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THE EFFECTS OF OVERFEEDING DURING SUCKLING ON THE DNA,
PROTEIN AND TRIGLYCERIDE CONTENT OF THE KIDNEYS
IN TWO STRAINS OF RATS

By

Marianne Stone

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1978

ABSTRACT
THE EFFECTS OF OVERFEEDING DURING SUCKLING ON THE DNA,

PROTEIN AND TRIGLYCERIDE CONTENT OF THE KIDNEYS
IN TWO STRAINS OF RATS

By

Marianne Stone

Male Osborne-Mendel and S SB/Pl rats were overfed from birth to
24 or 105 days. The effects of feeding a 44% or a 3% (w/w) fat diet,
supplementary feeding during the suckling period and reduced litter
size were observed. The overfeeding technique which exerted the
greatest effect on growth was feeding a high fat diet. In comparison
to rats fed the low fat diet, kidney DNA and protein were significantly
elevated in Osborne-Mendel rats of both ages fed the high fat diet.
The kidneys of these rats contained more cells than the controls,
although cell size was not different. The effect on growth of over-
feeding S 5B/Pl rats was negligible, although kidney DNA was elevated
at 24 days and triglyceride at both ages in the S SB/Pl experimental
group. The increase in kidney weight with age in both strains was due

to a doubling in the number of cells and in the protein/DNA ratio.

To Gary,
Gary Lee and
David

11

ACKNOWLEDGMENTS

My sincere appreciation is extended to the following people for
their contributions to this study:

Dr. Rachel Schemmel, my academic advisor, who provided the oppor-
tunity for me to complete my degree and whose professionalism and
optimism are much to be admired.

Dr. Hans Lillevik and Dr. Robert Merkel for their most helpful
advice and insight regarding this study.

Barbara Obst and Dr. Dorice Narins for their expertise in
delivering the supplemental feedings to the newborn animals.

Suwatana Sookpokakit for her valuable assistance as a computer
consultant and Jo McKenzie for typing several drafts and the final copy
of the thesis.

My family, for their support.

My appreciation is also extended to the Weight Watchers Foundation,

Inc. for funding this project.

iii

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . . .

REVIEW OF LITERATURE. . . . . . . . . . . . . . .

Growth of the Kidneys, in General . . . . . .
The Metanephric Kidney . . . . . . . . .

Indices of Growth . . . . . . . . . . .

Growth as Expressed by an Increase in Weight . .

Growth as Expressed by an Increase in Cell

and/or Cell Size . . . . . . . . . . .
The Accumulation of Protein an Lipid. .
Protein and Differentiation . . . .
Kidney Lipids . . . . . . . . . . .

Factors Which Affect Renal Growth . . . . . .

Number

Accelerated Growth, Obesity and Renal Development . .
Experimental Methods of Accelerating Growth. . .

Litter Size Reduction . . . . . . .
Supplemental Feeding. . . . . . . .
Feeding a High Fat Diet . . . . . .
Accelerated Growth and Renal Development

INTRODUCTION 0 O O O O O O O O O O O O O O O O O O
MTHODOLOGY O O O O O O O O O O O O O O O O O O 0

Conditions of the Experiment. . . . . . . . .
Sample Preparation. . . . . . . . . . . . . .
DNA Analysis. . . . . . . . . . . . . . . . .
Protein Analysis. . . . . . . . . . . . . . .
Triglyceride Analysis . . . . . . . . . . . .
Methods of Statistical Analysis . . . . . . .

RESULTS AND DISCUSSION. . . . . . . . . . . . . .
Total Body Weight . . . . . . . . . . . . . .
Total Kidney Weight . . . . . . . . . . . .

Kidney Weight/Body Weight Ratio . . . . . . .
Total Kidney DNA. . . . . . . . . . . . . . .

iv

Page
vi

vii

12
15
l6
17

18

19
19
19
20
21
23

25
27

27
29
31
33
35
37

38

38
39
41
42

Page

Total Kidney Protein. . . . . . . . . . . . . . . . . . . 44

Protein/100 grams Kidney Weight . . . . . . . . . . . . . . 46

Protein/DNA Ratio . . . . . . . . . . . . . . . . . . . . . 46

Total Kidney Triglyceride . . . . . . . . . . . . . . . . . 47
CONCLUSIONS . . .4. . . . . . . . . . . . . . . . . . . . . . . 69
APPENDIX

Solutions Used in the Analysis of Kidney,
DNA and Protein 0 O O O O O O O O O O O O O O O O O O I O 73

SELECTED BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 78

Table

LIST OF TABLES

Experimental Design. . . . . . . . . . . .
compOSition Of Diets O O O O O O O O O O O O 0

Live body weight, lean body mass, kidney(2) weight,
kidney(g)/100g. body weight, kidney(8)/100g. lean
body mass for 24 day old S 5B/Pl and Osborne-Mendel
rats exposed to various treatments . . . . . . . .

Live body weight, lean body mass, kidney(2) weight,
kidney(g)/100g. body weight, kidneY(8)/100g. lean
body mass for 105 day old 8 SB/Pl and Osborne-Mendel
rats exposed to various treatments . . . . . . . .

Results of the Multivariate Analysis of Age, Strain,
Diet, Supplementation and Litter Size on Body Weight,
Kidney(2) Weight, and Kidney DNA, Protein and
Triglyceride O O O O O O O O O O O O O O I O O 0
Total Kidney(2) DNA, protein and triglyceride of

24 and 105 day old S 5B/Pl and Osborne-Mendel

rats exposed to various treatments . . . . . .
Protein, g./100g. kidney . . . . . . . . . . . . . .
Protein/DNA (mg./mg.). . . . . . . . . . . . . .

Triglyceride, g./lOOg. kidney. . . . . . . . . . .

vi

Page
49

50

51

52

53

55
56
57

58

Figure

10.

LIST OF FIGURES
Page

Mean body weights of 24 and 105 day old S SB/Pl
and Osborne-Mendel rats fed either a high fat or
a low fat diet . . O O . O C C O O C O O O C O O O O C O 59

Total body weights of 24 and 105 day old S SB/Pl
and Osborne-Mendel rats exposed to various
treatments . . . . . . . . . . . . . . . . . . . . . . . 60

Mean kidney (2) weights of 24 and 105 day old
S SB/Pl and Osborne-Mendel rats fed either a
high fat or a low fat diet . . . . . . . . . . . . . . . 61

Total kidney weights of 24 and 105 day old
S 5B/Pl and Osborne-Mendel rats exposed to
various treatments . . . . . . . . . . . . . . . . . . . 62

Mean kidney (2) DNA of 24 and 105 day old
S SB/Pl and Osborne-Mendel rats fed either a
high fat or a low fat diet . . . . . . . . . . . . . . . 63

Total DNA in kidneys of 24 and 105 day old
S SB/Pl and Osborne-Mendel rats exposed to
various treatments . . . . . . . . . . . . . . . . . . . 64

Mean kidney (2) protein of 24 and 105 day old
S SB/Pl and Osborne-Mendel rats fed either a
high fat or a low fat diet . . . . . . . . . . . . . . . 65

Total protein in kidneys of 24 and 105 day old
S SB/Pl and Osborne-Mendel rats exposed to
various treatments . . . . . . . . . . . . . . . . . . . 66

Mean Kidney (2) triglyceride of 24 and 105 day old
8 SB/Pl and Osborne-Mendel rats fed either a
high fat or a low fat diet . . . . . . . . . . . . . . . 67

Triglyceride content of kidneys of 24 and 105 day old

S SB/Pl and Osborne-Mendel rats exposed to
various treatments . . . . . . . . . . . . . . . . . . . 68

vii

REVIEW OF LITERATURE

Growth of the Kidneys, in General

Embryologically, the mammalian kidney passes through three stages
of development; the pronephros, the mesanephros and the metanephros.
These kidneys develop successively, one to another, with the gradual
disappearance of the pronephros and mesanephros. The permanent kidney
of the adult mammal is the metanephros.

The tubules of all three kidneys arise from the mesoderm of the
intermediate cell mass or its extension, the nephrogenic cord (Allen,
1951). The vesicles of the pronephros begin to develop in human
embryos at about the twentieth day, and disappear nearly as soon as
they are formed. The mesanephros, or Wolffian Body, develops caudally
to the pronephros beginning about twenty-four days prenatally. It
attains its maximum number of sixty glomerular-tubular units by five
to six weeks (Osathanondh and Potter, 1963a or Potter and Osathanondh,
1966, p. l). About this time, a ureteral bud arises from each mesen-
phric duct. The buds grow and penetrate a mass of cells called the
metanephric blastema. Interaction between these two entities causes
the development of the metanephric kidney (Potter and Osathanondh,
1966). By the fourth prenatal month the mesanephros has degenerated.

The vesicles of all three kidneys contain glomeruli, tubules and
a main duct. The nephrons of the final kidney, however, are much more
highly organized and complex than the first two kidneys. In.man, there

1

 

 

 

DE

ti

2

is a time during the early development of the metanephros when the
nephrons of both the mesanephros and metanephros are active. The func-
tioning of the mesanephric kidney in various species appears to be
related to the efficiency of the placenta (Edelmann and Spitzer, 1969).
The pig has an inefficient placenta, but a very well functioning mesa-
nephros.

The rat has an efficient hemochorial placenta and a corresponding
non-functioning mesanephros. The situation in humans is similar to that
of the rat since, although the mesanephros is functional, it degenerates

quickly (Edelmann and Spitzer, 1969).

The Metanephric Kidney

The metanephric kidney develops from two types of cells; those of
the ureteric buds, and those of the metanephric blastema. The two
ureteric buds, which arise from the mesenphric ducts, give rise to the
ureter, renal pelvis, major and minor calyces, and collecting tubules.
The anterior portion of each bud, the ampulla, grows actively in length
and by dichotomous branching (Osathanondh and Potter, 19633, 1963b),
and is the end which induces nephrogenesis. Ampullary growth and the
formation of new nephrons is complete by 32-36 weeks in the human fetus.
The characteristics of ampullary growth are anterior extension, divi-
sion, induction of nephrons and establishment of communication with
nephrons (Potter, 1972).

The lower portion of the ureteric bud is the interstitial portion,
the cells of which do not highly differentiate. Growth in this section
continues until the full size of the kidney is attained, and is reflected

as an increase in the length of the tubules (Potter, 1972)

3

The cells of the metanephric blastema differentiate into either
nephrogenic or stromogenic cells which give rise to nephrons and con-
nective tissue, respectively. Differentiation is brought about when
the cells are penetrated by the ampulla. Those blastema cells near
the growing, dividing ampulla become the origin of the nephrons, while
those near the interstitial portion of the bud become connective tissue.

The dichotomous branching of the ampullae gives the kidney its
basic structural arrangement. The first few divisions are species
specific, and establish the number of lobes that each kidney will con-
tain. The kidneys of such mammals as rat, rabbit and cat have only one
lobe, in which a single papilla protrudes into a space which joins the
ureter (Potter, 1972). The kidneys of man and other primates are multi-
lobed. Potter (1972) has established that the first three to five
generations of tubules produced by ampullary division dilate to form
the renal pelvis, the next three to five generations dilate to form the
minor calcyces, papillae and cribiform plates, and the next six to nine
generations form the collecting tubules.

In the human fetus, the capacity of the ampullae for nephron induc-
tion is apparent from the eighth week until the thirty-second or thirty-
sixth week. Nephrogenesis first begins when the ureteral bud has from
twenty to forty terminal branches (Potter and Osathanondth, 1966). The
blastema cells near the end of the ampulla condense, or aggregate, and
become less mobile. Using time lapse photography, Saxen gt 31. (1965)
demonstrated the existence of a trapping effect of the aggregated cells
upon the more mobile blastema cells. The aggregate cells grow in this
way by trapping more cells, and by cell proliferation. Then the aggre-

gate assumes a spherical formation, with radially oriented wedge-shaped

 

 

 

 

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(I)

4
cells (Saxen 25 $1., 1968). A small cavity appears and the cells
elongate, forming a single layer. The structure then takes on a char-
acteristic S-shape. Upon further differentiation, one end of the
vesicle communicates with the ampulla, and the other end forms a spoon-
shaped double layer of cells. The cells of the outer layer become thin
and flat, and form Bowman's Capsule. The other layer of cells becomes
tall and narrow (columnar), and forms the epithelial layer of the glo-
merulus. When the capillary loop is evolved and grows close to these
columnar cells, it also moves a group of connective tissue cells into
the glomerulus. These connective tissue cells are thought to persist
as mesangial cells (Potter and Osathanondh, 1966, p. 3).

During its existence, the activity of the ampulla is variable.
Potter (1972, pp. 29-43) and Potter and Osathanondh (1966; Osathanondh
and Potter, 1963b) have defined four periods in the development of the
human kidney, based on changes in ampullary activity:

Period 1 extends from the fifth to the fourteenth or fifteenth

week prenatally. It begins when the ureteric bud arises from

the mesenphric duct, and includes the eighth week when nephro-

genesis begins. At the beginning of nephrogenesis, the ampulla

induces the formation of two nephrons. The ampulla divides and
each branch carries with it, one nephron. Upon dividing there-
after the ampulla is capable of inducing only one nephron.

