EFFECT or PRE- NATAL OVERNUTRmON 0N RENAL DEVELOPMENT IN THE RAT Thesis fer the Degree, of M. S. MICHIGAN STATE UNIVERSITY JUDY RAUCY' GUMBRECHT 1976 Agfif -_ .. M ‘ “4/ ABSTRACT EFFECT OF PRE-NATAL OVERNUTRITION ON RENAL DEVELOPMENT IN THE RAT By Judy Raucy Gumbrecht Uterine ligation was used to determine the effects of pre-natal overnutrition on renal development in the rat. Animals were used at 20 days gestation and day one after birth. Ligation of one uterine horn produced heavier offspring than those produced by sham or control dams. These offspring also had heavier kidneys. KW/BW ratios were also increased in pups from ligated dams. The ability of renal cortical slices from these animals to accumulate organic ions was quantified using a prototype acid and base, p-aminohippurate (PAH) and N~methy1nicotinamide (NMN), respectively. At 20 days gestation and one day old, there were no differences in PAH transport in kidneys of pups from ligated, sham, or control dams. However, PAH transport decreased between 20 days gestation and one day of age. Accumulation of NMN also showed no differences in the three groups of pups. NMN transport increased from 20 days gestation to day one in kidneys from all pups. Accumulation of amino acids into renal cortical slices was determined using a-aminoisobutyric acid (AIB), a nonmetabolizable amino acid. There were no differences in the ability of the kidneys to transport amino acids in all three groups of animals at day one. However, amino acid transport in kidneys of pups from ligated dams at 20 days gestation was less than in kidneys of pups from sham or control dams. Accumulation of a-methylglucoside was used as an index of glucose transport in renal cortical slices. There were no differences in glucoside transport in kidneys of pups from ligated, sham, or control dams at one day of age. There was less glucoside transport in kidneys of pups from ligated dams at 20 days gestation when compared to kidneys of pups from sham or control dams. There were no increases in glucose transport from 20 days gestation to day one. Gluconeogenic capacity of renal cortical slices was also deter- mined. There were no differences in the amount of glucose produced in kidneys of pups from ligated, sham, or control dams at 20 days gestation or day one. There were increases in glucose production in kidneys of pups at day one compared to kidneys of pups at 20 days gestation. Uterine ligation does not alter renal function at 20 days gestation or day one in the rat. However, renal development takes place after day one. EFFECT OF PRE-NATAL OVERNUTRITION ON RENAL DEVELOPMENT IN THE RAT By Judy Raucy Gumbrecht 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 1976 ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. J. T. Bond for her assistance and support throughout the course of this investi- gation. I would also like to thank Dr. J. B. Hook for his encourage- ment, interest, and constructive critism. The financial support of Dr. Leveille, Dr. R. Schemmel, and the Human Nutrition Department is gratefully acknowledge. My sincere appreciation also to my husband whose patients and understanding made this thesis and investigation possible. My parents, Mr. and Mrs. Pat Raucy, and Mr. and Mrs. Gumbrecht are gratefully acknowledged for their support. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . INTRODUCTION Renal Development . . Nutrition and Renal Development Infants of Diabetic MOthers Renal Tubular Transport Gluconeogenesis METHODS Animals . . Uterine Ligation Procedure . Timed Pregnancy Cesarean Section . . . Preparation of Renal Cortical Slices . Accumulation of Organic Ions by Renal Cortical Slices Transport of Amino Acids and Glucose By Renal Cortical Slices Gluconeogenesis . Protein and Water Composition of Renal Corical Slices Statistical Analysis . RESULTS Food Intake During Pregnancy . Length of Gestation . Body Weight . . Kidney Weight and Kidney Weight/Body Weight (KW/8W). ratio Protein Content of Renal Cortical Slices . Water Content of Renal Cortical Slices . Accumulation of PAH by Renal Cortical Slices . Accumulation of NMN by Renal Cortical Slices . Amino Acid Transport by Renal Cortical Slices Glucose Transport by Renal Cortical Slices . iv . 10 . 13 . 19 ' Page . vi . vii . 21 21 22 . 22 . 22 . 23 . 24 . 24 . 25 . 25 . 27 . 27 27 . 27 . 28 . 28 . 28 . 29 . 29 . 29 Page Glucose Production by Renal Cortical Slices . . . . . . . 30 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . 31 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . 37 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . 67 LIST OF TABLES Table Page 1. Food Intake of Rats During Gestation . . . . . . . 39 2. Gestational Period of Rats . . . . . . . . . . . . 40 vi Figure 10. 11. 