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KIDNEY FUNCTION OF THE PROGENY 0F RATS WITH
STREPTOZOTOCIN-INDUCED DIABETES

presented by

Ellen Christine Rolig

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M.S. Nutrition

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KIDNEY FUNCTION OF THE PROGENY 0F RATS WITH
STREPTOZOTOCIN-INDUCED DIABETES

By
Ellen Christine Rolig

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

1979

ABSTRACT

KIDNEY FUNCTION OF THE PROGENY OF RATS NITH
STREPTOZOTOCIN-INDUCED DIABETES
BY
Ellen Christine Rolig

Effects of gestational diabetes on development of
kidney function in the progeny were determined in vitro and

i vivo. Body and kidney weights of pups of diabetic

 

mothers (PDMs) were no different than those of control
mothers (PCMs) at l, 5, or 10, but were lower at 28 days of
age. Generally, the ability of renal cortical slices from
1, 5, lO, and 28 day old PDMs to accumulate p-aminohippurate
(PAH), n-methyinicotinamide (NMN), a-methylglucoside (aMG),
or aminoisobutyric acid (AIB) was similar to that of PCMs.
Renal ammoniagenesis was enhanced in l and 5 day old PDMs
but declined to control values at 10 and 28 days of age.
Renal gluconeogenesis was enhanced only in 5 day old PDMs.
Urinary response to a water or isotonic saline load by PDMs
was no different than that by PCMs at 5 or 10 days of age.
In contrast, 10 day old but not 5 day old PDMs excreted
more urine, total solute, and sodium in response to hyper-
tonic saline than age matched PCMs. These observations
indicate that gestational diabetes selectively modified

renal functional development.

Dedicated to my parents
HELEN AND GEORGE SHARP
and to my husband
STEVEN ROLIG

for unconditional love
and encouragement

ii

ACKNOWLEDGMENTS

I am deeply grateful to Dr. Jenny Bond for her advice,
encouragement, and constructive criticism during all phases
of this study. My sincere thanks are extended to Dr. Jerry
Hook for his invaluable guidance and his insistance on hard
work and excellence. I would also like to thank Dr. David
Rovner and Dr. Dale Romsos for their suggestions and
critical evaluation of this work. A special thanks are due
to my fellow graduate students, Bill Evers, Sue Ford, and
Robin Goldstein, for encouragement, advice, and personal
support throughout the course of this study.

I would like to acknowledge Dr. John Gill and Dr. Clyde
Anderson for advice concerning statistical analysis of the
data and Marty Bond for his assistance with graphic illus-
trations.

The financial support provided by Dr. Jenny Bond and
the Department of Food Science and Human Nutrition is

gratefully acknowledged.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES .

<
do
do

INTRODUCTION. .
Renal Development. . . .
Renal Tubular Transport Function .
Reabsorption of Glucose.
Reabsorption of Amino Acids.
Secretion of Organic Ions. . . . . . . 1
Renal Excretion of Salt and Water Loads. . . . . l
Renal Regulation of Acid- Base Balance. . . . l
Nutrition and Kidney Development . . . . . . . . 23
Infant of the Diabetic Mother (IDM). . . . . . . 26
Renal Function in the IDM. . . . . . . . . . . 28
Animal Models of the IDM . . . . . . . . . . . . 3l

m-bNtONU'lwd

METHODS . . . . . . . . . . . . . . . . 35
Rat Model of the IDM . . . . . . . . . . . . . . 35
Experiments. . . . . . . . . . . . . . . . . . 36

Series I . . . 37
Body Weight, Kidney Weight, and Kidney Cortex
Composition. . . . . . 37

In Vitro Renal Tubular Transport Capacity. . . . 37

Tj Vitro Renal Gluconeogenic and Ammoniagenic
—Capacity . . . . . . . . . . . . . . . . . . 40

Response to Acidosis . . . . . . . . . . . . . . 4l

Series II. . . . . . . . . . . . . . . . . . . . 4l
Animals. . . . . . . . . . . . . . . . . . . 4l
Urine Collection . . . . . . . . . . . . . . . 41
Salt and Water Loading . . . . . . . . . . . . . 42

Statistical Analysis . . . . . . . . . . . . . . 43
Series I . . . . . . . . . . . . . . . . . 43
Series II. . . . . . . . . . . . . . . . . . . . 43

RESULTS . . . . 44

Maternal Plasma Glucose and Infant Birth Weight. 44
Fetal and Neonatal Mortality . . . . . . . . . . 50

iv

Page

Body Weight and Kidney Weight. . 50
Percent of Water and Protein in the Renal
Cortex . . 50
Accumulation of Organic Acid (PAH) by Renal
Cortical Slices. 56
Accumulation of Organic Base (NMN) by Renal
Cortical Slices. . . 56
Accumulation of aAminoisobutyric Acid (AIB) by
Renal Cortical Slices. . 56
Accumulation of aMethylglucoside (aMG) by Renal
Cortical Slices. . . 6l
Gluconeogenic Capacity of Renal Cortical Slices. 6l
Ammoniagenic Capacity of Renal Cortical Slices 6l
Effect of Acidosis on Glucose and Ammonia Pro-
duction by Renal Cortical Slices . . 64
Effect of Gestational Diabetes on the Excretion
of Water, Isotonic and Hypertonic Saline Loads
by the Progeny . . . . . . . . . . . 64
Response of 5 and 10 Day Old PDMs and PCMs to a
Water Load . . . . . . . . . . . 92
Response of 5 and lO Day Old PDMs and PCMs to an
Isotonic Saline Load . . . . . . . . . . . 93
Response of 5 and lo Day Old PDMs and PCMs to a
Hypertonic Saline Load . . . . . . . . . . . 94
Response of 5 and 10 Day Old PDMs and PCMs to a
Water Load in the Presence of Exogenous ADH. 95
DISCUSSION. lOl
SUMMARY . 133
APPENDIX. T37
LIST OF REFERENCES. . l38

LIST OF TABLES

Table Page

l The relationship between maternal plasma glucose
concentration and birth weight of the progeny of
diabetic and control Sprague-Dawley rats. . . . . 45

2 Effect of gestational diabetes on fetal and neo-
natal mortality . . . . . . . . . . . . . . . . . 5l

3 Effect of acidosis on glucose and ammonia produc-
tion by renal cortical slices from ten day old
PDMs and PCMs . . . . . . . . . . . . . . . . . . 65

4 Response of five and ten day old PDMs and PCMs to
a water load: concentration of urinary constitu-
ents. . . . . . . . . . . . . . . . . . . . . . . 90

5 Response of five and ten day old PCMs and PDMs
to an isotonic saline load: concentration of
urinary constituents. . . . . . . . . . . . . . . 91

6 Response of five and ten day old PCMs and PDMs to
pa hypertonic saline load: concentration of uri-
nary constituents . . . . . . . . . . . . . . . . 96

7 Response of five and ten day old PDMs and PCMs to
a water load in the presence of exogenous ADH:
concentration of urinary constituents . . . . . . 98

8 Urinary solute concentration before salt and
water loading . . . . . . . . . . . . . . . . . . l37

vi

LIST OF FIGURES

Figure

I

TO

ll

The effect of gestational diabetes on the birth
weight of the progeny from Series I. . .

The effect of gestational diabetes on the birth
weight of the progeny from Series II

Effect of gestational diabetes on kidney weight
of the progeny . . . . . . . . . . .

Effect of gestational diabetes on body weight of
the progeny. . . . . . . .

Effect of gestational diabetes on the percent of
protein of cortical slices from kidneys of the
offspring. . . . . . . . .

Effect of gestational diabetes on the percent of
water of cortical slices from kidneys of the
offspring. . .

Effect of gestational diabetes on PAH accumula-
tion by cortical slices from kidneys of the
progeny (PAH S/M ratio).

Effect of gestational diabetes on NMN accumula-
tion by cortical slices from kidneys of the
progeny (NMN S/M ratio).

Effect of gestational diabetes on AIB accumula-
tion by cortical slices from kidneys of the
progeny (AIB S/M ratio). . . . . . . .

Effect of gestational diabetes on aMG accumula-
tion by cortical slices from kidneys of the
progeny (oMG S/M ratio). . . . . . . . . . .

Effect of gestational diabetes on gluconeogenic

capacity of kidney cortex slices from the
progeny. . . . . . . . . . . . . . . . . . . . .

vii

Page
47
49
53

53
55
55
58
T58
60
60

63

Figure

12

l3

T4

15

16

T7

18

I9

20

21

22

23

Effect of gestational diabetes on ammoniagenic
capacity of kidney cortex slices from the

progeny.

Effect of gestational diabetes on the excretion
of urine by the progeny during the first 4 hours
after salt and water loading . . . . . . .

Effect of gestational diabetes on the excretion
of urine by the progeny during the 0-2 and 2-4
hour periods after salt and water loading.

Effect of gestational diabetes on the excretion
of total solute by the progeny during the first
4 hours after salt and water loading . .

Effect of gestational diabetes On the excretion
of total solute by the progeny during the 0-2
and 2-4 hour periods after salt and water
loading. .

Effect of gestational diabetes on the excretion
of sodium by the progeny during the first 4
hours after salt and water loading .

Effect of gestational diabetes on the excretion
of sodium by the progeny during the 0-2 and 2-4
hour periods after salt and water loading.

Effect of gestational diabetes on the excretion
of chloride by the progeny during the first 4
hours after salt and water loading . . .

Effect of gestational diabetes on the excretion
of chloride by the progeny during the 0-2 and
2-4 hour periods after salt and water loading.

Effect of gestational diabetes on the excretion
of potassium by the progeny during the first 4
hours after salt and water loading . . . .

Effect of gestational diabetes on the excretion
of potassium by the progeny during the 0-2 and
2-4 hour periods after salt and water loading.

Effect of gestational diabetes on the excretion

of urea by the progeny during the first 4 hours
after salt and water loading . . . . . .

viii

Page

63

67

69

71

73

75

77

79

81

83

85

87

Figure

24

25

Effect of gestational diabetes on the excretion
of urea by the progeny during the O-2 and 2-4
hour periods after salt and water loading.

The effect of ADH (0.1 mU/g; s.c.) on urea

excretion by 5 and 10 day old PDMs and PCMs
after a water load (5% body weight). .

ix

Page

89

TOO

INTRODUCTION

Previous experiments have shown a direct relationship
between nutritional status and kidney development in the
fetus and neonate. Postnatal development of kidney struc-
ture and function in rats can be enhanced by pre- and post-
natal overnutrition (Hinick and Noble, l966; Solomon and
Capek, 1972; Bond _t‘_l., I977) and retarded by pre- and
postnatal malnutrition (Hall and Zeman, I968; Allen and
Zeman, l97l; l973a; l973b; Bond gt 31., 1977; Goldstein
£511.,1979).

The nutritional status of the fetus and the infant of
the diabetic mother appears to be different from that of a
normal infant. Evidence to date suggests that the fetus
of the diabetic mother is hyperinsulinemic (Steinke and
Driscoll, l965). Fetal hyperinsulinemia could be induced
by excess glucose and amino acids available to the fetus of
the pregnant diabetic (Osler and Pederson, 1960). The
resulting infant of the diabetic mother (IDM) has a number
of nutritional problems. Ten to thirty percent of IDMs
are large for gestational age and 40-50% develop neonatal

hypoglycemia (Persson, l975; Kitzmiller gt 11., T978).

Neonatal hypoglycemia appears to be caused by a combination

of high plasma insulin concentration (Block _t._l., T974)
and an inappropriately small pancreatic glucagon response
to the fall in blood glucose (Johnston and Bloom, l975).
The extent of this nutrient-hormonal imbalance in the fetus
depends on the severity of the maternal diabetes (Farquhar,
l976). Consequently, IDMs are a heterogeneous group of
infants with features of both over and under nutrition
(Farquhar, T976).

There is some indirect evidence that renal function is
impaired in IDMs. IDMs are more likely than normal infants
to develop metabolic acidosis (Lowery t T., T954; Segal

_t_al., l957; Kitzmiller gt 11., T978), to excrete greater
quantities of electrolytes, water and nitrogen (Osler,
T960; Cook gt_gl., T960), and to develop hypocalCemia and
hyperphosphatemia (Zetterstrom and Arnhold, T958; Kitz-
miller gt 11., T978). While the renal glomerulus appears
to develop normally in fetuses of diabetic women (Cassady
£5 11., T975; Naeye, T975), recent data suggest that the
renal tubules do not (Sokol and Hall, T977). To date the
direct effects of maternal diabetes on pre- and postnatal
development of kidney function have not been investigated.
Therefore, this study was undertaken to evaluate the

effects of gestational diabetes in rats on the renal func-

tional development of the progeny.

Renal Development

Anatomically, the kidneys of most mammals mature gradu-
ally throughout gestation and during early neonatal life.
Nephron development begins in the juxtamedullary region and
progresses toward the capsule (Baxter and Yoffey, T948;
Potter, T965; Speller and Moffat, l977). In humans, the
number of new nephrons increases between six and thirty-
six weeks of gestation. Maturation of the kidney in the
newborn consists of growth and development of nephrons
present at birth (Potter, T965). Human fetal kidneys have
nephrons at various stages of development up until 36
weeks of gestation when all nephrons have morphological
characteristics of the adult type (Potter, l965). Although
full term human newborns have the same number of glomeruli
and tubules as the adult, these are not structurally
mature. Fetterman and co-workers (T965) examined the
development of glomeruli and proximal tubules from birth
to adulthood in human kidneys and found that all parts of
the nephron increase in size and complexity with increasing
age. The most striking of these changes involves the
development of the convolutions of the proximal tubules
and the formation and elongation of the loop of Henle. In
contrast, kidneys of newborn rats have one third the number
of nephrons present in adult kidneys and these are primar-
ily juxtamedullary nephrons (McCrory, T972; Speller and

Moffat, l977). Postnatal nephrogenesis in the rat occurs

primarily in the cortex by differentiation of successive
layers of nephrons from the peripheral nephrogenic zone
(Speller and Moffat, 1977). By 2 weeks after birth the
number of nephrons has doubled and by four weeks, it has
tripled (Kittelson, 1917). As the number of nephrons in-
creases, the tubules lengthen and the interlobular arteries
elongate to supply glomeruli (Speller and Moffat, 1977).

The development of kidney function is closely associ-
ated with, though not entirely dependent on, anatomical
maturation. While kidney function in the healthy newborn of
most species is adequate for survival and growth, it may be
inadequate under stress (McCance and Wilkinson, T947;
McCance gt 11., 1954; Falk, 1955). Newborns when compared
with adults have less ability to excrete a water, solute,
acid or alkali load (Falk, 1955; Goldstein, 1970), less
ability to concentrate urine in the presence of dehydration
(Heller, 1947; Falk, 1955), a decreased response to anti-
diuretic hormone (ADH) (Heller, 1944; 1952; Falk, 1955),
and both a decreased extraction of p-aminohippurate (PAH)
(Horster and Lewy, 1970) and a decreased maximal tubular
capacity for PAH (Hook _t _l., 1970). In addition, glomerular
filtration rate (GFR) and renal blood flow (RBF) are directly
related to age; both are lowest at birth and progressively
increase to adult values (Potter t 1., 1969; Horster and

Valtin, 1971; Leake £3 11., 1977).

Renal blood supply and distribution influence kidney
function and development. The neonatal kidney receives a
lower percent of the cardiac output than the adult kidney
(Kleinman and Reuter, 1973). Additionally, the distribu-
tion of blood within the neonatal kidney is different than
that observed in the adult (Jose gt 11., 1971). This is
due, in part, to a high renal vascular resistance present
at birth (Assali _t _l., 1968; Gruskin gt 11., 1970). After
birth there is a decrease in renal vascular resistance
followed by an increase in renal blood flow associated with
changes in the distribution of flow within the kidney. The
greatest flow is supplied to the mature nephrons. Thus, in
the immature kidney, the nephrons located in the inner
cortex would receive the highest flow while the flow to
the outer cortex would be low at birth and would increase
with age (Jose t 1., 1971; Kleinman and Reuter, 1973;

Olbing t al., 1973).

Renal Tubular Transport Function

Renal tubular transport functions such as reabsorption
and secretion depend on both the morphological and bio-
chemical development of the specific cells involved, in
particular, proximal tubular cells. In the adult kidney,
these processes have been elucidated largely with the use
of micropuncture techniques (Pitts, 1976). Since the

immature kidney contains nephrons at various stages of

development (Fetterman _t__l,, 1965), micropuncture tech-
niques are inappropriate for the study of the development
of renal tubular transport functions. For this reason,
development of tubular function must be analyzed with more
integrative techniques. One such technique is the kidney
slice technique.

The slice technique as first described by Cross and
Taggart (1950) has been used for many in_11£rg_studies of
renal transport processes and metabolism. Thin renal cor-
tical slices incubated in an appropriate medium in an
oxygen atmosphere will accumulate a number of compounds.
Accumulation of compounds by renal cortical slices is
expressed as the tissue concentration of material divided
by the medium concentration or as the slice/medium (S/M)
ratio or tissue/medium (T/M) ratio. If the SIM ratio
exceeds one, it is thought to indicate an active transport
process. Alternatively, renal cortical slices incubated
with precursors will synthesize products such as glucose
and ammonia. The kidney slice technique has both advan-
tages and disadvantages. Advantages include the elimina-
tion of unwanted changes in GFR, RBF, and extra-renal
factors, the precise control over incubation conditions,
and the presence of many intact kidney cells. Disadvantages
include the presence of both proximal and distal tubules
in renal cortical slices and the absence of a continuous

filtration process in the slice preparation. Additionally,

S/M ratios indicate the net accumulation resulting from the
influx, the efflux, and nonspecific binding. Thus S/M
ratios may indicate nonspecific binding and/or peritubular
transport in conjunction with or rather than luminal trans-
port (Berndt, 1976).

Experimental data from both animals and humans indi-
cate renal tubular function in the newborn is different
than in the adult. The immature kidney when compared to
the adult kidney has a lower percent of the glomerular
filtrate reabsorbed by the tubules (McCrory, 1972), a lower
intrinsic sodium transport capacity (McCrory, 1972), a
lower threshold for bicarbonate reabsorption (Edelmann
gt 11., 1967), a lower capacity to reabsorb sugar (Segal
gt__l,, 1973a), and a lower capacity to secrete organic

acids and bases (Rennick gt al., 1961; Kim gt_gl., 1972;

Hook, 1974).

Reabsorption of Glucose

The renal mechanism for glucose reabsorption has been
characterized as an active transport system of limited
capacity located in the proximal tubule. It is sodium
dependent, phlorizin inhibited, and highly specific for
sugar substrates such as glucose, galactose, xylose, fruc—
tose, and non-metabolizable sugars such as amethyl-D-
glucopyranoside (aMG) (Pitts, 1976). The studies on renal

glucose reabsorption during development are limited.