During Period 1, an ampulla which already has an attached

nephron cannot induce the formation of another. Only a freshly

divided ampulla can induce nephrogenesis. This period may be
identified microscopically by the observation that the nephron

attaches to the ampulla before the ampulla grows very far from

the point of branching.

Period 2 extends from week fourteen or fifteen until week
twenty or twenty-two of fetal growth. It is known as the
period of arcade formation. An arcade is a succession of

from two to eight (usually four) nephrons which drain into

a single collecting tubule. During this time there is little
ampullary branching, and the function of nephron induction is
carried on by ampullae which already have an attached nephron.
Unlike Period 1, ampullae with attached nephrons can induce
nephrogenesis. The result is a succession, or arcade, of
nephrons only the youngest of which is attached to the ampulla.
The connecting tubules of each nephron in the arcade merge
into that of the youngest nephron. The forward growth of the
ampulla, at this time, is due to growth in the interstitial

portion of the ureteral bud.

Period 3 extends from twenty or twenty-two weeks until thirty-
two to thirty-six weeks prenatally. It is responsible for the
formation of the unbranched terminal portions of the collecting
tubules, and for the nephrons whose glomeruli are located in
the outer half of the cortex. The ampullae again grow by local
cell proliferation, although they seldom branch. They grow
beyond the arcade formations, and beyond any nephrons formed
during this period. The ampulla induces nephrogenesis only
when not carrying an attached nephron, and the nephrons which
are formed become attached to the interstitial portion of the

tubule.

6
Period 4 extends from the thirty-second to thirty-sixth week
prenatally until adult life. The ampullae disappear at this
time, and there is no further terminal growth or branching of
the collecting tubules. The induction of nephrons ceases.
Any changes which occur are due to interstitial growth and

cellular differentiation.

By the time of birth, then, the kidneys of the neonate contain an
extensive tubular system, and essentially all of their nephrons. From
thirty-two to thirty-six weeks prenatally until adulthood the ureters
and collecting tubules grow in length, the cells of the metanephric
blastema continue to differentiate into connective tissue, the vascular
system elaborates, and the nephrons grow and differentiate. The
nephrons grow primarily by an increase in the length of their tubules,
especially in the areas of the proximal tubule and the Loop of Henle.
The glomeruli of the adult have only a slightly greater diameter than
those of the infant or fetus (Allen, 1951). As growth continues the
nephric tubule becomes more coiled, or tortuous, especially in the
proximal portion.

The cortex of the mature kidney is composed of terminal branches
of collecting tubules, glomeruli, proximal and distal tubules and the
portions of the Loops of Henle which lie near the collecting tubules.
The medula contains collecting tubules and the remaining portions of

the Loops of Henle (Potter and Osathanondh, 1966).

Indices of Growth

 

There was considerable interest early in history regarding the

growth and development of the kidneys. It had been noticed that in

7
persons born with one kidney, the organ was considerably enlarged. In
the same context, Nowinski and G033 (1969) have given an interesting
account of the historical development of the unilateral nephrectomy and
research that has evolved concerning the growth of the remaining kidney.
In 1871, Rosenstein (as cited in Nowinski and Goss, 1969) noted that a
difference normally exists in the weight of the left and right kidney.
This has been a controversial topic for several years with some investi-
gators reporting that the right kidney in rats is larger than the left
(Arataki, 1926; Coe and Korty, 1967; Halliburton, 1969), and others
finding no difference (Smith and Moise, 1927; Zumoff and Pachter, 1964;
Mason and Ewald, 1965).

Not trusting weight to be the only criterion of growth, Ribbert,
in 1882 (as cited in Nowinski and Goss, 1969), measured kidney volume
by its displacement of water in a measuring cylinder and also measured
the diameters of glomeruli microscopically. In 1917, Kittelson studied
the relative growth of the cortex and medulla at different ages in rat
kidney by tracing serial sections of kidney on paper and then cutting
and weighing them. The weight of the paper was converted to surface
area, and then to volume. Kittelson also used tracings to count the
number of glomeruli and to calculate their volume. In the late 1920's
and early 1930's, MacKay §£_§1, (1927a, 1927b, 1931) used weight and
surface area as criteria of growth when experimenting with the effects
of age and diet on the kidney. Donaldson (1924) and Dunn £5 a1. (1947)
measured individual organ and total body weight and length in studies
of normal growth in the rat.

Although weight gain is a valuable parameter in the assessment

of growth, it is disadvantageous in that weight can increase in the

8
absence of growth, such as with the accumulation of fat and water. In
1960, Widdowson and Dickerson studied the growth of organs and tissues
by using another approach. Chemical maturity was assessed at various
ages in pig and man by analyzing organs and tissues for water, total
nitrogen, sodium, potassium, chloride, phosphorus, magnesium and
calcium. Later, Widdowson and coworkers (1971, 1972) expanded their
chemical composition studies to include the analysis of deoxyribon-
ucleic acid (DNA).

It is now possible to use weight gain data in conjunction with more
specific growth parameters at the cellular level. An organ or tissue
grows by increasing its number of cells, by enlarging its existing cells
and by accumulating more intercellular material. In 1948, Boivin gt a},
published figures for the DNA content of the cell nuclei of various
vertebrates. They established that the DNA content of the diploid cells
within a given species is constant. This was confirmed by Mirsky and
Ris (1949), and by Thomson.g£_§1, (1953) in the rat. Thomson found that
such factors as age, strain, sex, body weight, fasting, a protein free
diet, a thiamin deficient diet and a high fat diet did not affect the
DNA content of rat nuclei. In 1962, Enesco and Leblond utilized the
constancy of DNA to measure cell proliferation during the growth of
various organs and tissues in the young male rat. Total organ or tissue
DNA was divided by the constant, 6.2 pg. (Enesco, 1957) in order to
determine the number of nuclei. The total weight of the organ was
divided by the number of nuclei in order to determine the amount of
material associated with each nucleus. This gave a rough estimate of
cell size, although it included intercellular material. In 1965, Winick

and Noble determined cell number and cell size in the tissues and organs

9
of the rat in 35359 and postnatally. Weight/DNA and protein/DNA ratios
were used as an expression of cell size, and the RNA/DNA ratio as an
index of protein synthesis. Later the authors expressed results as
total DNA, RNA, and protein when reporting the effects of underfeeding
(Winick and Noble, 1966) and overfeeding (Winick and Noble, 1967), on
the cellularity of the rat.

The measurement of cell number and cell size has added a valuable
dimension to the study of kidney growth. Calculating weight/DNA, pro—
tein/DNA, RNA/DNA and lipid/DNA ratios gives an approximation of the
average size and composition of the cells. Some cells are more highly
differentiated than others, however, and have more protein, RNA or lipid
associated with them. The figure representing cell number is also some-
what arbitrary. The chemical methods for measuring DNA do not differen-
tiate between cells of the parenchyma and those of such fractions as
blood vessels, nerves and connective tissue. It is known that the
parenchymal cells of an organ make up the majority of the cells, and
the connective tissue cells almost all of the rest (Enesco and Leblond,
1962). In reference to liver, Munro and Fleck (1969) have concluded
that most of its mass and most of its DNA content can be attributed to
hepatocytes and only a "tolerably low error" is introduced from the
presence of other cells. Enesco and Puddy (1964) showed that 60 to 70%
of the nuclei of muscle fibers in rat, or 85% of muscle mass was
attributable to the contractile cells. Control group mice in the
simulated high altitude experiments of Naeye (1966) were shown to have
the following approximate percentages of parenchymal cells in selected

organs: heart, 87%; liver, 78%; and adrenal cortex, 83%.

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10
Growth as Expressed by an Increase in Weight

Growth may be defined simply as an increase in size or weight.
Organ and tissue weight increase progressively from early prenatal life
until maturity. During this time the chemical composition of the organs
and tissues is not static, but is changing in a manner which leads to
chemical and functional maturity. Rahill and Subramanian (1973) have
stressed that the fetal kidney is characteristically different from
the postnatal kidney in that it has a high water content and thrives in
an hypoxic environment. Widdowson and Dickerson (1960) found that in
kidney, as well as in other soft tissues, there is a proportionate
decrease in water and an increase in the proportion of nitrogen from
early gestation until adulthood. Fetal kidney, heart and liver were
found to reach their adult chemical composition before skin and skeletal
muscle, and it was suggested that this is related to their earlier func-
tional development.

Each organ and tissue contributes a certain proportion to the body
as a whole and the proportion is not the same at all ages (Widdowson
and Dickerson, 1960). In general, kidney weight in relation to body
weight decreases from birth to maturity, unlike an organ such as the
liver in which the relationship remains relatively constant. Widdowson
and Dickerson (1960) found human kidneys to form a higher proportion of
body weight at birth than at 13 to 14 or 20 to 22 weeks of gestation,
and in adulthood. In the pig, the kidneys contributed most to the total
weight of the body at 46 days of gestation. The weight of the kidneys
of the newborn infant, relative to body weight, is three times greater
than in the adult (Allen, 1951). The kidneys of the adult comprise

about 0.4% of the total body weight (Pitts, 1968).

the

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Solomon and Bengele (1973) state that the relationship between
organ weight and body weight as a function of development has received
little attention, with very few reports referring to the kidney. The
majority of the studies of the kidney show that following birth there
is an increase in the ratio to a peak level, followed by a decline
(Jackson, 1913; Kittelson, 1917; Arataki, 1926). In the mature animal,
the ratio remains constant. Solomon and Bengele (1973) suggest that
the organ weight to body weight ratios for kidney, heart, liver and
spleen peak at different times, experience a different rate of decline,
and are not determined exclusively by a common body weight factor such
as amount of fat or change in skeletal or muscle mass. Each of these
organs has its own intrinsic growth pattern relative to body weight.

In 1913, Jackson showed that the kidney weight to body weight
ratio of the albino rat increased from 0.96 at birth to a maximum of
1.44 at twenty days. The ratio decreased thereafter to 1.03 at ten
weeks and 0.93 at five months. Kittelson (1917) attributed this early,
rapid increase in the ratio, as well as its subsequent decline, to the
growth of the medulla. He found that the growth index of the kidney
cortex remains quite constant and corresponds to that of the body as a
whole, while the medulla shows a more varied relative growth rate. The
medulla was found to double its growth index (relative to body weight)
between birth and two to three weeks of age. Thereafter it showed a
continuous, marked decrease, indicating that its growth lagged behind
that of the whole body. The volumetric ratio of medulla to cortex was
found to vary in the following manner: 1:3.05 at birth; 1:2.02 at
at two weeks; 1:1.50 at three weeks; 1:2.10 at twelve weeks and 1:2.87

in adulthood.

 

 

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12

Growth as Expressed by an Increase
in Cell Number and/or Cell Size

Enesco and Leblond (1962) were the first to explore the relative
contribution of cell division and cell enlargement to growth. In a com-
prehensive study of the male Sherman rat, they assessed DNA and weight/
nucleus of whole body, tissues and selected organs including kidney,
lung, pancreas, adrenal, testes, thymus and liver. The rats were sacri-
ficed and examined at 0, 7, 17, 34, 48 and 95 days of age. Growth of
the organs and tissues, in general, was characterized by three phases:
a) from birth until 17 days of age, growth occurred almost exclusively
by cell division, b) between 17 and 34 to 48 days of age, cell division
proceeded at a reduced rate while cell size increased, especially in the
tissues, c) after 48 days of age, the addition of new cells slowed down.
The cells of such tissue as muscle and adipose continued to enlarge
whereas in the organs there was no further increase in cell size. In
regard to the kidneys specifically, the number of cells were found to
increase rapidly after birth and then more slowly until a constant rate
of cell division was reached. There was no change in weight/nucleus,
or cell size, between 7 and 17 days. There was however, a rapid increase
in weight/nucleus between 17 and 48 days. After this time, cell size
remained constant. Between 7 and 95 days of age, the number of nuclei
in the kidneys of the rats increased 6.5 times while the weight/nucleus
doubled. (Enesco and Leblond, 1962; Enesco, 1956).

Winick and Noble (1965) confirmed the finding of Enesco and Leblond
that cell division plays a major role in the growth of organs. The same
conclusion had been drawn earlier by Kurnick (1951) in reference to the

kidneys. Winick and Noble. expanded their study of the cellular growth

13
of the rat to include the gestational period where they found growth to
occur only by cell division. It was found that from 10 days after con-
ception until 13 days postnatally growth is characterized by rapid cell
division. Following this time, the rate of increase declines at dif-
ferent times in different organs. As assessed by thymidine - l4C
incorporation, DNA synthesis was found to have ended by the forty-fourth
day postnatally in the kidneys, and had been the highest at four days.

Regarding cell size, Winick and Noble (1965) plotted weight/DNA and
protein/DNA ratios for whole rat and individual organs. A discrepancy
existed between the ratios during the neonatal period, when the weight/
DNA ratios decreased and the protein/DNA ratios either remained constant
or increased. This was attributed to water loss during the neonatal
period. The authors found that there is a steady increase in organ
protein, beyond the cessation of DNA synthesis, until a steady state is
reached between protein synthesis and degradation at which time growth
is complete. In a fashion similar to Enesco and Leblond, they have
classified the growth of whole body and individual organs from ten days
after conception until maturity into three phases:

a) Growth by rapid cell division

b) Growth by cell division and cell hypertrophy

c) Growth by hypertrophy alone.

