suggesting to Calcagno and
Lowe (1963) that diet could induce renal tubular
maturation. No changes were observed in glomerular
filtration rate (GFR) or renal plasma flow (RPF) in these

infants, so that the organic acid transport system was

apparently selectively stimulated.

ES}.
Most workers find that sex differences affect the
accumulation of PAH by renal cortical slices. Renal
cortical Slices from mature male rats had greater PAH
uptake than did those from females (Huang and McIntosh,
1955; Ferguson and Matthews, 1963). These workers demon-
strated that the sex differences in the PAH S/M ratio is
due to the stimulatory effect of testosterone and not to
the inhibitory effect of estrogen. Farah et al. (1956)
observed no difference between males and females in
ability of kidney slices to accumulate PAH. Kleinman
at al. (1966), however, reported that transport of PAH

in viva and in vitra was higher in males than females.

Bowman (1970) also reported increased PAH transport in

162

renal cortical Slices from male rats. S/M ratios for NMN
and TEA, however, were the same for male and female rats.
Apparently the increased S/M ratios obtained with kidneys
from male rats is a result of increased uptake and

decreased runout of PAH (Bowman, 1970).

Effects of Manipulations of Transport System

on 0% Consumption by Renal Cortical Slices

Copenhaver and Davis (1965) found no significant
changes in oxygen consumption when the pH of the media
ranged from 6.5 to 8.5. Taggart et al. (1953) reported
similar respiration values for slices developing S/M
ratios from 3.8 to 12.6. Although strophanthidin inter-
ferred with PAH transport, presumably by affecting the K+
concentration in the medium, no concomitant change in
oxygen consumption was observed (Burg and Orloff, 1962b).
Bourke et al. (1967) found nephrectomized serum depressed
PAH transport in kidney Slices, but did not affect slice
oxygen consumption. Chung at al. (1970) did not find
changes in oxygen consumption with changes in PAH and PSP

+ in the

transport produced by using Na+, K+, and Ca+
medium in varying concentrations.

Huang and McIntosh (1955) reported a marked decrease
in PAH uptake and oxygen consumption in kidney slices
from hyposectomized rats, but suggested these were second-

ary to changes in testosterone and estrogen as a result

of the hyp0physectomy.

163

New eL ai. (1959) reported that oxygen consumption
based on the nitrogen content of slices was lowest in
the newborn, the age group which also accumulated the
least PAH. These workers suggested that perhaps oxygen
consumption and PAH transport were related.

The purpose of this study was to demonstrate that
renal tranSport capacity is different in animals made
obese by feeding a high fat diet. The in vitra slice
preparation (Cross and Taggart, 1950) was used since it
excludes factors that may alter tubular secretion in the
intact animal. The Specific objectives of this study
were (1) to demonstrate the effect of obesity on the
renal secretion of the organic acid, PAH and (2) to
determine factors affecting the accumulation of PAH by

renal cortical slices from these animals.

164

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METHODS

Accumulation of Organic Ions by Renal Cortical Slices

 

The Cross and Taggart (1950) slice technique was
used to study the ability of renal cortical slices to
actively accumulate organic ions. PAH was used as the
prototype to study organic anion transport while NMN was
used as the prototype for studying organic cation trans-
port.

Osborne-Mendel male rats of NIH stock and bred in the
Human Nutrition Laboratory were fed either a grain ration
(GR) (Appendix A; Campbell et al., 1966) or a 60% fat
ration (HF) (Appendix B; Mickelsen at al., 1955) which
has been shown to produce obesity in this strain (Schemmel
at al., 1969). The animals were housed in a temperature
controlled room with a 12-12 hour light-dark cycle. Food
and tap water were available ad Zibitum.

Animals were killed by cervical dislocation or by
guillotine after obtaining the body weight. The kidneys
were rapidly removed, trimmed of fat and capsule, and
placed in iced normal saline. Kidney weights were obtained
and renal cortical slices of 0.3-0.4 mm thickness were
prepared free hand. Approximately 100 mg of tissue was
added to the incubation media. The latter was a phosphate
buffer media as devised by Cross and Taggart (1950) con-

5 6 14

taining 7.4 x 10- M PAH and 6.0 x 10- M NMN-C

(4.6 mc/mmol) and adjusted to pH 7.4.

168

169

Incubations were carried out in a Dubnoff Metabolic
Shaker at 25°C under a gas phase of 100% oxygen. Incuba-
tions were for a 90-minute period except during initial
uptake experiments. After incubation, slices were
quickly removed from the media, blotted, and weighed. A
2 m1 aliquot of the medium was taken from each beaker.
Three ml of 10% trichloroacetic acid (TCA) were added to
graduated cylinders containing tissue or medium. The
tissue was macerated with a glass stirring rod. Slice
and medium samples were brought to a final volume of
10 ml with distilled water and centrifuged at 1400 rpm
for 10 minutes. After centrifuging, 2.5 ml of the super-
natant were used to determine PAH Spectrophotometrically
as described by Smith et al. (1945). When NMN-C14 was
used to study base transport, 1 ml of slice or media
supernatant was added to scintillation vials containing
10 m1 of modified Bray's solution (6 g of 2,5-diphenyl-
oxazole and 100 g of napthalene per liter of dioxane).
Radioactivity was determined using a Beckman LS-lOO liquid
scintillation counter, employing external standardization.
All samples were counted to an accuracy level of i2.00%.

TranSport was expressed as the slice to medium (S/M)
ratio which is equal to the concentration of PAH per
gram of tissue (wet weight) divided by the concentration

of PAH per ml of media or in the case of NMN-€14,

 

170

disintegrations per minute per gram of tissue (wet
weight) divided by the disintegrations per minute per ml

of medium.

The Effect of Serum on Accumulation of PAH by Renal Cortical

 

Slices

To study the effect of serum on the accumulation of
PAH by renal cortical slices, 0.5 or 1.0 ml of serum was
added to 2.5 ml of medium prior to incubation. Blood was
obtained from animals at the time of sacrifice. This was
allowed to clot and the serum was harvested after centri-
fugation. S/M ratios obtained with the addition of serum
to the media were compared with those obtained when
saline in the same volume as serum was added. Kidneys
from control animals (GR) were incubated in serum from
GR- and from HF-fed rats. Serum from HF-fed rats were

used with renal cortical slices from GR- and HF-fed rats.

Kinetic Analysis of PAH gptake

 

Slices were incubated for 2 and 12 minutes at PAH

4 M in order to estimate

concentrations of 2,4 and 8 x 10-
the rate of PAH transport. PAH was estimated spectro-
photometrically as previously described. Uptake was
calculated as the amount of PAH accumulated per gram of
slice per minute between 2 and 12 minutes incubation.