Tudvad (1949) compared sugar reabsorption among premature
babies, full term babies, and adults and found that the
reabsorption is low at birth and increases proportionately

with age. I vitro experiments suggest that the active

 

transport process for glucose in the proximal tubular cell
in the immature kidney is different from that in the mature
kidney (Kolinska, 1970; Segal gt 31,, 1971; 1973a; 1973b).
Since glucose uptake is an active transport process,
proximal tubular cells can accumulate glucose against a
concentration gradient and can produce intracellular]
extracellular glucose concentration ratios greater than one.
Accordingly, the uptake and efflux of aMG by renal cortical
slices have been characterized in rats (Segal gt 11.,

1971; l973a), rabbits (Kolinska, 1970), and humans (Segal
g£_al,, 1973b). Until recently, the interpretation of
these data has been hampered by limitations inherent to the
in vitro slice technique. Specifically, it was nut known
whether the uptake of oMG occurred at the peritubular or
the luminal membrane. Current evidence indicates that oMG
does not interact with the peritubular side but that it is
transported into the proximal tubular cell via a glucose
receptor at the brush border (Naftalin, 1970). Further-
more, the kinetics of oMG transport in the renal cortex
slices reflect the kinetics of the glucose carrier at the

brush border (Turner and Silverman, l977).

Immature kidneys appear to have a lower capacity to
reabsorb sugars than adult kidneys (Segal gt_al., 1973a).
Cortical slices from kidneys of one day old rats were
unable to actively transport aMG as indicated by aMG S/M
ratios less than one. Slices from five day old rats
concentrated aMG slightly and as the rats increased in
age, the ability of kidney slices to accumulate aMG also
increased. Renal cortical slices from fifteen day old rats
accumulated the same amount of aMG as those from adults

(Segal £3 11., l973a).

Reabsorption of Amino Acids

The renal reabsorptive mechanisms for amino acids
similarly involves active transport into proximal tubular
cells. There are at least three renal mechanisms for the
reabsorption of amino acids. One reabsorbs basic amino
acids: lysine, arginine, ornithine, and cystine, a second
reabsorbs glutamic and aspartic acids and a third reabsorbs
un-ionized amino acids (Pitts, 1976). Experiments comparing
the net tubular reabsorption of seventeen free amino acids
indicated that while the percentage of amino acids reab-
isorbed was lower in infancy than childhood, when GFR was
considered, the differences disappeared (Brodehl and Gellis-
sen, 1968).

The accumulation of amino acids by renal cortical

slices has been used to characterize the cellular mechanisms

10

for amino acid uptake in both mature and immature kidneys.
Evidence indicates, however, that accumulation of amino
acids by renal cortical slices reflects the uptake at the
peritubular cell membrane (Foulkes, 1971). Uptake at the
luminal membrane would reflect tubular reabsorption whereas
uptake at the peritubular side probably has a nutritive
role (Silbernagal gt 11., 1975). Despite the fact that
uptake of amino acids by kidney slices does not represent
renal reabsorption of amino acids, good correlations have
been established repeatedly between the properties of amino
acid reabsorption and the accumulation of amino acids by
kidney slices. For example, L-lysine and L-ornithine
inhibit both reabsorption of L-arginine from the tubular
lumen (Bergeron and Morel, 1969) and its accumulation by
slices (Rosenberg gt _l., 1962). These correlations justify
the continued use of the slice technique to study amino acid
transport systems of the kidney, particularly with immature
kidneys where micropuncture studies are inappropriate.
Aminoisobutyric acid (AIB) is a non-metaboliZable amino
acid frequently used to study kidney slice uptake of amino
acids. It is actively transported in the same way as
metabolizable amino acids. Kidney slice accumulation of
A18 is expressed as the ratio of the amount of A18 in the
slices divided by the amount in the medium or the A18 S/M

ratio.

11

Kidney slice accumulation of AIB has been determined
in both immature and mature rats (Webber and Cairns, T968;
Segal gt_gl., 1971), sheep (Scharrer gt_gl., 197T), and
humans (Solomon gt_gl., 1976). In general, the rate of AIB
uptake is slower in kidney slices from immature animals but
the SIM ratios or the concentration gradients which can be
achieved are greater (Webber and Cairns, 1968; Segal gt gl.,
1971). This is partly explained by a slower rate of efflux
of AIB from immature kidney slices (Webber, 1968). In
rats, neonatal kidney slices have the highest AIB S/M ratio
and these gradually decline to adult values by 15 days of
age (Webber and Cairns, 1968; Segal gt gt., 1971). In
humans this pattern of development occurs prenatally. The
ability of kidney slices from seven to seventeen week old
human fetuses to accumulate AIB decreased with increasing
fetal age (Solomon gt gl., 1976). The characteristics of
AIB accumulation by kidney slices from immature animals are
different than those observed for adults. While in both,
the uptake of A18 is sodium dependent and occurs as an
electrogenic co-transport with sodium, only uptake by
immature kidney slices can berenhanced by the addition of
insulin to the incubation medium (Scharrer gt gl., 1971).
Additionally, AIB uptake by kidney slices from immature
animals is enhanced by preincubating slices in a Krebs-
Ringer buffer for 30 minutes at 37°C (Reynolds and Segal,
1976). This is not true for kidney slices from adults.

12

The enhanced uptake of AIB produced by preincubation is
abolished by anaerobioses during the preincubation period.
It is also abolished by the presence of cycloheximide or
other protein synthesis inhibitors in the preincubation
medium (Reynolds and Segal, 1976). Based on these findings,
the authors suggested that a new protein, responsible for
enhanced AIB transport, is synthesized during the preincu-

bation period.

Secretion of Organic Ions

Active renal secretion of organic ions is another major
proximal tubular function. Many substances are added to
the urine by tubular secretion. Organic compounds such as
sulfonic acids and carboxylic acids are secreted by a non-
specific transport mechanism for organic acids. Compounds
secreted by this transport system include phenol red, p-
aminohippurate (PAH), penicillin, chlorothiazide, and glu-
curonides. A second mechanism transports a group of organic
bases such as guanidine, thiamine, choline, histamine,
tetraethylammonium (TEA) and n-methylnicotinamide (NMN)
(Pitts, 1976). Secretion serves as a mechanism for excre-
ting toxic metabolic products and foreign substances.
Since most drugs are bound to plasma proteins and not
filtered at the glomerulus, secretion is a major mechanism
for drug excretion (Pitts, 1976). Of the two systems, the

organic anion transport system has been most extensively

13

studied. This active transport system is relatively effi-
cient. In man, the maximum secretory rate of PAH averages
80 mg/minute/1.73 m2 of surface area (Pitts, 1976). The
secretion of PAH by infants (8 days to 3 months of age) is
approximately 30% below the secretion of PAH by adults
(Weil, 1955).

Renal secretory mechanisms can be studied by the 1g
gjttg slice technique (Cross and Taggart, 1950). Active
uptake of PAH by renal cortical slices reflects the 1g
111g tubular secretion of organic acids (Cross and Tag-
gart, 1950). Similarly, the net accumulation of NMN or

TEA by slices reflects the i vivo tubular secretion of

 

organic bases (Farah and Rennick, 1956). Using the slice
technique, the renal secretory capacity of immature rats
(Kim gt_gl,, 1972), dogs (Rennick, 1961; Hook _t gl.,
1970), and rabbits (Hirsch and Hook, l970a,b) has been
determined. In these species, PAH S/M ratios are low at
birth and progressively increase with age. Peak values are
reached from one to four weeks of age depending on the
species, and thereafter decline to adult values. NMN S/M
ratios are low at birth and gradually increase to adult
values.

The organic anion secretory process can be induced.
Pretreatment of pregnant or immature animals with organic

acids such as penicillin causes a rapid development of

this transport process in the fetus and the newborn.

14

Hirsch and Hook (T970) pretreated pregnant rabbits with
penicillin during the last half of gestation. Kidneys
from rabbits born of treated does developed increased PAH
S/M ratios (Hirsch and Hook, 1969). Similarly, pretreat-
ment of neonatal rats with penicillin enhanced PAH accumu-
lation but did not alter NMN uptake (Kim gt_g1,, 1972).
Pretreatment with penicillin also increased the clearance
and transport maximum (Tm) of PAH in both infant rats

a1

fl

(Noordewier and Withrow, 1976) and infant dogs (Bond g____,,
al.,

1976) and the extraction of PAH in infant dogs (Bond

"

1976) and infant rabbits (Kaplan and Lewy, 1978). Penicillin
pretreatment had no effect on the secretory capacity of

adult animals (Hirsch and Hook, T970a). Since the adminis-
tration of substrate to immature animals can stimulate the
capacity to secrete organic acids, the stimulus to normal
maturation may be an endogenous substrate load (Hirsch and

Hook, 1970a).

Renal Excretion of Salt and Water Loads

The ability to excrete salt and water loads adminis-
tered intragastrally or intravenously promptly and completely
is characteristic in adult animals (Pitts, 1976). This
ability is limited in the newborn of most species. In
newborn rats, administration of a water load (5% of the body
weight) does not produce a prompt water diuresis (Heller,

1947; Falk, 1955). Instead, increase in urine flow is

15

slight and delayed and there is little change in the osmotic
pressure of the urine (McCance and Wilkinson, 1947). In
4-6 day old rats, water loading produces a small but sig-
nificant water diuresis and a dilute urine (McCance and
Wilkinson, 1947; Falk, 1955). In 10-12 day old rats, the
response to a water load is not significantly different
from that seen in adult rats (McCance and Wilkinson, 1947;
Falk, 1955). In contrast, human infants do respond to a
water load with a prompt water diuresis (McCance gt 11.,
1954). However, the water diuresis produced in the human
infant is less than that produced in the adult. Human
infants and adults were given a water load (6% of body
weight). After 4 hours, the adults excreted 90% of the
load whereas the infants excreted only 50% of the load
(McCance gt gl., 1954). As previously mentioned, the pro-
cess of nephron formation is complete in the kidneys of
newborn humans but not complete in the kidneys of newborn
rats. Therefore, kidneys of newborn rats resemble those
of fetuses of humans. This anatomical difference partly
explains the more mature response to a water load observed
in human infants when compared to rat infants, however,
reasons for the smaller response to water loading by new-
born compared to adult humans are not fully understood.
The ability to excrete a water load depends on the amount
of water presented to the ascending limb of the loop of

Henle and the early convoluted tubule and the ability of

16

this segment to dilute the urine. The amount of urine
delivered to the loop of Henle depends on GFR and proximal
tubular water reabsorption (Loggie gt gl., l975). GFR is
lower in the kidneys of newborn animals of all species
studied and therefore is partly responsible for the imma-
ture response to a water load. Since human newborn infants
can dilute their urine to the same degree as adults (McCance
gt gl., 1954) it is probable that the relatively low GFR
observed in human infants is one major factor responsible
for the immature response to a water load (Loggie gt_gl.,
1975).

Similarly, when an adult animal is given a large
sodium load, the extracellular fluid (ECF) space imme-
diately expands. The kidney responds by increasing GFR
and decreasing the fractional sodium reabsorption in both
the proximal and distal tubule (Davis gt_gl,, 1969). This
results in a rapid excretion of sodium and return of ECF
space to pre-load conditions. In contrast, when a newborn
animal is given a large sodium load, serum sodium concen-
tration increases, body weight increases, and a generalized
(edema develops (McCance and Widdowson, T957). Administra-
tion of hypertonic saline solution to rats less than 3
weeks of age produced an osmotic diuresis that was smaller
and delayed when compared to the adult response (McCance
and Wilkinson, 1947; Falk, 1955). Similarly, newborn dogs

excreted 5% of a sodium load administered intravenously

17

after 2 hours compared to 30% excretion by adult dogs given
an equivalent load despite equivalent increases in GFR
following the salt load (Kleinman and Hsieh, 1974). Factors
that influence the relatively small renal response by
immature animals to salt loading appear to be related to
the distal rather than the proximal tubule. Kleinman and
Hsieh (1974) reported that after administration of i.v.
saline loads to newborn dogs, fractional sodium reabsorp-
tion was decreased in the proximal tubules but increased
in the distal tubules. Since aldosterone is one factor
that influences distal tubular sodium reabsorption, it
could be partially responsible for the immature response.
The concentration of aldosterone in the blood is higher in
newborn infants than in adults (Siegel gt gt., 1974). This
appears to be related in part to a lower excretory or
metabolic breakdown rate of aldosterone in the newborn
(Weldon _t _l,, 1967).

Immature animals cannot concentrate the urine to the
same degree as adults during dehydration (Heller, 1947;
Falk, 1955). The ability to produce concentrated urine is
primarily dependent on a high osmolar gradient between the
medullary interstitium and cortical region. Kidneys of
immature animals have lower osmolar gradients than those of
adults (Stanier, 1972). As the animal matures, there is
little change in the medullary sodium gradient but the urea

gradient increases with age (Forrest and Stanier, 1966).

18

Infants fed a high protein diet or urea supplements concen-
trated their urine to a greater degree than infants on a
low protein diet (Edelmann gt gt., 1960). Thus it appears
that the maintenance of a urea gradient is a major factor
in the ability to concentrate urine. A number of other
factors may theoretically contribute to a low renal medul-
lary osmolar gradient, however, more data are needed to

determine their actual role.

Renal Regulation of Acid-Base Balance

The kidneys, the lungs and the blood buffers regulate
the acid base balance in mammals. The role of the kidney
involves both proximal and distal tubular hydrogen ion
secretion and proximal tubular bicarbonate reabsorption.
Hydrogen ion secretion is dependent on its concentration
gradient across the luminal membrane. Renal tubular fluid
is buffered by phosphates, ammonia, and other substances
which associate with the proton to maintain a concentra-
tion gradient from tubular cells to luminal fluid (Pitts,
1976). Approximately 50% of the hydrogen ion is buffered
by phosphates and salts of weak acids and 50% is buffered
by ammonia (Pitts, 1976). The maximal gradient is reached
when the urinary pH equals approximately 4.5 (Pitts, 1976).
Therefore the ability of the immature kidney to secrete
hydrogen ions depends on the following: 1) the capacity of

proximal and distal tubules to secrete hydrogen ions,

19

2) the capacity of the proximal tubular cells to produce
ammonia and 3) the availability of buffers in the tubular
fluid. Studies with immature animals indicate the ability
to cope with an acid-base imbalance is reduced during the
neonatal period (McCance, 1961; Goldstein, 1970).

McCance (1961) administered ammonium chloride to human
infants and adults to induce a metabolic acidosis. The
infants produced less urinary titratable acid (T.A.) and
less urinary ammonia than adults. Infants have lower
levels of serum phosphate than adults. This may account
for their lower T.A. excretion. McCance and Widdowson
(1960) administered buffered neutral phosphate with ammonium
chloride to human infants. This treatment tripled the
amount of T.A. excreted by the infants. Edelmann 33.21-
(1967) found that full term human infants and adults have
the same capacity to secrete hydrogen ions. Thus the de-
creased capacity to cope with an acid-base imbalance ob-
served in the human infant is probably due to the decreased
availability of buffers in the tubular fluid or a decreased
capacity to produce ammonia. Preterm human infants, how-
ever, secrete less protons than full term infants (Sven-
ningsen, 1974).

Similarly the ability to respond to an acid-base
imbalance is lower in infant than adult rats (Goldstein,
1970). Goldstein (1970) administered ammonium chloride to

infant and adult rats. The adults responded with a rapid

20

increase in ammonia excretion, but did not significantly
increase T.A. excretion. The response of the infant rat

was significantly less. In rats, the normal adult response
to acidosis develops between 14 and 21 days of age (Gold-
stein, 1970). This maturational process appears to depend
on a supply of acid since chronic administration of ammonium
chloride to ten day old rats enhanced the rate of both
ammonia and T.A. excretion (Goldstein, 1970).

The rate of renal excretion of ammonia is determined
by two factors: 1) the diffusion of preformed ammonia from
the blood, through the tubular cells, and into the luminal
fluid and 2) ammoniagenesis in the proximal tubular cells
(Pitts, 1976). The diffusion of ammonia, which is lipid
soluble and easily passes through membranes, is determined
primarily by urinary pH. The lower the pH of the tubular
fluid, the more ammonia will diffuse from the tubular cell.
Renal ammonia is produced primarily in the mitochondria of
proximal tubular cells. These cells contain a number of
enzymes that could be involved in the production of ammonia
from amino acids. These include transaminases, glutamin-
.ase, and glutamate dehydrogenase (GDH). Generally,
ammonia is produced from glutamine; glutamine is hydrolyzed
to glutamate and ammonia by glutaminase. Glutamate is then
deaminated by GDH to aketoglutarate and ammonia, a reaction
which requires the reduction of NAD+ to NADH. However, a

number of amino acids can be transaminated to glutamate and

21

consequently be used for ammonia production (Pitts, 1976).
Experiments in mature dogs and rats indicate that under
normal conditions 73% of the urinary ammonia is derived
from plasma amino acids, 43% from glutamine and 30% from
alanine, glycine, and glutamic acid. Arterial ammonia
provides the remaining 27%. In the presence of acidosis
there is an increased renal extraction of glutamine from
the circulation (Pitts, 1976). Using radioactive-labeled
tracers, Pitts (1976) found that the major fraction of
glutamine was converted to (xketoglutarate and ammonia.
Most of the ammonia was excreted to buffer the protons.
Eighty percent of the cxketoglutarate went to carbon di-
oxide while twenty percent was converted to glucose (Pitts,
1976).

All of the following biochemical factors are thought
to affect renal ammonia synthesis: 1) glutaminase concen-
tration (Janicki and Goldstein, 1969), 2) glutamate concen-
tration (Goldstein, 1966), 3) gluconeogenic capacity (Gold-
stein, 1966), and 4) mitochondrial NADTINADH ratio (Preuss,
1968; Preuss, 1969).

In the rat, renal glutaminase concentration is low in
the first two weeks of life and increases to adult levels
between the second and third week of life (Benyajati and
Goldstein, 1975). The activity of this enzyme is therefore
associated with development of the ability to increase

ammonia excretion in response to acidosis.

22

Renal gluconeogenesis may influence ammonia production
by regulating the concentration of glutamate (Goldstein and
Harley-DeWitt, 1973). A rate determining enzyme in the
gluconeogenic pathway is phospho-enol-pyruvate carboxy-
kinase (PEPCK). Since the activity of this enzyme increases
during metabolic acidosis (Goodman gthl,, 1966; Alleyne
and Scullard, 1969), an increased rate of renal gluconeo-
genesis is associated with the increased rate of ammonia
excretion observed in response to acidosis.

The mitochondrial NADTINADH ratio is thought to control
the direction of the GDH reaction, thereby regulating glu-
tamate concentration (Preuss, 1968; 1969). The Tower the
ratio, the lower the conversion of glutamate to ammonia and
aketoglutaratea

Previous studies using adult rats have shown that
cortical slices from kidneys of acidotic animals utilized
more glutamine and formed more ammonia, glucose, and glu-
tamate than kidney slices from nonacidotic rats (Paglaira
and Goodman, 1970; Roobol and Alleyne, 1974). Goldstein
and Harley-DeWitt (1973) determined the gluconeogenic
capacity of kidney cortical slices from 10, 12, l4, l6,
and 21 day old rats. Renal gluconeogenic capacity was
significantly lower in 10 day old rats than in adults, but
rose to adult levels by 12 days of age. The effect of
metabolic acidosis on lg 11312 renal gluconeogenesis was

measured in 21 and 36 day old rats (Fraser and Alleyne,

23

1974). In all rats with acidosis, there was a significant
increase in renal gluconeogenesis. The effect of metabolic
acidosis on renal gluconeogenesis has not been determined
in rats less than 21 days old. However, it has been esti-
mated by the determination of the effect of medium pH on
the capacity of renal cortical slices from 10, 12, 14, 16,
and 21 day old rats to produce glucose (Goldstein and
Harley-DeWitt, 1973). The response of gluconeogenesis to
decreasing medium prrom 7.7 to 7.0 was 56% lower in 10
day old rats than in adults but increased to adult levels
by 14 days of age. The NADTINADH ratios were significantly
lower in kidneys from 10-21 day old rats than in those from
adults (Goldstein and Harley-DeWitt, 1973). This study
indicates that both a low gluconeogenic capacity and a low
NAD+/NADH ratio may contribute to the decreased renal
ammoniagenic capacity observed in rats less than 14 days

old.