The cellular growth pattern of the kidneys has been fairly well
defined, and the literature (Kurnick, 1951; Zumoff and Pachter, 1964;
Widdowson £5 31., 1972; Potter 33 él-» 1969) has supported and expanded
the findings of Enesco and Leblond (1962) and Winick and Noble (1965).
By total nuclear count, Zumoff and Pachter (1964) verified that there

is a diminution of nuclear division in the kidney during the mid portion

 

 

of
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for:
is :

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of D

to b:
33‘ f1

twice

 

 

 

 

sharp
incre
Weigh

time

14
of its active growth phase. They suggest that the sex hormones play a
role in inhibiting cell division at this time. Potter g£_§1, (1969)
found that more than half of the increase in kidney weight with growth
is reflected by an increase in cell number, while increasing cell size
plays its major role after the rat has attained a body weight of 200 g.

Priestley and Malt (1968) traced changes in nucleic acid and pro-
tein synthesis in mouse kidney from early gestation (16 days after
mating of parents) until adulthood. They found that the concentrations
of DNA and RNA reached a peak within the first week of life, declined
to a stable level by 40 days and persisted at this level until 200 days
of age.

In regard to protein synthesis, Priestley and Malt found the rate
to be twenty times as great in the fetal kidney as in the adult kidney.
By five to six days after birth the rate of synthesis had decreased to
twice that of the adult kidney. The authors found that along with a
sharp decrease in kidney fluid just before birth, there was more of an
increase in protein than could be accounted for by the change in dry
weight. They attributed the greater content of kidney protein at this
time to a high rate of fetal protein synthesis. This speculation is in
contrast to that of McCrory (1972) and Winick and Noble (1965) who
attribute the increase to alterations in body fluid volume compartments
relative to the birth process. Priestley and Malt (1968) found that
from five to six days after birth the total protein content of the
kidney increased slowly and gradually to a plateau at 45 days of age.
There was no appreciable decrease in the rate of protein synthesis up
to 200 days. This adds credence to the view of Winick and Noble (1965)

that the experimentally observed increase in the protein/DNA ratio with

 

 

.2. .lrll . ll 5'} ..

ifvll‘

15
age is not a consequence of increased rates of protein synthesis but
of decreased rates of cell division.

The cellular pattern of growth in rat and mouse kidney then, is
similar. Studies of the cellular growth pattern of human kidneys are
limited. By examining the kidneys, heart, gastrocnemii and liver of
human fetuses and newborn infants Widdowson 2£.§l- (1972) observed two
distinct phases of prenatal organ growth. One was a period of rapid
cell division between the earliest observed week (week 13) and the 25th
week, when total organ DNA doubled weekly. The other period was char-
acterized by a rapid increase in the protein/DNA ratio and was observed
during the last 10 weeks of intrauterine life. Although, the cells were
still dividing during the last ten weeks, the organs had only 20% of
the adult number of cells at birth. By birth, the cells of the kidneys
had nearly achieved their final size. Winick gt a1. (1972) concur that
cell division increases in human fetal organs from 13 to 14 weeks of
gestation through term, and into the first year of life. They maintain,
however, that cell size in the kidney remains unchanged throughout
gestation. There is a discrepancy as to this finding and that of
Widdowson g£_§1, (1972). Winick and Noble suggest that in the kidney,

there is no change in cell size until after the first year of life.

The Accumulation of Protein and Lipid
An organ increases in size due to the accretion of protein, water,
lipids and nucleic acids. Most of the protein is packaged within cells,
and that outside the cells is primarily collagen. The lipids of the
kidney are mainly triglyceride and the phospholipids and cholesterol of

membranes. Lipid, in the form of osmiophilic droplets, is evident in

16
the interstitial cells of the cortex and especially in those of the

medulla (Osvaldo and Latta, 1966; Moffat, 1975).

Protein and Differentiation

The development of an organ involves not only growth in size or
mass, but includes the process of differentiation. Differentiation
involves primarily the synthesis of specific protein molecules, and
refers to the production of the stable, structural elements of a cell
(Goss, 1964). The process leads to diversification of cell structure
and function, and in the kidney results in the development of at least
sixteen different cell types (Nowinski and Goss, 1969).

Nephron induction is complete by three or four weeks after birth
in the rat (Kittelson, 1917; Arataki, 1926; Boss g£H§1., 1963; Bengele
and Solomon, 1974) and, in the human, by the time the fetus weighs
between 2100 and 2500 g. (Potter and Thierstein, 1943). Differentia-
tion of the cells then continues, and the nephrons change in size and
configuration as they assume proper positioning in the kidneys.

Each segment of the nephron is lined with a specific type of
epithelium. Bowman's Capsule is a double walled cup of squamous
epithelium, the visceral layer of which becomes modified as it comes
into contact with the glomerulus. At this junction, specialized cells
called podocytes develop and send processes which surround the capil-
laries. The squamous epithelium of Bowman's Capsule changes to cuboidal
epithelium at the neck of the proximal tubule. This tubule includes
microvilli, and a brush border which McCrory (1972) considers to be a
structurally integrated subcellular organele, not unlike the mito-

chondria. The brush border ends at the Loop of Henle where squamous

l7
epithelium again becomes apparent. The epithelium continues throughout
the distal and collecting tubules where it becomes more cuboidal and
then columnar (Bloom and Fawcett, 1975).

Types of cells which are found in the glomerulus are endothelial
cells, mesangial cells, and smooth muscle cells along with the previously
mentioned podocytes. The basement membrane contains collagen as mea-
sured by hydroxproline analysis (Goodman SE a1., 1955; Bonting gt 31.,
1961; Latta, 1961), and in man increases in thickness from birth to
three years of age.

Other cell types in the kidney include the specialized cells of the
juxtaglomerular apparatus, and the fibroblasts, chondrocytes and

osteoblasts of connective tissue.

KidneyiLipids

 

Several investigators have attempted to define the lipid composi-
tion of kidneys, and in particular, to establish whether there is a
divergence in the lipid content of the cortical and medullary zones.
Morgan 23 a1. (1963), Gold (1970) and Muller g£_§1, (1976) have found
the phospholipid content of the cortex to be greater than that of the
medulla in the rabbit, dog and rat, respectively. In the same studies,
triglyceride and cholesterol were found to a greater extent in medulla
than cortex in rabbit and dog kidney while the opposite was true for
the rat. Muller £5 a1. (1976) found a corticomedullary concentration
gradient (cortex, outer medulla, inner medulla) in adult rat kidney
for total lipids, cholesterol, phospholipids and free fatty acids.

There appears to be little zonal difference in the fatty acid com-

position of the various lipid fractions of the kidney (Muller §£_§1,,

18
1976). Several investigators (Gold, 1970; Muller g£_a1,, 1976) have
found a high concentration of arachidonic acid in the phospholipids of
kidney. This is probably related to its role in the synthesis of

prostaglandins.

Factors Which Affect Renal Growth

Growth of the kidneys is limited at the histological level by the
number of nephrons which are induced. Although no new nephrons are pro-
duced after the kidney matures, the organ is capable of adjusting its
size in response to an increased work load. In this context, renal
growth may be stimulated by such experimental methods as partial
nephrectomy, nephrectomy serum, ureteral ligation, protein and folic
acid injection, diets high in protein or sodium and low in potassium,
NH4C1 acidosis, thyroxine, testosterone, cold exposure, growth hormones,
and mineralocorticoids (Nowinski and Goss, 1969).

During early development, before differentiation is complete,
several factors are known to influence cell size, and cell and glomerular
number. Naeye (1966) found subnormal kidney weights in undernourished
mice, and in mice raised in an hypoxic environment. Growth retardation
took different forms with each stress. The kidneys of the hypoxic mice
were small because they had a deficient number of cells, while both
cell number and cell size were affected in the undernourished mice. In
both groups there were significantly fewer glomeruli than in the con-
trols. Zeman (1966, 1967, 1970) and Zeman and Stanbrough (1969) found
that the offspring of undernourished rats showed a reduction in kidney
weight (relative to body weight), fewer nephrons, decreased kidney DNA,
RNA, and protein and immature renal function. Accordingly, Winick and

Noble (1966) established that caloric restriction during the suckling

19
period results in retarded organ growth as represented by a decrease in
cell number. Having ascertained that undernutrition early in develop-
ment influences renal growth, attention has been focused on the effect

that accelerated growth might have on renal weight and cellularity.

Accelerated Growth, Obesity and Renal Development

Experimental Methods of Accelerating Growth

With the current interest in obesity being centered on prevention,
numerous studies have investigated the influence of early infant feeding
practices on the accumulation of excess body fat. Several research
models have been devised for studying overnutrition in the suckling rat.
This period in life is critical in determining the subsequent weight and
food intake pattern of the animal (Kennedy, 1957a, 1957b; Widdowson and

Kennedy, 1962; Widdowson and McCance, 1960).

Litter Size Reduction

 

The redistribution of newly born mammals into smaller litters has
proven to be an effective means of overfeeding. (In theory, each pup
then receives a greater proportion of the dam's milk, and is able to
more fully "achieve its growth potential.") Normally rats are born in
litters of eight to twelve. Reducing the litter to three should pro-
vide a model of overnutrition, while raising the animals eighteen per
litter would have the opposite effect. Using these extremes in litter
size, Widdowson and McCance (1960) found that rats raised in small
litters weighed two to four times as much at weaning as did those from
large litters, and that the former became much larger adults.

Parks (1926) was one of the first to observed the effect of litter

20
size on the growth of rodents, when he traced growth curves of mice
born in litters of one to ten. He found that mice born three, four and
five per litter showed better growth than those born in litters of six
through ten, and concluded that growth was inversely proportionate to
the size of the litter. Heggeness g£_§l, (1961) has also assumed that
rate of growth is inversely related to litter size, due to the avail-
ability of milk. In a study by Winick and Noble (1967), however, rats
raised three per litter grew no differently than those raised six per
litter, although the three per litter and six per litter groups did
weigh significantly more at weaning and in adulthood than the twelve
per litter controls. In contrast, wurtman and Miller (1976a) were un-
able to produce accelerated weight gain and lipid storage of a persistent

nature in rats raised in litters under twelve (2, 4 or 8 per litter).

Supplemental Feeding

A method of overfeeding in which the quantity and quality of milk
can be modified during the suckling period is supplemental force feed-
ing. The two procedures which have been employed include repeated tube
feeding and gastrostomy with continuous infusion of liquid (Winick,
3.; fl- , 1972) .

Repeated tube feeding has been carried out successfully either as
a complete substitute for dam delivered milk (Dymsza £5 31,, 1964;
Miller and Dymsza, 1963) or as supplemental feedings (Czajka-Narins and
Hirsch, 1974; wurtman and Miller, 1976b). The technique has proven to
be time consuming and technically difficult. Particular attention must
be given to the osmolarity (Aprille and Rulfs, 1976) and particle size

of the supplement. The pups sometimes regurgitate or aspirate the

21
feeding into their lungs (Czajka-Narins and Hirsch, 1974). The sucking
reflex is apparently essential for proper gastric emptying of the load
(Miller and Dymsza, 1963).

Bull and Pitts (1971) have found that force feeding a supplement
exceeding 35% of stomach capacity does not cause alterations in the
absorption of dietary energy. With this in mind, Czajka-Narins and
Hirsch (1974) developed a model for overfeeding suckling rats, in which
a supplemental nonfat dried skim milk formula was delivered four times
daily by bulbous tipped syringe. The supplemented animals showed
accelerated growth with significantly more body fat at an early age
(14 to 20 days), and more body protein throughout the 105 days of the
study. Somewhat different results were attained by Wurtman and Miller
(1976b) when they supplemented suckling rats with Dymsza's (1964)
formula, and with commercial baby food. The experimental animals in
this study had persistently more body fat from the seventeenth to the
sixtieth day than did their non—supplemented controls, but carcass
protein was not different. In the former study, supplementation was
extended from the first to the twenty-first day of life, while in the

latter the rats received feedings from the tenth to the sixteenth day.

Feeding a High Fat Diet

 

Obesity has been produced experimentally by feeding a high fat
diet to weanling rats. Greater weight gains occur when Crisco and
lard furnish the calories than when vegetable oils are fed (Barboriak,
33 11., 1957). In 1955, Mickelsen £5 51. gave Osborne-Mendel rats a
diet, §g_libitum, in which 85% of the calories came from Crisco.

Seventy percent of the experimental male rats attained weights close

'm
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Ex
SC
ma
St:
wit

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22

to, or over 1,000 g. Similar results were achieved by Barboriak,
§£_al, (1957) when he fed rats a diet in which 81% of the calories came
from lard.

Fenton and Carr (1951) noted a strain difference in the rate of
fat deposition when four strains of mice were fed a high fat diet.
Exploring the response of rats to nutritionally induced obesity,
Schemmel g£_al, (1970) reported that the Osborne-Mendel strain responded
maximally to high fat feeding, while S 5B/P1 rats showed minimal response.
Strain and ration were equally influential in the development of obesity
with strain most influencing body weight, and diet the accumulation of
body fat. Peckham £5 31. (1962) also found the latter to be true.