12. LIST OF FIGURES Schematic diagram of the in vivo and the in vitro systems . Schematic diagram of the p- slice incubation system under several conditions . Effect of uterine ligation on body weight in rats at 20 days gestation and day one . Effect of uterine ligation at 20 days gestation and day one . Effect of uterine ligation at 20 days gestation and day one . Effect of uterine ligation renal cortical slices from and day one ..... Effect of uterine ligation renal cortical slices from and day one Effect of uterine ligation renal cortical slices from and day one Effect of uterine ligation renal cortical slices from Effect of uterine ligation renal cortical slices from and day one Effect of uterine ligation renal cortical slices from and day one Effect of uterine ligation Page . 41 aminohippurate (PAH) . 43 . 45 on kidney weight in rats . 47 on KW/BW ratio in rats . 49 on protein content of rats at 20 days gestation on water content of rats at 20 days gestation . 51 . 53 on PAH accumulation in rats at 20 days gestation . 55 on NMN accumulation in rats at 20 days gestation on AIB accumulation in rats at 20 days gestation S7 . 59 on a-MG accumulation in rats at 20 days gestation . 61 on gluconeogenic capacity of renal cortical slices with and without exogenous substrate at 20 days gestation . vii . 63 Page 13. Effect of uterine ligation on gluconeogenic capacity of renal cortical slices with and without exogenous substrate at day one . . . 6S viii INTRODUCTION Increasing evidence is accumulating that nutritional status pre- and postnatally, even for short periods of time, can produce long lasting effects on physical, biochemical and behavioral characteris- tics in growing and adult animals. Investigations concerned with the development of renal function in various nutritional states have been limited. The normal newborn is not a miniature adult. Although he may have the physiological and biochemical capability to maintain homeo- statis in normal situations, he has little or no reserve for stress. It is well established that the kidneys of the normal newborn of most species are anatomically and functionally immature. Whether the kidneys of newborns subjected to alterations in nutritional status are able to respond to stress is not known. This series of experiments was specifically designed to test the hypotheSis that overnutrition in utero alters renal function in the laboratory rat. RENAL DEVELOPMENT Development In the human fetus about 20% of the nephrons are formed by three months gestation and by five months gestation 30% are formed. Formation of new nephrons ceases when the fetus reaches a length of 46 to 49 centimeters and a weight of 2100 to 2500 grams (Edelmann, 1972). Maturity of the kidney as far as production of new glomeruli is concerned is almost always attained during the time the fetus or in- fant weighs between 2100 and 2500 grams. In the rat, nephron develop- ment continues during the first and the second week after birth so that the number of nephrons at birth is more than doubled by two weeks of age (Falk, 1955). \ Between the fourteenth and twenty-fifth weeks of intrauterine life of the fetus the cells of the kidney are dividing rapidly. In the last ten weeks there is a rapid growth in cell size. The cells continue to divide but much more slowly (Widdowson et al., 1972). The protein to DNA ratio in the kidneys increases rapidly in the last 10 weeks of intrauterine life. Widdowson et a1. (1972) observed large increases in the amount of DNA in the kidneys after birth because the total DNA at term in human fetal kidneys was less than 20% of the adult values. Renal cell hyperplasia also predominates before birth in the rat and mouse. Shortly after birth, cell hyperplasia diminishes and cell hypertrophy becomes the mechanism of renal growth in these two species. The rat fetal kidney has a higher water content than the post- natal kidney (Rahill et al., 1973). At birth there is still anatomical immaturity of the kidneys. The kidney develops in a centrifugal pattern. The deeper structures are formed first. When full-term gestation is reached in the human about 20% of the loops of Henle are still within the cortex (Edelmann, 1972). Functional Development During intrauterine life the kidney is able to perfbrm most of the functions which characterize the adult organ. Renal plasma flow is 3 low during intrauterine life and renal vascular resistance is high. Formation of urine by the human fetal kidneys begins toward the end of the first trimester (Edelmann, 1972). Bennett (1975) reported that the fetal kidney has excretory capacity since it has the ability to clear inulin: from amniotic fluid. Fetal urine is hypotonic to plasma because of low concentrations of electrolytes, urea and