A.Lineweaver-Burk plot (Clark, 1964) was used to plot the

results. The reciprocal of the rate of PAH uptake per

171

minute was plotted against the reciprocal of the PAH

concentration.

Oxygen Consumption of Renal Cortical Slices

 

Oxygen consumption of slices was determined using a
multiple-unit constant pressure microreSpirometer developed
by Reineke (1961). Reaction vessels were incubated in a
37° water bath and shaken at 60-100 cycles/minute. Pure
oxygen was used to gas all reaction vessels. After 30 1

minutes equilibration, the system was closed and oxygen

 

consumption recorded at 15 minute intervals for 60-75
minutes. The kidney slices were prepared from rats that
were killed by a sharp blow on the head. Kidneys were
rapidly removed and placed in iced Ringer's-Phosphate
(R-P) solution. Cortical slices 0.4-0.5 mm thick were
cut with a Stadie-Riggs microtome and placed in R-P
solution. Slices were blotted and approximately 100 mg
weighed and placed in each reaction vessel in a R-P-
200 mg % glucose solution. Triplicate samples were used
for 02 consumption studies. In addition triplicate
samples from each kidney were blotted, weighed, and
dried to constant weight at 95°C to determine dry matter
content. These samples were then ether-extracted using
:3 Goldfisch apparatus to determine fat content.

Oxygen consumption was calculated on wet weight (w)

and.dry weight (d).

172

002W and QO2D were used to express the ul of OZ/mg wet
or dry tissue/hour respectively. All values were corrected

to standard temperature and pressure (STP).

Renal Cortical Slice Composition

Slices were prepared as described previously for
accumulation studies and kept in iced saline until blotted,
weighed and placed in a drying oven for 24 hours at
100°C. Total water content of cortical slices was deter-
mined as the difference between the wet and dry weights
and tissue water was expressed as a percent of the wet
weight.

After drying, the total ether-extractable fat content
of the renal cortical slices was determined for the dried
sample using the Goldfisch apparatus. In some cases, it
was necessary to pool dried tissue samples for fat
determinations. The content of fat was expressed on a
wet or dry basis as mg per 100 mg of wet or dry tissue

weight, respectively.

Plasma Free Fatty Acids

Blood samples from nonfasted animals were obtained
‘bY heart puncture from animals fed the GR or HP ration
for 4_5 weeks postweaning. These were stored for free
fatty acid determinations at a later time by the color-

imetric method of MacKenzie at al. (1967).

 

173

Histological Study of Renal Cortical Slices

Slices were prepared as described previously for
accumulation studies. Some slices were used in a standard
90-minute accumulation study. In addition, several
slices from the same beaker were placed in Zenker's
solution (Armed Forces Institute of Pathology, 1968)
(50 g potassium dichromate, 70 g mercuric chloride, and
water to 2000 ml) for 10-12 hours, washed overnight in
cold running tap water to remove excess mercury salts
and placed in 70% ethanol in coded vials. A double—blind
study was made to determine if histological differences
between renal cortical slices from GR and HF-fed rats
could be demonstrated. Kidneys were imbedded in paraffin
and sections 2 u thick prepared. Hemotoxylin and eosin
and PAS stains were used (Armed Forces Institute of

PatholOQY, 1968; Lillie, 1965).

Statistical Analyses

All data were analyzed statistically using Student's
"t" test, paired or group comparison unless otherwise
rusted (Steel and Torrie, 1960). For those studies
involving the effect of different PAH concentrations on
'the velocity of uptake, a kinetic analysis employing a
15ineweaver-Burk plot (Clark, 1964) was used. Linear
:negression analysis (method of least squares, Goldstein,
11964) was used in determining the effect of age, body

vneight, and kidney weight on PAH and NMN accumulation and

174

the effect of body weight on kidney weight. In all
statistical tests, the 0.05 level of probability was

used as the criterion of significance.

‘PWcj‘Tia—u _" “‘fi 1 7: WV

RESULTS

Accumulation of the Organic Acid, PAH, by Renal Cortical
Slices
Organic acid tranSport by renal cortical Slices was

depressed in HF animals (Figure 16). When kidney cortical

slices from these animals, at different ages, were

incubated for 90 minutes in the Cross and Taggart medium,

a PAH S/M ratio of 4.81:0.29 (mean i S.E.) developed

(Figure 16). Animals of comparable ages fed the GR diet
had kidneys which developed PAH S/M ratios of 6.96:0.41
under the same incubation conditions (Figure 16). These
values are significantly different from those obtained

from HF animals (P<0.001).

Factors Affecting the Accumulation of PAH by Renal

Cortical Slices

 

The ability of renal cortical slices to accumulate
PAH was inversely correlated with age (Figure l7),body
‘weight (Figure IUD,and kidney weight (Figure 19). As
either of these variables increased, the transport of
PAH decreased. This was true for both groups of rats fed
either HF or GR. The inverse relationships were signifi-
cant.(P<.Ol) with GR, HF, or all data pooled.

Kidney weight was positively correlated with body

vweight (Figure 20),independently of the diet (P<.Ol).

175

176

Rate of Initial Uptake of PAH

The rate of PAH uptake by renal cortical slices from
lZ-week old GR rats was greater than that of slices from
HP rats of the same age (Figure 21). The results are
plotted on a double reciprocal plot (Lineweaver—Burk plot)
and are expressed in Figure 21. The lepes of lines for
GR and HF animals were 0.391 and 0.360 respectively. The
plot for the GR group exhibited a VMax of 39.97 (ug/g/min)
while the VMax for the HF group was 14.92 (Table 13). Km
values for GR and HF animals were 15.62 and 5.37 (10"4
moles/L) respectively (Table 13).

The rate of PAH uptake by renal cortical slices from
60-week old GR rats was greater than that for Slices from
HF rats of the same age (Figure 22). The results are
again plotted on a double reciprocal plot. The lepes of
the lines for GR and HF animals were 0.490 and 0.318
respectively. The plot for the GR group exhibited a
VMax of 12.45 (pg/g/min) while the VMax for the HF group

was 4.63 (Table 13). Km values for GR and HF animals

were 6.10 and 1.47 (10"4 moles/L), reSpectively (Table 13).

Accumulation of the Organic Base, NMN, by Renal Cortical
Slices

To study the specificity of the inhibition of the
anionic transport system, the accumulation of the organic
base, NMN, was determined. In some cases, simultaneous

accumulation of PAH and NMN was measured.

177

The accumulation of NMN by renal cortical slices from
HF animals was no different from that of GR animals
(Figure 23). Slices from animals fed GR incubated under
the conditions as described previously (90 minutes, Cross
and Taggart medium, 100% oxygen) deve10ped NMN S/M ratios

of 6.75:0.33 (Figure 23). This was not significantly

‘ —L A"!

different (P>.05) from the NMN S/M ratios (6.55:0.40)
obtained when slices from HF animals were incubated

similarly.