Accelerated growth may be implemented in the suckling rat by
feeding a high fat ration to the lactating dam. Emery g£_a1, (1971) and
Schemmel g£_§l, (1973) have shown that this dietary regimen increases
the caloric density of the milk. Dams fed a high fat diet secrete milk
containing almost twice as much fat as that of grain-fed dams. In
Emery's study, overfed pups from the Sprague Dawley strain showed
accelerated weight gains at two weeks of age. Schemmel gt $1., using
the obesity-susceptible Osborne-Mendel strain, produced overfed rats
which by ten days of age were 50% heavier than the controls. In both
studies, weight gain and the accumulation of body fat by the time of
weaning were significantly greater in those animals that had received
milk from high fat-fed dams than in those whose dams had been fed
grain. The experimental Osborne-Mendel rats had almost four times as

much body fat as did the controls.

23
Accelerated Growth and Renal Development

One of the most prominent manifestations of obesity is cardio-
vascular renal disease. Weil pointed out in 1955, that excessive
perirenal fat surrounding the kidneys could be detrimental to their
function. Kennedy (1957c) found that kidney lesions, similar to those
of advancing age, were apparent earlier in rats made obese by hypo-
thalamic lesion. Renal damage to the degree which caused death in rats
over two years of age was evident in obese rats from one year to fifteen
months of age. Accordingly, Johnson (1972) found significantly more
perirenal fat, a greater number of kidney lesions and lesions of a more
severe nature in 45 week old Osborne-Mendel rats made obese by feeding
a high fat diet. Aprille and Rulfs (1976) also described large storage
depots of fat around the kidneys of ten day old rats that had been tube-
fed a 78% fat diet. Widdowson and Kennedy (1962) found an increased
incidence of kidney disease in fast growing (3/litter vs. 15-20 litter)
male rats, whereas Barboriak (1957) found no systematic influence of an
81% fat or oil diet on kidney damage up to two years of age.

From comprehensive studies on growth and development (Widdowson
and McCance, 1960; McCance, 1962; McCance and Widdowson, 1962; Widdowson
and Kennedy, 1962) have come to the following observations:

1. During normal growth, all parts of the body do not develop

equally fast or at the same time.

2. Undernutrition alters the shape and form of the growing

animal because those parts which develop before others
retain priority of growth and may continue to increase
in size at the expense of others (undernutrition affects

growth of the skeleton less than it does growth of the adipose).

24
3. A very high plane of nutrition, by saturating the growth
requirements of the animal, accelerates the development
of all tissues, but particularly those with little struc-
tural stability and low priority (i.e., adipose tissue).
Accelerating body weight gains early in development results in a
proportionate increase in the size of the kidneys. Thus, kidney growth
is related to body size rather than to age (Moment, 1933; Widdowson
and McCance, 1960; Rakusan, 1975). The young overfed rats in experi-
ments by Solomon and Capek (1972a) and Solomon and Bengele (1973) had
lower kidney weight/body weight ratios than did the controls, regardless
of differences in body fat. The authors surmized that the kidney
weight/body weight ratio in these animals had already peaked and was
on the decline, indicating greater organ maturity. Tests as to func-
tional development substantiated this theory (Solomon and Capek, 1972b).
Winick and Noble (1967) studied the cellular aspects of acceler-
ated growth in the organs of neonatal rats. Raising animals three and
six per litter resulted in kidneys which weighed 14% more at weaning
than did those of the controls. Through this study it was established
that increasing calories during the suckling period accelerates organ

growth exclusively by increasing the rate of cell division.

INTRODUCTION

In 1970, Schemmel £3 al. reported a difference among seven strains
of rats in their susceptibility toward obesity when fed a high fat (60%
w/w) diet from the time of weaning. The Osborne-Mendel rats responded
by accumulating up to 50% body weight as fat, while the S SB/Pl rats
maintained only 14% fat in their bodies. As a result of this study
there was interest as to how these two strains of rats would respond
to aggressive overfeeding earlier than 21 days of age. Introducing
more calories during the suckling period might simulate the human situ-
ation where infants in our society are often exposed to calorically
dense foods earlier in life than is necessary. The overall experiment
was designed to study the effects of overnutrition during the suckling
period on the growth and development of whole body and individual organs
and tissues in two strains of rats. Three overfeeding techniques were
utilized in an attempt, particularly, to force the development of
obesity in the S SB/Pl animal. These techniques included a) feeding a
high fat (44% w/w) diet b) supplemental force-feeding and c) litter
size reduction.

In 1972, Johnson used the Osborne-Mendel rat and an experimental
60% fat (w/w) diet in studying kidney development and function in the
(obese animal. She reported total kidney weights to be significantly
greater at 25, 35, and 45 weeks of age in those Osborne-Mendel rats

fed.the high fat diet from weaning than in those fed a grain ration.

25

26
The purpose of the present investigation was to ascertain whether early
overfeeding practices would cause an increase in the weight of the kid-
neys of obesity-susceptible (Osborne—Mendel) and obesity-resistant
(S 5B/Pl) rats. If such an increase in weight occurred, an attempt
would be made to determine whether it was due to an increase in cell
number, cell size or both. Observation would also be made of the
changes in the number and size of the cells between 24 and 105 days

of age.

METHODOLOGY
Conditions of the Experiment

Eighty newborn, male rats from the S 5B/P11 strain and eighty from
the Osborne-Mendel strain2 were assigned to the experimental design
depicted in Table l. The rats were organized into litters of three or
six, and were assigned either to receive, or not to receive supplemental
force-feeding. A high fat diet was fed to one-half of the dams in each
strain from partuition until the pups were weaned. The other half
received a low fat diet. One-half of the suckling rats from each strain
was raised to weaning (24 days) and the other half to 105 days of age.
Upon weaning, those rats not sacrificed were maintained on the respec-
tive high or low fat diet of their dams until 105 days. The variables
in this experiment then, were Age, Strain, Diet, Supplementation and
Litter Size. There were 32 different treatments, with 5 rats in each
category.

The composition of the high and low fat diets that were fed to

the dams and weaned pups is shown in Table 2. Forty-four percent

 

1The pedigreed S 5B/P1 Cr breeding stock was secured through the
generosity of Samuel L. Poiley, former Head, Mammalian Genetics and
‘Animal Production Section D R and D, Chemotherapy, National Cancer

Institute, DHEW.

2Originally, ninety-six rats from each strain were included in
the study. For statistical purposes, due to the death of several
‘rats, the number was randomly cut to 80 per strain; 5 rats per

treatment, rather than 6.

27

28
Crisco, by weight, was used in the high fat ration, comprising 70% of
the total calories. Seven percent of the calories in the low fat
ration were from fat, in the form of corn oil. Carbohydrate, as cere-
lose, contributed 7% of the calories in the high fat diet, and 70% in
the low fat ration. In both rations, 23% of the calories came from
protein, as casein. Each gram of the high fat ration contained 5.67
kcal., and each gram of the low fat ration, 3.81 kcal. In both rations
there were 17.2 kcal/g. protein. The rations and drinking water were
available ad libitum.

The supplementary feeding formula simulated the fat content of
milk produced by auxillary dams of the same strains fed high and low
fat diets. The milk fat was analyzed by the Roese-Gottlieb Method as
outlined by the Association of Official Analytical Chemists (Horwitz,
1970).

The mean fat content of milk from S SB/Pl dams fed the low fat
ration was 15%, while that from dams fed the high fat ration was 20%.
Respective percentages in the milk of Osborne-Mendel dams were 18 and
24%. This was based on the analysis for fat of three to five samples
for each group of rats. Four supplemental milk mixtures were prepared
incorporating corn oil in the above percentages. Fifteen, 20, 18,
and 24 ml. of corn 011 plus, in each case, 15 g. of nonfat dry milk
solids were made to volumes of 100 ml. with distilled water. The mix-
tures were shaken well before use. The method for force-feeding the
pups was that of Czajka-Narins and Hirsch (1974). The pups were force-
fed between 8:00 and 9:00 a.m. and 4:00 and 5:00 p.m. each day from
birth until weaning. The nonsupplemented pups were handled each day

in like manner, but not force-fed.

29
The experimental animals were housed in wire screen cages
(18x18x25 cm.). A temperature of 23:1 degree centigrade was maintained
in the room, and conditions were such that each 12 hour period of light
was followed by 12 hours of darkness. Disturbances were kept at a min-

imum during cage cleaning, feeding and handling.

Sample Preparation

 

Upon sacrifice, the animals were weighed, anesthesized with ether,
and decapitated. The kidneys of each animal were quickly removed,
decapsulated, placed on aluminum foil and set on dry ice. Then the
organ was weighed, wrapped in aluminum foil, placed in a Zip-lock plastic
bag3 on dry ice and later stored in a freezer at -18 degrees centigrade.

The day before each organ in a series of samples was ground in
preparation for DNA, protein and triglyceride analysis, the amount of
distilled deionized4 water which would dilute the kidneys to a volume
ten times their weight (w/v) was calculated:

Kidney weight at
sacrifice (g.)

Kidney weight at

sacrifice (g.) = Water to dilute

x 10

This amount of water was measured by pipette, and equally divided between
two labelled 55 ml. test tubes. The tubes for each sample were capped
with parafilm5 and refrigerated overnight at 0 degrees centigrade. For

each sample, 5 ml. of 10% trichloroacetic acid, 10 g./100ml. (w/v), was

 

3Lab Apparatus Company, 18901 Cranwood Parkway, Cleveland, Ohio.

4Illco-Way Ion X Changer, Research Model 1, Illinois Water Treat-
ment Co., 840 Cedar Street, Rockford, 111.

5American Can Company, Neenah, Wisconsin.

30
pipetted into a centrifuge tube in preparation for protein analysis.
In similar fashion, 5 ml. of 20% trichloroacetic acid, 20 g./100ml.
(w/v), was pipetted into a centrifuge tube for DNA analysis. All of
the tubes were then capped with marbles and refrigerated overnight.

On the day of sample preparation, the tubes containing water, and
those containing 10 and 20% trichloroacetic acid were taken from the
refrigerator and placed in a bed of crushed ice. The samples to be
ground were taken from the freezer and placed on dry ice. As each
organ was processed, it was unwrapped and placed in the first tube of
premeasured cold water. The tube with water and sample was placed in
a 400 ml. beaker filled with crushed ice. The sample was then homog-
enized at speeds 5 and 6 of a Brinkman polytron6 for a time necessary
to insure complete dispersion of the sample. Tweezers were used as
needed for removing fibers which had caught in the blades. The tube
was then removed and placed on ice, and the second tube of water was
used to rinse the blade of the polytron. This portion was then added
to that of the first tube. The blade of the polytron was then rinsed
with distilled deionized water, ethyl alcohol and acetone, in prepara-
tion for the next sample.

While swirling the contents of the ice-clad tube, 1 ml. of homog-
enate was transferred by pipette into the chilled centrifuge tubes for
protein analysis (10% TCA). For DNA analysis, the amount of homogenate
that was transferred toznxfl.tubes (20% TCA) was 0.5 ml. for the 24 day
old rats and 1.0 ml. for the 105 day old rats. The remainder of the

homogenate was topped with parafilm and stored in the freezer until

 

6Brinkman Instruments Inc., Cantiague Rd., Westbury, N.Y.

31

triglyceride analysis could be performed. The contents of the centri-
fuge tubes were mixed on a Vortex test tube mixer7 for several minutes.
The tubes were then placed in a Sorval Model RCZB refrigerated centri-
fuge8 and spun at -15 degrees centigrade and 3,000 r.p.m. for twenty
minutes. At the end of this time, the supernatant was decanted and
discarded from both protein and DNA tubes. The tubes containing the
portion for protein analysis were capped with marbles and refrigerated
at 0 degrees centigrade for analysis within the week. The samples

for DNA assay were processed immediately.

DNA Analysis

 

The method which was employed for DNA analysis was Burton's Modi-
fication of the Diphenylamine Reaction (Burton, 1956). This reaction
is between the deoxypentose of DNA and diphenylamine in a mixture of
glacial acidic acid and sulfuric acid. Acetaldehyde potentiates the
color development. The solutions that were used in the analysis were
prepared as outlined in the Appendix.

To the pellets which remained on ice in the centrifuge tubes
(20% TCA), 6 m1. of room temperature, 5% trichloroacetic acid,
5g./100 m1. (w/v), were added by pipette. The samples were placed in
a hot water bath9 at 90 degrees centigrade for 30 minutes. Any pre-

cipitate adhering to the sides of the tubes was scraped down by

stirring rod into the TCA solution. The samples were then cooled on

 

7Model K-500-2, Scientific Industries Inc., Springfield, Mass.

8Ivan Sorvall Inc., Newtown, Conn.

9Precision Scientific Co., Chicago, Ill.

32
ice, and centrifuged at 3,000 r.p.m. and -15 degrees centigrade, for
10 minutes.

Two mililiter duplicates of the supernatant of each DNA sample
were transferred by pipette into labelled 25 ml. test tubes. To the
tubes were added by pipette, 4 ml. of freshly made diphenylamine-
acetaldehyde solution (Appendix). The contents of the tubes were
mixed on the Vortex test tube mixer, capped with glass marbles and
allowed to stand overnight (16-20 hours) at room temperature.