Nutrition and Kidney Development

Alterations in pre- and postnatal nutrition can influ-
ence the normal morphological and physiological development
of the kidney. The degree of influence depends on the type
and duration of the nutritional insult as well as the
species involved. Many studies investigating the effects
of nutrition on renal development have used rats. Since

the rat kidney is relatively immature at birth, one can

24

extrapolate to other species only with great care.

. Postnatal protein calorie over and under nutrition can
be produced in young rats by adjusting litter size (Winick
and Noble, 1966). A normal litter contains 8-12 pups.
Winich and Noble (1966) found that rats raised in 18-pup
litters in comparison to those from normal litters had a
reduced kidney cell number; kidney cell size was unaffected.
This decrease in cell number was thought to be permanent
since it persisted at 133 days of age. Similarly, rats
raised in 3-6 pup litters had an increase in kidney cell
number; again, kidney cell size was not affected. To per-
manently alter kidney cell number, the postnatal nutritional
modification must occur early in neonatal life since these
authors found that post weaning malnutrition had no effect
on kidney cell number (Winick and Noble, 1966).

In addition to kidney cell number, kidney function is
affected by postnatal nutritional alterations. Bond and
co-workers (T977) adjusted rat litters to 5, 10 and 20 pups
and measured the accumulation of PAH and NMN by renal
cortical from both 5 and 10 day old rats. These data indi-
cate that renal secretion of organic acids and bases is
significantly lower in pups reared in large litters when
compared to pups reared in normal or small litters.

Experimentally, prenatal malnutrition is produced
primarily by restricting an essential dietary component

during pregnancy. Protein restricted diets fed to rats

25

during pregnancy caused a number of morphological and func-
tional changes in the kidneys of the offspring (Hall and
Zeman, 1968; Zeman, 1968; Allen and Zeman, 1971; l973a;
l973b). These offspring had smaller kidneys, with less
total DNA and less total protein, fewer and less well dif-
ferentiated glomeruli, and fewer collecting ducts when
compared to controls (Zeman, 1968). Functionally, protein
deprived offspring had a reduction in GFR, a depressed
ability to handle a water or osmotic load, and a depressed
response to ADH when compared to age matched controls (Hall
and Zeman, 1968; Allen and Zeman, 1971; 1973a; 1973b).
Renal tubular transport functions, as measured by the
accumulation of PAH, NMN, and AIB, by renal cortical
slices, were not affected by prenatal protein restriction
(Goldstein gt_gl., 1979). However, young rats nursed by
protein deprived dams showed a decrease in body weight and
kidney weight. Additionally, accumulation of both PAH

and NMN by kidney slices was depressed in rats nursed by
protein deprived dams (Goldstein gt gl., 1979). Since
maternal protein deprivation during lactation decreases
the quantity of milk available for nursing (Venkatachalam
and Ramanathan, 1964), this is, in effect, another model
for protein-calorie malnutrition and the effects observed
on kidney development in the progeny are similar to those
observed in progeny raised in very large litters (Bond

t 1., 1977).

26

Thus far, studies on the effects of pre- and postnatal
overnutrition on renal development are few. As previously
mentioned, early postnatal overnutrition produced a perma-
nent increase in renal cell number (Winick and Noble, 1966),
enhanced tubular secretory capacity (Bond gt,gl,, T977),
and has been related to an increase in single nephron GFR
(Solomon and capek, 1972). Prenatal overnutrition can be
produced by a simple operation in the rat. One uterine
horn is ligated before the female is bred. Rats manipulated
in this way gave birth to a small number of heavier pups
(Van Marthens gt gl., 1972). Utilizing this model, Gumbrecht
and co-workers (1977) examined the effects of prenatal
overnutrition on kidney composition and function at 20 days
of gestation and one day after birth. Bodies and kidneys
of pups from ligated dams weighed significantly more than
those of age matched controls, but kidney function and com-
position were the same for the two groups. In contrast,
Bond and co-workers (1977) observed that kidney slices from
,5 and 10 day old rats from ligated dams accumulated sig-
nificantly more PAH and NMN than kidney slices from controls.
These data suggest that development of the renal secretory

function was enhanced by prenatal overnutrition.

Infant of the Diabetic Mother (IDM)
The newborn infant of the diabetic mother (IDM) has a

number of metabolic problems resulting from an alteration

27

in prenatal nutritional status. Throughout gestation, the
fetus of the diabetic receives more glucose, amino acids,
and ketones than does the fetus of a normal infant (Felig,
1977). The quantity of excess nutrients available to the
fetus depends on the severity of the maternal diabetes.
While a direct relationship between maternal blood glucose
concentration and infant birth weight has not been estab-
lished in humans (Persson, 1975), evidence from animal
studies indicates that mild to moderate maternal diabetes
produces neonates that are normal or large for gestational
age while severe maternal diabetes produces infants that
are small for gestational age (Kervran gthl., 1978).
Similarly, some human IDMs are small for gestational age
(Persson, 1975; Farquhar, 1976). While these are more
commonly born to women with diabetic complications, some
mothers with apparently uncomplicated diabetes produce such
infants (Farquhar, 1976).

Approximately 20-30% of IDMs are born large for ges-
tational age (LGA) (Persson, T975; Farquhar, 1976; Kitz-
miller _t _l., 1978). These infants have a higher percent
fat and glycogen but a lower percent extracellular and
total water than infants of normal mothers (INM) of com-
parable maturity (Osler and Pederson, 1960; Cheek _t gl,,
1961). This difference in body composition could not occur

unless there was a simultaneous increase in fetal insulin

secretion. In fact, evidence to date indicates that the

28

fetus and infant of the diabetic mother are hyperinsulinemic
(Block gthl,, 1974).

The major clinical problems of the IDM are associated
with hyperinsulinemia. In a recent study, 47% of IDMs
Ideveloped hypoglycemia, while 36% were LGA. Although
macrosomia was associated with hypoglycemia, a significant
number of TONS of average weight developed hypoglycemia
(Kitzmiller gt_gl., 1978). Furthermore, fetal hyperinsu-
linemia appears to alter the maturation of other organ
systems. For example, approximately 8% of IDMs suffer from
respiratory distress syndrome (RDS) (Kitzmiller 2£.£l-9
1978). Excess insulin may depress plasma cortisol levels
in the fetal circulation (Sosenko gt_gl., 1979). Cortisol
stimulates the synthesis of lecithin, the principle compo-
nent of surfactant (Sosenko gt_gl., 1979). A decrease in
the synthesis of surfactant could be responsible for the
increased incidence of ROS in IDMs.

Other forms of neonatal morbidity observed in IDMs
include hypocalcemia (22%), hyperbilirubinemia (19%) and
congenital anomalies (9%). Interestingly, these are
problems commonly observed in premature infants and some
investigators have suggested that IDMs are premature for

their size (Farquhar, 1976).

Renal Function in the IDM
The chemical homeostasis in newborn IDMs is not normal.

Calculations of the corrected bromide space by Cheek gt_gl,

29

(1961) showed that IDMs have less extracellular fluid and
less total body water than normal infants. Since sodium
and chloride are major electrolytes in the extracellular
fluid, it is not surprising that Fee and Weil (1963) found
total body sodium and chloride low in IDMs in comparison to
normal infants. IDMs tend to develop an uncompensated
metabolic acidosis with decreased plasma pH, increased pCOz
and some decreases in plasma protein, chloride, and total
base (Lowery gt_gl,, l954; Segal gt_gl., 1957). In addition,
intrapartum fetal distress was diagnosed in 25% of the
diabetic women who had spontaneous or induced labor. This
diagnosis was based partly on scalp pH below 7.25 (Kitz-
miller gtht., 1978).

Both normal and abnormal electrolyte excretion in IDMs
have been observed. Osler (1960) and Cook gt gt. (1960)
found that IDMs excrete proportionately more sodium, potas-
sium, chloride and nitrogen than infants of comparable
weight and slightly more than infants of comparable gesta-
tion. Osler and Pederson (1960) reported that the mean
electrolyte excretion in IDMs was higher than what is
considered as normal for gestational age. Stapleton (1956),
however, found electrolyte excretion in IDMs the same as in
normal infants. Abnormal electrolyte excretion may reflect
abnormalities in body composition, endocrine function and/or

kidney function.

3O

IDMs have been reported to have low normal and high
‘plasma potassium levels (Farquhar, 1976), raised serum
phosphate levels (Zetterstrom and Arnhold, 1958; Farquhar,
1976), and low plasma calcium levels (Zetterstrom and
Arnhold, 1958; Tsang gt gt., 1972; 1975; Kitzmiller _t gt.,
1978).

Maturation and function of the renal glomerulus appears
to be normal in fetuses and infants of diabetic mothers.
GFR, estimated by thiosulphate clearance, was the same in
IDMs and normal infants (Osler, 1960). Naeye (1975)
examined the kidneys of fetuses from diabetic women and
found no histological changes in the glomeruli. The
increase in amniotic fluid creatinine concentration through-
out gestation indicates a normal development of fetal
glomeruli. This pattern was observed in amniotic fluid of
pregnant diabetic women (Cassady gt gt., 1975).

Recent evidence indicates, however, that fetal renal
tubules may not mature normally during a diabetic pregnancy.
Sokol and Hall (1977) examined the disappearance of betaz
microglobulin (82M) from the amniotic fluid of pregnant
diabetic women. This protein is normally reabsorbed by
fetal proximal tubules and amniotic fluid 82M concentration
normally decreases as the proximal tubules mature and can
reabsorb this protein. In pregnant diabetics, the amniotic
fluid 82M did not decrease. This led the authors to suggest

that the fetal renal tubules do not mature properly during

31

diabetic pregnancy.

(Animal Models of the IDM

The two most frequently used diabetogenic agents are.
alloxan and streptozotocin (Rerup, 1970). Both drugs are
cytotoxic to the beta cells of the pancreas. Both drugs
have undesirable side effects, particularly relative to
nephrotoxicity. Streptozotocin, however, is less nephro-
toxic than alloxan (Rakieten gt gl,, T963) and acts by
reducing the NAD+/NADH content of the islet cells. This
is followed by beta cell necrosis and diabetes. The changes
can be prevented by the simultaneous administration of
nicotinamide (Dulin and Wyse, 1969).

Experimental maternal diabetes has been induced in
Rhesus monkeys (Mintz _t gt., 1972; Cheek gt_gt., 1974),
guinea pigs (Junod gt 11., 1967), and rats (Solomon, 1959;
Pitkin ££.11-- 1971; Szalay and Gaal, 1975; Aerts and
Van Assche, 1977; Kervran _t _l., 1978).

Solomon (1959) induced diabetes midgestationally in
rats with alloxan and measured body weight, number of still-
born, and percent moisture, protein and fat in the fetuses.
Mildly diabetic rats delivered young with birth weights
greater than those of controls; there was also an increased
incidence of stillborn young. Severely diabetic rats could

not maintain pregnancy and produced no progeny. The pro-

portions of body moisture, protein, and fat observed in

32

fetuses of mildly diabetic rats were no different than

'those of control offspring. In these experiments, pregnant
diabetic rats did not receive insulin.

Pitkin _£.El- (1971) induced diabetes in virgin rats
with streptozotocin, bred them and maintained three groups:
pregnant diabetics that received no insulin, pregnant dia-
betics that received a daily injection of insulin and a
nondiabetic control group. Fetal and placental composition
wereexamined on day 21 of pregnancy. Fetuses of untreated
diabetic rats were larger and had significantly more fat,
less water, and more DNA than those of controls. Fetuses
of insulin treated diabetics were no different than those
of control rats.

Aerts and Van Assche (T977) induced diabetes in rats
on day one of gestation to study the endocrine pancreas of
the fetuses. Rats were injected with 30, 40, or 50 mg/kg
streptozotocin to produce "mild" and "severe" diabetes.
Increased neonatal loss was observed in progeny of severely
diabetic rats but birth weights of the progeny of severely
and mildly diabetic rats were no different than those of
control offspring. The endocrine pancreas of fetuses and
newborn rats from mildly diabetic dams showed islet hyper-
trophy and beta cell hyperplasia. Beta cells of fetuses of
severely diabetic dams were degranulated. In these experi-

ments, pregnant diabetic rats did not receive insulin.

33

Kervran gt g1, (1978) induced diabetes with strepto-
zotocin in rats prior to mating. Pregnant rats were divided
into "severe" diabetics (blood glucose concentration above
300 mg/dl) and "mild" diabetics (blood glucose concentration
ranging from 100 to 200 mg/dl). When compared to control
fetuses, fetuses from severely diabetic dams showed a slight
decrease in body weight, a decrease in pancreatic insulin
stores and a decrease in plasma insulin concentration. In
contrast, fetuses from mildly diabetic rats compared to
those from controls showed no change in body weight, an
increase in pancreatic insulin, and an increase in plasma
insulin concentrations. In these experiments, pregnant
diabetic rats did not receive insulin.

These data show that rats born from diabetic dams can
be considered animal models of the IDM. Additionally, rats
born of dams with mild diabetes represent a model for pre-
natal overnutrition. Since alterations in prenatal nutri-
tional status have been shown to effect the structural and
functional development of the kidneys in the progeny (Hall
and Zeman, 1968; Allen and Zeman, 1971; 1973a; l973b;

Bond gt_gl., 1977) it was of interest to determine the
effects of maternal diabetes on the development of kidney
function in the offspring. Therefore, the first series of
experiments was designed to determine the effects of maternal
diabetes on 1) kidney weight and composition in the offspring

and 2) the development of kidney function in the offspring

34

in such a way as to eliminate extra-renal factors. The
second series of experiments was designed to determine the
effect of maternal diabetes on the ability of the progeny
to excrete a water, an isotonic saline, or a hypertonic

saline load.

METHODS

Rat Model of the IDM

Female Sprague-Dawley rats were obtained from Spartan
Research Inc., Haslett, MI or Harlan Industries Inc.,
Indianapolis, IN on day one of their first gestation. Rats
were randomly separated into two groups. Rats from one
group were injected i.p. with streptozotocin (50 mg/kg)
(courtesy of Dr. W.E. Dulin, Upjohn Co.) dissolved in 0.1 M
citric acid (pH adjusted to 3.8-4.2). Rats from the remain-
ing group were injected with 0.1 M citric acid solution (pH
adjusted to 3.8-4.2) and were considered controls. Two to
four days after injections, animals were placed in meta-
bolism cages and urine samples were collected and analyzed
for glucose (Tes-tape, Eli Lilly and Co.). Animals showing
0.1% glucose in their urine were considered diabetic.
Diabetic animals were injected s.c. each afternoon between
4 and 8 PM with 2-4 units of NPH Iletin (isophane insulin
suspension, Eli Lilly Co.). In the first series, diabetic
animals received a set dose of 2 units of insulin per day
throughout pregnancy. In the second series, the insulin
dose was adjusted between 2 and 4 units per day according

to urinary glucose concentration. Controls received

35

36

subcutaneous injections of saline. On the 18th day of
gestation, blood samples were collected 24 hours after the
daily insulin or saline injection. Approximately one m1 of
blood was collected from the orbital sinus into a small
testtube pretreated with sodium fluoride (to inhibit glyco-
lysis) and heparin (to inhibit clot formation). This was
centrifuged and the plasma was separated from the red cells.
The plasma was frozen and later deproteinized and analyzed
for glucose by an enzymatic colorimetric assay (Sigma Chemi-
cal Co.).

Animals were housed in a temperature controlled room
with a 12-12 hour light-dark cycle. All animals received
a standard rat chow (Wayne Lab Blox; 24% crude protein;
4.0% crude fat; 4.0% crude fiber; and 68% carbohydrate by
weight) and water ad libitum. Rats were allowed to deliver
normally. At birth, live pups from diabetic mothers (PDMs)
and live pups from control mothers (PCMs) were weighed,
adjusted to 10 per litter, and crossfostered to lactating
controls. Lactating controls used as foster mothers

delivered litters at the same time as diabetic mothers.

Experiments

Two series of experiments were completed. In the first
series, kidney composition and function were evaluated in l,
5, 10, and 28 day old PDMs and PCMs using jg_gttgg kidney

slice techniques. In the second series,ability of PDMs and

37

PCMs to excrete water and salt loads was determined using

in vivo techniques.

 

Series 1

Body Wgtght, Kidney ngght, and Kidney Cortex Composition
One, 5, 10, and 28 day old progeny were weighed and
decapitated; the kidneys were removed, decapsulated, and
weighed. Total water content of kidney cortex slices was
determined as the difference between wet and dry weight.
To obtain a dry weight, slices were placed in pre-weighed
crucibles and dried at 100°C for 24 hours. Total protein
content of renal cortical slices was determined by dissol-
ving the slices in 3 N KOH, diluting each sample and
assaying for protein according to the Lowry procedure

(Lowry gt gl., 1951).

In Vitro Renal Tubular Transport Capacity

Animals were killed by cervical dislocation. Kidneys
were immediately removed, decapsulated, and placed in cold
isotonic saline (0.9% sodium chloride).

The renal slice preparation described by Cross and
Taggart (1950) was used. Renal cortical slices 0.2-0.3 mm
thick were prepared freehand. The accumulation of the
prototype organic acid, p-aminohippuric acid (PAH) and the
prototype organic base, N-methylnicotinamide (NMN) by the

slices was used as an index of renal intrinsic secretory

38

function. Approximately 50 mg of renal cortical slices was
incubated in 2.7 mls of Cross and Taggart medium (1950)
containing 7.4 x 10"5 M PAH (Eastman Kodak) and 6.0 x 10'6 M
(2.5 x 102 uCi/ml) 14C-NMN (New England Nuclear) and adjusted
to pH 7.4. Incubations were carried out in a Dubnoff
Metabolic Shaker at 25°C under 100% oxygen for 90 minutes
(Cross and Taggart, 1950).

The accumulation of the non-metabolizable sugar,
«methyl glucoside (oMG) by renal cortical slices was used
to determine the intrinsic renal transport capacity for
sugars. Approximately 50 mg of kidney cortex slices was
incubated in 3 ml of Krebs-Ringer Bicarbonate Buffer (Krebs
gt_gl,, 1963) containing 2 mM aMG (Sigma Chemical Co.) and
0.2 uCi/ml 14C aMG (New England Nuclear). Incubations were
carried out in stoppered reaction flasks filled with 95%
oxygen 5% carbon dioxide in a Dubnoff Metabolic Shaker at
37°C for 90 minutes (Segal gt gl., l973a).