 

 

Factors Affecting the Accumulation of NMN by Renal Cortical
Slices

The accumulation of NMN by renal cortical slices
was significantly correlated with age (P<0.05) (Figure 24)
and kidney weight (P<0.01) (Figure 25) in the HF group,
but not in the GR group. When all data were pooled
regardless of diet, NMN accumulation was significantly
correlated with age (P<.05) (Figure 24) and kidney weight
(P<.01) (Figure 25). NMN accumulation was not correlated

with body weight (Figure 26) for any group (P>.05).

Effect of Changing Diets on Accumulation of Organic Acid

and Base

 

Since the previous results suggested that accumulation
of PAH by renal cortical slices was depressed in animals
fed HF when compared to those on GR, the effect of the
diet per 32 was investigated. A summary of the effect of

diet.switching on PAH accumulation by kidney cortical

178

slices and its comparison with the standard dietary
regimens are given in Table 10. Animals fed HF for any
period of time had depressed accumulation of PAH when
compared to animals fed GR for any period of time
(Figure 16,Figure 27, Table 10). Animals fed GR and
switched to HF at least two days prior to sacrificing I
had renal cortical slices which accumulated PAH as if
the animals had been on the HF ration throughout the ;

experiment (Figure 27, Table 10). The accumulation of

 

.9... ,. .

PAH by renal cortical slices from HF animals was signifi—
cantly less than that by slices from GR animals
(Figure 16,Figure 27).

When animals were fed HF and switched to GR at least
two days prior to being sacrificed, kidney slices from
these animals developed PAH S/M ratios not significantly
different (P>.05) from those obtained with kidneys from
animals fed GR continuously (Figure 16,Figure 27). The
accumulation of PAH by kidney slices from animals fed GR
or HF and switched to GR.was significantly greater than
that.by kidney cortical Slices from animals fed HF or GR
and.switched to HF (Figure 16,Figure 27, Table 10).

NMN accumulation by renal cortical slices was
lnmaffected by diet switching (Figure 28, Table 11). The
«effect of diet switching on the accumulation of organic
base by renal cortical slices as measured by NMN S/M
:natios is summarized in Table ll.‘When animals were fed

(SR and switched to HF, accumulation of NMN was not

179

significantly different (P>.05) from that by renal corti-

cal slices from animals fed HF and switched to GR

(Figure 28, Table 11).

Effect of Serum on Accumulation of PAH by Renal Cortical
Slices

Since the previous results indicated that the
depressed accumulation of PAH by renal cortical slices was
due in large part to the diet, the effect of serum on
accumulation of PAH was determined. To test this, kidney
slices from animals fed GR or HF were incubated with the
normal media plus serum from GR or HF animals (Table 12).
Accumulation of PAH by renal cortical slices incubated
with media plus saline was used as a control against
dilution effects.

In all cases, the accumulation of PAH by renal
cortical slices was enhanced by the addition of serum
independent of the source of serum or kidneys (Table 12).
.Accumulation was unaffected by the addition of an equal
volume of saline (Table 12). The PAH S/M ratios deve10ped
.by renal cortical slices from HF or GR.animals incubated
in HF'serum were not different (P>0.05). Those developed
by renal cortical slices from HF or GR animals with the
addition of GR serum were not different (P>0.05) .
Ihxrthermore, there were no differences in the PAH S/M

IHICiOS deve10ped when HF serum was used when compared

with GR serum (P>0.05) .

 

180

The S/M ratios obtained with all serum-kidney com-
binations were significantly greater (P<0.05) than those
with the dilution control (2.5 ml medium + 0.5 m1 saline)
or with the control medium (2.7 m1). There were no
significant differences in the PAH S/M ratios obtained in
2.5 ml medium plus 0.5 ml saline when compared with 2.7

ml medium (P>0.05).

Histological Study of Renal Cortical Slices

The histological sections revealed kidneys in both
groups that were normal for rats of the age used (30
weeks old) (Figure 29, Figure 30). No apparent morpho-
logical differences were distinguishable between kidney
slices from HF and GR animals in histological sections by
light microsc0py (Figure 29, Figure 30). In both groups,
tubules were normal except for slight dilation. Infre-
quent casts were present in kidneys from both groups. No
evidence of kidney infection or inflammation was seen.
Glomerular changes observed in kidneys from both dietary
groups included focal hypercellularity, increased

Inesangial tissue, and limited metaplasia.

Kinetic Analysis of PAH Uptake
The rate of PAH uptake by renal cortical slices from
:12 and 60 week old animals on GR and HF was determined.
PAH uptake by renal cortical slices from HF animals

xmas less than that from GR animals independent of age

181

(Figure 21, Figure 22, Table 13). Kidneys from young
animals on either diet had greater uptakes than those
of older animals (Figure 21, Figure 22, Table 13). A

summary of the kinetic analysis is given in Table 13.

Oxygen Consumption by Renal Cortical Slices

The oxygen consumption by renal cortical slices from
kidneys of rats fed GR and HF is given in Table 16. There
were no significant differences in the oxygen consumption

on a dry or a wet weight basis (P>0.05).

Renal Cortical Slice Composition

 

The fat and water composition of renal cortical slices
from kidneys of rats fed GR and HF is given in Table 17.
There were no significant differences in the percent of

water and total ether-extractable fat of the slices from

the two groups (P>0.05).

 

 

‘4'...—

182

Figure 16. Accumulation of PAH (S/M ratio) in renal cortical

slices from male rats of different ages fed the
control grain ration (GR) or the experimental
high fat ration (HF). Each bar represents the
mean i (S.E.) obtained from duplicate deter-
minations on the number of animals shown in
parentheses. The Value obtained from the HF
group is significantly less than that of the

GR group (P<0.001).

 

IO

O~

PAH S/M RATIO
3:

I?
21

 

 

C)
:0

DIET

Figure 16

 

 

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196

Accumulation of NMN (S/M ratio) in renal
cortical slices from male rats Of different
ages fed GR or HF. Each bar represents

the mean : (S.E.) Obtained with duplicate
determinations on the number Of animals in
parentheses. The values Obtained are not
significantly different (P>0.05).

197

10

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DIET

Figure 23

 

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Figure 27.

204

Accumulation Of PAH (S/M ratio) in renal
cortical slices from male rats fed GR and
switched to HF or fed HF and switched to GR.
Each bar represents the mean i (S.E.)
obtained from duplicate determinations on
the number of animals in parentheses. These
values are significantly different from each
other (P<0.05).

 

 

 

205

10

 

 

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a»

0
an
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23

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DIET

Figure 27

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Figure 28.