Two sets of standards were treated similarly. The standard was
calf thymus deoxyribonucleic acid,10 and the solution was made as
described in the Appendix. Appropriate increments of the standard
were diluted with 5% trichloroacetic acid in order to attain a volume
of 2 m1., and concentrations of 0, 10, 20, 3o, 70 and 100 ug/ml. DNA.
The standards were treated with 4 ml. of the diphenylamine-acetaldehyde
solution, mixed, capped with glass marbles and allowed to stand over-
night along with the treated samples.

The next day, absorbance of the samples and standards was read
on a Beckman, Model DB spectrophotometer11 at 700 and 595 nm. The
blank was 2 ml. of 5% trichloroacetic acid which had been treated
along with the standards. If any reading was observed at 700 nm., it
‘was subtracted from the absorbance at 595 nm. There was found to be
little, if any, interference at 700 nm. Duplicates of the samples

agreed within 5%. Absorptivity was calculated for each concentration

 

10Type 1, D 1501, Sigma Chemical Corporation, P.0. Box 4508
St. Louis, Mo.

11Beckman Instruments Inc., 2500 Harbor Blvd., Fullerton, Cal.

33

of the standard. When the mean absorptivity of one set of standards
was not the same as the other, the value of the set which gave the
best standard curve was used. The mean absorptivity was used to
calculate the amount of DNA in the kidneys as follows:

Absorbance, unknown
Mean absorptivity

 

= Concentration DNA, unknown

Concentration DNA, unknown x Total volume 5% TCA Total volume
Aliquot size (supernatant) Sample size (homogenate) x of homogenate

Protein Analysis

Total kidney protein was assessed by the method of Lowry (1951).
The reaction is between Folin—Ciocalteau reagent (molybdate, tungstate,
phosphoric acid) and the proteins, tryptophan and tyrosine. Cupric ion
intensifies the development of the blue color. The solutions used in
the analysis were prepared as outlined in the Appendix.

To the previously refrigerated centrifuge tubes containing the
pellets (10% TCA) for protein analysis was added 5 ml. of 1N NaOH.12
The contents of each tube was mixed on the Vortex mixer until all of
the pellet was in solution.

Into a series of 55 m1. test tubes was pipetted 0.95 ml. of dis-
‘tilled deionized water. After thoroughly redmixing the samples, 0.05
rnl. duplicate aliquots were removed by pipette and added to the tubes
«containing the water. The contents of all of the tubes were then

ttuoroughly mixed. Using the same vortex mixer, 5 m1. of copper sulfate-

sodium carbonate-sodium tartrate solution (Appendix) was added by

 

12USP, J. T. Baker Chemical Co., Phillipsburg, N.J.

34

pipette to each constantly agitated tube. After ten minutes and as
each tube was constantly agitated, 0.5 m1. of 1N Folin-Ciocalteaul3
solution (Appendix) was added to each tube by micropipette. The sam-
ples were allowed to sit at room temperature for 30 minutes, after
which absorbance was read at 740 nm. on the Beckman DB spectrophotometer.

Two sets of standards were run with the samples. The standard was
crystalline bovine albumin,14 and it was prepared as outlined in the
Appendix. Just before use, 1 ml. of the standard stock solution was
pipetted into a 10 m1. volumetric flask. The solution was made to
volume with 1N NaOH. The contents were mixed by inverting the flask
several times. Appropriate increments of the standard were combined
with distilled deionized water to a volume of 1 ml. The set contained
0, 50, 100, 150, 200, 250 and 300 ug/ml. The standards were mixed and
treated with the copper sulfate-sodium carbonate-sodium tartrate and
Folin-Ciocalteau solutions along with the samples. After observing
the absorbance at 740 nm., absorptivity was calculated for each concen-
tration of the standards. Again, if the mean absorptivity of one set
of standards was not the same as the other, the one from the set that
gave the best standard curve was used. The amount of protein in the

samples was calculated by the use of the mean absorptivity, in the

following manner:

 

13Phenol Reagent Solution 2N (Folin—Ciocalteau), SO-P-24, Fisher
Scientific Co., Fair Lawn, N.J.

14
Ohio.

No. 7285, Nutritional Biochemicals Corporation, Cleveland,

35

Absorbance, unknown

Mean absorptivity = Concentration protein, unknown

 

 

Concentration protein, unknown x Total volume NaOH Total
Aliquot size Sample size (homog- x volume of
enate) homogenate

Triglyceride Analysis

Triglyceride was measured by acid hydrolysis according to the
method described by the Association of Official Analytical Chemists
(Horwitz, 1970).

A series of Mojonnier extraction flasks,15 housed in 150 ml.
beakers, was set up on trays. Each flask and beaker was tared on a
Mettler analytical balance,16 and approximately 30 mg. of each kidney
homogenate sample was added to the flask by pasteur pipette. The
homogenate was subjected to constant mixing at speeds 5 and 6 of the
polytron, in between sampling. The blade of the polytron was rinsed
with distilled deionized water, ethyl alcohol, and acetone between
samples. After all of the samples had been weighed, any homogenate
adhering to the walls of the flasks was rinsed down by pipette with
10 ml. of hydrochloric acid (4+1).17 A blank was included in the
series, containing only 10 m1. of hydrochloric acid (4+1). The con-
tents of each flask was mixed by shaking, and the flask was placed

in.a hot water bath18 at 70 degrees centigrade. Duplicate samples were

 

15Model G3, Mojonnier Brothers Co., 4601 West Ohio Street,
Chicago 44, 111.

16Type H 15, Mettler Instrument Corporation Hightstown, N. J.
17Four parts 37% HCl to one part distilled deionized water.

18The hot water baths were 3,000 m1. pyrex, water-filled beakers,
each heated on a Corning, No. PC-35 hot plate, Corning Glass Works,

Corning, N.Y.

36

analyzed where quantity permitted. The hot water bath was brought to
a boil, and the samples and blank were timed for 30 minutes. After this
time the contents of the flasks were cooled to room temperature, and
distilled deionized water was added to the midpoint of the constricted
lower portion of the flask. Twenty-five mililiters of anhydrous ethyl
either19 was added to each flask by graduated cylinder. The flasks
were closed with No. 1 rubber stoppers which had been previously soaked
in acetone. The contents of the flasks were shaken by hand for one
minute.20 Then 25 m1. of petroleum ether21 was added, the stopper for
each flask applied, and the contents shaken for another one minute.
After the flasks had sat unstOppered at room temperature for 30 minutes,
the t0p, clear layer of ether and triglyceride was decanted into tared
Goldfisch extraction beakers.22 The beakers had previously been washed
in hot soapy water, rinsed in tap and distilled water and acetone, and
dried in an oven23 for at least one hour. Thirty minutes before
weighing, the beakers were placed in a desicator for cooling and drying.

The extraction with ethyl and petroleum ether was repeated two more
times, with intermittent shaking and standing at room temperature for
30 minutes. Before the last extraction, distilled water was added where
needed in order to bring the contents to the same level at the bend

of the flask. After each extraction, the decanted solvent was added

 

19Analytic reagent, Mallinckrodt Chemical Works, St. Louis, Mo.

20In lieu of a centrifuge.

21(30-60)0 C, Analytic reagent, Mallinckrodt Chemical Works,
St. Louis, Mo.

22Corning Glass Works, Corning N.J.
23Precision Scientific Co., Chicago, Ill.

37
to that of the first extraction.
The ether from the extraction was allowed to evaporate from the
beakers overnight24 under the hood. The next day the beakers were
placed in an oven at 70 degrees centigrade for one hour. They were then
transferred to desicators where they remained for 30 minutes. The
beakers were weighed, and the total triglyceride and percent triglyceride

were calculated by difference, in the following manner:

Beaker wt.final - Beaker wt.initial = Wt. triglyceride, aliquot

and
Blank

Wt. triglyceride, aliquot

Wt. aliquot + 10 (dilution) = A trlglyceride

% triglyceride x Kidney wt. = Total kidney triglyceride

Methods of Statistical Analysis

The differences in the data brought about by the variables Age,
Strain, Diet, Supplementation and Litter Size as well as interactions
between those variables were analyzed by use of the five-way multivariate
analysis of variance and univariate analysis of variance. These statis-
tics along with group means and standard deviations were compiled at the
IMichigan State University Computer Center. There were no S-way nor
any 4-way interactions which significantly influenced the results, and

the majority of 3-way interactions involved the variables Age, Strain
and Diet. For this reason, the data was grouped by Age and Strain, and
the differences brought about by Diet tested for significance by use of

the Student T Test.

 

24Or solvents could have been evaporated over a steam bath.

RESULTS AND DISCUSSION

Total Body Weight

 

Tables 3 and 4 show the mean live weights of rats from each treat-
ment of the study at 24 and 105 days of age. There was a significant
(P<0.01) increase in body weight with age for all groups within each
strain, as recorded in Table 5. S 5B/Pl rats weighed approximately six
times as much at 105 days as at 24 days, while Osborne-Mendel rats
weighed approximately seven times as much. The weight gain of the
Osborne-Mendel rats fed the high fat diet was greater than in those fed
the low fat diet for all treatments. In the S 5B/Pl strain, however,
feeding a high fat diet was responsible for a greater gain in weight in
only one out of the four treatments (HF, Su, 6/L).

Total body weights of the Osborne-Mendel rats were significantly
greater (P<0.01) than those of the S 5B/Pl rats in all similar treat-
ments (Table 5). At 24 days, mean body weights of the S SB/Pl rats
ranged from 47 to 55 g., while those of the Osborne-Mendel strain
ranged from 66 to 90 g. The range in body weights at 105 days was 259
to 329 g. for the S SB/Pl rats, and 433 to 634 g. for the Osborne-Mendel
rats. The lightest rat at 105 days of the study weighed 234 g. and
was from the S 5B/Pl strain (LF/6/L, Su), while the heaviest rat was
an Osborne-Mendel (HF, 6/L, Su) and weighed 721 g.

There was a significant main effect of diet (P<0.01) on body weight

in the Osborne-Mendel strain at both 24 and 105 days of age (Figure l).

38

39

Osborne-Mendel weanling rats that had been nursed by dams fed the high
fat diet weighed 13 g. more than Osborne-Mendel offspring nursed from
dams fed the low fat diet. By 105 days of age, Osborne—Mendel rats
fed the high fat diet had a mean body weight 128 g. greater than those
fed the low fat diet (461 vs. 589 g.). There was no significant effect
of diet on body weight in the S 5B/Pl strain at either 24 or 105 days.
The differences in mean body weight between S 5B/Pl rats maintained on
high and low fat diets were 3 g. at 24 days and 18 g. at 105 days.

Supplementation or changing litter size from six to three had
little effect on body weight (Figure 2). There was no five-way inter-
action of Age, Strain, Diet, Supplementation and Litter Size which
significantly increased values for body weight, nor were there any sig-
nificant four-way interactions (Table 5). The three-way interactions
of Age, Strain and Diet, and Diet, Supplementation, and Litter Size
were found to be significant. The former three-way interaction, how-

ever, was the most relevant in terms of this study (Figure 1).

Total Kidney Weight

 

Mean weights of the kidneys of the various groups of rats are
presented in Tables 3 and 4. Kidney weights increased significantly
(P<0.01) with age (Table 5). The weights of the kidneys of all rats
at 105 days were about four times the weights at 24 days. Although
the kidneys of the S 5B/P1 rats weighed less than those of the Osborne—
Mendel, the increase in weight between 24 and 105 days was of the same
magnitude in both strains. Feeding Osborne-Mendel rats a high fat diet
led to greater gains in kidney weight with age than feeding a low fat

diet in all treatments except 6/L, NS, where the weight gain was the

40
same. In the S SB/Pl strain, all groups of rats fed the high fat diet
experienced a greater kidney weight gain with age than did those fed
the low fat diet.

There was a significant (P<0.01) strain difference in kidney weight
(Table 5). The mean weights of the kidneys of the S SB/Pl rats ranged
from 0.48 to 0.58 g. at 24 days and 1.74 to 2.38 g. at 105 days. The
range of weights in the Osborne-Mendel strain was 0.63 to 0.85 g. at
24 days and 2.51 to 3.51 g. at 105 days. For comparable groups, the
kidney weights of the S SB/Pl rats were 70-75% as great as those in the
Osborne-Mendel strain.

There was a significant effect (P<0.01) of diet on the kidney
weights of the animals in this study (Table 5 and Figure 3). Kidneys
weighed significantly more in rats fed the high fat diet than in rats
fed the low fat diet, regardless of age or strain. At weaning, the
mean kidney weight of the S 5B/Pl rats nursed from dams fed the high
fat diet was 12% greater than that of S 5B/Pl rats nursed from dams
fed the low fat diet (0.56 vs. 0.50 g.). In the Osborne-Mendel strain
the difference was 16% (0.78 vs. 0.67 g.). By 105 days, S 5B/Pl and
Osborne-Mendel rats maintained on the high fat diet had mean kidney
‘weights l9 and 21% greater, respectively, than those maintained on the
low fat diet. The mean weights, in grams were 2.22 vs. 1.88 for the
S SB/Pl strain, and 3.19 vs. 2.61 for the OsborneeMendel.