The accumulation of the non-metabolizable amino acid,
aminoisobutyric acid (AIB) by renal cortical slices was used
to determine the capacity of kidney slices to accumulate
amino acids. Approximately 50 mg of tissue was preincubated
in 3 ml of Kreb-Ringer Bicarbonate Buffer (Krebs gt gl,,
1963). Preincubations were carried out in stoppered reac-
tion flasks filled with 95% oxygen-5% carbon dioxide in a
Dubnoff Metabolic Shaker at 37°C for 30 minutes. After

preincubation, slices were transferred to a similar medium

39

containing 0.065 mM AIB (Sigma Chemical Co.) and 0.1 uCi/ml
14C-AIB (New England Nuclear) and were incubated in stop-
pered reaction flasks filled with 95% oxygen-5% carbon
dioxide in a Dubnoff Metabolic Shaker at 37°C for 60
minutes (Reynolds and Segal, 1976).

After incubation, slices were removed from the medium,
blotted, weighed and placed in tissue homogenizers with
3 mls of trichloroacetic acid (TCA, 10%). Similarly, 2 mls
of medium were mixed with 3 mls of TCA. Both medium and
tissue homogenates were brought to a final volume of 10 mls
with distilled water and centrifuged at 1400 rpm for 10
minutes. A 1.0 m1 aliquot of supernatant was used to deter-
mine PAH spectrophotometrically as described by Smith gt g1.
(1945). Additionally, a 1.0 ml aliquot was added to scin-
tillation vials containing 10 mls of modified Bray's Solu-
tion (6 gm of 2,5-diphenyloxazole and 100 gm of naphtha-

lene per liter of dioxane). Radioactivity of 14

14C-aMG, or 14C-AIB in the supernatants was determined

C-NMN,

using a Beckman LS-250 liquid scintillation counter employ-
ing internal standards. Transport was expressed as the
slice to medium (S/M) ratio. This was calculated as the
concentration of PAH per gram of tissue (wet weight)
divided by the concentration of PAH per ml of media.

14 14

Similarly, in the case of C-NMN, C-aAIB, or 14C oMG,

disintegrations per minute (dpm) per gram of tissue (wet

40

weight) was divided by dpm per ml of medium.

In Vitro Renal Gluconepggnic and Ammoniggenic Capacity
Slices were prepared as previously described. The
technique described by Krebs _t g1. (1963) was used to
quantify renal gluconeogenic and ammoniagenic capacity.
Approximately 20-40 mg of kidney cortex slices was incubated
in 5 ml of Krebs-Ringer Bicarbonate Buffer both in the
presence and absence of substrate, 10 mM sodium glutamate
(Sigma Chemical Co.). Incubation flasks were filled with
95% oxygen- 5% carbon dioxide, stoppered, and incubated in a
Dubnoff Metabolic Shaker at 37°C for 90 minutes. Slices
were removed from the flasks, blotted and dried in pre-
weighed crucibles at 100°C for 24 hours to determine dry
weights. After removal of slices, 0.5 ml 10% (V/V) HCTO4
was mixed with the medium and the sample was centrifuged to
precipitate protein. The supernatant was assayed for ammonia
(Kaplan, 1965) and glucose (Glucostat, Worthington Biochemi-
cal Corp.). Net production of glucose or ammonia was calcu-
lated as the difference between glucose or ammonia produced
in the presence of substrate and that produced in the
absence of substrate. Results were expressed as micromoles

of glucose or ammonia produced per hour per gram dry slice

weight (umol/hr/gm).

41

Response to Acidosis

Ten day old PDMs and PCMs were gavaged via polyethylene
tube (PE-10) with ammonium chloride (Sigma Chemical Co.)
(5 mM/kg; 2.5% of the body weight) or water (2.5% of the
body weight. After 2.5-3.5 hours the rats were decapitated,
the kidneys removed, decapsulated and weighed. lg liELQ
renal gluconeogenic and ammoniagenic capacities were deter-

mined as previously described.

Series II

 

Animals

Pregnant control and diabetic rats were treated as pre-
viously described. Rats were allowed to deliver normally.
At birth, PDMs and PCMs were weighed and transferred to lac-
tating controls. Each lactating control received 5 PDMs and
5 PCMs. 0n the day of the experiment, 5 and 10 day old rats
were taken from the dams and placed in beakers lined with
cage bedding. Throughout the experiment, pups were warmed

to 30-32°c by a light bulb (McCance and Wilkinson, 1947).

Urine Collection

 

The bladder of each infant rat was emptied before each
treatment at 2 hour intervals by gentle suprapubic pressure
and stroking of the perineum. This method takes advantage
of the fact that infant rats cannot urinate spontaneously,

but instead urinate in response to a perineal stimulus

42

(McCance and Wilkinson, 1947). The urine was collected by

a syringe and transferred to preweighed vials.

Salt and Water Loading

Five and ten day old PDMs and PCMs were removed from
their mothers and placed in beakers under an electric light
bulb. The ambient temperature was maintained at 30-32°C.
Each pup was weighed and the bladder was emptied. Each rat
was gavaged via polyethylene tube (PE-10) with 5% of its
body weight of one of the following solutions: distilled
water, 0.15N sodium chloride (isotonic saline), or 0.5N
sodium chloride (hypertonic saline). Animals in the fourth
group received 5% of their body weight of distilled water
and a s.c. injection of ADH (0.1mU/g; Pitressin, Parke
Davis Co.). Two to four drops of green food color (U.S.
Certified, Kroger) were added to 100 mls of each solution.
This allowed easy detection of regurgitation or accidental
esophogeal perforation. PCMs and PDMs receiving the same
treatment were suckled by the sam dam. Bladders were
emptied 2 and 4 hours after treatment. Urinary volume was
determined by weighing the urine. Urinary sodium and potas-
sium concentrations were determined with a flame photometer
(Instrumentation Laboratory Inc.). Urinary chloride con-
centration was determined with a Buchler-Cotlove chlorido-
meter (Buchler Instruments Inc.). Urinary nitrogen was

determined colorimetrically with a modified urease Berthelot

43

reaction (Sigma Chemical Co.). Urinary osmolality was
determined with a vapor pressure osmometer (Wescor, Inc.).
Results are expressed in terms of concentration (mOSm,

mM or mEq/liter) and excretion (mOSm, mM, or mEq/hour/weight).
StatisticalfAnalysis

Series I

Except for the frequency distribution (Figures 1 and 2)
all data were reported as means i SEM. Differences between
the means were analyzed using the Least Significant Difference
(LSD) test after analysis of variance (completely random
design). A paired-t-test was used to analyze the effect of
acidosis on renal glucose and ammonia production between
littermates (Table 3). The .05 level of probability was used

as the criterion of significance (Steel and Torrie, 1960).

Series II

 

All data were reported as means i SEM. Differences
between the means were analyzed using the "Improved" Bonfer-
roni-t-test (Games, 1977) after analysis of variance (fac-
torial design and split plot design). The .05 level of
probability was used as the criterion of significance

(Steel and Torrie, 1960).

RESULTS

Maternal Plasma Glucose and Infant Birth Weight

Plasma glucose concentration was significantly greater
in diabetic than control rats in both series of experiments
(Table 1). The average birth weight of live pups from dia-
betic mothers (PDMs) (6.23 i 0.17) was not significantly
different than that of pups from control mothers (PCMs)
(6.5 i 0.11) from Series I (Table 1). In Series I, 55% of
the PDMs had birth weights below the grand mean (the average
birth weight for all rats: 6.34), while 33% of PCMs had
birth weights below the grand mean (Figure 1). Similarly,
the average birth weight of live PDMs (7.12 i 0.21) was no
different than that of PCMs (6.85 i 0.15) from Series II
(Table 1). In Series II, when litters included at least 5
pups, 78% of PDMs had birth weights greater than the grand
mean (6.98) while only 42% of PCMs had birth weights greater
than the grand mean (Figure 2; Top). In addition, to correct
for small litter size, a frequency distribution of average
birth weights for litters containing 9 to 12 pups is shown
on the bottom of Figure 2. Seventy-one percent of the PDMs
had birth weights above the grand mean while only 22% of the
PCMs had birth weights above the grand mean (Figure 2;

44

45

Table l. The relationship between maternal plasma glucose
concentration and birth weight of the progeny of
diabetic and control Sprague-Dawley rats

 

 

Maternal
Plasmaa Birthb Correlation
Glucose Weight Coefficient
(mg/d1) (9) (r)
Series I
Control 96.7 : 4°(22) 6.50 e o.11(22)
-0.192(44)
Diabetic 321.9 1 33(22)* 6.23 i O.17(22)
Series II
Control 97.1 i 5(10) 6.85 t 0.15(8)
0.220(18)
Diabetic 435.4 1 22(11)* 7.12 i 0.21(10)

 

aBlood samples were collected on the 18th day of gestation
from the orbital sinus 24 hours after the daily insulin or
saline injection.

.°Birth weight of live pups was determined by dividing litter

weight by the number of pups.
cEach value represents the mean 1 SEM (number of litters).

*Signicicantly different from control (student's t; p<.05).

Figure 1.

46

The effect of gestational diabetes on the

birth weight of the progeny from Series I.
Each bar represents the number of litters

with the average pup weight falling in the
indicated range. All litters contained at
least 8 pups.

Number of litters

47

      

'2 IPCM

11

10

9

3

7

6

5

4

3 ‘ V
2 \ s
I \ l . .\ \1 * \‘

 

(5.5 5.5 6.0- 6.5- 7.0- 7.5-
5.9 6.4 6.9 7.4 8.0

MEAN BIRTH WEIGHT (9)

Figure 2.

48

The effect of gestational diabetes on the
birth weight of the progeny from Series II.
Each bar represents the number of litters
with the average pup weight falling in the
indicated range.

Top: Each litter contained at least 5 pups.

Bottom: Each litter contained 9 to 12 pups.

NO. OF lITTERS

NO OF lITTERS

49

 

 

PCM -
PDM l\\‘
3 5 pups/litter
5
4 .L.
3 ‘1
5.5-5.9 6.0-6.4 6.5-6.9 7.0 -7.4 7.5-8.0
BIRTH WEIGHT(g)
9.12 pups/ litter
5 1'
4
3
2
T

 

    

5.5-5.9 6.0-6.4 6.5-6.9 7.0-7.4 7.5-8.0
BIRTH WEIGHT(g)

50

Bottom).

Fetal and Neonatal Mortality

The number of fetal and neonatal deaths is an indicator
of the severity of maternal diabetes (Solomon, 1959). In
the first series of experiments, 13% of the PDMs were born
dead and 7.4% died within 24 hours after birth (Table 2).
These rates were significantly greater than those for con-
trols in the same series of experiments (1.5% and 0% respec-
tively) (Table 2). In the second series of experiments,
however, the stillbirth rate and neonatal death rate were

comparable in the two groups (Table 2).

Body Weight and Kidney Weight

Body weight and kidney weight of PDMs were no different
than those of PCMs at 1, 5, or 10 days of age (Figures 3 and
4). However, by 28 days of age, PDMs weighed 29% less than
PCMs (66 i 6 g verses 83 i 2 9). Similarly, the kidneys
from PDMs weighed 20% less than those from PCMs (0.63 i 0.05
verses 0.78 i 0.40). The kidney weight/body weight ratios
for PDMs were no different than those for PCMs at any age
studied.

Percent of Water and Protein in the Renal Cortex
The percentage of water (Figure 5) and protein (Figure
6) in renal cortical slices from PDMs was no different than

that of PCMs at any age.

51

Table 2. Effect of gestational diabetes on fetal and neo-
natal mortality

 

Series I Series II
Control Diabetic Control Diabetic

 

 

Number of litters 22 18 10 10

Number of young

(average per litter) 11.7 11.9 7.9 9.8
Total number of young 263 215 83 98
Live born young 259 187 79 92
Still born younga 4 28 4 6
Still born rate (%) 1.52 13* 4.8 6.1
Neonatal deathsb o 16 o 0
Neonatal death rate (%) 0 7.4* 0 0

 

aDoes not include loss of entire litters.
°Neonata1 deaths include those rats that were alive at
birth but died within 24 hours.

cSignificantly different from control ( (Chi)2 test; p<.05).

Figure 3.

Figure 4.

52

Effect of gestational diabetes on kidney
weight of the progeny. Each bar represents
the mean i SEM obtained from at least 5
observations. One observation consisted

of kidneys pooled from one liter. Asterisks
denote a statistical difference between PCM
and PDM within an age group (p<.05).

Effect of gestational diabetes on body weight
of the progeny. Each bar represents the mean
t SEM obtained from at least 5 observations.
One observation consisted of kidneys pooled
from one liter. Asterisks denote a statisti-
cal difference between PCM and PDM within an
age group (p<.05).

53

' .°
an

  

.0
o~

- PCM
\\\‘ PDM

  

KIDNEY WEIGHT(g)
.0 .0
N h

8

o-

20

 
  

 

\‘
\\

 

aoov WEIGHT(g)

O

\\
5

AGE(days)

 

10

Figure 5.

Figure 6.

54

Effect of gestational diabetes on the percent
of protein of cortical slices from kidneys of
the offspring. Each bar represents the mean
i SEM obtained from at least 5 observations.
One observation consisted of pooled slices
from one liter.

Effect of gestational diabetes on the percent
of water of cortical slices from kidneys of
the offspring. Each bar represents the mean
obtained from at least 5 observations. One
observation considted of pooled slices from
one liter. The SEM was too small to be

shown with the bars.

55

- PCM
s\\\ PDM

 

% WATER
KIDNEY CORTEX

 

1o
AGE (days)

56

Accumulation of Organic Acid (PAH) py Renal Cortical Slices

Accumulation of PAH by renal cortical slices (PAH S/M
ratios) increased gradually from 1 to 28 days of age in
PCMs. In PDMs, PAH S/M ratios increased from 1 to 5 days of
age, decreased at 10 days of age, and increased to control
values by 28 days of age (Figure 7). PAH S/M ratios of
PDMs were no different than those of PCMs at l, 5, or 28
days of age. However, at 10 days of age, PAH S/M ratios of
PDMs were 32% lower than those of PCMs (6.41 t 0.8 verses
9.75 i 1.4) (Figure 7). This difference was statistically
different.

Accumulation of Orggpic Base (NMleby Renal Cortical Slices
Accumulation of NMN by renal cortical slices (NMN S/M

ratios) from PDMs was no different from that of PCMs at any

age (Figure 8). In both groups, NMN S/M ratios gradually

increased from 1 to 28 days of age (Figure 8).

Accumulation of aAminoisobutyric Acid (AIB) py Renal Corti-
cal Slices

Accumulation of AIB by renal cortical slices (AIB S/M
ratios) from PDMs was no different than that of PCMs at any
age (Figure 9). In both groups, AIB S/M ratios decreased

with increasing age (Figure 9).

Figure 7.

Figure 8.

57

Effect of gestational diabetes on PAH accumu-
lation by cortical slices from kidneys of the
progeny (PAH S/M ratio). Each bar represents
the mean 1 SEM of at least 6 determinations.
Asterisks denote a statistical difference
between PCM and PDM within an age group
(p<.05).

Effect of gestational diabetes on NMN accumu-
lation by cortical slices from kidneys of the
progeny (NMN S/M ratio). Each bar represents
the mean 1 SEM of at least 6 determinations.

PAH S/M

.b

NMN S/M
IQ -b 0‘ on

—a
N

on

 

 

- PCM
\\\‘ PDM

N
10

AGE(days)

5

 

28

Figure 9.

Figure 10.

59

Effect of gestational diabetes on AIB accumu-
lation by cortical slices from kidneys of the
progeny (AIB S/M ratio). Each bar represents
the mean i SEM of at least 7 determinations.

Effect of gestational diabetes on aMG accu-
mulation by cortical slices from kidneys of
the progeny (aMG S/M ratio). Each bar
represents the mean 1 SEM of 4 determina-
tions. Asterisks denote a statistical
difference between PCM and PDM within an
age group (p<.05).

AIMS/M)

a-MG (S/M)

6O

 
     

1.1
._ :zzi

 

 

 

AGE (days)

61

Accumulation of aMethylglucoside (aMG) by Renal Cortical
11.15.11

Accumulation of aMG by renal cortical slices (aMG S/M
ratio) from PDMs was no different from that of PCMs at 1,
5, or 28 days of age (Figure 10). However, at 10 days of
age, aMG S/M ratios from PDMs were 31% lower than those
from PCMs (1.58 1 0.1 verses 2.3 1 0.3) (Figure 10).

Gluconeogggic Capacity_of Renal Cortical Slices

The gluconeogenic capacity of renal cortical slices
from PDMs was no different than that of PCMs at 1, 10, or
28 days of age (Figure 11). However, at 5 days of age, the
gluconeogenic capacity of kidney slices from PDMs was 50%
greater than that of PCMs (64 t 11 verses 43 t 3.4 umol/
hour/gram dry weight).

Ammoniggenic Cgpacity of Renal Cortical Slices

The ammoniagenic capacity of renal cortical slices from
PDMs was 300% that of PCMs at one day of age (456 1 130
versus 182 1 33 pmol/hour/gram dry weight) and 200% that of
PCMs at 5 days of age (380 1 72 verses 187 1 24 umol/hour/
gram dry weight) (Figure 12). Although this trend persisted,
the renal ammoniagenic capacity of PDMs was not significantly
different than that of PCMs at 10 days of age. Similarly, at
.28 days of age, renal ammoniagenic capacity of PDMs was no

different than that of PCMs (Figure 12).

Figure 11.

Figure 12.

62

Effect of gestational diabetes on gluco-
neogenic capacity of kidney cortex slices
from the progeny. Each bar represents the
mean 1 SEM of at least 7 determinations.
Asterisks denote a statistical difference
between PCM and PDM within an age group
(p<.05).

Effect of gestational diabetes on ammonia-
genic capacity of kidney cortex slices from
the progeny. Each bar represents the mean
1 SEM of at least 7 determinations.
Asterisks denote a statistical difference
between PCM and PDM within an age group
(p<.05).

GLUCOSE SYNTHESIS

(pmoI/hr/g)

AMMONIA SYNTHESIS

(pmoI/hr/g)

O
O

.h
0

N
O

.8

a:
9

    

- PCM

’ 11

1

 

 

600 ‘

 

\\
\\ 1 1 1

5 10 28

 

 

1 1
111

5 10 28
AGE(da ays )

64

gffect of Acidosis on Glucose and Ammonia Production by
Renal Cortical Slices

Kidney slices from 10 day old PDMs and PCMs treated
with ammonium chloride (SmM/kg) produced slightly more glu-
cose but no more ammonia than those from controls (Table 3).
Renal gluconeogenic and ammoniagenic capacities of kidney
slices from PDMs was no different than that of kidney
slices from PCMs both in the presence and absence of acido-

sis (Table 3).