206

Accumulation Of NMN (S/M ratio) in renal
cortical slices from male rats fed GR and
switched to HP or fed HF and switched to GR.
Each bar represents the mean i (S.E.)
obtained from duplicate determinations on the
number of animals in parentheses. These
values are not significantly different from

each other (P>0.05).

 

 

207

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8 6 4 2 0

0:5. 2} 222

HF-vGR

GR-- HF

DIET

Figure 28

Figure 29.

208

Histological sections of kidneys from HF-fed
rats.

A&B: Kidneys from typical HF rats 30 weeks of
age. Tubules are slightly dilated and lumens
contain some proteinaceous debris. Glomeruli
have normal basement membranes; Bowman's
spaces are empty. Occasional glomeruli Show
metaplasia.

Measured PAH S/M Ratio: A - 3.75; B - 3.77.
PAS stain, X256.

 

209

   

Figure 29

 

 

210

Figure 30. Histological sections of kidneys from GR-fed
rats.

A&B: Kidneys from typical GR animals 30 weeks
of age. Tubules are slightly dilated and
contain some debris. Glomeruli have
normal basement membranes and empty
Bowman's spaces. Occasional glomeruli
exhibit metaplasia, focal hypercellularity,
and increased mesangial tissue.

Measured PAH S/M Ratio: A - 7.39; B - 8.51.
PAS stain, X256.

211

   

, Figure 30

212

Table 10. Effect Of diet on the accumulation (S/M ratio)a
Of PAH by slices Of rat renal cortex.

 

 

Dietb NumberC PAH S/M Ratiod
GR 23 6.96:0.41e
HF 18 4.81:0.29e
GR+HF 9 4.63:0.30
HF+GR 5 6.48:0.73

 

aRatios were determined after incubation at 90 minutes, at
25°C under 100% oxygen atmosphere.

bDiets used were Grain Ration (GR) and High Fat Ration

(HF). GR and HF were fed throughout the study. GRfiHF
indicates the animal was fed GR and switched to HF prior
to assay. HF+GR animals were fed HF and switched to GR
prior to determining PAH accumulation. HF+GR and GReHF
animals were on the first diet from 1 tO 45 weeks and on
the second diet from 2 days to 3 weeks. PAH accumulation
was unaffected by the length Of time the first and second
diets were fed; therefore, all values are pooled.

C O O I 0
Total number of animals on any regimen lrreSpectlve Of
age.

d . . . .
For each animal dupllcate determinations were made.
These values were averaged and used to compute means
and standard errors.

eSignificantly different from GR (P<0.001).

 

-L

erg-altruism

213

Table 11. Effect Of diet on the accumulation (S/M ratio)a
Of NMN by slices Of rat renal cortex.

 

 

Dietb NumberC NMN S/M Ratiod
GR 19 6.75:0.33
HF 15 6.55:0.40
GR+HF 7 7.03:0.34
HF+GR 3 8.36:0.65

 

aRatios were determined after incubation for 90 minutes at
25°C under 100% oxygen atmosphere.

bThe Grain Ration (GR) and High Fat Ration (HF) were fed

throughout the study. GRfiHF indicates the animal was fed
GR and switched to HF prior to assay. HF+GR animals were
fed HF and switched to GR prior to determining NMN accumu-
lation. HF+GR and GRfiHF animals were on the first diet
from 1 to 45 weeks and on the second diet from 2 days to

3 weeks. NMN accumulation was unaffected by the length

Of time the first and second diets were fed; therefore,
all values were pooled.

c . . . .
Total number Of animals on any regimen irrespective Of
age.

dFrom each animal duplicate determinations were made.

These values were averaged and used to compute means and
standard errors.

 

214

Table 12. Accumulation of PAH (S/M ratio)a by slices of
rat renal cortex in the presence of serum.

 

 

 

Incubatéon Serum Kidney Numberd PAH S/M
Medium Source Source Ratio
fr.
2.5 ml medium HFC GRC 5 15.38:0.Zl: r
+ 0.5 ml serum HF HF 4 13.45il.27e
GR GR 3 l4.73:0.02e .
GR HF 3 15.0811.65
2.5 ml medium -- HF 2 4.25:0.02e
+ 0.5 ml saline -- GR 2 7.28:0.25
2.7 ml medium -- HF 18 4.81:0.29e L
-- GR 23 6.96:0.41

 

aRatios were determined after incubation for 90 minutes at
25°C under 100% oxygen atmosphere.

bCross and Taggart (1950) medium with PAH concentration of
7.4 x 10"5 M was used.

CDiets used were Grain Ration (GR) and High Fat Ration
(HF) .

d . . .
The number Of experiments each representing one animal as

a serum source and another as kidney source.

eSignificantly different from control (GR kidneys in
2.5 ml medium + 0.5 ml saline) and from GR kidneys in
2.7 ml standard media (P<0.05).

215

Table 13. Kinetic analysis Of PAH uptake in rat renal
cortical slicesa.

 

 

 

 

Dietb Age SlopeC Km c Vm max
(wks.) (10"4 moles/L) (ug/g/min)C
GR 12 .391 15.62 39.92
HF 12 .360 5.37 14.92
GR 60 .490 6.10 12.45
HF 60 .318 1.47 4.63
a

Rate Of PAH uptake (pg PAH/g kidney slice/min) at PAH
concentrations Of 2, 4 and x 10 M was determined by
measuring the difference in 8accumulation after 2 and 12
minutes of incubation. Triplicate determinations Of
uptake for each diet and age were made.

bDiets used were Grain Ration (GR) and High Fat (HF) and

were fed throughout the study.

CValues were Obtained from the Lineweaver-Burk plot shown
in Figures and .

216

Table 14. Effect of diet on pH Of 24-hour urine samples
from lO-week old male rats.

 

 

Dieta Number 1 2 3

0—24 hrs 72-96 hrs 96-120 hrs
GR+GR 9 8.22:0.42 8.08:0.37c 8.09:0.38C
GR+HF 9 8.13:0.39 6.64:0.54 6.39iO.45

 

aDiets used were Grain Ration (GR) and High Fat Ration
(HF).

GR+GR indicates GR was fed throughout the study.

GRfiHF indicates GR was fed 0-24 hrs and HF afterwards.

bThe time the initial urine collection was started was

designated as 0 hr. Subsequent collections were timed
accordingly.

CValues are significantly different (P<0.05) from those
Obtained from all animals in period 1 (0-24 hrs).