There were no significant main effects of supplementation or

litter size on kidney weight (Table 5 and Figure 4).

41

Kidney Weight/Body Weight Ratio

 

Tables 3 and 4 list the mean kidney weight/100 grams body weight
(KW/BW) ratios for rats exposed to each of the thirty-two treatments of
the study. The KW/BW ratios decreased in both strains with age. This
is consistent with the develOpmental relationship that has been observed
between organ weight and body weight in which, following birth, the
KW/BW ratio increases to a peak level, and then declines with age
(Solomon and Bengele, 1973). At weaning, the mean KW/BW ratio for all
S SB/Pl rats was 1.04 compared to 0.94 for the Osborne-Mendel rats. At
105 days, the ratios were 0.71 and 0.56, respectively. When the kidney
weights are expressed on the basis of lean body mass1 (Tables 3 and 4),
there is no difference in the relative size of the kidneys in the two
strains at 24 days. Thus, greater body fat accounts for the lower KW/BW
ratio in the 24 day old, Osborne-Mendel rats. There may, however, be a
strain difference in kidney weight by adulthood. The kidney weight/lean
body mass ratio for 105 day old S 5B/Pl rats is 0.92 compared to 0.87
for the Osborne-Mendel strain. The adult S SB/Pl rats have relatively
larger kidneys than the Osborne-Mendel rats. The greater KW/BW ratio
in this strain is not entirely due to the Osborne-Mendel animals
having a greater amount of body fat than the S 5B/P1 rats.

Osborne-Mendel rats maintained on the high fat diet had the same
KW/BW ratios, at both ages, as did the Osborne-Mendel rats maintained
on the low fat diet. This finding is in disagreement with those of

other overfeeding studies (Johnson, 1972; Solomon and Bengele, 1973)

 

1As determined by carcass weight minus ether extracted fat.
Heart, brain, liver, kidneys, gastrocnemius muscle and three fat
depots had been removed before lipid extraction.

42

and can be explained by the fact that the control diet in this study
was a semi—purified low fat ration, rather than a grain ration as used
by Johnson (1972) and Solomon, etc. It has been found in our laboratory
that those rats maintained on a semi-purified ration show greater weight
gain than those fed grain (Winnie EE.El°’ 1973). The body weights of
the Osborne-Mendel control rats in this study are conspicuously greater
at 24 and 105 days than are those reported by Johnson (1972). On the
other hand, the kidneys are comparatively of the same weight.

The KW/BW ratio in the S SB/Pl rats was slightly higher in the
high fat-fed animals. At 24 days, the ratio was 1.07 for rats weaned
from high fat dams and 1.02 for those weaned from low fat dams. At 105
days, the ratios were 0.75 and 0.68 respectively.

From the data in Tables 3 and 4, it may be seen that kidney weight
relative to lean body mass was greater in the high fat-fed rats regard-
less of age or strain. These rats had proportionately larger kidneys

than did those fed the low fat diet.

Total Kidney DNA

 

The mean values for kidney DNA are shown in Table 6. There was
a significant (P<0.01) increase in DNA with age (Table 5). In both
strains, kidneys contained approximately two times the amount of DNA
at 105 days as at 24 days. The mean total DNA content of all S 5B/P1
rats at 24 days was 2.18 mg. and at 105 days, 4.06 mg. In the Osborne-
Mendel strain, the mean total DNA content was 2.54 mg. and 5.87 mg.,
respectively. In all treatments, the Osborne-Mendel rats fed the high
fat diet gained more kidney DNA between 24 and 105 days than did those

fed the low fat diet. In only one of the four treatments (6/L, Su) in

43
the S 5B/Pl strain was the gain in DNA with age greater on the high
fat diet.

There was a significant (P<0.01) main effect of strain on total
kidney DNA in this study (Table 5). At 105 days, mean values for DNA
ranged from 4.91 mg. to 7.25 mg. in the Osborne-Mendel strain, and
3.75 mg. to 4.31 mg. in the S SB/Pl. At 24 days, the kidneys of
Osborne-Mendel rats contained 0.36 mg., or 17% more DNA than those of
the S SB/Pl rats.

There was neither a five-way interaction, nor any four—way inter-
actions which caused significantly greater scores for DNA. There was
a significant (P<0.01) main effect of Diet, as well as Age and Strain
as discussed above.

The three-way interaction of Age, Strain and Diet significantly
(P<0.01) affected DNA values in all but the 105 day old S 5B/P1 rats
(Figure 5). The kidneys of rats that had nursed from high fat dams
contained significantly more cells at weaning than those rats that had
been nursed from low fat dams. This was true for both strains, although
the difference was only 8% in the S 5B/Pl rats. The greater cell
number in the Osborne-Mendel strain was a reflection of 2.75 mg. DNA
for the high fat animals and 2.32 mg. for the low fat animals. In the
S 5B/P1 strain, the mean DNA content was 2.27 mg. and 2.09 mg., respec-
tively.

By the time the low fat, S 5B/Pl rats reached 105 days, they had
demonstrated catch-up growth in regard to cell number. The rats that
had been maintained on a high fat diet had 4.11 mg. DNA, and those
maintained on low fat, 4.00 mg. DNA.

The Diet effect on DNA was greatest in the Osborne-Mendel strain

44

by 105 days. There was 27% more DNA in the kidneys of the Osborne—
Mendel, high fat rats than in those of the low fat rats at this age.
This is represented by mean values of 5.17 mg. DNA for Osborne-Mendel,
low fat-fed rats and 6.57 mg. for those fed the high fat diet. Between
24 and 105 days of age, Osborne-Mendel rats fed the high fat diet
gained 3.82 mg. kidney DNA and those fed the low fat diet, 2.85 mg. DNA.

The greatest amount of kidney DNA measured was 7.25 mg. in the
Osborne-Mendel, high fat, 3/litter, nonsupplemented group. Supplementa-
tion or changing litter size from 6 to 3 rats had no significant effect

on kidney DNA (Table 5 and Figure 6).

Total Kidney Protein

The protein content of the kidneys of all rats in this study in-
creased significantly (P<0.01) between 24 and 105 days of age (Table 5).
The gain by 105 days represented four times the kidney protein at 24
days in both strains. The kidneys of 24 day old S 5B/P1 rats contained
a mean of 58 mg. protein and the 105 day old 8 SB/Pl, a mean of 239 mg.
Values for the Osborne-Mendel rats were 81 mg. and 359 mg. respectively.
In all comparable treatments, in both strains, those rats maintained on
a high fat diet had greater gains in kidney protein than did those
maintained on low fat. The mean gain in kidney protein for the Osborne-
Mendel, low fat rats was 252 mg. while that of the Osborne-Mendel high
fat was 304 mg. Gains for similar groups in the S 5B/P1 strain were
167 mg. and 196 mg., respectively.

There was a significant (P<0.01) main effect of strain on the
protein content of the kidneys (Table 5). For all comparable treat-

ments, higher values were observed in the Osborne-Mendel strain. At

45
weaning, mean total kidney protein in the S SB/Pl rats ranged from
53 to 73 mg., while the range in the Osborne-Mendel rats was 58 to
94 mg. (Table 6). At 105 days, the respective ranges were 204 to 308
mg. and 296 to 418 mg.

The variable, Diet, significantly (P<0.01) affected kidney protein
(Table 5). The kidneys of rats that had been weaned from, or maintained
on, the high fat diet had 15 to 20% more protein than those on the low
fat diet. This may be illustrated by considering the interaction of
Age, Strain and Diet (Figure 7). The kidneys of the rats fed the high
fat diet contained more protein than those of rats fed the low fat diet.
This was significant at 24 days and at 105 days in the Osborne-Mendel
strain. The significance was at the 5% level in the S 5B/Pl, 24 day
old rats. Although there appeared to be a significant difference
between the mean protein content of the kidneys of 105 day old,

S 5B/Pl rats fed high and low fat diets, the significance was con-
tributed by only one of the four treatments (6/L, NS). Because the
mean value for this group of rats is considerably out of line, for no
apparent reason, the significance is questionable. The mean values
'which the graph represents at 24 days are: S 5B/P1, low fat, 55 mg.;
S SB/Pl, high fat, 62 mg.; Osborne-Mendel, low fat, 74 mg.; Osborne-
iMendel, high fat, 88 mg. The respective mean values at 105 days are:
S 5B/Pl, 222 mg. and 257 mg.; Osborne-Mendel, 326 mg. and 392 mg.

There was no significant main effect of Litter Size on kidney
‘protein (Table 5 and Figure 8). The main effect which surfaced for
Supplementation was the result of nonsupplemented rats having more
kidney protein than supplemented. At 24 days, supplemented rats of

‘both.strains had a mean of 66 mg. of kidney protein compared to 71 mg.

46
for those nonsupplemented. At 105 days, the mean values were 288 and
309 mg., respectively. Obviously, supplementing the diet exerted no
positive effect on kidney protein. There was no significant five-way
interaction apparent, nor were there any significant four-way inter-

actions.

Protein/lOO grams Kidney Weight
Values for protein/100 grams kidney weight are presented in Table
7. At 24 days kidneys in both strains contained approximately 11%
protein, and at 105 days, 12%. These percentages are lower than those
reported by others (Winnick and Noble, 1966, 1967; Spector, 1956).
Nowinski and G033 (1969) report kidney protein content in rats of

around 14%.

Protein/DNA Ratio

 

The protein/DNA ratios for all groups of rats are shown in Table 8.
The ratios doubled in both strains between 24 and 105 days of age. The
ratio in the S SB/Pl strain increased from a mean of 27 to 59 while
the increase in the Osborne-Mendel rats was from 32 to 61. The cells
in the kidneys of S SB/Pl rats were smaller at both ages than in com-
parable Osborne-Mendel rats. There was no difference in the protein/DNA
ratio of the low fat and high fat rats within each strain and age, indi-
cating that the cells were of the same size. The exception, again,
was in the S 5B/P1, 105 day old, high fat, nonsupplemented group where

the unexpected higher protein value influenced the ratio.

47

Total Kidney Triglyceride

 

Results of the ether extraction of kidney triglyceride are pre-
sented in Table 6. The kidney triglyceride content in all groups of
rats increased significantly with age (P<0.01), while triglyceride as
a percent of kidney weight remained relatively constant at about three
percent (Tables 5 and 9). The kidney triglyceride of Osborne-Mendel
and S SB/Pl rats at 105 days was three to four times that at 24 days.

There was a significant (P<0.01) main effect of strain on kidney
triglyceride (Table 5). At 24 days of age the kidneys of S SB/Pl rats
contained a mean of 18 mg. of triglyceride, while those of the Osborne-
Mendel rats contained 21 mg. By 105 days, the triglyceride content
was 62 and 83 mg., respectively.

The effect of feeding a high fat diet is most clearly shown by the
three-way interaction of Age, Strain and Diet (Figure 9). The trigly-
ceride content of the kidneys of rats fed the high fat diet was signifi-
cantly greater than in those fed the low fat diet in both strains at
24 days, and in the S 5B/P1 strain at 105 days of age. At weaning,
kidneys of S SB/Pl rats that had suckled from high fat dams had a mean
triglyceride content of 21 mg. compared to 15 mg. for those suckled from
low fat dams. At this age, in the Osborne-Mendel strain, the values
were 24 mg. and 17 mg., respectively. By 105 days, mean triglyceride
in the kidneys of S 5B/Pl rats fed the high fat diet was 70 mg. in
contrast to 55 mg. in those rats fed the low fat diet. The kidneys of
105 day old Osborne-Mendel rats contained 88 mg. and 78 mg. for rats
fed high and low fat diets, respectively. Although the difference in
triglyceride between the two diets was significant for both strains at

weaning and for the S SB/Pl rats at 105 days, the range of triglyceride

48
in the kidney was only 2 to 4%. Whether the differences are of any
biological significance is questionable. In any event, triglyceride
was the one substance measured which most contributed to the increased
kidney weight of the S 5B/P1 rats fed the high fat diet. Also, S SB/Pl
rats had equally as great a percentage of kidney triglyceride as did
the Osborne-Mendel rats (Table 9).

There was no main effect of supplementation on kidney triglyceride
(Table 5 and Figure 10). In regard to litter size, 24 day old rats of
both strains combined that were raised six per litter had a mean kidney
triglyceride value of 19.1 mg., or 3.12%. Rats of both strains com-
bined that were raised three per litter had 19 mg. kidney triglyceride,
or 3.08%. While litter size had exerted no effect at 24 days, there
was a slight effect at 105 days. Rats of both strains raised six per
litter had a mean triglyceride content of 64.6 mg. (3.06%), and those
raised in litters of three, 56.2 mg. (2.57%). There was no significant
five-way interaction of variables, nor were there any significant four-

way interactions.