Effect of Gestational Diabetes on the Excretion of water,
Isotonic and Hypertonic Saline Loads by the Progeny

In general, PDMs and PCMs demonstrated a similar
ability to compensate for water and isotonic saline loads
(Figures 13 to 24; Tables 4 and 5). The urine, total
solute, sodium; potassium, chloride and urea excreted in
4 hours relative to body weight was the same in 5 or 10 day
old PDMs treated with isotonic saline or water when compared
to age matched PCMs receiving the same treatment (Figures
l3, l5, l7, l9, 23). While the ability to compensate for
water and isotonic saline loads was similar in PDMs and
PCMs, the ability to compensate for a hypertonic saline load
appears to be different between the two groups. After a
hypertonic saline load, total solute excretion by both 5
and lo day old PDMs was significantly greater than that by
age matched PCMs (Figure 15). Additionally, 10 day old PDMs

65

Table 3. Effect of acidosis on glucose and ammonia produc-
tion by renal cortical slices from ten day old
PDMs and PCMs

 

 

a b Percent
Hater NH4CL change
PCM
Glucose c d
(umol/hr/g ) 67.2 t 1.9 72.8 t 2.0* 8.4
Ammonia
(umol/hr/g) 438 i 18 403 t 3] -
PDM
Glucose
(umol/hr/g) 67.1 1 6.6 7l.8 i 7.3* 6.3
Ammonia
(umol/hr/g) 406 t 32 395 i 35 -

 

aRats were gavaged with water (2.5% of body weight).

bRats were gavaged with ammonium chloride (SmM/kg; 2.5% of
body weight).

cDry weight.
dEach value represents the mean i SEM obtained from 4 rats.

*Significantly different from control (p<.05).

Figure 13.

66

Effect of gestational diabetes on the
excretion of urine by the progeny during
the first 4 hours after salt and water
loading. Rats were gavaged with water,
isotonic saline (0.lSN sodium chloride) or
hypertonic saline (0.5N sodium chloride)

at 5% of the body weight. A fourth group
received the same dose of water and a
subcutaneous injection of ADH (0.l mU/g).
Bladders were emptied 2 and 4 hours after
treatment. Each bar represents the mean

i SEM obtained from an "n" of 4. For 5 day
old animals, urine from 2-5 rats was pooled
for an "n" of 1. For l0 day old animals,
urine from one rat was used for an "n" of
l. Means not sharing a common superscript
differ significantly (p<.05).

00
guns

.0

URINE excnenon (ml/k52/4hrs)

 

 

 
 

||||||||||||||||||||||||||||||||||lllllllllllllllllllllllf

H20

||||||||||||||||||||||||||||||||||||||||||||—°

 
 

O.I5N NaCI 0.5 N NaCl H2O +ADH

||||||||||||||||||||||||||||||||||||||I|||||||||||||||||||l|||)—°

|||||||||||||||||||f r

OJSN NaCI 0.5 N NaCl H20 +ADH

Figure 14.

68

Effect of gestational diabetes on the
excretion of urine by the progeny during
the 0-2 and 2-4 hour periods after salt

and water loading. Rats were gavaged with
water, isotonic saline (0.l5N sodium
chloride; or hypertonic saline (0.5N sodium
chloride at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.l mU/g). Bladders were emptied 2 and 4
hours after treatment. Each bar represents
the mean i SEM obtained from an "n" of 4.
For 5 day old animals, urine from 2-5 rats
was pooled for an "n" of 1. For 10 day old
animals, urine from 1 rat was used for an
"n" of 1. Means not sharing a common
superscript differ significantly (p<.05).

(pl/ lit/gram)

URINE excnenow

0.15%

0.10-*

0.05 '4

 

 

 

 

 

 

 

OJ 5-

0.10 -

0.05-i

 

 

 

 

 

 

L
"2

F
l

 

 

.HmHlHIIHIHHHIHHH”HHIHHIIIHHHIIHHIIIHIIIHHHHHHHIIHHIHHHIWHMill

llHlIlllllllllHIIIHHHIHHHIHIlllllllllIIHHHHHIIHIHIHHIHHHHHI

69

O—Zhr
PCM 2-4Iw

 

 

 

 

 

 

 

 

 

 

 

 

 

 

, __J h
OJSN N00 0. N NaCI

 

 

 

 

 

 

 

 

 

 

 

 

Hl-l

3.5 N

   

Figure l5.

70

Effect of gestational diabetes on the
excretion of total solute by the progeny
during the first 4 hours after salt and
water loading. Rats were gavaged with
water, isotonic saline (0.l5N sodium
chloride) or hypertonic saline (0.5N sodium
chloride) at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.l mU/g). Bladders were emptied 2 and

4 hours after treatment. Each bar repre-
sents the mean 1 SEM obtained from an "n"
of 4. For 5 day old animals, urine from
2-5 rats was pooled for an "n" of 1. For
10 day old animals, urine from 1 rat was
used for an "n" of 1. Means not sharing a
common superscript differ significantly
(p<.05).

71

24
22
20
IO
I6
I4

IO

|||||||||||||||||||||||||||||||||||||||||||||||||||l||I- "

 

Illllllllllllllf °
lllllllllllllllllf- °

w

 

 

(mOsIn/kg I4 I")

||||||||||||||f °

H20 OJSN NoCI 0.5N

-PCM
EPOM

SOLUTE EXCRETDN
E
§

N¥OOON§OOON§

ll|I|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||= "

||||||||||||||||||I °

C
.—_'I=

N20 OJSN NaCI 0.5N NaCI H20+ADH

Figure l6.

72

Effect of gestational diabetes on the
excretion of total solute by the progeny
during the 0-2 and 2-4 hour periods after
salt and water loading. Rats were gavaged
with water, isotonic saline (0.15n sodium
chloride) or hypertonic saline (0.5" sodium
chloride) at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.l mU/g). Bladders were emptied 2 and 4
hours after treatment. Each bar represents
the mean i SEM obtained from an "n" of 4.
For 5 day old animals, urine from 2-5 rats
was pooled for an "n" of 1. For 10 day old
animals, urine from l rat was used for an
"n" of 1. Means not sharing a common
superscript differ significantly (p<.05).

SOLUTE EXCRETION (mOsm/ hr/ kg)

 

 

 

 

 

 

 

 

 

 

 

 

73

O-2hr

 

 

PCM 2-4hr c
O-2hr
PDM -4hr c
A995 0
E J?

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

HHHHHHHH!HHHWIE7

__ L. LL
OJSN NoCl 0.5N NaCI

 

 

AgeIO

ab

 

 

.-_—
——
—_..—
.—
.—
.—
.—
.—
—_
.—
.——-
——
.—
—.—
——
—
——
_—
.—
-__—
__
.—
__——
.—
_———-
_——
——
_—
.—
.___
—_
.—
—._-
.—.——
_.———
——
—_
..__—o
_—
_—
__———
_—
_——
__———
_——-
——
——
_—
_—
_—
__—-—
.._.—_-
_——
__—
.—
—_
—_
——
‘—
_—
__—
.————
.—_——-—
_‘_—--
_——
——
.—
_—
.—
._——
__———-
——
——
_—
_—
..._———
.—
.___.—
...——

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L.
OJSN NaCI 0.5N NaCI

__._

 

Figure 17.

74

Effect of gestational diabetes on the
excretion of sodium by the progeny during
the first 4 hours after salt and water
loading. Rats were gavaged with water,
isotonic saline (0.15N sodium chloride) or
hypertonic saline (0.5N sodium chloride) at
5% of the body weight. A fourth group
received the same dose of water and a
subcutaneous injection of ADH (0.l mU/g).
Bladders were emptied 2 and 4 hours after
the treatment. Each bar represents the
mean i SEM obtained from an "n" of 4. For
5 day old animals, urine from 2-5 rats was
pooled for an "n" of l. For 10 day old
animals, urine from 1 rat was used for an
"n" of 1. Means not sharing a common
superscript differ significantly (p<.05).

75

ll|||||||||||||||||||||||||||||||||||||||||||||||)— °'

 

 

SODIUM excnsnoN (qu/kg/4hrs)

.L A9010

 

O
é

H20 O.l5NNoCl 0.5N NaCl H20+ADH

Figure 18.

76

Effect of gestational diabetes on the
excretion of sodium by the progeny during
the 0-2 and 2-4 hour periods after salt
and water loading. Rats were gavaged with
water, isotonic saline (0.15N sodium
chloride) or hypertonic saline (0.5N sodium
chloride) at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.1 mU/g). Bladders were emptied 2 and

4 hours after treatment. Each bar repre-
sents the mean t SEM obtained from an "n"
of 4. For 5 day old animals, urine from
2-5 rats was pooled for an "n" of 1. For
10 day old animals, urine from 1 rat was
used for an "n" of 1. Means not sharing a
common superscript differ significantly
(p<.05).

77

0'21"

 

 

 

 

 

 

 

 

 

 

 

‘ PCM 2-4hr
J_ O-2I1r
PDM 2-4hr
30--
20--
b b 5
io-- n In a: [—311

 

a a a 0
H20 O.15N NaCl 0.5N NaCl

SODIUM stcmsnon (qu/Iir/ kg-2)

 

 

 

 

 

 

 

 

 

 

 

 

.11—
30--
-— AgeIO
zo—r E;
io—- g;
__ b b b b E
Ame. 20.0 HI I El 2

 

 

 

 

 

 

 

 

Figure 19.

78

Effect of gestational diabetes on the
excretion of chloride by the progeny during
the first 4 hours after salt and water
loading. Rats were gavaged with water,
isotonic saline (0.15N sodium chloride) or
hypertonic saline (0.5N sodium chloride)
at 5% of the body weight. A fourth group
received the same dose of water and a .
subcutaneous injection of ADH (0.1 mU/g).
Bladders were emptied 2 and 4 hours after
treatment. Each bar represents the mean

2 SEM obtained from an "n" of 4. For 5
day old animals, urine from 2-5 rats was
pooled for an "n" of 1. For 10 day old
animals, urine from 1 rat was used for an
“n" of 1. Means not sharing a common
superscript differ significantly (p<.05).

79

#10
OO

||||||||||||||||||||||||||| ||||||||||1— °

 

 

 

H20 O.15N NoCI 0.5N NaCl H20+ADH

b b

CHLORIDE excnenou (qu /k§2/4 hr)

so - g
70- 2
so - 2
so - =2:
.0 .. g
30 .. g
20 g
m _ :

 

H20 O.15N NaCl 0.5N NaCI H20+ADH

Figure 20.

80

Effect of gestational diabetes on the
excretion of chloride by the progeny during
the 0-2 and 2-4 hour periods after salt and
water loading. Rats were gavaged with
water, isotonic saline (0.15N sodium
chloride) or hypertonic saline (0.5N sodium
chloride) at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.1 mU/g). Bladders were emptied 2 and

4 hours after treatment. Each bar repre-
sents the mean i SEM obtained from an "n"
of 4. For the 5 day old animals, urine
from 2-5 rats was pooled from an "n" of

1. For 10 day old animals, urine from 1
rat was used for an "n" of 1. Means not
sharing a common superscript differ signi-
ficantly (p<.05).

81

0'21"

 

PCM 2-4hr
30-- O-2hr
PDM 2-4I'Ir
+-
20 Ages
10‘-
a a 0
L534: Hm a“; H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CHLORIDE EXCRETION (qu/Iir/ kg-2)

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

H20 O.15N NaCl 0.5N NOCI
ao-- 5
2°" AgelO
1m— g g
0 °_ 0 0 a g
M HI ll g. 2
H20 O.15N NOCI 0.5 N NOCI

Figure 21.

82

Effect of gestational diabetes on the
excretion of potassium by the progeny
during the first 4 hours after salt and
water loading. Rats were gavaged with
water, isotonic saline (0.15N sodium
chloride) or hypertonic saline (0.5N sodium
chloride) at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.1 mU/g). Bladders were emptied 2 and 4
hours after treatment. Each bar represents
the mean i SEM obtained from an "n" of 4.
For 5 day old animals, urine from 2-5 rats
was pooled for an "n" of 1. For 10 day old
animals, urine from 1 rat was used for an
"n" of 1. Means not sharing a common
superscript differ significantly (p<.05).

83

0‘

- PCM
PDM ab

ON-b

. 1? |||||

whoop

|||l|||||||||||||||||||||||||||||||||||||||||||||||||||||h-—

||||||||||||||||||- °
||||||||||||||1 °

 

 

 

92.

H O own I 0.5NNOCI H20+ADH

N

b

 

5'
1

d
A
I

AgeO

:5
T

POTASSIUM EXCRETION ( qu/kgilm hr)
8
T

   

Nham

   

 

|||||||||||||||||||||||||||||||||||l|||||||||||||1—a-

||||||||||||||||||||||||1 °

 

|||||||||||||||f °
lllllllllllllllll- °

H O O.15N NaCI 0.5NNOCI H20+ADH

N

Figure 22.

84

Effect of gestational diabetes on the
excretion of potassium by the progeny
during the 0-2 and 2-4 hour periods after
salt and water loading. Rats were gavaged
with water, isotonic saline (0.15N sodium
chloride) or hy ertonic saline (0.5N

sodium chloride) at 5% of the body weight.
A fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.1 mU/g). B1adders were emptied 2 and 4
hours after treatment. Each bar represents
the mean i SEM obtained from an "n" of 4.
For 5 day old animals, urine from 2-5 rats
was pooled for an "n" of 1. For 10 day old
animals, urine from 1 rat was used for an
"n" of one. Means not sharing a common
superscript differ significantly (p<.05).

85

0'21"

 

 

 

 
   

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7 H 0.15 N NaCI

0.5N NOCI

PCM 2-4hr
__ O-2hr
4’0 PDM 2-4I1r
ab g
3.0“*' A995 2
ab 2
A 2.0-- :—
~ g
19 I) E
E LO“. 0 E
-= T;
\ g
g L-— 1.1 g
3- H20 O.15N NOCI 0.5N NOCI
2
Q
.—
III
5
X
Ill
2 4.0-,—
D
a AgeIO
< 3.0-—
5 .a
m g
2.0-“ 2%
111: a a g
.49 2
° :-
20

Figure 23.

86

Effect of gestational diabetes on the
excretion of urea by the progeny during the
first 4 hours after salt and water loading.
Rats were gavaged with water, isotonic
saline {O.TSN sodium chloride) or hypertonic
saline 0.5M sodium chloride) at 5% of the
body weight. A fourth group received the
same dose of water and a subcutaneous
injection of ADH (0.1 mU/g). Bladders were
emptied 2 and 4 hours after treatment.

Each bar represents the mean : SEM obtained
from an "n" of 4. For 5 day old animals,
urine from 2-5 rats was pooled for an "n"
of 1. For 10 day old animals, urine from 1
rat was used for an "n" of l.

UREA EXCRETION (Pmol/kg'2/4Iir)

 

 

87

    
    

ge5

IIIIF

|||||||l|||||||— ,,

||||||||||||||1|||1|||||||||||||

H O.15N NaCI

”O

AgeIO

|||||||||||||||||||||||||||||||||||||||||||||||||1-
||||||||||||II|||||||||||||||||||||||||||||||||l-

H20 O.15N NOCI

o
3

P

0.5N

2
Q

||||||||||||||||||||||||||||||||||||||||1—

|||||l|||l|||||l||||||||||||||||||III||||||||||||||||||||||lllllll—

—-

'D
n
3

0.5N NaCl

|||||||||||||||||||||l|||||||||||1

1120+ ADH

é

H20+ADH

Figure 24.

88

Effect of gestational diabetes on the
excretion of urea by the progeny during
the 0-2 and 2-4 hour periods after salt
and water loading. Rats were gavaged with
water, isotonic saline (0.15N sodium
chloride; or hypertonic saline (0.5N sodium
chloride at 5% of the body weight. A
fourth group received the same dose of
water and a subcutaneous injection of ADH
(0.l mU/g). Bladders were emptied 2 and

4 hours after treatment. Each bar repre-
sents the mean 1 SEM obtained from an "n"
of 4. For 5 day old animals, urine from
2-5 rats was pooled for an "n" of 1. For
10 day old animals, urine from 1 rat was
used for an "n" of l.

( meI/ I'Ir/ kg'2 )

UREA EXCRETION

 

 

 

 

 

 

89

0'21"
. PCM 2-4Iir

O-2Iir
PDM 2-4Iir

 

 

 

 

 

 

 

l|llIlll|||||||||||Ill||l|Illllllllllllllllllllllllllll

lllllllllllllllllllllllllllllllllllllllllllllllllll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N::
0

OJSN NOCI

AgeIO

 

_—
—
——
—.
——
—
.—
——
—
—.
.—
_
c—n—u-
n—I—u—u—
_—
.—
.—
_———
—_
_
——
_
_
——
_
.—
—
—_
_—
—
_
—
—
_
—
—_
_
—.
—_
—
_
.—
_—
up-
.—
_
—
-—
__
———
-—-—
—_
-—
—
——
_
——
.—
—_
—_
_
—

 

——
_
—_
——
~—
.—
nan—nau—
.—
-—
~—
—
~—
—
——
—
—
—
—
_—
—_
—
_
—_
——
.—
_—
——
—
—_
-——-
~—
—_
—_
——
——
——
Inn—u—
-—-—.
_
—
—
_-
__
—
-
—
——
—
——
——
——
——
——
—
.—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

H20 O.15N NOCI

 

'1“ 1 Ages

 

 

._——.—
_—
__
.—
.—
—
——
-—
—
-———
a__——
«II—u..—
_
——
—-
__
_
.—
_
—_
——
——
—_
_
.—
—_
——
——
_
.—
_
.—
_
—_
—_
_
—
—
——
.—
*—
_
_
—
—_
.—
.—

 

 

 

 

 

 

9':
0'

 

L

N NOCI

 

 

 

 

 

 

 

Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll

 

 

 

 

 

 

 

0.5N NOCI

90

Table 4. Response of five and ten day old PDMs and PCMs to
a water load: concentration of urinary constituents

 

Urinary 5 Day Olds 10 Day Dlds
6°"5t‘tuent o-z Hour 2-4 Hour 0-2 Hour 2-4 Hour

Osmolality (m05m/1iter)
PCM 145128a 307173 13114 274128
PDM 140:35 186:31 129:8 165:24
Urea (mM/liter)
PCM 40:11 99:21 33:4 77:9
PDM 30:4 67:20 29:4 44:9
Sodium (mEq/liter)
PCM 2.911.6 7.8i0.7 5.6:1.5 2.5:].8
PDM 4.6:2.2 2.8:2.2 3.8:0.7 0.8:0.6
Chloride (mEq/liter)
PCM: 10.215 10.312 21.0111 8.115
PDM 7.213 13.815 21.014 17.418
Potassium (mEq/liter)
PCM 16.8:5.5 22.0:6 9.0:1 37.0:11
PDM I3.0:4.7 12.0:4 12.1:0.9 15.312.4

aEach value represents the mean 1 SEM obtained from an "n" of
4. For 5 day old animals, urine from 2-5 rats was pooled
for an "n" of one. For 10 day old animals, urine from one
rat was used for an "n" of one. Rats were gavaged with
water at 5% of the body weight. Bladders were emptied
after 2 and 4 hours.