. -—r' "1!
lean

 

217

Table 15. Effect of giet on the pH Of the medium after
incubation Of renal cortical slices

 

 

 

 

91.-
. b
Diet Number pH g
GR 4 7.41:.01 :
HF 4 7.36:.01C
L2,.

aIncubations were for 90 minutes at 25° under 100% oxygen
atmOSphere. Immediately after removing the renal cortical
slices, the pH Of the medium was determined.

bDiets used were Grain Ration (GR) and High Fat Ration
(HF) and were fed throughout the study.

cSignificantly different from the control (GR) value
(P<0.05).

218

Table 16. Oxygen consumption Of renal cortical slices
from rats fed the grain ration or high fat

 

 

ration.
Diet Number ul/hr/mg tissue ul/hr/mg
(wet) (dry)
GRa 4 2.20:0.24 12.84iO.99
HF 4 2.52i0.09 11.97i1.32

 

Diets used were Grain Ration (GR) and High Fat Ration
(HF).

Fe
1;

 

‘HZL

219

Table 1?. Approximate composition Of renal cortical
slices from rats fed the grain ration or high
fat ration.

 

 

Diet Number % Moisture % Fat

(Dry Wgt Basis)
GRa 9 86.56i0.69 8.28:0.60
HF 7 88.46i1.32 9.85i2.33

 

 

aDiets used were the Grain Ration (GR) and the High Fat
Ration (HF).

DISCUSSION

Osborne-Mendel rats fed a high fat diet become grossly
Obese as determined by increased body weight or body fat
(Schemmel et al., 1969). They are useful as an experi-
mental model for investigating the physiological effects
of obesity which is a problem Of major proportion in the
United States. Armstrong et al. (1951), employing the
tables of the Metropolitan Life Insurance Company, esti-
mated about 15 million persons in the country were 10%
overweight and at least 5 million are 20% or more above
normal weight. Obesity has been called the most common
nutritional disorder in the United States by Braunstein
(1971), who estimates that 30% Of the adult pOpulation is
greater than 20% overweight. Recent reports linked Obesity
with an increased incidence or severity Of various cardio—
vascular, skeletal, metabolic and organ abnormalities
(Mayer, 1968; Armstrong et al., 1951). The association
Of abnormal kidney function in obese humans was reported
by several workers (Bittnerova et al., 1968; Ross, 1960;
Mayer, 1968).

An association Of kidney damage with increased body
weight was reported in hypothalamically Obese rats
(Brobeck et al., 1943; Stevenson et al., 1950 and Kennedy,
1957), in rats with unrestricted feed intakes (Berg and
Simms, 1960; Bras and Ross, 1964) and in genetically Obese

rats (Zucker, 1965). Studies in our laboratory, reported

220

 

221

in this thesis, suggest that there are alterations in a
number of kidney functions measured in viva and as seen
in renal histology.

Renal transport of organic acids is an important
homeostatic mechanism as it is a means Of excretion Of
potentially toxic by-products Of metabolism (Pitts, 1968).
Selleck and Cohen (1965) suggested that the primary
function Of the organic acid transport system is to move
specific products Of intermediary metabolism (nonesterified

fatty acids, a-ketoglutarate, citrate, etc.) to sites Of

 

dissimilation in the kidney and liver. According to
Goldberg et al. (1970), the organic acids (other than
amino acids) in the urine are primarily derived from
bacterial metabolism in the gastrointestinal tract.
These include hippuric acids (Asatoor, 1965) and indolic
compounds (Milne at al., 1960). Endogenous production
from phenylalanine accounts for a small portion of the
hippuric acid in the urine (Armstrong et al., 1955); how-
ever, the major portion is derived from dietary
precursors (Armstrong et al., 1955). The intestinal
bacterial flora play an important role in the production
Of hippuric acid from dietary precursors (as benzoic
acid and sodium benzoate) (Asatoor, 1965).

Organic acid tranSport is a specific function Of the
renal proximal tubule. This is in contrast to the renal
handling of sodium which involves the entire length Of

the nephron. Sodium reabsorption is a general function

222

of the nephron and as such requires considerable expendi-
ture Of metabolic energy. Inasmuch as transport of

organic acids is limited to the proximal tubule, it probably
requires Significantly less energy than sodium handling.
Normally the sodium reabsorption mechanism is not Operating
at full capacity and is not challenged by the filtered ,gfi
sodium load. Thus a small decrement in energy availa- h
bility might not affect overall sodium transport. In

these experiments, it was possible to challenge the organic

 

 

acid transport system by measuring uptake in the steady 1;
state. The rationale behind this was that when so
challenged, subtle changes in this function might be Ob-
served prior tO the appearance Of physiological or
biochemical lesions Of the renal parenchyma. TranSport
of the organic acid, PAH, in vitra was determined in a
Cross and Taggart incubation System. Thin renal cortical
slices in a salt buffered, PAH-containing medium were
incubated for 90 minutes under 100% oxygen at 25°C.
Accumulation of PAH was expressed as the final slice
concentration/medium concentration ratio (S/M ratio).

The accumulation Of PAH (S/M ratio) was significantly
depressed in HF animals when compared to GR animals
(Figure 16). This was in agreement with PSP excretion
in viva in these animals. PAH transport was inversely
correlated with age (Figure l7),body weight (Figure 18)

and kidney weight (Figure l9)for both diets. Animals fed

 

223

HF were significantly heavier and had a higher percentage
Of body fat than GR animals Of the same age (Schemmel

et al., 1969). Kidney weights were positively correlated
with body weight in all animals (Figure 20). The
depression Of PAH accumulation by renal cortical slices
with age confirms work Of Barrows and co-workers (Adams
and Barrows, 1963; Beauchene at al., 1965). NO reports
regarding the effect of body weight on PAH transport were
available in the literature. Numerous workers previously
reported an association of a kidney weight with body
weight (Hatai, 1913; Webster et al., 1947; Widdowson and
McCance, 1960).

Since PAH transport was depressed in the kidney
slices from the HF animals, it became Of interest tO
determine whether other tubular transport mechanisms were
similarly altered.

Parallel systems for transport Of organic acids and
bases are located in the proximal tubules. The importance
Of the organic base transport system is less apparent,
although a number Of urine and plasma constituents are
secreted by this system (Peters, 1960). Thiamine,
choline, NMN (a nicotonic acid metabolite), guanidine,
piperidine and methyl guanidine are naturally occurring
compounds tranSported by the organic base system (Peters,

1960). This system might exist to secrete some unknown

 

224

substance(s), which because of high toxicity, must be main-
tained at very low plasma concentrations (Pitts, 1968).