other nitrogenous-end products. The urine is also acid with a pH of about 6 (Edelmann, 1972). Human fetal urine during the last few hours or days of fetal life has a lower specific gravity than adult urine (McCance, 1948). Other in- vestigators have shown that fetal lamb's urine is low in phosphates. In the lamb there is a relatively low glomerular filtration rate (GFR) and a high urine output. Tubular reabsorption of sodium is lower in fetal lamb than mature lamb kidneys which may be due to the immature loops of Henle (Smith et al., 1969). Following placental separation, there is an increase in renal blood flow and functional capacity of the kidneys. Functional capacity can be approximated from the measurement of GFR.which is low in neonates compared to adults. Not all glomeruli are mature and some are nonfunc- tional. Barnett et al. (1952) found that the GFR (inulin clearance) in infants was only 30-50% of adult values. At the end of the first year the GFR was closer to those found in adults because of the steady progression of increased GFR. GFR is also proportional to kidney weight during growth (Potter et al,l 1969). Barnett et a1. (1952) also measured effective renal plasma flow (ERPF) by para—aminohippuric acid (PAH) clearance and found that the ERPF in infants is 20-40% of the ERPF values in adults. 4 Reabsorptive capacity in man is different for adult kidneys than for newborn kidneys. However, balance between filtration and reabsorption is present at all stages of postnatal development (Arant et al., 1974). Sodium reabsorption increases in proportion to the in- crease in nephron mass, or an increase in kidney protein (Potter et al., 1969). The newborn is unable to excrete excessive loads of sodium and becomes edematous when fed these loads (Edelmann et al., 1960). The tubular reabsorptive capacity for glucose in infants might be more highly developed than that for other substances (Smith, 1951). Arant et a1. (1974) showed that transport maximum for glucose (TmG) increased in puppies during the first three weeks of life. This suggests that there may be changes due to an increase in tubular mass or number of functional units. In young puppies the ratio of TmG to inulin clearance (Gin) was high. There is a more rapid increase in Cin than TmG. This suggests that in puppies at 18 hours of age there is a pre- ponderance of tubular function rather than glomerular function (Arant et al., 1974). There is also a slight increase in glucose threshold with age. Although the threshold was similar to that observed in adult dogs. The renal threshold for bicarbonate in infants is also lower than in adults. This low bicarbonate threshold in puppies is not due to a reduced capacity of the nephron to reabsorb bicarbonate, but is related to the amount of sodium and water reabsorbed in different segments of the nephron (Edelmann, 1973). Blood pH and bicarbonate values in infants are lower than in older humans. This is the normal state at this age. There is apparently no impairment or limitation in acidification mechanisms in infants. This was deduced from a study in which human 5 infants were given ammonium chloride and no impairment was seen (Edelmann, 1973). Almost 99% of the water filtered by the glomeruli is reabsorbed passively by the tubules as a result of active transport of sodium. The mature kidney is able to produce a urine as dilute as 50 milliosmoles per liter under conditions of water diuresis and as concentrated as 1300 milliosmoles per liter during antidiuresis. Infants are unable to con- centrate their urine to more than 700 mosm/l compared to 1300 in the adult. This is probably because of the shortness of the loops of Henle (Edelmann, 1972). Newborn rats are also unable to produce a highly concentrated urine. Trimble (1970) showed that ten day old rats were unable to enhance their concentrating ability when given an acute urea load. However, twenty day old rats can enhance concentrating ability. Enzyme Development Certain enzyme activities present in the tissues of the adult liver cannot be detected in the liver of the fetus. The hippurate synthe- sizing system is made up of three well defined enzyme activities located in the mitochondria of the liver in the adult rat (Brandt, 1960). There was no detectable enzyme activity in whole homogenates of liver from late fetal or newborn rats to five days of age (Brandt, 1960). In the new- born and fetal kidney the activity of certain enzymes is not fully developed. Wacher et a1. (1961) demonstrated that at birth the kidney concentrations of alkaline phosphatase, glutaminase, and carbonic an- hydrase were low compared to adult rat kidneys. The concentration of glutaminase and carbonic anhydrase did not show any significant change before twenty-one days