91

Table 5. Response of five and ten day old PCMs and PDMs to
an isotonic saline load: concentration of urinary
constituents

 

 

Urinary 5 Day 01ds 10 Day 01ds
Constituent 0-2 Hour 2-4 Hour 0-2 Hour 2-4 Hour
Osmolality
(mOSm/liter)
PCM 433159a 524113 489127 757142 F

PDM 407136 504120 475147 579136*
Urea (mM/liter) '
PCM 88.3:5.6 93.015.9 99.419.3 134.1125.4

 

PDM 86.6:12.9 92.0:7.3 97.5:13.6 94.5:10.9
Sodium (mEq/liter) '

PCM 61.1i18 122118 10116.9 165.5:8.6

PDM 62.5114 92:16 67:7.5* 138.0116
Chloride (mEq/liter)

PCM 64.3:9.9 99.417.3 102.817.6 145133
PDM 53.5:6.8 68.517.6 93.718.1 111112
Potassium
(mEq/liter)
PCM 39.0:11.0 34.417.3 22.513.5 55.3111.8
PDM 38.018.8 28.816.1 23.012.2 16.414.0*

 

aEach value represents the mean 1 SEM obtained from an "n" of
4. For 5 day old animals, urine from 2-5 rats was pooled
for an “n" of one. For 10 day old animals, urine from one
rat was used for an "n" of one. Rats were gavaged with
isotonic saline (0.15 N sodium chloride) at 5% of the body
weight. Bladders were emptied after 2 and 4 hours.

*Significantly different from PCM (p<.05).

92

excreted significantly more sodium (Figure 17) and urine
(FigUre 13) after a hypertonic saline load than age matched

PCMs.

Response of 5 and 10 DayAOld PDMs and PCMs to a Water Load
Five and ten day old rats responded to a water load (5%
of the body weight) with a diuresis, excreting significantly
more urine than rats given an isotonic saline load (Figures I
13 and 14). Both 5 and 10 day old PDMs and PCMs excreted J
59% or more of the water load within the first two hours

(Figure 14). Four hours after the water load, both 5 and

 

10 day old PCMs and PDMs excreted approximately 70% of the :
load (Figure 14). Two to four hours after the water load,
10 day old PDMs excreted 36% of the load while age matched
PCMs excreted only 18% of the load. This was significantly
different (p<.05).

The urinary excretion relative to body weight of total
solute (Figures 15 and 16), sodium (Figures 17 and 18),
chloride (Figures 19 and 20), potassium (Figures 21 and 22),
and urea (Figures 23 and 24) by 5 and 10 day old PDMs after
a water load was no different than that by age matched
PCMs. Similarly, the concentration of solutes in the urine
from 5 and 10 day old PDMs after a water load was no dif-
ferent than that of urine from age matthed PCMs (Table 4).

93

Response of 5 and 10 Day Old PDMs and PCMs to an Isotonic
Saline Load

In contrast to the water load, the isotonic saline load
(0.15N sodium chloride, 5% of the body weight) did not
produce a diuresis. A relatively small pencent of the total
load was excreted after 2 or 4 hours (Figure 14). Five and
ten day old PDMs and PCMs excreted approximately 16-18% of
the load within the first two hours and approximately 30% of
the load during the entire 4 hour period (Figures 13 and 14).
The excretion relative to body weight of total solute
(Figures 15 and 16), sodium (Figures 17 and 18), chloride
(Figures 19 and 20), potassium (Figures 21 and 22) and urea
(Figures 23 and 24) by 5 and 10 day old PDMs after an iso-
tonic saline load was no different than that by age matched
PCMs.

Similarly, the concentrations of solutes in urine from
5 and 10 day old PDMs collected 2 and 4 hours after adminis-
tration of isotonic saline was no different than that of
urine from age matched PCMs with the exceptions of potassium
and osmolar concentrations (Table 5). Urine from 10 day
old PCMs, collected 2-4 hours after treatment, had a greater
potassium concentration as well as a greater osmolality than
urine from age matched PDMs (Table 5).

An isotonic saline load did not produce a natriuresis
in 5 day old PDMs or PCMs but did produce one in 10 day old
PDMs and PCMs (Figure 18).

 

94

Response of 5 and 10 Day Old PDMs and PCMs to a Hypertonic
Saline Load

The osmotic diuresis produced by gavaging rats with
hypertonic saline (0.5N sodium chloride; 5% of the body
weight) was delayed when compared to the diuresis produced
by administration of water (Figure 14). Five day old PCMs
excreted 14% of the load by 2 hours and 48% of the load by
4 hours (Figure 14). Age matched PDMs excreted 22% of the ’
load by 2 hours and 66% of the load by 4 hours (Figure 14).
These were not significantly different. Ten day old PCMs

 

excreted 18% of the hypertonic saline load after 2 hours and
66% after 4 hours (Figure 14). Age matched PDMs excreted
40% of the load after 2 hours and 95% of the load after 4
hours (Figure 14). After administration of hypertonic
saline, ten day old PDMs excreted significantly more urine
in the first 2 hours (Figure 14), a similar amount of urine
in the second two hours (Figure 14), and significantly more
urine in the entire four hour period (Figure 13) when com-
pared to age matched PCMs. In addition, both 5 and 10 day
old PDMs excreted more total solute than age matched PCMs
(Figure 15). The excretion of sodium (Figures 17 and 18),
chloride (Figures 19 and 20), potassium (Figures 21 and 22),
and urea (Figures 23 and 24) by 5 day old PDMs was no
different than that by age matched PCMs treated similarly.
Ten day old PDMs treated with hypertonic saline excreted

the same amount of potassium (Figure 21), chloride

95

(Figure 19) and urea (Figure 23) but significantly more
sodium (Figure 17) within the 4 hour period than age matched
PCMs treated similarly.

With the onset of the osmotic diuresis, urinary concen-
trations of sodium, chloride, and urinary osmolality
increased while urea concentrations decreased (Table 6).
Urinary potassium concentration remained unchanged in 5 day
old PCMs but decreased in 5 day old PDMs with the onset of
the osmotic diuresis (Table 6). The potassium concentration
of urine collected from 10 day old PDMs 2 hours after the
administration of hypertonic saline was significantly lower
than that of urine from age matched PCMs (Table 6). In
contrast, urine collected from ten day old PDMs 4 hours
after treatment had the same potassium concentration as
that collected from PCMs (Table 6).

In the first 2 hours after the administration of hyper-
tonic saline ten day old PDMs excreted more urine than age
matched PCMs (Figure 14) but the urine from PCMs had a
significantly greater osmolality than that of PCMs (Table 6).

Response of 5 and 10 Day Old PDMs and PCMs to a Water Load

in the Presence of Exogenous ADH

 

The excretion of urine (Figure 13), total solute
(Figure 15), sodium (Figure 17), chloride (Figure 19),
potassium (Figure 21), and urea (Figure 23) by 5 and 10 day

old PDMs and PCMs given a subcutaneous injection of ADH

96

Table 6. Response of five and ten day old PCMs and PDMs
to a hypertonic saline load: concentration of
urinary constituents

 

 

Urinary 5 Day 01ds 10 Day Olds
Constituent 0-2 Hour 2-4 Hour 0-2 Hour 2-4 Hour
Osmolality
(m05m/1iter)
PCM 526i6a 550191 574132 555127
PDM 481i22 485128 52519* 50113

Urea (mM/liter)
PCM 61.8i8.5 33.838.1 81.8:10.6 36.6154
PDM 50.0i5.6 28.814.7 57.3:3.4 30.312.1
Sodium (mEq/liter)
PCM 140323 188i18 20514.3 23718.2
PDM 158:29 210i15 20414.6 23615.3
Chloride (mEq/liter)

PCM 107116 138i19 167125 225117
PDM 118316 152123 176135 183121
Potassium
(mEq/liter)
PCM 87:27 54:29 62.618.4 35.5125
PDM 52113 3619 24.913.8* 27.712.2

 

aEach value represents the mean 1 SEM obtained from an "n" of
4. For 5 day old animals, urine from 2-5 rats was pooled
for an "n" of one. For 10 day old animals, urine from one
rat was used for an "n" of one. Rats were gavaged with
hypertonic saline (0.5N sodium chloride) at 5% of the body
weight. Bladders were emptied after 2 and 4 hours.

*Significantly different from PCM (p<.05).

97

immediately after a water load (5% of the body weight) was
no different than that of age matched PDMs and PCMs given
only water.

Similarly, urinary concentrations of sodium, chloride
potassium, urea, and urinary osmolality were no different
in animals in the presence and absence of exogenous ADH
(Table 7 verses Table 4).

The ADH injection did, however, affect the pattern of
urea excretion (Figure 25). When given exogenoUs ADH, both
5 and 10 day old PDMs and PCMs excreted significantly less
urea during the 2-4 hour period than during the 0-2 hour
period (Figure 25). In contrast, both groups of animals
treated with water alone excreted the same amount of urea

in the 0-2 hour and the 2-4 hour periods (Figure 25).

98

Table 7. Response of five and ten day old PDMs and PCMs
to a water load in the presence of exogenous ADH:
concentration of urinary constituents

 

Urinary 5 Day 01ds 10 Day Olds
Constituent 0-2 Hour 2-4 Hour 0-2 Hour 2-4 Hour

 

Osmolality

(m05m/liter)
PCM 155149a 201132 12815 274120

PDM . 128i18 202:17 137i14 230i61
Urea (mM/liter)

PCM 31.615.2 67i11 32.0i3.6 61.6i8.7

PDM 32.6i5.2 42i3 37.9111.7 73.0i18
Sodium (mEq/liter)

PCM 5.010.8 1.510.8 11.8i2.3 5.0i1.2

PDM 4.0i1.1 1.4i1.4 12.6i1.9 9.5i2.5
Chloride (mEq/liter)

PCM 8.313.5 11.615.9 7.412.8 6.214.5
.PDM 3.4tl.6 3.712.3 15.5i6.1 1.1i1.1
Potassium
(mEq/1iter)
PCM 8.5:1.4 11.114.4 5.0i0.9 39.514.6
PDM 11.212.7 16.115.l 6.311.l 29.1:6.0

 

aEach value represents the mean 1 SEM obtained from an "n"
of 4. For 5 day old animals, urine from 2-5 rats was
pooled for an "n" of one. For 10 day old animals, urine
from one rat was used for an "n" of one. Rats were
gavaged with water at 5% of the body weight and injected
subcutaneously with ADH (0.l mU/g). Bladders were
emptied after 2 and 4 hours.

Figure 25.

99

The effect of ADH (0.l mU/g; s.c.) on urea
excretion by 5 and 10 day old PDMs and PCMs
after a water load (5% body weight).
Bladders were emptied 2 and 4 hours after
treatment. Each bar represents the mean

1 SEM obtained from an "n" of 4. For 5 day
old animals, urine from 2-5 rats was pooled
for an “n" of 1. For 10 day old animals,
urine from 1 rat was used for an "n? of one.
Asterisks denote a statistical difference
from the 0-2 hour collection (p<.05).

 

 

 

(mel/hr/ 119-2 )

UREA EXCRETION

 

 

 

 

 

 

 

 

 

 

 

100

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DISCUSSION

Newborn infants of diabetic mothers (IDMs) have a
number of significant problems. As a population, they are
a heterogeneous group of infants whose chemical homeostasis
at birth is altered by varying degrees of hyperinsulinemia
(Steinke and Driscoll, 1965; Block gt gl., 1974), hypoglu-
cagonemia (Johnston and Bloom, 1975), hypocalcemia (Zetter-
strom and Arnhold, 1958; Tsang gt gl., 1972; 1975), hypo-
glycemia (Farquhar, 1976; Kitzmiller gt gl., 1978), R05
(Farquhar, 1976; Kitzmiller gt 91., 1978), altered electro-
lyte and nitrogen excretion (Osler and Pederson, 1960;
Osler, 1960; Cook gt gl., 1960), and metabolic acidosis
(Lowery gt gl., l954; Segal _t gl., l957; Kitzmiller _t gl.,
1978).

The use of rats made diabetic by alloxan or streptozoto-
cin as an animal model for human diabetic pregnancy has led
to conflicting results with respect to fetal and neonatal
body weight. Aerts and Van Assche (1977) injected Nister
rats on day one of pregnancy with 30, 40, or 50 mg/kg of
streptozotocin to produce both mild and severe gestational
diabetes. They found fetal weight of severely diabetic

animals lower when compared to that of controls or mildly

101

102

diabetic animals; however, at birth there were no significant
differences in body weight among rats born of control, mildly
diabetic, or severely diabetic dams. Similarly, Kervran

gt g1. (1978) produced "mild" diabetes (blood glucose rang-
ing from 100 to 200 mg/dl) and "severe" diabetes (glucose
concentration above 300 mg/dl) in pregnant rats to study the
fetal pancreas. At term, fetuses from severely diabetic
‘rats weighed significantly less than those from controls

but fetuses from mildly diabetic rats weighed the same as
those from controls.

Solomon (1959) injected Sprague-Dawley rats on the
8-l4th day of gestation with different doses of alloxan.

The mildly diabetic rats prodUced overweight progeny while
the severely diabetic rats were unable to maintain pregnancy.
In contrast, other studies have reported that untreated
maternal diabetes in rats produced significantly heavier
neonates while diabetic rats treated with daily injections
of insulin produced normal weight progeny (Pitkin gt gl.,
1971).

In the present study, Sprague-Dawley rats were injected
with streptozotocin (50 mg/kg) on day one of gestation and
treated with insulin throughout gestation in an attempt to
maintain a "mild" diabetic state. Blood samples were col-
lected on the 18th day of gestation, 24 hours after the
daily insulin injection,to verify the presence of diabetes

and to ascertain the severity of diabetes in the absence of

103

insulin control. The birth weight of the progeny did not
correlate with maternal plasma glucose levels (Table 1).
This finding supports clinical studies showing no direct
relationship between maternal plasma glucose levels and
infant birth weights (Persson, 1975). Since plasma glucose
concentrations reported in the present study were determined
from blood collected approximately 24 hours after insulin or
saline injections, they probably do not represent the
average daily plasma glucose concentration. For this

reason plasma glucose levels of the dams can not be used as
a means of classifying the severity of the diabetes. Since
previous studies with rats have shown positive correlations
between the severity of maternal diabetes, birth weight of
the progeny and fetal and neonatal mortality (Solomon,

1959; Szalay and Gaal, 1975), it was of interest to examine
these parameters in the present study.

The birth weights of the progeny from diabetic dams
were not significantly different from those of controls
(Table 1). This observation confirms some previous reports
(Pitkin and Van Ordin, 1974; Aerts and Van Assche, 1977;
Kervran gt_gl., 1978) but disagrees with others observing
increased weight in fetuses from mildly diabetic pregnant
rats (Solomon, 1959; Pitkin gt g1., 1971). The reason(s)
for this discrepancy remain unclear. However, a similar
situation exists in the population of human IDMs. Persson

(1975) reported that of 81 insulin-dependent diabetics, 56

104

gave birth to normal weight infants, 17 gave birth to over-

weight infants (greater than the 90th percentile) and 8 gave
birth to underweight infants (smaller than the 10th percen-

tile). Since mean litter birth weight for PDMs ranged from

low to high (Figures 1 and 2), diabetic rats in the present

study were most likely a combination of both severe and mild
diabetics.

Alternatively, fetal and neonatal mortality reflect the
severity of the maternal diabetes (Solomon, 1959). In the
present study, the stillbirth rate from the diabetic group
in Series I was 13% (Table 2). This was significantly
greater than that of the control group (1.5%) and agrees
with the stillbirth rate for diabetic rats reported by
Solomon (1959) and by Szalay and Gaal (1975). Additionally,
the neonatal death rate (percentage of pups dying within 24
hours after birth) for the diabetic group (7.5%) in Series I
was greater than that of the control group (0%). Since both
Solomon (1959) and Szalay and Gaal (1975) reported a higher
incidence of fetal and neonatal deaths from severely dia-
betic rats, it is likely that the hyperglycemia in diabetic
rats from Series I was not as well controlled as that in
Series II. Neonatal and stillbirth rates of the diabetic
group in Series II were comparable to those of control.
Furthermore, there were fewer pups with low birth weights
from diabetics in Series 11 (Figure 2) than from diabetics

in Series I (Figure 1). These data suggest that the

105

diabetic rats in Series I represent the severely diabetic
model while those in Series 11 represent the mildly diabetic
model. This is further supported by an analysis of the
frequency distribution of the body weights at birth
(Figures 1 and 2). Fifty-five percent of the PDMs in
Series I weighed below the grand mean (Figure 1) while 77%
of PDMs weighed above the grand mean in Series II (Figure
2).

Regardless of birthweight, IDMs suffer from altered
insulin metabolism. Approximately 50% of IDMs are hypo-

glycemic (Farquhar, 1976; Kitzmiller gt 1., 1978) as a

result of hyperinsulinemia (Block gt gl., 1974). Fetuses
of mildly diabetic rats showed marked hyperinsulinemia
which increased in magnitude with fetal age (Kervran gt gl.,
1978). At birth, these fetuses had significantly higher
plasma insulin and glucose concentrations; birth weights,
however, were not significantly different. 0n the other
hand, neonates from severely diabetic animals had signifi-
cantly depressed insulin levels, elevated blood glucose
levels, and weighed significantly less than control infants
(Kervran gt gl., 1978).

In Series I, body weights and kidney weights of rats
born from diabetic dams were no different than those born
of control dams at one, five, or ten days of age. However,

by 28 days of age, both the body and kidney weights of PDMs

were significantly less than those of age matched controls

106

(Figure 3 and 4). It is likely that a majority of the pups
used in this set of experiments (Series I) were born from
dams with severe diabetes (Figure 1, Table 2). Since neo-
nates from severely diabetic animals have degranulated beta
cells (Aerts and Van Assche, 1977) and significantly
depressed plasma insulin concentrations (Kervran gt gl.,
1978) it is possible that PDMs from Series I lacked suffi-
cient insulin for normal growth. The observation that

PDMs and PCMs weighed the same at l, 5, and 10 days of age
indicates that as long as the animals were suckling, they
grew normally. Hhen rats are approximately 14 days of age,
they begin to consume the high carbohydrate rat chow (Wayne
Lab Blox; 24% protein; 4% fat; 4% fiber; and 68% carbohydrate
by weight) in addition to suckling. It could be that pups
from severely diabetic dams could not produce sufficient
insulin for use and storage of nutrients and thus, by 28 days
of age, weighed significantly less than controls. Although
kidney weight was lower in PDMs at 28 days of age, the
protein and water content of kidney slices was no different
in PDMs than PCMs at any age (Figures 5 and 6). These
findings agree with data reported by Solomon (1959) and
Pitkin gt _1. (1971), who analyzed fetal composition and
found the same percentage of protein and water in fetuses
born of diabetic dams as those born of control dams.

Evidence to date suggests that PDMs suffer from an

altered postnatal nutritional status (Aerts and Van Assche,

107

1977; Kervran _£._l-1 1978). Since alterations in postnatal
nutritional status have been reported to both enhance (Bond
gt gl., 1977) and retard (Goldstein _t _l., 1979) postnatal
development of renal tubular transport functions in rats, it
was of interest to determine the effects of gestational
diabetes on the postnatal development of renal tubular
transport functions in the progeny.

The intrinsic renal transport capacity of PAH from
control rats 1 to 28 days of age showed a similar pattern
of functional development as that reported by Kim gt_gl.
(1972) and Goldstein gt_gl, (1979). The accumulation of PAH
by renal cortical slices from control litters was low at
1 day of age and progressively increased to maximum values
at 28 days of age (Figure 7). A similar pattern was
observed for PDMs except at 10 days of age. At this age,
renal cortical slices from PDMs accumulated significantly
less PAH than those from PCMs (Figure 7).