The Specificity Of the depression of transport of
organic acids in the HF group was determined using NMN
as a prototype to study base transport. Workers have

shown that the accumulation of PAH by renal cortical

r:
slices in the Cross and Taggart incubation system may be r,
specifically depressed by a number Of factors without any
change in NMN tranSport (Hook and Munro, 1968; Hirsch and
Hook, 1969b). Accumulation Of NMN by renal cortical hJ

 

slices from animals fed GR and HF was not different. NMN
transport was inversely related to age (Figure 24) and
kidney weight (Figure 25) in the HF group. Body weight,
however, was not significantly related tO base transport
(Figure 26). NO explanation for the differences Observed
between the effect Of age, body weight and kidney weight
on transport of PAH and NMN in the animals is known.
Other workers have suggested that the NMN tranSport system
is more resistant to manipulation than the organic acid
tranSport system (Hirsch, 1970; Bowman, 1970). These
results suggested that the diet per se affects the
accumulation of PAH by rat renal cortical slices.

When diets were switched, accumulation Of PAH by renal
cortical slices from animals fed HF for any period of time
and switched to GR for any period Of time just prior to

sacrifice (Figure 27, Table 10) was similar to that by

225

kidneys from animals fed GR throughout (Table 10).
Animals fed GR and switched to HF for any period Of time
had kidneys in which transport of PAH was significantly
less than that for animals fed GR prior to sacrifice.
Thus diet per se appeared to be a primary factor
influencing the final S/M ratio, although age, body FT
weight and kidney weight influenced the accumulation Of :3
PAH by renal cortical slices. These Observations suggested E

that the effect Of diet on the accumulation of PAH was

 

easily reversible and the time required for this change ii
was less than 2 days.

Since the effect of diet appeared readily reversible,
it was reasoned that a dietary metabolite in the serum
might be directly affecting the accumulation of organic
acid in the in vitra system. However, when serum was
incubated with the kidney slices, PAH accumulation was
enhanced. Serum from HF animals stimulated the accumula-
tion Of PAH by kidney slices from both GR and HF animals
(Table 12) Similarly, GR serum added to the incubation
media significantly increased the accumulation Of PAH
by kidney slices from both groups (Table 12). These
results confirm the effect of normal serum in enhancing
PAH accumulation by renal cortical slices reported by
Orringer at al. (1971).

Thus, if some factor Of dietary origin in the serum

of the HF animals is depressing PAH accumulation by renal

226

cortical slices, its concentration is insufficient to
produce an inhibitory effect when added to the incubation
system. Apparently, the factor(s) responsible for the
enhancement of PAH accumulation is in adequate concentra-
tion to overcome any inhibitor that might be present in

the HF serum. This is in contrast tO reports that

"uremic" serum (White, 1966; Preuss et al., 1966) or serum
from nephrectomized animals (Hook and Munro, 1968), when
added to this incubation system, depressed the accumulation
Of PAH by renal cortical slices.

The depressed PAH accumulation Of kidney slices from
HF rats could be increased to a level equal to that Of
slices from GR rats by the addition Of the serum. This
suggests that there is no difference in the inherent
functional capacities Of the kidneys from the Obese and
control rats for PAH transport. Since differences in
other kidney functions are reported in HF animals, this
is a significant result.

The factor(s) in the serum enhancing the transport
has not been elucidated. Conceivably, the effect could
be mediated by an increased or preferential energy source
(glucose, acetate, etc.) and/or a more favorable electro-
lyte balance. In addition, the protein in the serum
could possibly aid in maintaining the cellular integrity
of the Slice during the incubation period.

The depressed PAH accumulation in kidneys from HF

animals could result from a number Of contributing factors.

A "‘1'!

1“ ""

 

227

These include differences in entry, accumulation and/or
runout Of PAH, in non—specific binding Of PAH tO protein,
and in extracellular water, intracellular water or
protein content of the renal cortical slice.

The renal transport system as studied is not a pure
system and renal organic acid transport probably does not
follow true Michales-Menten (M-M) kinetics. Nevertheless,
the Lineweaver-Burk plot remains one Of the most useful
tools available to study these transport kinetics
(Farah et al., 1959; Nagwekar and Unnikrishnan, 1971;
Orringer at al., 1971). An analysis Of the inhibition Of
the tranSport system using a Lineweaver-Burk plot is
useful in that its findings are consistent with results
obtained from the intact animal. Also, it aids in
predicting how a new inhibitor may function.

The S/M ratio reported in this study was measured in
a steady state system and thus represents binding and
runout capacity Of the renal tissue as well as transport
capacity. Therefore, the S/M ratio is a measure of the
ability Of the tissue to maintain a concentration
gradient. During short periods Of incubation (as between
2-12 minutes) uptake of PAH is linear, suggesting that
intracellular accumulation Of the compound is not
sufficiently high to alter the rate of influx. The uptake
over short periods of time indicates the rate Of tranSport

of a material into the tissue, according to Ross et al.

T3.

 

RF..-

228

(1968b). In the Lineweaver-Burk plot, the x-intercept
(l/Km) could reflect the relative affinity Of a carrier

substance for PAH and the y-intercept (l/Vh , the maximal

ax)
velocity Of the PAH accumulation process. On the basis

Of these assumptions the data presented in Figure 17 and
Tab1e13.suggest that the apparent affinity and maximal
velocity are both different when values for Older GR
animals are compared with older HF animals. These changes
in kinetics suggest non-competitive inhibition. Com-
petitive inhibition was suggested when PAH S/M ratios

were depressed after adding fasting serum to the incuba-
tion media (Hook and Munro, 1968). Another interpretation
and probably the more likely is that the inhibitor-
carrier interaction is nonreversible or slowly reversible
and that M-M kinetics are not applicable. This inhibitor
may be in such low concentrations that no effect is seen
in acute studies. Apparently the inhibitor was not
released even after the renal cortical Slices remained in
saline or the Cross and Taggart medium for two hours.

In the intact animal, an inhibitor could accumulate and
eventually impede the transport system. Such a possibility
was suggested by Balagura-Baruch and Stone (1969) in dOgs.
These workers reported that a-ketoglutarate inhibited

PAH secretion by a noncompetitive or mixed mechanism.

The possibility that the acidity of the urine could

influence PAH accumulation was also considered. This was

 

229

based on the fact that when the kidneys were removed from
the animals, they contained small amounts of urine, the
composition Of which was similar to that examined for
acidity. Several workers have reported the susceptibility
Of the acid tranSport system to the pH Of the incubation
medium (Bowman, 1970; Forster and Copenhaver, 1956). A
group of lO-week Old animals fed GR for 7 weeks had 24
hour urinary samples with a pH of 8.18:0.37 (Table 18).
Nine of the original group after switching to HF for one
day had urine samples for the 24 hour collection with a
pH Of 6.64:0.54 (Table 18). Recognizing that diets of
different composition may produce acid, neutral or
alkaline ash, and that urinary pH is not as valid a
measure Of H+ secretion as are others (i.e., titratable
acidity), the contribution Of urinary pH to the depression
Of PAH accumulation in kidneys from HF animals was
doubtful. Also, despite these differences in urine pH

Of the animals fed the different rations, it is doubtful
whether these are responsible for the Observed changes

in the S/M when the rations were switched. Furthermore,
if tubular urinary pH is important, perhaps the pH of

the incubation media is different after incubating HF
slices compared to GR. The pH Of the medium immediately
after 90 minutes incubation was measured and found to be
significantly different (Table 19). However, the

physiOlOgical significance Of this difference in pH is

230

questionable. Copenhaver and Davis (1965) reported the
accumulation of PAH by rat renal slices was not different
between pH 7.3 and 7.4.