of age. At 21 days there was a 20% increase above newborn concentrations of both glutaminase and carbonic anhydrase. Alkaline 6 phosphatase decreased between birth and 14 days. At 21 days, however, it returned to newborn concentrations and by 33 days the concentration was 300% of newborn levels. These observations in enzyme concentration made by Wacher et a1. (1961) may explain the differences in renal acid- base regulation between young and adult rats. NUTRITION AND RENAL DEVELOPMENT Undernutrition and the kidney Despite the kidney's requirement for protein and nutrients to carry on varied metabolic activities, they appear to be relatively resistant to the effects of nutritional deprivation. During prolonged starvation in adult man, oxygen consumption in the kidneys increases along with an increase in carbon dioxide production (Owen et al., 1969). There is also an increase in renal production of glucose and aceto- acetate. Since ketone bodies in the urine are higher than normal during starvation, there is also an increase in ammonium excretion. Polyuria and nocturia are the most prevalent results of starvation. With poly- uria there is also an output of very dilute urine. This is probably due to the decrease in blood urea nitrogen (BUN). Development of the kidney in rats is also altered by protein restriction (Allen et al., 1973). Allen et a1. (1973) showed that kidneys from protein restricted rats have fewer and less well differen- tiated glomeruli. They also showed fewer collecting tubules and proximal tubules with fewer convolutions. Reduced activity of acid and alkaline phosphatases were seen as well as low urine flow rate and a limited capacity to concentrate urine. Dams fed a protein deficient diet (6% protein) during pregnancy produced offspring with altered kidney function, 7 i. e. reduced GFR (Allen et al., 1973). Edelmann (1972) showed that in premature human infants who were fed diets with a high protein content (5 grams %) and low protein content (2.5 grams %) there were no differences in rates of growth or concentration of electrolytes in the extracellular fluid. However, inulin and PAH clearances in the high protein group were doubled over those in the low protein group. The effects of undernutrition are also seen in rat pups. In- creasing the litter size after birth will subsequently alter nutritional status. This alteration of nutritional status depresses and/or delays the maturation of renal organic ion transport systems. Johnson et a1. (1974) showed that PAH accumulation was greater in kidneys from rat pups of five or ten pup litters compared to pups from twenty pup litters at five and ten days of age. Furthermore, accumulation of NMN by kidneys of pups from five or ten pup litters was significantly greater than NMN accumulation in kidneys from pups of twenty pup litters at ten days of age. Overnutrition and the kidneys Several investigators have shown that altering litter size of pre-weaning rats will alter the amount of milk each pup receives. Heggeness et a1. (1961) showed that in small litters the increased availability of milk influenced the rate of weight gain in pre- weaning animals. Animals from small litters accumulated more body fat than animals from larger litters. The nutritional status, however, did not influence the rate of maturation measured by eye—opening, hair appearance, and percentage of water in the fat free carcass. Winick et a1. (1967) found that rats nursed in large groups grew more slowly 8 and attained a smaller final size than rats in normal size litters, suggesting a reduced cell number in all organs. Rats that were suckled in small groups grew more rapidly and attained a larger final size than controls. During prenatal and early postnatal development all organs, with the exception of adipose tissue, grow by cell division. When caloric intake is increased growth is increased by an increase rate of cell division. Cell size is unchanged. Animals from small litters have a larger number of cells in their organs than normal animals (Winick et al., 1967). The neonatal rat is less effective in regulating water and electrolyte balance than the adult rat. In the rat from a normal litter, the regulatory response to blood volume expansion develops at 35 to 40 days, about the time nephrons appear fully developed. However, in rats from a small litter this regulatory response is developed by 25 days of age (Bengele et al., 1974). Solomon et a1. (1972) showed that there was a definite relationship between food intake and the rate of functional development of the kidney. They found that increasing the food availability postnatally by altering litter size resulted in an increase in superficial single nephron glomerular filtration rate (SNGFR). The SNGFR is more