49

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no.onom.H so.onmo.a mo.onem.o Newm snmm g\m .:m .m: .m
so.onms.fl qo.oaoa.a mo.oaqm.o Hahn «was q\o .:m .m: .m
«H.0nom.a NH.oneo.H so.onmm.o «Ham snmm q\m .mz .mm .m
oa.onmq.a mo.on~o.fi No.OHsm.o mnwm maom g\o .mz .mm .m
mm.oan.H eo.oaso.a so.onmm.o when mnHm a\m .sm .mg .m
«H.0nqm.a no.oaqo.a mo.oams.o neon mans q\c .sm .mg .m
Ho.onmm.fi Ho.onmm.o mo.onas.o whom mass q\m .m2 .m4 .m
mo.oafim.a so.oa~o.fi so.oan.o Hahn «nee A\e .mz .mg .m
2mg mooa\w 3m .wooH\w va .us swoon va .us Amy .63 uamaumoue

.us Amv amaeax 2mg w>HH .umm sum camuum

 

mucoEuoouu msowum> ou womoaxo mumu HoodoZIoauonmo pom Hm\mm m mac awe «N you mmma moon coma
.wooa\va zocwfix .usmfioz zoos .wooa\va hoaufix .ustoB ANV %o=ufix .mmma keen coma .unwwos upon o>fiq

52

 

 

 

mH. chew. o oo.onmm.o Hq.onmo.m omaosm mmamom H\m .am .m: .zo
mo. onso. H mo. onsm. o mm.oaHs.m omHmNm onnsmo H\e .:m .m: .zo
mo. came. H so.onoo.o um.oHHm.m quaoqm NNHmmm H\m .mz .m: .20
HH. onHw. o so. ones. o Hm.onmn.~ amassm oeHHhm H\o .mz .m: .20
mo. one“. o m9 onmm. o Hm.onHm.~ NHHomm Hsawma H\m .sm .mH .2o
09 once. o 49 onmm. o m~.onom.~ oNHmmm «HHst H\o .sm .mH .zo
m9 chem. o 09 on9 o «H.ono~.~ NNHHNM Nmnmms H\m .mz .mH .zo
NH. oHNm. o 59 chum. o o~.onwe.~ aunmmm omHoes H\o .mz .mH .zo
aH.onma.o e9 ones. o m~.onou.~ canemm omamom H\m .sm .mm .m
mo.onHo.H mo.oH-.o os.onwm.~ omnemm mmnmmm H\o .:m .m: .m
mo.onom.o Ho.onen.o H~.oH~H.~ oHHmHN oNHNNN H\m .mz .mm .m
mo.onam o No.onwa.o oH.onH.~ «HHNNN mHHNmN H\o .mz .m: .m
mo.onow.o No.oHso.o eH.OHom.H mHHHNN omnwam H\m .sm .mH .m
MH.onHw.o mo.onno.o AH.onse.H msHmHN NHHmmN H\o .sm .mH .m
mo.oamw.o so.onme.o mm.onoa.H OHHmHN HNHowN H\m .mz .mH .m
«c.0H~¢.o ~o.oHH~.o OH.onmm.H mHmHN enmem H\e .mz .mH .m
zmH.wOOH\w 3m .wooH\w va .63 gauge Ame .63 Hmv .ua unmaummue

.63 HNV.NuceHM zmH m>HH .umm ecu aHmuum

 

muomaumouu moowum> cu ommooxo moon HopaozIocuonmo can Hm\mm m uao hop moa you name moon coma
.wooH\va xoopax .unwwm3 anon .wooa\awv moccwx .unwfios ANV hoovfix .mmma Amos coma .unwaoa moon m>Hq

q mqm<e

53

TABLE 5

RESULTS OF THE MULTIVARIATE ANALYSIS OF AGE, STRAIN, DIET,
SUPPLEMENTATION AND LITTER SIZE ON BODY WEIGHT, KIDNEY (2)
WEIGHT, AND KIDNEY DNA, PROTEIN AND TRIGLYCERIDE

 

 

 

 

Source of Mean sq. of F P-less
variation df hypothesis value than
Body weight
Age 1 4465073.6465 6245.4069 0.0001*
Strain 1 586041.7649 819.7108 0.0001*
Diet 1 42524.8615 59.4806 0.0001*
Supplementation l 1387.6920 1.9410 0.1662
Litter size 1 9877.5679 13.8160 0.0004*
Interactions:
2 way 10 50421.7199 70.5261 0.0001*
3 way 10 3557.2903 4.9757 0.0001*
4 way 5 1381.4075 1.9322 0.0939
5 way 1 229.9793 0.3217 0.5717
Kidney weight
Age 1 130.5062 3510.4294 0.0001*
Strain 1 8.7419 235.1439 0.0001*
Diet 1 2.0462 55.0409 0.0001*
Supplementation 1 0.0058 0.1565 0.6931
Litter size 1 0.0437 1.1752 0.2805
Interactions:
2 way 10 0.5856 15.7524 0.0001*
3 way 10 0.1136 3.0549 0.0018*
4 way 5 0.1213 3.2641 0.0085*
5 way 1 0.0613 1.6476 0.2018
22!:
Age 1 189.8173 970.9182 0.0001*
Strain 1 28.3639 145.0817 0.0001*
Diet 1 6.0126 30.7543 0.0001*
Supplementation 1 0.1413 0.7225 0.3970
Litter size 1 0.0060 0.0304 0.8618
Interactions:
2 way 10 2.0558 10.5152 0.0001*
3 way 10 0.4954 2.5339 0.0083*
4 way 5 0.4832 2.4718 0.0361
5 way 1 0.2564 1.3114 0.2545

54

TABLE 5--Continued

 

 

 

Source of Mean sq. of F P-less
variation df hypothesis value than
Protein
Age 1 1400176.0018 1568.1193 0.0001*
Strain l 116127.7961 130.0567 0.0001*
Diet 1 16648.0414 18.6449 0.0001*
Supplementation l 6520.0996 7.3021 0.0079*
Litter size 1 1623.8295 1.8186 0.1800
Interactions:
2 way 10 8069.4217 9.0373 0.0001*
3 way 10 1461.4565 1.6368 0.1041
4 way 5 1304.4736 1.4609 0.2077
5 way 1 361.6080 0.4050 0.5258
Triglyceride
Age 1 109680.2168 642.5258 0.0001*
Strain l 4296.5223 25.1698 0.0001*
Diet 1 2595.2805 15.2036 0.0002*
Supplementation 1 162.2318 0.9504 0.3316
Litter size 1 1401.1060 8.2079 0.0050*
Interactions:
2 way 10 646.2842 3.7860 0.0002*
3 way 10 231.2761 1.3549 0.2096
4 way 5 208.6060 1.2221 0.3030
5 way 1 445.6258 2.6106 0.1088

 

55

 

 

 

 

 

9 m~+9 mm «AHmam oe.on-.e ~.~H~.- NHHmm an. OHNH. H H\m .am .mm .26
o.a~Hw.mw maHmHs ~N.HHma.e o.~nm.o~ mnso HH. onem. H H\o .am .m: .zo
s. OH+9 ea smasmm mo.~HmN.e o.oHH.m~ anom wH. case. N H\m .mz .mm .26
N. m Ha. om mmnmem em.onm~.m s.MHHm.- «HHHw om. onm9 N H\o .mz .mm .zc
m.a Hm.oo wmneam so.OHHa.s H.mH~.eH oHHmH o9 onw9 ~ H\m .am .aH .zc
9 mHH9 mm mmnoom oo.ono.m ~.HHw.sH aamm he. sham. H H\o .sm .mH .zo
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H.omHm.Hm «NHMNN ms.oHHm.s ~.HHN.H~ «Hon oH.onH.~ H\o .:m .m: .m
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meme moH meme «N
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hmcowx Hmuoe wmcpfix HmuOH mam :fimuum

 

mHzmze<mme mDOHm<> OH numomxm mH<M AMQZMZImzmommO Qz<
Hm\mm m 040 w<m mOH Qz< «N we mnHmmowduHMB Qz< szeomm .<ZQ an VMZQHM A<HOH

o mqde

56

 

 

 

 

 

 

 

 

 

 

«n.0H om.NH mo.HH mm.HH oh.~a oo.HH em.HH ww.NH
wq.m mo.¢H qw.HH Nw.HH 0H.~H m~.ma MH.NH 0H.MH
am .Hma. am .mmm. am .Nmm. am .mmm.
mm MA mm mu
Hm\mm m Housmanocuonmo
meme eHo.Nwo moH
sq.m om.NH oo.HH co.HH «N.HH oo.HH mn.oa HQ.HH
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am .mmm. am .mmw. am .mmm. am .mmm.
mm mA mm ma
Hm\mm m HmvanImGHonmo

 

 

 

 

 

 

 

 

 

 

 

 

 

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57

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mm no mm om no em co mm
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58

 

 

 

 

 

 

 

 

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59

FIGURE I

MEAN BODY WEIGHTS OF 24 AND 105 DAY OLD S SB/Pl AND OSBORNE-MENDEL
RATS FED EITHER A HIGH FAT OR A LOW FAT DIET

BODY WEIGHT (9)

moo

A00

N00

 

Nb o><m

ESE—um
D1...

2.." No «on 8.. 9.96

mm w\_u_.

 

Zmbz moo< $991.3
5w 03$

0.2. mm m\ u—

0.2..

60

FIGURE 2

TOTAL BODY WEIGHTS OF 24 AND 105 DAY OLD S SB/Pl AND OSBORNE-MENDEL
RATS EXPOSED TO VARIOUS TREATMENTS

moo

BODY WEIGHT (9)
J:
O
0

N00

 

ZN): 83 $391.3.
2:.- ammagmzam

NA g<w

Earn
Dim.

AI. .58:
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2
IE

   

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is

 

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61

FIGURE 3

MEAN KIDNEY (2) WEIGHTS OF 24 AND 105 DAY OLD S SB/Pl AND OSBORNE-MENDEL
RATS FED EITHER A HIGH FAT OR A LOW FAT DIET

ZMDZ x52m< E Smaxfim

Wm NA U><m
Ea. Cu
ab _H_ _.__u
z... No 33 3.. 9.96
M. Na
T
w No
a
W
Y. _.m
E
N
D
m _.0
00

mm m\_u_ 0.2..

.00 0><w

mm m}:

 

0.2..

 

62

FIGURE 4

TOTAL KIDNEY WEIGHTS 0F 24 AND 105 DAY OLD s SB/Pl AND
OSBORNE-MENDEL RATS EXPOSED TO VARIOUS TREATMENTS

KIDNEY WEIGHT (9)

ob v

uh ..

_.m..

R

Oh

 

 

NA 96

ES E"
U 3."

4| :3...
z u a 33 3.. 035

  

__:___ ______ ______
Ea

mmm\_u_

SET;

Zm>z Eczmim vim—@149
2...: 439...!ng

 

 

 

.00 9a

 

 

 

 

 

 

63

FIGURE 5

MEAN KIDNEY (2) DNA OF 24 AND 105 DAY OLD S SB/Pl AND OSBORNE-
MENDEL RATS FED EITHER A HIGH FAT OR A LOW FAT DIET

 

KIDNEY DNA (mg)

Zm>z x_ozm< Amy 02>

NO NA o><m
as: .- m.

D ID
zumo «3m 3.. 9.9.6

5m U><m

    
  

.05
O

 

5"
O

:15
O

9‘

N
O

6

mm m\_u_ 0?. mm m\ 2 OZ.

64

FIGURE 6

TOTAL DNA IN KIDNEYS OF 24 AND 105 DAY OLD S SB/Pl AND
OSBORNE-MENDEL RATS EXPOSED TO VARIOUS TREATMENTS

gmbz .2on «NV 02).

Z... 3m>43m24w

 

  

  

 

 

 

 

 

 

 

 

_. no 96 .8 95a]
.3 II
EE— Cu
mo- .U :m
Imam. II. 33:
M a z u 033 2: 93.5
WAC l_ ......... .222
E
m
uIn. u .
II __________ II I
N .
o i. EE$E SEER SEES
mum)”.— 02. www\ v. 0.2.

 

65

FIGURE 7

MEAN KIDNEY (2) PROTEIN OF 24 AND 105 DAY OLD S SB/Pl AND OSBORNE-
MENDEL RATS FED EITHER A HIGH FAT OR A LOW FAT DIET

 

KIDNEY PROTEIN (mg)

Amm

umm

NNm

.mm

mm

Zm>z 502m<amv .um0._.m_z
NA 0.940 .00 0><m

Saar...
DI...

z u mo 33. 6a.. 9.96

an 0.00

mum}: 0.2. mm m):

 

0.2..

66

FIGURE 8

TOTAL PROTEIN IN KIDNEYS OF 24 AND 105 DAY OLD S SB/Pl AND
OSBORNE-MENDEL RATS EXPOSED TO VARIOUS TREATMENTS

 

 

     

    

 

 

 

 

 

 

 

 

 

i. 47. .42. W 4. +1 mm
693$ 666$ 936$ ...............
.. 8.
I
I I DNN
266 6a 29. n u z
I. :8: I.
II I mun
E: D
I A... 99
[I 93 no. 93 E I mmc

 

mhzwstkwmk .._I_<
.z_w._.0ma “N v rwsz. z<w2

(6w) NIBIOHd A3NOI>I

67

FIGURE 9

MEAN KIDNEY (2) TRIGLYCERIDE OF 24 AND 105 DAY OLD S SB/Pl AND
OSBORNE-MENDEL RATS FED EITHER A HIGH FAT OR A LOW FAT DIET

§m>z x.02m< AN. ....m.0.:<0mw_0m

.00 NA 0.940 .00 0><m

EEC...