Differences in accumulation of PAH by renal cortical
slices from HF and GR rats was not reflected in changes
in oxygen consumption (Table 20). This is in agreement
with a number Of reports (Taggart at al., 1953; Burg and
Orloff, 1962a; COpenhaver and Davis, 1965; Bourke et al.,
1967; Chung at al., 1970). These results are in contrast
to those reported by Huang and McIntosh (1955).

The fat and moisture percentage compositional changes
were not significantly different in the two groups
(Table 21) .

NO apparent morphological differences were Observed
in renal cortical sections from HF and GR animals by
light microscopy (Figure 29, Figure 30). Although renal
cortical slices from GR animals Obtained PAH S/M ratios
which were significantly greater than those developed by
Slices from HF animals, the slices could not be distin-
guished histologically (Figure 29, Figure 30). Similarly,
Hirsch et al. (1971) Observed no histological differences
between kidneys from penicillin-treated and control rabbits
although penicillin treatment resulted in a significant
increase in PAH accumulation. There may, however, be pre-
cise differences in ultrastructure or enzyme activity which
could be determined by electron microscopy or histo-

chemically.

SPECULATION

Intuitively one would consider that the high fat content

of the diet Of the obese animals was the factor responsible
for the depression Of PAH accumulation by rat renal
cortical slices. Before such an assumption is accepted,

it should be recognized that the HF and GR rations differ
in other reSpectS. The HF ration is composed primarily

Of purified ingredients and a hydrogenated vegetable fat,
whereas the GR ration contains primarily corn and soybean
meal.

Discrete subtle differences in the diets and their
effects on accumulation of PAH as determined in these
experiments must be considered before the dietary
component(s) or metabolite(s) contributing to depression
of PAH tranSport can be determined. This could be of
special significance if in fact the component(s) is
irreversibly binding the sites involved in PAH transport.
One Of the problems in attempts to isolate the PAH carrier
system has been a lack Of a specific inhibitor which
irreversibly blocks PAH transport (as dibenamine blocks
NMN tranSport). Characterization of the blocker in the
HF diet could conceivably provide a compound useful in
the isolation of the carrier system.

Nonesterified fatty acids (NEFA) from the HF diet
could also be affecting accumulation of PAH in renal

cortical slices in this incubation system. Inhibitors of

231

232

organic acid transport, probenicid (Huang and Lin, 1965)
and Chlorothiazide (Beyer and Baer, 1961), also inhibit
net renal NEFA utilization (Barac-Nieto and Cohen, 1968).
Cohen (1964) reported that a-ketoglutarate, a inhibitor
of organic acid transport (Cross and Taggart, 1950)
inhibits net renal NEFA uptake. NEFA are bound tO
proteins (Goodman, 1958a), to cell membranes, (Goodman,
1958b), and to intracellular particles (Reshef, 1966).
Conceivably, NEFA could be an inhibitor in this study.
This should be further investigated.

The factor(s) in the serum which enhance the accumu-
lation Of PAH by renal cortical slices are unknown. The
serum contains energy sources, acetate ions, and electro-
lytes known to be involved in the transport process or
to stimulate it. Also, whether the addition Of serum or
metabolites (as ATP) would overcome the depressed PAH
accumulation in slices from Older animals or anoxic

Slices is not known.

SUMMARY

The effect Of Obesity and a high fat diet (HF) on
the accumulation Of PAH by renal cortical slices was
determined using the in vitra slice technique Of Cross
and Taggart (1950). The accumulation Of PAH was
significantly depressed in animals fed the HF diet used to
produce Obesity. Accumulation of PAH decreased with
increasing age, body weight and kidney weight independent
Of diet. Similarly, kidney weight was Significantly
correlated with body weight in HF and GR animals.

The rate Of PAH uptake was determined and analyzed
kinetically using a Lineweaver-Burk plot. GR anbmals
at 12 weeks Of age exhibited a higher Ymax and Km indica-
tive Of greater velocity and apparent affinity. In Older

GR animals (60 weeks), the V5 and Km were greater than

ax
that of the younger and older HF animals, but less than
that Of the young GR animals.

Organic base accumulation was determined in order to
demonstrate the specificity Of the effect Of the HF diet
and Obesity on organic acid transport. Accumulation Of
NMN was not different in GR and HF animals. There was no
correlation Of NMN accumulation with body weight. In HF
animals NMN accumulation by renal cortical slices decreased
with increased age and kidney weight. Age and kidney

weight in the GR animals were not related to NMN accumula-

tion.

233

234

Exchanging the diets for a few days or a few weeks
affected the transport Of PAH, but not NMN transport.
Animals fed GR for any period Of time immediately prior
tO sacrifice had kidneys which accumulated PAH signifi-
cantly more than those fed HF just prior to sacrifice.

Addition Of HF or GR serum to the incubation medium
enhanced PAH accumulation by kidney cortical slices from
GR or HF animals. The differences Observed in PAH
transport in the HF animals did not result in differences
in oxygen consumption by renal cortical slices. Histo-
logically HF kidney slices were not different from GR
kidney slices although PAH S/M ratios were significantly
different. Although composition of slices from GR and HF
animals were slightly different, the extent that these
differences would affect the PAH transport system is not
known.

These data suggest that PAH tranSport is depressed in
animals with increased age, body weight and kidney weight.
Furthermore, PAH transport may be depressed by dietary
manipulation, feeding a 60% fat diet. These data
emphasize the importance Of carefully considering the
diet, age, and body weight in studies of renal function

in rats.

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131:447-458.

APPENDICES

APPENDIX A

Grain Ration (Campbell et al., 1966)

 

Component

Amount/100 gm.

 

ground corn
soybean meal
alfalfa meal
fish meal
dried whey
limestone

dicalcium phOSphatel

iodized sal
mineral mix
vitamin mix3
penicillin
streptomycin
arsenilic acid
vitamin A
vitamin D2

60.70
28.00
2.00
2.50
2.50
1.60
1.75
0.50
.116
.127
0.20
0.80
96.80
801 I.U.
75 I.U.

gisaaaaaaiaai

 

lFeed grade CaHPO4-
22-25% Ca.