The accumulation of PAH by renal cortical slices is
age related. As the rat increases in age, so does the
ability of kidney slices to accumulate PAH. Recently,
Stopp and Braunlich (1977) quantified the kinetic parameters
of PAH uptake by renal cortical slices using the Lineweaver-
Burke analysis. These data suggest that the number of
transport sites increases during postnatal development of

the rat kidney. Insulin production may be abnormal in PDMs

(Aerts and Van Assche, 1977; Kervran gt gl., 1978): Since

108

insulin promotes protein synthesis and since a protein
carrier is responsible for PAH transport, it is possible
that the depressed plasma insulin concentrations have
delayed the synthesis of these protein transport sites.
Thus, kidneys from 10 day old PDMs have no more transport
sites than those from 5 day old PDMs. In contrast, the
number of transport sites in kidney slices from PCMs
increased as the animal increased from 5 to 10 days of age
(Figure 7). However, as PDMs mature between 10 and 28 days
of age, transport proteins are synthesized such that kidney
slices from 28 day old PDMs accumulate the same amount of
PAH as those from age matched controls (Figure 7).
Alternatively, the accumulation of PAH by renal corti-
cal slices is sensitive to sodium or potassium concentration
in the medium but the accumulation of NMN is not (Kim gt gl.,
1972). Kidney slices required an ionic environment similar

to the i vivo situation to accumulate PAH. This was not

 

true for the accumulation of NMN. Since IDMs have been
reported to have low tissue levels of potassium (Fee and
Neil, 1963) it may be that the electrolyte composition of
kidney slices from 10 day old PDMs was sufficiently altered
to depress the transport of PAH.

The accumulation of the organic base, NMN, by renal
corticai slices from both PDMs and PCMs l to 28 days of age
showed the characteristic developmental pattern observed by

Kim gt g1. (1972), and appeared to follow that of organ

109

growth. The ability of renal cortical slices from PDMs to
accumulate NMN was the same as that of slices from PCMs
(Figure 8). The accumulation of NMN by renal cortical
slices appears to be less sensitive to environmental changes
than that of PAH. As mentioned previously, kidney slices
accumulate NMN regardless of the sodium or potassium concen-
tration of the medium (Kim gt gl., 1972). Therefore, NMN
S/M ratios may not represent active transport of NMN, but
indiscriminate tissue binding. If this were so, the accu-
mulation of NMN by renal cortical slices would not be
altered by either changes in the electrolyte composition of
kidney slices or by a lower number of functional transport
proteins for NMN.

In contrast to the developmental patterns of kidney
slice accumulation of PAH and NMN, the accumulation of AIB
by renal cortical slices is highest at birth and progres-
sively declines to adult values (Figure 9). These data
support observations by Webber and Cairns (1968) and Segal
gt g1. (1971). The observation that renal cortical slices
from both PDMs and PCMs accumulated similar amounts of AIB
probably indicates that renal tubular cells have a similar
nutritive need for amino acids. AIB accumulation by renal
cortical slices does not represent active reabsorption of
amino acids by the tubular lumen but rather active uptake
at the peritubular membrane. The fact that kidney slices

from immature animals can form higher concentration gradients

110

or higher S/M ratios than those from adults is partly
explained by a slower rate of efflux of AIB from immature
kidney slices (Hebber, 1968).

Unlike the accumulation of AIB, renal cortical slice
accumulation of OMB does reflect active transport of sugar
at the luminal membrane and thus represents the active
reabsorption of sugar by the kidneys. In the present study,
renal cortical slices from 1 and 5 day old controls exhibi-
ted a slight ability to concentrate aMG (Figure 10). By
10 days of age, renal cortical slices from control rats
were able to accumulate aMG to maintain S/M ratios greater
than 2. These data are consistent with those reported by
Segal _t _l. (l973a) who found that renal cortical slices
from newborn rats were unable to maintain a concentration
gradient. Adult aMG S/M ratios of approximately 2.5 were
attained by 14 days of age. The aMG S/M ratios from PDMs
were similar to those of PCMs except at 10 days of age.

At this age, aMG S/M ratios from PDMs were significantly
less than those for PCMs (Figure 10). The accumulation of
OMB, like that of PAH, reflects the number of functional
transport sites (Turner and Silverman, 1977). A lower
number of transport proteins resulting from alterations in
insulin production by PDMs may account for low aMG S/M
ratios observed in 10 day old PDMs (Figure 10).

Human IDMs have been reported to develop metabolic

acidosis (Lowery gt gl., l954; Segal gt gl., l957;

111

1., 1978). The kidneys play a major role in

Kitzmiller gt
the regulation of acid-base balance. Immature animals in-
cluding humans have less ability to compensate for acid-base
disturbances than adults (McCance, 1961; Goldstein, 1970).
The availability of urinary buffers, such as ammonia or
phosphates, determines the amount of hydrogen ion that can
be excreted. The availability of urinary phosphate is
limited by the serum level; however, the availability of
ammonia is enhanced during metabolic acidosis by renal
ammoniagenesis in the proximal tubular cells (Pitts, 1976).
Thus, it was of interest to determine the ammoniagenic
capacity of renal cortical slices of the progeny of diabetic
dams.

Results from the present study indicate that the pro-
duction of ammonia by renal cortical slices from PCMs was
low (less than 200 pmollhour/gram tissue, dry weight) at
1 day of age and remained at this level at 5 and 10 days of
age. At 28 days of age, there was a marked increase in
ammoniagenic capacity (400 umol/hour/gram) (Figure 12).

This pattern of development is comparable to that reported
by Goldstein gt g1. (1979) and supports other studies in
rats reporting low rates of ammonia excretion during the
first 2 weeks of age and a rapid increase between 14 and 21
days of age (Goldstein, 1970; Goldstein, 1971). Similarly
in the rat, renal glutaminase and glutamine synthetase

levels were low, approximately one third of the adult

112

levels, at 9 days of age but increased progressively from
10 to 22 days of age to 70% of the adult value (Goldstein,
1971). Therefore, the dramatic increase in renal ammonia-
genic capacity observed in 28 day old rats coincides with
an increase in the activity of ammoniagenic enzymes (Gold-
stein, 1971). In the present experiments, young rats born
of diabetic dams had a greatly enhanced renal ammoniagenic
capacity at l and 5 days of age (Figure 12). Although the
trend persisted at 10 days of age, the differenfie between
PDMs and PCMs was not significantly different (Figure 12).
By 28 days of age, renal cortical slices from PDMs produced
the same amount of ammonia as those from PCMs.

It has been demonstrated that chronic administration
of ammonium chloride to 10 day old rats enhanced the rate
of ammonia excretion (Goldstein, 1970). Furthermore, it
is well documented that metabolic acidosis causes an
increase in both renal ammoniagenesis and gluconeogenesis
in adult rats (Goodman gt gl., 1966; Pitts, 1976). There-
fore, it is possible that prenatal and neonatal metabolic
acidosis stimulated the production of ammonia by kidneys of
PDMs. Fetuses of diabetic animals can develop metabolic
acidosis in two ways. The first is in conjunction with the
mother during diabetic ketoacidosis (Farquhar, 1976; Felig,
1977). The second occurs when fetal blood glucose concen-
trations increase in association with maternal hyperglycemia.

Robillard gt g1. (1978) infused hypertonic glucose solutions

113

into well oxygenated fetal lambs. Hhen fetal blood glucose
concentration rose above 150 mg/dl there was a simultaneous
increase in fetal plasma lactate levels and a decrease in
plasma pH. Hhen fetal plasma glucose concentrations rose
and pC0

above 300 mg/dl, fetal DO 2 decreased and a severe

2
metabolic acidosis ensued. Since ketonuria was not observed
in any of the pregnant diabetic rats in the present study,
it is unlikely that fetuses of diabetics developed ketoacido-
sis. However, it is probable that fetal hyperglycemia
occurred and caused a lactate acidosis. A chronic acidosis
throughout gestation could have increased the capacity of
renal cortical slices from PDMs to produce ammonia.

Renal gluconeogenesis, like renal ammoniagenesis, is
stimulated by metabolic acidosis in adult rats (Goodman
gt_gl., 1966). It is thought that renal gluconeogenesis may
influence ammonia production by regulating the concentration
of glutamate (Goldstein and Harley-Dewitt, 1973). In view
of the effect maternal diabetes had on the renal ammonia-
genic capacity of the progeny, it is interesting to consider
the effects of maternal diabetes on renal gluconeogenic
capacity of the progeny.

The developmental pattern for renal gluconeogenic
capacity in control rats was similar to that for ammonia-
genic capacity (Figure 11). That is, gluconeogenic capacity
was relatively low at l, 5, and 10 days of age but showed a

marked increase at 28 days of age (Figure 11). This agrees

114

with the developmental pattern reported by Goldstein gt g1.
(1979) for rats. Similarly, other studies have reported
that the ability of renal cortical slices to produce glucose
was significantly lower in 10 day old rats than in adults,
but rose to adult levels by 12 days of age (Goldstein and
Harley-Dewitt, 1973). The increase in renal gluconeogenic
capacity observed to occur with increasing age does not
appear to be associated with the development of gluconeo-
genic enzymes. Zorzoli gt g1. (1969) measured the activity
of renal glucose-6-phosphatase (G-6-P) and PEPCK and found
that adult levels were reached by 3-4 days of age. PEPCK
reached a maximum of 140% of adult values at 7 days of age
and thereafter declined to adult values. Therefore it
appears that the increase in renal gluconeogenic capacity
that occurs between 10 and 28 days of age is more closely
associated with the increase in renal ammoniagenic capacity
than with the development of gluconeogenic enzymes.

Renal gluconeogenic capacity in one day old PDMs was
no different than that in age matched PCMs. Zorzoli gt g1.
(1969) found that the activity of G-6-P in newborn rats was
only 25% of the adult value. Similarly, the activity of
PEPCK was about 50% of the adult value. Thus it could be
that in one day old rats, metabolic acidosis increased renal
ammoniagenesis but did not increase renal gluconeogenesis
due to the limited activity of gluconeogenic enzymes at this

age. However, by 5 days of age, renal cortical slices from

115

PDMs produced significantly more glucose from glutamate than
those from age matched PCMs (Figure 11). It could be that
with the increase in the activities of G-6-P and PEPCK that
occursfrom 2-4 days of age, these enzymes now responded to
the chemical signals known to stimulate gluconeogenesis (an
increase in aketoglutarate or a decrease in cytosolic pH).
Since glucose production by renal cortical slices from PDMs
at 10 and 28 days of age was no different than that for age
matched controls (Figure 11) it appears that thé stimulus
that is present at 5 days of age is gone by 10 days of age.

Since it was hypothesized that fetal and neonatal aci-
dosis induced renal ammoniagenic capacity in neonatal PDMs,
it was of interest to induce an acute acidosis with intra-
gastric administration of ammonium chloride to 10 day old
PDMs and PCMs and measure subsequent renal ammoniagenic and
gluconeogenic capacities.

Acute acidosis did produce a small but significant
increase in gluconeogenesis (Table 3) by renal cortical
slices from both PDMs and PCMs but it had no effect on renal
ammoniagenesis. Goldstein and Harley-Dewitt (1973) measured
the gluconeogenic capacity of renal cortical slices from 10
day old rats. In their studies, medium pH was altered to

simulate 1 vivo acidosis (medium pH of 7.1). They reported

 

that slices incubated in acidotic medium produced 22% more
glucose than those incubated at pH 7.7. Slices incubated

at pH 7.1 produced 61.4 1 4.5 pmol/g dry wt x hr whereas

116

those incubated at pH 7.7 produced 50.3 1 3.4. In the
present experiments, all slices were incubated in medium
with a pH of 7.4. Slices from acidotic animals produced
8.4% more glucose than those from controls (Table 3). Renal
cortical slices from acidotic animals produced 72.8 1 2
umol/hr/gram dry wt while those from littermates produced
67.2 1 1.9. Thus the values reported in this study are
consistent with those reported by others (Goldstein and
Harley-DeWitt, 1973) and provide evidence that alterations
in medium pH, the method used by Goldstein and Harley-Dewitt
(1973) did indeed simulate the i vivo acidotic state.

 

Renal cortical slices from PDMs produced the same amount of
glucose and ammonia as those from PCMs both in the presence
and absence of aCidosis (Table 3). Additionally, ammonia
production by kidney slices was not enhanced by acidosis
(Table 3). This observation supports data reported by many
workers (Preuss gt 1., 1974; Roobol and Alleyne, 1974;

Cartier gt gl., 1975) showing that a reduction in the pH

of the medium either reduces ammonia production by kidney
slices or fails to affect it, while simultaneously increasing
glucose production. However, the amount of glucose and
ammonia produced by both acidotic and control animals in

this experiment appears higher than that produced by 10 day
old rats in previous experiments (Figure 11 and 12 compared

to Table 3). It is possible that all the pups were in a

mild state of acidosis from lack of food. In previous

117

experiments, pups were taken from their mothers and imme-
diately killed. In contrast, in this experiment, pups were
kept from their mothers for 3 to 4 hours before they were
killed. While this mild state of acidosis may mask the
effects of the ammonium chloride on renal ammoniagenesis
and gluconeogenesis, it probably would not mask differences
between PDMs and PCMs. Acidosis remains a possible cause
for the increased ammoniagenic capacity observed in one and
five day old PDMs. However, renal ammonia prodUction can
be influenced by other factors. Recent studies have shown
that ifl.!i££2 renal ammoniagenesis appears to be regulated
by substrate oxidation by kidney slices. When kidney slices
are incubated in a medium containing lactate alone or com-
bined with other fuels, renal ammoniagenesis is markedly
inhibited. In addition, the presence of lactate causes an
increased accumulation of glutamate. In contrast, the inhi-
bition of ammoniagenesis or the accumulation of glutamate
does not occur when kidney slices come from acidotic rats
(Preuss gt gl., 1978a). Thus it appears that lactate alone
or combined with other substrates decreases ammoniagenesis
primarily at the GDH step and that slices from acidotic rats
are resistant to substrate mediated inhibition.
Alternatively, potassium depletion is associated with
high rates of ammonia excretion (Tannen, 1977). Since low
plasma potassium concentrations have been reported for some

IDMs (Farquhar, 1976), potassium depletion may also be a

118

factor in the enhanced ammoniagenic capacity observed in
PDMs.

IDMs have been reported to excrete abnormally large
amounts of urine, potassium, chloride, and nitrogen (Osler,
1960; Cook gt gl., 1960; Osler and Pederson, 1960). Since
abnormal electrolyte excretion may reflect abnormal kidney
function among other things, it was of interest to determine
the ability of PDMs to compensate for salt and water
excesses. '

The results indicate that 5 and 10 day old PDMs and
PCMs responded in a similar way to a water load. PDMs and
PCMs gavaged with water (5% of body weight) responded by
increasing volume (Figure 13 and 14) and decreasing concen-
tration (Table 4) of urine. Approximately 50% of the water
load was excreted by 2 hours and 70% by 4 hours after treat-
ment. The results for the control group agree with those
reported by McCance and Wilkinson (1947) and Falk (1955)
and Allen and Zeman (1973). Similarly, the concentrations
of urea and the osmolality of urine during a water diuresis
reported in the present study agree with those reported by
McCance and Wilkinson (1947) for 4 to 12 day old rats.

Since the total excretion of urine by PDMs was no
different than that by PCMs (Figure 13) in response to a
water load, it is difficult to explain the observation that
10 day old PDMs excreted more urine in the 2-4 hour period

than age matched PCMs. Since Falk (1955) and McCance and

119

Wilkinson (1947) reported that the older the rat, the greater
the percent of the water load it was able to excrete, it is
possible that the continuation of a water diuresis into the
2-4 hour period by PDMs indicates that PDMs have a more
mature response to a water load than PCMs. Alternatively,
the secretion of or response to ADH can affect water excre-
tion.

ADH increases the permeability of the distal tubular
and collecting duct membranes to water and thus increases
water reabsorption. The degree to which water is reabsorbed
is, in turn, dependent on the hypertonicity of the medullary
interstitum. Since IDMs have been reported to excrete more
urine than INMs (Osler, 1960) a decreased production of ADH
or a decreased sensitivity to ADH by the kidneys would
result in a greater urine flow. In this light, it is
interesting to examine the effects of exogenous ADH on the
ability of PDMs and PCMs to excrete a water load.

A subcutaneous injection of ADH (0.l mU/g body weight)
given in conjunction with water (5% of body weight) adminis-
tered by gavage had no effect on the subsequent water
diuresis in 5 and 10 day old PDMs and PCMs (Figures 13, 15,
l7, 19, 21, 23). This is in contrast to data reported by
Falk (1955) who found that rats treated with ADH as young as
3 days of age, excreted half as much urine as nontreated
animals after a water load. Similarly, Falk found that

exogenous ADH caused an increase in urinary chloride

120

concentration and urinary osmolality. Alternatively, the
results reported in the present study agree with those
reported by Heller (1947) who did not show an inhibitory
effect of ADH on a water diuresis until rats were 4 weeks
old. Falk injected the ADH one-half hour after the adminis-
tration of water and collected urine samples every half hour
while Heller measured the cumulative excretion over a period
of hours. Allen and Zeman (1973) found that ADH depressed
urine flow in 6 day old rats between 30 and 60 minutes after
a water load but ADH did not reduce urine flow from 60

to 180 minutes after the water load.

The results reported in the present study represent the
cumulative urine collection over 2 hours. Therefore it
could be that ADH depressed urine flow in the first hour, a
water diuresis ensued in the second hour, and when bladders
were emptied at 2 hours, the initial urine was diluted by
the subsequent diuresis. Thus the final concentration was
no different than if the animal had not received ADH.
Interestingly, all animals injected with ADH showed an
altered pattern of urea excretion (Figure 25). This is
especially intriguing since previous studies with ADH show
that it acts primarily during the first hour after the
injection (Allen and Zeman, 1973). The excretion of urea
was no different in the first 2 hours, but was significantly
depressed in the second two hours (Figure 25). Although the

concentration of urinary urea varied significantly among the

121

treatments, the excretion of urea was no different among the
treatments, indicating that the excretion of urea was not
affected by the diluting mechanism of the kidney. The
reason for a depressed urea excretion 2-4 hours after the
administration of ADH is not known.