dependent on kidney weight than body weight. Total GFR/unit/kidney weight from animals of reduced litters is less than GFR/unit/kidney weight from intact litters. They also showed that kidneys from animals of reduced litters are smaller relative to body weight than kidneys from intact litters. This led the investigators to conclude that when there is an increased food intake this would lead to an increase in fluid intake which may result in volume 9 expansion. This increased volume expansion leads to an increased filtration by superficial nephrons and an increased GFR which can eventually lead to renal hypertrophy of superficial elements (Solomon et al., 1972). Solomon et al. (1973) in another study on organ weight found that the only organ to be affected by increased fbod availability to young rats was the kidney. During early development only kidney weight to body weight (KW/8W) ratios were lower for animals from reduced litters than those animals from intact litters. KW/BW is inversely related to the rate of growth in these overnourished animals. Litter size can also be altered prenatally to increase the avail- ability of nutrients to the fetus. Prenatal growth is largely controlled by the intra-uterine environment (McLaren et al., 1960). Both the total number of fetuses and their distribution between the uterine horns are controlled within a narrow range. There is an association between fetal weight and intrauterine position, as well. McLaren et al. (1960) fbund that the lightest fetus was at the cervical end of the horn with increasing weights toward the ovarian ends. They also observed that when the range of litter sizes in mice increased, the differences in the mean weights of full-term fetuses were seen. There is a limited pool of nutrients in the maternal blood for which the fetuses compete. Unilateral ligation of one uterine horn of female rats will increase the nutrient supply to the remaining horn. This suggests that dams with a ligated uterine horn have the potential for nourishing twice as many fetuses than they actually produce so each fetus receives more nutrients (Van Marthens et al., 1972). Van Marthens et al. (1969) showed that ligation of one uterine horn caused a decrease in litter size. 10 However, body weight (BW) of the offspring was increased. Pups at birth were heavier, had a higher cerebral brain weight, higher total cerebral protein, and increased cerebral DNA than the control pups. Van Marthens et a1 (1972) used the same technique in rabbits and pro- duced offspring that were 50% heavier and placental weights were in- creased 105%. The offspring also had an increased cerebral DNA (21% above controls). In naturally occuring reduced litter size, there are no significant increases in BW or placental weight (Van Marthens et al., 1972). Rat pups from ligated dams are larger at birth and at five and ten days of age than sham operated dams. The development of renal transport for both organic acids and bases were also altered by this accelerated growth in utero. Johnson et a1. (1974) showed that accumulation of NMN at five and ten days of age was increased in kidneys of rat pups from dams with one uterine horn ligated. They also showed that kidneys in five pup and ten pup litters accumulated more PAH than controls at five days of age. PAH accumulation was also increased in kidneys from ten pup litters at ten days of age. INFANTS OF DIABETIC MOTHERS Fetal overnutrition is frequently seen in infants of diabetic mothers (IDM). The increased nutrient supply to both pups of ligated dams and IDMs results in an increase in body weight at birth. Excessive weight in IDMs is caused by an increase in body fat (Baird et al., 1962). The increased fat deposition is due to the baby's endogenous insulin, stimulated by fetal hyperglycemia. This fetal hyperglycemia is due to the maternal hyperglycemia which stimulates an increased secretion of ll insulin by the fetus (Hultquist, 1950). There is a positive correlation between the blood glucose concentration of the mother and the baby at birth. Hyperinsulinism may also be caused by an antagonist to insulin that is transmitted across the placenta (Baird et al., 1962). Excessive insulin activity continues for at least two hours after birth in response to an unknown factor (Farquhar, 1965). The best evidence for this hyperinsulinism is the morphological change in IDMs. There is hyper- trophy of the pancreatic islets due to hyperplasia of the beta cells. The cells are functionally active and the quantity of islet tissue is related to the infants birth weight (Baird et al., 1962). IDMs have an improved glucose tolerance and a high plasma insulin activity when compared to normal infants. According to Farquhar (1965) large babies tend to lose more