D I...
2» N0 33 63.. 9.96

m
0

O)
O

A
O

 

KIDNEY TRIGLYCERIDE (mg)

N
0

mm m\_u_ 0...... mm m\.u_. 0.2..

68

FIGURE 10

TRIGLYCERIDE CONTENT OF KIDNEYS 0F 24 AND 105 DAY OLD S SB/Pl
AND OSBORNE-MENDEL RATS EXPOSED TO VARIOUS TREATMENTS

KIDNEY TRIGLYCERIDE (m9 )

 

 

£m>z x.02m<a N. a.m.—Romaom .
>5- 3m>42mzflw

NA 0.96 .00 0><0

E... Cu :55]
D 3."

Al :00: .II..
2 u 033 62636

T

1

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r
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.99 9999Fl
0.;

_ .3391. . . :02... . 3331

 

CONCLUSIONS

Age
Although the ages of 24 and 105 days are not ideal for the study

of renal growth exclusively, they are acceptable in light of the fact
that the original research was designed with several purposes in mind.
The overall growth of whole body and individual organs and tissues was
to be assessed in relation to the influence of early overfeeding prac-
tices. One must remember when reviewing these data, that nephrogenesis
in the rat is complete by three or four weeks of age (Kittelson, 1917;
Arataki, 1926; Boss g£_§l., 1963; Bengele and Solomon, 1974). Because
cell division is complete on or around 44 days (Winick and Noble, 1965)
it must be realized that any increase in DNA occurs before then. In
reference to protein, its accumulation begins to plateau at about 45
or 50 days of age (Priestley and Malt, 1968).

The variable, Age, led to an increase in all indices measured.
All animals in both strains had kidneys at 105 days which weighed four
times as much as they had.at 24 days. The increase in weight was
reflected by a doubling in the number of cells, and in the protein/DNA
ratio. These findings are similar to those of Winick and Noble (1965,

1967).

Strain
The S SB/Pl and Osborne-Mendel rats responded differently to

exPerimentally induced overnutrition begun in the suckling period.

69

70

By weaning, Osborne-Mendel pups that had been nursed by dams fed a

high fat diet weighed 13 g. more than those nursed by dams fed a low
fat diet. The respective difference in the S SB/Pl strain was 3 g.

The strain difference was even more apparent by 105 days when the
difference between the experimental and control Osborne-Mendel rats

was 128 g. and between the S 5B/Pl rats, 18 g. Clearly the Osborne-
Mendel rat is a good model for studying the ramifications of overnutri-
tion. In this species of animal there appears to be a genetic differ—
ence in the propensity toward obesity, as evidenced by the response of
the S 5B/Pl rat. Providing this strain of rat with a more calorically
dense milk during the suckling period and a post-weaning calorically
dense diet, to say nothing of force—feeding and reducing litter size,
had no effect on the rate of growth. Schemmel g£_§l. (1970) did, in
fact, find that body weight gain in seven strains of rats fed a high
fat (60% w/w) diet was influenced by genetics. The S SB/Pl rat showed
minimal response. In a later study, Schemmel g£_§l. (1972) showed that
the S SB/Pl rat eats less food than the Osborne-Mendel, and converts
food calories to body energy less effectively. Fenton and Carr (1951)
also found in.mice that the effect of the fat content of the ration on

weight gain was dependent upon the strain of animal employed.

Diet
The overfeeding technique which exerted the greatest influence on
growth was feeding a high fat diet. Osborne-Mendel rats maintained on
a high fat diet had significantly greater body and kidney weights at
both ages than did their controls. The increased kidney weight at 24

days was primarily the result of the organ containing a greater number

71

of cells, although triglyceride was also increased. The greater kidney
weight at 105 days was also due to the acquisition of more cells, the
organs of the experimental group containing 27% more DNA than those of
the controls. There was no difference at either age in the protein/DNA
ratio between Osborne-Mendel rats fed high fat or low fat diets, indi-
cating that the cells were of the same size.

It may be concluded that feeding the Osborne—Mendel rat a high fat
diet from birth caused accelerated growth of the kidney as well as the
whole body. The more pronounced growth of the kidney was a function of
cell division. These findings are in agreement with those of Winick and
Noble (1967), who concluded that increasing the number of calories
available to the young rat accelerates growth by increasing the rate of
cell division.

Supplemental Feeding and
Litter Size Reduction

 

 

Supplemental force-feeding and litter size reduction have each been
effective in accelerating growth, when used independently. Whether
either of these techniques was influential to the outcome of this study
is questionable (see also, Harris g£_§l., 1977). The over-riding effect
of feeding a high fat diet may have masked the contribution of either
technique.

The technical details of force feeding, in this experiment, were
implemented and supervised by one who had perfected the method (Czajka-
Narins, 1974), ruling out the possibility of faulty delivery of the
supplement. Giving more than two supplemental feedings a day, or using
a random schedule may have been helpful. The latter adjustment would

rule out the possibility that the pups anticipated the feedings and

72

decreased their food intake beforehand (WUrtman and Miller, 1976b).
Supplemental force-feeding may have also indirectly affected the amount
of milk available from the dams by causing the satiated pups to suck
less and thus diminish the stimulus for increased milk production.

It has not been clearly confirmed that growth in the rat is
inversely related to litter size due to the availability of milk. To
the contrary, Winick and Noble (1967) found no difference in growth
parameters of Sprague Dawley rats raised three or six per litter.
Clearly growth rate may be influenced by using extremes in litter size
(Widdowson and McCance, 1960). Also, the work of Winick and Noble
(1967) shows that there are differences in the growth of rats raised
twelve and six per litter. Perhaps raising rats in litters of three
diminishes the suckling stimulus needed for milk production and makes

less milk available to them than to those raised in litters of six.

APPENDIX

APPENDIX

SOLUTIONS USED IN THE ANALYSIS OF
KIDNEY DNA AND PROTEIN

APPENDIX

SOLUTIONS USED IN THE ANALYSIS OF
KIDNEY DNA AND PROTEIN

Solutions Used in the Analysis of Kidney DNA

 

l. 20% TCA: 200 g. of trichloroacetic acid1 were weighed on a
Mettler tOp loading balance,2 and transferred to a 1,000 ml.
volumetric flask. Enough distilled deionized3 water was added
for thorough mixing, and then for making the solution to
volume. The solution was mixed by inversion and stored in the
refrigerator in 1 liter glass bottles.

2. 10% TCA: 100 g. of trichloroacetic acid were weighed on a
Mettler top loading balance, and the solution was made to
volume and stored as above.

3. 5% TCA: 50 g. of trichloroacetic acid were weighed and made
to volume as above. The solution was stored at room temperature
in 1 liter glass bottles.

4. NaOH, 1 pellet/liter: One pellet of sodium hydroxide4 was

placed in a 1,000 ml. volumetric flask. A small amount of

 

1Analytic reagent, Mallincrodt Chemical WOrks, St. Louis, MO.

2Mettler P 1200, Mettler Instrument Corporation, Hightstown, N.J.

3Illco-W’ay Ion XChanger, Research.Model 1, Illinois Water Treat-
ment Co., 840 Cedar Street, Rockford, Ill.

AUSP, J. T. Baker Chemical Co., Philipsburg, N.J.

73

74

distilled deionized water was added, and the flask shaken until
the pellet dissolved. The solution was made to volume with dis-
tilled deionized water, mixed, and stored at room temperature in
a 1 liter glass bottle.

Diphenylamine-acetaldehyde solution: 100 ml. of glacial acetic
acid5 was measured into a graduated cylinder. To this was

added 1.5 g. of crystalline diphenylamine.6 The cylinder was
stOppered, and the contents shaken by hand until all of the
diphenylamine was in solution. Then 1.5 m1. of concentrated
H28047 was added by pipette. The contents were shaken, and

100 ml. was transferred into a 100 ml. volumetric flask. The
solution was made fresh daily, and was kept out of the light
until it was used. Just before using, 0.5 ml. of refrigerated
acetaldehyde8 solution (aqueous, l6 mg./m1.) was added to the

volumetric flask containing the diphenylamine solution, and the

DNA standard: 10 mg. of calf thymus DNA9 was cut, weighed and

transferred into a 100 ml. volumetric flask. 50 ml. of a "one

10 was added in order to dissolve

 

5ACS reagent, A38—C, Fisher Scientific Co., Fair Lawn, N.J.

No. 4938, Analytical reagent, Mallinckrodt Chemical Works,

5.
contents were shaken.

6.
pellet/liter" solution of NaOH
6

St. Louis, Mo.

7No. 3-9681, J. T. Baker Chemical Co., Phillipsburg, N.J.

8N0. 2401, Mallinckrodt Chemical WOrks, St. Louis, Mo.

9Type 1, No. D 1501, Sigma Chemical Corporation, P.O. Box 4508,

St. Louis, Mo.

10

USP, J. T. Baker Chemical Co., Phillipsburg, N.J.

75

the DNA. After several hours and intermittent mixing with a
stirring rod, the DNA was dissolved. The solution was then

made to volume with 50 ml. of 10% TCA. The white precipitate
which formed, disappeared upon gently heating the flask in an
asbestos heating mantle. The final solution had a concentration
of 100 ug./ml. It was stored in a plastic container at 0 degrees
centigrade. The following protocol was followed in diluting the

standard for preparation of the standard curve:

ug, DNA/ml. m1. DNA Standard ml. 5% TCA

 

 

0 0
10 0 2
20 O 4
30 0.6
70 1 4
100 2 0

Solutions Used in the Analysis of Kidney Protein

 

10% TCA: The solution was prepared as under Solutions Used in

the Analysis of Kidney DNA.

 

l N NaOH: 40 g. of sodium hydroxide were weighed on the Mettler
tOp loading balance, and transferred to a 1,000 ml. volumetric
flask. Enough distilled deionized water was added for thorough
mixing, and then for making the solution to volume. The solution
was mixed by inversion and stored at room temperature in 1 liter
plastic bottles.

’SH 011 was weighed on the.Mettler

0.5% CuSO 4 2

4: 0.5 g. of CuSO

 

llAnalytical Reagent, No. 4844, Mallinckrodt Chemical Works,

St. Louis, Mo.

76
analytical balance12 and transferred to a 100 ml. volumetric flask.
Enough distilled deionized water was added for thorough mixing,
and then for making the solution to volume. The solution was
mixed by inversion, and stored at room temperature in 100 ml.
plastic bottles.
2% Na2C03--0.02% Na2C4H406-2H20: 20 g. of sodium carbonate13
was weighed on the Mettler top loading balance, and transferred
to a 1,000 ml. volumetric flask. 0.2 g. of sodium tartrate14
was weighed on the Mettler analytical balance, and transferred
to the same flask. Enough distilled deionized water was added
to the flask for thorough mixing, and then for making the solu-
tion to volume. The solution was mixed by inversion, and stored
at room temperature in 1 liter bottles.
Copper sulfate-sodium carbonate-sodium tartrate solution: Just
before treating the samples and standards, 2 ml. of 0.5% CuSOa'
H20 stock solution was transferred by pipette to a 100 ml.
volumetric flask. The solution was made to volume with the 2%
sodium carbonate--0.02% sodium tartrate solution. The final
solution was used for one day only.
Folin—Ciocalteau solution, 1N: Just prior to the treatment of
the samples and standards, 10 ml. of 2N Folin—Ciocalteau reagent15

was transferred by volumetric pipette into a 25 ml. beaker. Ten

 

12Type H 15, Mettler Instrument Corporation, Hightstown, N.J.

13Anhydrous, No. 3602, J. T. Baker Chemical Co., Phillipsburg, N.J.

14Crystalline, J. T. Baker Chemical Co., Phillipsburg, N.J.

lSPhenol Reagent Solution 2N (Folin-Ciocalteau), SO-P-24, Fisher

Scientific Co., Fair Lawn, N.J.

77

mililiters of distilled deionized water was in like manner,
transferred to the beaker. The addants were mixed well and
added to the samples and standards at the appropriate time.

7. Protein standard stock solution: Two hundred and fifty mili-
grams of crystalline bovine albumin16 was weighed and trans-
ferred to a 50 ml. volumetric flask. Approximately 20 ml. of
distilled deionized water was added and the flask inverted until
the albumin was dissolved. The solution was made nearly to
volume with more distilled deionized water, and then the flask
was stoppered and refrigerated overnight. By the next day the
foam which had formed during dilution and mixing had disappeared,
and the solution was made to volume with distilled deionized
water. The standard was stored in a plastic bottle at 0 degrees
centigrade, and was diluted 1:10 with 1N NaOH before use.

The following protocol was followed in diluting the standard for

preparation of the standard curve:

 

 

 

ug. Protein/ml. ml. Protein Standard ml. Water
50 0.1 0.9
100 0.2 0.8
150 0.3 0.7
200 0.4 0.6
250 0.5 0.5
300 0.6 0.4

16

No. 7285, Nutritional Biochemicals Corporation, Cleveland, Ohio.

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"IIIIIIIIIIIIIII“