A water load distributes into all the cellular compart-
ments, decreasing the osmolality of the tissues and increas-
ing the volume. Both baroreceptors and osmoreceptors are
stimulated and cause a decrease in ADH secretion. This
causes a decrease in the permeability of the membranes of
the distal tubules and collecting ducts to water and results
in a water diuresis. An isotonic saline load, on the other
hand, distributes only to the extracellular fluid compart-
ment. There is no change in osmolality of the fluid but
there is an increase in volume. In response to an isotonic
saline load, it has been demonstrated that renal tubular
sodium reabsorption is decreased and that this decrease is
not affected by aldosterone, ADH, or a decreased GFR (Pitts,
l976). Micropuncture studies have demonstrated that the
natriuresis observed in adult rats infused with isotonic
saline is a result of reduction in both proximal and distal

1., 1969).

tubular sodium reabsorption (Davis gt

The results of the present study indicate that an iso-
tonic saline load (0.15N NaCl; 5% body weight) did not pro-
duce a diuresis in 5 or 10 day old PDMs or PCMs. Four

hours after the administration of an isotonic saline load,

122

only 30% of the load had been excreted (Figure 14). Urine
excretion 0-2 and 2-4 hours after administration of isotonic
saline was significantly less than that after water or
hypertonic saline loads (Figure 14). These results are
similar to those reported by Hoy (1966) who administered
water, hypertonic saline, and isotonic saline intravenously
and intragastrally to newborn rats and measured cumulative
urine flow and urinary chloride concentrations over 2 hours.
The administration of isotonic saline had no effect on
urinary chloride concentration after 2 hours. The urinary
chloride concentrations reported by Hoy (1966) are similar
to those reported in the present experiments.

Although the administration of isotonic saline did not
produce a diuresis, it appeared to affect the concentration
of urinary solutes 4 hours after treatment (Table 5).
Compared to pretreatment values (Appendix A) urinary osmo-
lality, sodium, and chloride concentrations appeared to
increase, urea concentration remained unchanged, and potas-
sium concentration appeared to decrease. The fact that urea
concentration remained the same as pretreatment values
(Appendix A) indicates that urine flow after an isotonic
saline load was no different than it was before the load.

In contrast, after a hypertonic saline or water load, the
urea concentration decreased as the water or osmotic diure-

sis ensued.

123

Five day old PDMs responded to an isotonic saline load
in the same manner as 5 day old PCMs (Figures 13 to 24).

Ten day old PDMs excreted the same amount of urine (Figures
13 and 14) and urinary solutes (Figures 15 to 24) in response
to isotonic saline as age matched PCMs; however, the osmo-
lality as well as the potassium concentration of urine from
PDMs collected during the 2-4 hour period after the adminis-
tration of isotonic saline was significantly lower than that
of urine from PCMs (Table 5). Similarly, the sOdium concen-
tration of urine collected 0-2 hours after treatment from
PCMs was significantly greater than that from PDMs. Since
the excretion of these solutes is no different between the
two groups, urine flow must be slightly increased in PDMs
during these periods (Figure 14).

According to Pitts (1976), a water diuresis unrelated
to change in osmolality of the body fluids can be induced in
adults by infusion of large volumes of isotonic saline.
Supposedly, volume and pressure receptors are stimulated by
the excess of extracellular volume. The excretion of water
is compensatory in that it reduces extracellular fluid
volume. Unless accompanied by the excretion of sodium,
loss of volume is limited by increasing osmolality of the
body fluids. In adults, enhanced sodium excretion (natri-
uresis) is readily induced by infusions of isotonic saline
(Pitts, l976). Kleinman and Hsieh (1974) found that saline

loading produced a decrease in proximal tubular sodium

124

reabsorption in both adult and newborn dogs and a decrease
in distal tubular sodium reabsorption in adults. In con-
trast, there was an increase in distal tubular sodium
reabsorption in newborn dogs. This may partly explain the
observation that newborn animals do not excrete a saline
load to the same extent as adults.

Although the urinary sodium concentration rose after
isotonic saline administration a natriuresis did not occur
in 5 day old rats since the excretion of sodium was no
different than that of animals receiving water. By 10 days
of age, however, an isotonic saline load produced a natri-
uresis in both PDMs and PCMs (Figure 14).

A hypertonic saline load administered by gavage to 5
and 10 day old rats produced an osmotic diuresis. Compared
with a water diuresis, this was delayed since maximum urine
excretion occured 2-4 hours after treatment rather than 0-2
hours after treatment (Figure 14). Additionally, when com-
pared to the water or isotonic saline load, the hypertonic
saline load produced a significant increase in the total
excretion of sodium (Figure 17) and chloride (Figure 19) in
all rats and the total excretion of potassium (Figure 21)
and total solute (Figure 15) in 10 day old rats. Urea
excretion was no different after a hypertonic saline load
than after a isotonic saline or water load (Figure 23).

The response by 5 and 10 day old rats to a hypertonic

saline solution reported in the present study is similar to

125

that reported by Falk (1955) for 4—8 day old rats. Falk
gavaged 4-8 and ll-13 day old rats with 5% of the body
weight of hypertonic saline. He found no significant dif-
ference in the ability to excrete a hypertonic saline load
between the 4-8 and 11-13 day olds. Furthermore, he found
that urine flow and urinary chloride concentration increased
rapidly in all groups, however, the increase in urine flow
by rats less than 3 weeks of age was delayed when compared
to adults. The urinary chloride concentrations (Table 6) in
response to hypertonic saline agree with those reported by
Falk (1955) and Hoy (1966). These researchers did not
report concentrations of other urinary electrolytes.

Ten day old PDMs and PCMs excreted significantly more
urine in response to hypertonic saline than to isotonic
saline (Figures 13 and 14). Ten day old PDMs excreted
significantly more urine than age matched PCMs. At 5 days
of age, PDMs treated with hypertonic saline excreted more
urine than those treated with isotonic saline but PCMs did
not. Furthermore, PDMs excreted significantly more urine
during the first 2 hours after treatment than PCMs. Since
Falk (1955) found that the older the rat, the more rapidly
it responded to a hypertonic saline load, these data could
indicate that 10 day old PDMs excreted a hypertonic saline
load in a more mature fashion than PCMs. Similarly, both
5 and 10 day old PDMs excreted more total solutes than age

matched PCMs in response to hypertonic saline (Figure 15).

126

In 10 day old PDMs, this was due to the significant increase
in urine flow during the first 2 hours since the urine from
PDMs had lower osmolality than that of PCMs (Table 6). In
view of the fact that solute excretion by PDMs after a
hypertonic saline load was greater than that by PCMs, it was
interesting to examine the major urinary constituents that
are osmotically active. These are sodium, chloride, potas-
sium and urea.

As with solute excretion, total sodium excretion by
both 5 and 10 day old rats was greater in animals treated
with hypertonic saline when compared to those treated with
isotonic saline or water (Figure 17). At 10 days of age,
the response by both PDMs and PCMs was greater than that of
the 5 day old counterparts. Ten day old PDMs excreted more
sodium than age matched PCMs. Since there was no signifi-
cant difference in the urinary sodium concentration, the
greater excretion of sodium by PDMs is probably due to
increased urine flow (Figure 13). This is apparent when
the pattern of urine flow is examined. PDMs excreted 2
times more urine in the first 2 hours after treatment than
PCMs (Figure 14). Similarly, PDMs excreted 2 times more
sodium in the first 2 hours than PCMs (Figure 18). Urine
excretion as well as sodium excretion by PDMs and PCMs
during the 2-4 hour period after treatment were not sig-
nificantly different. Therefore, one possible reason that

PDMs excreted more sodium over the 4 hour period than PCMs

127

(Figure 17) is a more rapid onset of an osmotic diuresis.
The chloride excretion by 10 day old rats in response
to a hypertonic saline load was similar to sodium excretion
(Figure 20, Table 6). This was not true for 5 day old
animals. At 5 days of age there was no difference in the
total chloride excreted by animals gavaged with water, iso-
tonic saline, or hypertonic saline. Chloride excretion
after the first two hours was the same regardless of treat-
ment (Figure 20). However, by 2-4 hours after treatment, 5
day old rats excreted significantly more chloride in res-
ponse to hypertonic saline than in response to isotonic
saline or water (Figure 20). The increase in chloride
excretion is caused by the significant increase in urine
flow which occurred during this period (Figure 14). These
data agree with those reported by Falk (1955) who found
that urinary chloride concentration in 4-8 day old rats
treated with hypertonic saline was less than 100 mEq/liter
by 1 hour, approximately 200 mEq/liter by 2 hours, and
maintained at 200 mEq/liter until 4 hours after treatment.
Chloride excretion by 10 day old rats was similar to that
described for sodium. PDMs excreted significantly more
chloride in the first two hours after treatment with hyper-
tonic saline than PCMs (Figure 20). Again, since urinary
chloride concentration was not significantly different
between PDMs and PCMs (Table 6) this increased excretion can

be explained by the increased urine excretion observed

128

during this period (Figure 14).

Total potassium excretion by 10 day old rats was sig-
nificantly greater in animals treated with hypertonic saline
than those treated with isotonic saline or water (Figure 21).
It is well established that an osmotic diuresis in adult
animals increases the excretion of potassium, but a water
diuresis does not (Pitts, 1976). The results from the
present experiments indicate that the response of an in-
creased excretion of potassium in conjunction with an
osmotic diuresis develops sometime between 5 and 10 days of
age in rats (Figure 21). This response, however, appears to
have developed earlier in PDMs. Five day old PDMs treated
with hypertonic saline excreted more potassium than those
treated with water whereas age matched PCMs treated simi-
larly excreted the same amount of potassium as those treated
with water (Figure 21).

Filtered potassium is largely reabsorbed in the proxi-
mal tubule and the amount of potassium presented to the
tubule varies only slightly despite variation in urinary
potassium. Changes in potassium secretion by the distal
tubules determine changes in potassium excretion. Potassium
secretion by the distal tubule is dependent on the delivery
of sodium to the distal nephron. The more sodium ions that
diffuse across the luminal membrane into the cell, the
higher will be the distal transepithelial potential differ-

ence. It is the reabsorption of sodium that is responsible

129

for the electronegativity of the tubular fluid and this is
responsible for maintaining the electrochemical driving
force for potassium ions to enter the tubular lumen (Pitts,
1976). Thus, during an osmotic diuresis, delivery of sodium
to the distal tubule is increased, sodium reabsorption is
increased and the transepithelial potential is increased
causing an increase in potassium secretion and an increase
in potassium excretion.

While there were no differences in potassiUm excretion
by PCMs and PDMs gavaged with hypertonic saline, the con-
centration of potassium in urine collected from PDMs 2
hours after treatment was significantly less than that from
PCMs (Table 6). This can be explained by the fact that
urine flow of PDMs during this time was significantly
greater than that of PCMs and indicates that the onset of
the osmotic diuresis began earlier in PDMs than PCMs
(Figure 14).

Urea excretion by animals treated with hypertonic
saline was no different than that by animals treated with
isotonic saline or water (Figures 23 and 24). Both during
the water diuresis and the osmotic diuresis, urea concen-
tration of the urine decreased (Tables 4 and 6). After an
isotonic saline load, the urea concentration of the urine
remained the same as pretreatment values (Appendix A).

This supports previous evidence concerning the renal handling

of urea in adult rats (Lassiter gt gl., 1961; 1964). Urea

 

130

is the main end product of nitrogen metabolism and its
excretion can be influenced by the amount of protein inges-
ted (Addis and Drury, 1923). It undergoes 2 processes in
the kidney, glomerular filtration and tubular reabsorption.
Micropuncture studies have demonstrated that tubular reab-
sorption of urea is passive and dependent on the concen-
tration gradients established by water reabsorption (Las-

siter gt 1., 1961; 1964). Therefore, during either a water

or osmotic diuresis in mature rats, the urea excretion
remains constant while its urinary concentration decreases.
The results of the present study indicate that 10 day
old PDMs excreted a greater percent of the hypertonic saline
load than age matched PCMs (Figures 13 and 14). In response
to such a load, PDMs excreted a greater quantity of total
solute (Figure 15) which may be explained by an increase in
sodium excretion (Figure 17) relative to body weight, when
compared to age matched PCMs. Sodium excretion is the net
result of the sodium filtered minus the sodium reabsorbed.
Therefore, PDMs could have a greater GFR or a lower tubular
reabsorption of sodium. Adult rats can excrete greater
quantities of hypertonic saline more rapidly than can infants
(Falk, 1955). Therefore it appears that PDMs have demonstra-
ted a more mature renal response to a hypertonic saline load
than PCMs. It is unlikely that this enhanced renal function
is due to postnatal nutrition since PDMs and PCMs receiving

the same treatments were suckled by the same dam. Since a

 

131

majority of rats used in this series of experiments (Series
II) had birth weights above the grand mean (Figure 2), it is
possible that the enhanced renal function observed is a
result of prenatal overnutrition. Bond gt _1. (1977) showed
that 5 and 10 day old rats born of dams with one ligated
uterine horn had enhanced renal secretory capacities for or-
ganic ions. Alternatively, Hall and Zeman (1968) and Allen
and Zeman (1973) demonstrated that prenatal protein malnu-
trition severely depressed GFR as well as the ability to

excrete both water and osmotic loads in the progeny. There-

fore, it is possible that a mild gestational diabetes in

 

rats enhanced renal development in the progeny.

The results of these experiments indicate that ges-
tational diabetes modified the development of certain kidney
functions in rats. Compared to normal development, the
development of intrinsic renal tubular transport functions
appeared to be relatively unaffected, the development of
renal gluconeogenic and ammoniagenic capacities were altered,
but not permanently, and the development of the ability to
excrete a water or isotonic saline load was not affected.
The ability to excrete a hypertonic saline load appeared to
be enhanced by gestational diabetes.

In light of this information, many questions can be
raised. What changes in body chemistry caused the increased
renal ammoniagenic capacity observed in neonatal PDMs? What

hormonal or renal tubular changes are involved with the

132

enhanced ability to excrete hypertonic saline?

To avoid complications, IDMs are frequently delivered
prematurely (Farquhar, 1976). According to a recent survey
(Kitzmiller _t _l., 1978), IDMs were delivered from the 28th
to the 39th week of gestation. The kidneys of fetuses
delivered before 36 weeks of gestation would be in the
process of nephrogenesis and, in that sense, similar to the
kidneys of neonatal rats. Therefore, studies concerning
the development of renal function in PDMs may help to more
fully understand the ability of human IDMs to cope with the

alterations in the chemical homeostasis that occur shortly

after birth.

 

SUMMARY

The effect of gestational diabetes on the development
of kidney function in the progeny was determined using 3g

vitro slice techniques and i vivo excretion studies.

 

Female rats were injected on day one of gestation with
streptozotocin (50 mg/kg) and were given daily insulin
injections throughout pregnancy. At birth, pups from dia-
betic mothers (PDMs) and pups from control mothers (PCMs)
were cross fostered to lactating controls.

Two series of experiments were completed. In Series I,
body weight, kidney weight, kidney cortex composition and
kidney function were assessed at l, 5, 10, and 28 days of
age. Renal function was quantified using the 1g tittg
kidney slice technique. In Series II, ability to excrete
water, isotonic saline and hypertonic saline loads was
assessed at 5 and 10 days of age.

In Series I, significantly more PDMs were born dead or
died within 24 hours after birth than PCMs. Body weight
and kidney weight of PDMs were no different than those of
PCMs at l, 5, or 10 days of age, but were significantly
lower at 28 days of age. Newborn rats of severely diabetic

dams have low blood insulin levels (Kervran gt gl., 1978)

133

134

and beta cell degranulation (Aerts and Van Assche, 1977).
Thus growth retardation observed in PDMs may be related to
depressed insulin secretion.

The percentage of water or protein in the kidney cortex
from PDMs was no different than that of PCMs at l, 5, 10,
or 28 days of age.

Renal tubular transport functions were quantified
utilizing the slice technique described by Cross and Taggart
(1950). Accumulation of a prototype organic ac1d (PAH),
organic base (NMN), amino acid (AIB) or sugar (aMG) by renal
cortical slices was expressed as a slice/medium (S/M) ratio.

Accumulation of PAH by renal cortical slices from PCMs
was lowest at day one of age and increased from 1 to 28 days
of age. For PDMs, PAH S/M ratios were similar to those
observed for PCMs except at 10 days of age when they were
significantly lower. Similarly, accumulation of aMG by
renal cortical slices from PCMs was lowest at birth and
increased with age. For PDMs, aMG S/M ratios were similar
to those reported for PCMs, except at 10 days of age, when
they were significantly depressed. It was suggested that
lower insulin levels in PDMs compromised protein synthesis
and that this resulted in a lower number of functional
transport sites for sugars or organic acids in ten day old
PDMs when compared to age matched PCMs.

Accumulation of NMN by renal cortical slices from PDMs

was similar to that of PCMs. NMN S/M ratios were lowest at

 

135

birth and increased with age. Accumulation of NMN by renal
cortical slices is not sensitive to ionic changes in the
medium (Kim gt gt., 1972) and may represent indiscriminate
tissue binding.

Accumulation of AIB by renal cortical slices from PDMs
was no different than that from PCMs. AIB S/M ratios were
highest at birth and decreased with age.

Renal ammoniagenic or gluconeogenic capacity of PCMs
was low at l, 5, and 10 days of age but significantly in-
creased by 28 days of age. Renal ammoniagenic capacity of
PDMs was significantly greater than that of PCMs at l and 5
days of age but declined to normal values by 10 and 28 days
of age. Renal gluconeogenic capacity of PDMs was the same
as that for PCMs at 1, 10, and 28 days of age. ‘At 5 days
of age, renal cortical slices from PDMs produced signifi-
cantly more glucose than those from PCMs. Since fetal
hyperglycemia leads to a lactate acidosis (Robillard gt gl.,
1978) it was suggested that a mild chronic acidosis in the
fetus or neonate enhanced renal ammoniagenesis in newborn
PDMs.

To produce acidosis, ten day old PDMs and PCMs were
gavaged with ammonium chloride (5 mM/kg; 2.5% of body
weight). Approximately 3 hours later, renal cortical slices
from acidotic rats produced slightly more glucose but no

more ammonia than those from non-acidotic rats. Renal cor-

tical slices from PDMs produced the same amount of glucose

 

136

and ammonia as those from PCMs.

In Series II the stillbirth and neonatal death rates
were similar for PDMs and PCMs. To assess kidney function,
the ability to excrete salt and water loads was determined
in 5 and 10 day old PDMs and PCMs. Rats were given one of
4 treatments (5% body weight of water; 5% body weight of
isotonic saline; 5% body weight of hypertonic saline; and
5% body weight of water in the presence of exogenous ADH)
and bladders were emptied by perineal stimulation after 2
and 4 hours. Urine volume, electrolytes, nitrogen, and
osmolality were determined. The urinary response to a water
or isotonic saline load was similar in 5 or 10 day old PDMs
and PCMs. In contrast, 10 day old PDMs excreted more urine,
total solute, and sodium in response to a hypertonic saline
load than PCMs. Since the ability to excrete hypertonic
saline increases as the animal matures, these data may indi-
cate that PDMs have a more mature renal response to hyper-

-tonic saline than PCMs.

I

 

APPENDIX

137

APPENDIX A

Table 8. Urinary solute concentration before salt and
water loading

 

 

PCM PDM
Osmolality (mOSm/liter) 406 1 16 (4)?“b 376 1 42 (4)
Urea (mM/liter) 89.4 1 5.1(4) 80.0 1 ll (4)
Sodium (mEq/liter) 19.3 1 5.0(8) 26:0 1 6.0(8)
Chloride (mEq/liter) 59 1 12 (8) 57 1 15 (8)
Potassium (mEq/liter) 62.3 1 4.5(8) 64.5 1 6.9(8)

 

aEach value represents the mean 1 SEM (n).

bUrine samples were collected from 5-6 days old rats.

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