2H 0, proximate analysis 18.5% P,

2

2 . . . .
Percentage compOSltion Of mineral mix:

FeSO4 7H , 40.83;
2.45; Coélz-enzo,

CaCO
0.90;

I
3K1

15.72;

' 0.43.

ZnCO3,

3Percentage composition Of vitamin mix:
55.13; calcium pantothenate, 0.43; riboflavin, 0.26;
(0.1% mannitol triturition),

niacin, 2.60; vitamin B

0.52; a-tocOpherOl

acet

lie

MnSO4°H20, 32.06;

7.62; CuSOq'SHZO,

choline chloride,

(250 I.U.P. per gm.), 1.53;

menadione, 0.17; DL methionine,

244

39.36.

APPENDIX B

High Fat Ration (Mickelsen et al., 1955;
Schemmel et al., 1969a)

 

 

 

Component Amount/100 gm.

ea
shortening (Crisco) 60.00 gm. :1
casein 25.00 gm. '
non-nutritive fiber 2.00 gm.
aureomycin 0.01 gm. T
liver mix 2.00 gm. .
DL methionine 0.25 gm.
sucrose 3.54 gm. 1
mineral mix 5.00 gm. 9
vitamin mix 2.20 gm. T

 

1Percentage composition of mineral mix: CaCO 21.0;
Ca (p04 ), 14.9; CuSO 5H 20, 0. 039; FePO 4H ”8 l. 47;

MgSO 04, 9. 00; MnSO 8.0 K 2A1 (504) 24H 20, 0. 009;
KCl, 12.0; KI, 0. 805; KH ”9 31. §a01,210. 5; NaF,
0. 057.

2 . . . . . . .
Percentage compOSltion Of Vitamin mix: Vitamin A
concentrate (200,000 U.S.P./gm.), 0.45; vitamin D con-
centrate (400,000 U.S.P./gm.), 0.025; a-tOpocerhOl,
0.50; ascorbic acid, 4.50; i-inositol, 0.50; choline
chloride, 7.50; menadione, 0.225; para-amino benzoic
acid, 0.50; niacin, 0.45; riboflavin, 0.10; pyridoxine
HCl, 0.10; calcium pantothenate, 0.30; biotin, 0.002;
folic acid, 0.009; vitamin B, 0.0135; sucrose, 84.75.

245

APPENDIX C

Modified Grain Ration (Smith, 1969)

 

. 15% II p"

 

 

Component Amount/100 gm.
ground corn 61.6 gm.
soybean meal 28.4 gm.
alfalfa meal 2.0 gm.
fish meal 2.6 gm.
dried whey 2.6 gm.
limestone l 0.17 gm.
dicalcium phOSphate 1.75 gm.
iodized salt 0.5 gm.
mineral mix2 116 mg.
vitamin mix3 127 mg.
penicillin 0.2 mg.
streptomycin 0.8 mg.
arsenilic acid 96.8 mg.
Vitamin A 801 I.U.
vitamin D2 75 I.U.

 

lFeed grade CaHPO4-2H20, proximate analysis 18.5% P, 22-

25% Ca.

2Percentage composition of mineral mix: MnSO 'H
FeSO '7H 0, 40.83; CaCO3, 15.72; ZnC03,

2.45? CoClZ'GH 0, 0.90; KI, 0.43.

2

4 0, 32.06;
7.62; c3504;5H20,

3Percentage composition of vitamin mix: choline chloride,
55.13; calcium pantothenate, 0.43; riboflavin, 0.26;
niacin, 2.60; vitamin Bl (0.1% mannitol triturition),
0.52; a-tocOpherol acetafie (250 I.U.P. per gm), 1.53;
menadione, 0.17; DL methionine, 39.36.

246

APPENDIX D
Modified High Fat Ration (Smith, 1969)

 

 

 

Component Amount/100 gm.
shortening 56.6 gm.
casein 23.6 gm.
non-nutritive fiber 2.0 gm.
aureomycin 0.01 gm.
liver mix 1.89 gm.
DL methionine 0.240 gm.
sucrose 1 3.34 gm.
dicalcium phosphate 2.97 gm.
mineral mix 5.0 gm.
vitamin mix3 2.2 gm.
metaphosphoric acid 0.27 gm.
magnesium carbonate 1.96 gm.
1

Feed grade CaHPO4'2H20, proximate analysis 18.5% P, 22
25% Ca.

2Percentage composition of mineral mix: CaCO 21.0;

Ca3(PO4), 14.9; CuSO4 5H20, 0.039; FePO4 4H20, 1. 47;
MgSO4, 9. 00; MnSO4, 0. 02; K 22141 (30) H-24H20 0. 009; KCl,
12.0; x1, 0. 005; KH2P04, 31. 0; 2NaC1,410.5; NaF, 0.057.

3 . . . . . . .
Percentage compOSltion Of Vitamin mix: Vitamin A concen-

trate (200,000 U.S.P./gm.), 0.45; vitamin D concentrate
(400,000 U.S.P./gm.), 0.025; a-tocoperhol, 0.50; ascorbic
acid, 4.50; i-inositol, 0.50; choline chloride, 7.50;
menadione, 0.225; para-amino benzoic acid, 0.50; niacin,
0.45; riboflavin, 0.10; pyridoxine HCl, 0.10; calcium
pantothenate, 0.30; biotin, 0.002; folic acid, 0.009;
vitamin B, 0.0135; sucrose, 84.75.

247

_.___...-
t

 

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1
J

APPENDIX E

Parameters Evaluated in Histological
Sections of Kidneys

Ca sule
Thickened

Irregular

Glomerulus
Bowman's Capsule

Thickened
Fibrosis
Hyaline
Crescent
Necrosis
Irregular
Glomerular Tuft
Broken
Thickened
Atrophic
Adhesions
Fibrosis
Degeneration
Bowman's Space
Fibrosis
Blood
Protein
Enlarged

Proximal Tubule

 

Epithelium
Hyperplasia
Degeneration
Necrosis
Cystic with Filtrate
Swelling
Cellular Casts
Fibrosis
Basement Membrane Thickened

Henle's Logp_
Cells and Protein Casts
Hyaline Casts
Hemoglobin Casts

 

Corticomedullary Junction
Cystic Tubules with Filtrate
Protein Debris in Tubules
Tubular Necrosis

 

CollectingTubules
_—Casts
Epithelium Degeneration
Hemoglobin and Protein in
Lumen

 

Renal Papillae
Protein in Tubules
Necrosis
Hemorrhage

 

Renal Pelvis
Hemorrhage

 

Miscellaneous
CaléificatIOn
Nephritis
Hemorrhage, Focal
Fibrosis
Infarction

 

’.‘.'|
.r

~03 z

 

NeutrOphils

Lymphocytes

248