weight in the first week of life than do those infants in the normal weight range. Most infants exhibit a slight neonatal weight loss, increased by lack of food intake after birth. Premature and IDMs do not receive food until one to several days after birth. The infants are required to use stored nutrients or their own body protein to cover the energy requirements during the period of starvation (Osler, 1960). Normal infants metabolize glycogen, fat, and little protein for energy so they have a low excretion of water, electrolytes and nitrogen during the first few days of life. The respiratory quotient (RQ) in normal infants is 1.00 indicating carbohydrate consumption as the energy source. IDMs first utilize glycogen which increases water excretion and then mainly protein. This burning of protein accounts for the increased nitrogen excretion (Osler, 1960). IDMs are premature but they have large fat 12 depots and increased glycogen content. The glycogen is consumed soon after birth but the fat consumption of IDMs during the first few days of extrauterine life is unknown (Osler, 1960). At birth IDMs have reduced total and extracellular water content compared to normal infants and a larger intracellular water content (Osler, 1960). IDMs pass more urine than normal infants during their first few days of life. They also excrete more sodium chloride, and potassium than normal infants (Farquhar, 1965). This may be due to an increased break down of protein or glycogen or to undeveloped enzymatic processes in the kidneys. 'For example: when carbonic anhydrase is absent, the kidneys excrete more sodium and potassium (Osler, 1960). Farquhar, (1965) found that total body sodium and chloride were also reduced compared to normal babies, but potassium concentrations were unchanged at birth. They also reported an increased serum phosphate concentration, but a decreased serum calcium concentration (Farquhar, 1965). The plasma pH was similar in both normal and IDMs during the first hour after birth and then the pH in IDMs decreased significantly and the pCOZ values were raised (Farquhar, 1965). There appeared to be a metabolic acidosis. The GFR is lower in IDMs after birth and the kidneys also fail to produce a concentrated urine which may be because of a reduction in antidiuretic hormone (ADH) production or an inability of the kidneys to react to ADH. Edema is also increased in IDMs during the first few days of life (Osler, 1960). 13 RENAL TUBULAR TRANSPORT Secretion of Organic Compounds Renal tubular secretion is the mechanism of transporting material from the blood or peritubular fluid into the tubule cell and from the cell into the lumen (Taggart, 1958). It can be an active or a passive process. Active transport involves the transport of a substance across a biological membrane against a concentration gradient utilizing cellular energy to complete the process. The rate of transport will increase as the concentration of material present for transport is increased. Eventually, saturation of the mechanism occurs and a fixed maximal transfer rate is attained. This maximal rate of transport in the kidney is called Tm or Transport Maximum (Taggart, 1958). The first substance known to be secreted by the kidneys was phenol red (Marshall et al., 1923). The same mechanism that transports other organic acids. Another mechanism actively transports organic bases. Secretion also serves as a mechanism for rapid excretion of ingested foreign substances such as hippurates, penicillin, and sulfates (Pitts, 1963). Secretion of these substances takes place in the proximal tubule of the kidney in all mammals. Many substances are added to the urine by tubular secretion. Maximal rates of transport have been shown for para-aminohippurate (PAH), phenol red, and diodrast. They share a common secretory mechanism (Taggart, 1958). This secretory mechanism is dependent on aerobic metabolism. ATP as an energy source is also required (Taggart, 1958). The secretory mechanisms for organic compounds can be studied in vitro using the slice technique devised by Cross and Taggart (1950). 14 This technique permits the tubular transport of organic substances and can be used to measure transport as well as metabolic activities. Cross and Taggart (1950) showed that accumulation of PAH using thin slices of renal cortex is closely related to the tubular excretion of PAH in the intact animal (Fig. 1). In the intact animal, PAH is actively secreted from the blood into the proximal tubular cell and then diffuses down a concentration gradient into the tubular lumen (Tune et al., 1969). Active transport is much greater than back diffusion out of the cell with the net result being accumulation of PAH in the tubular cell. PAH is not bound within the tubular cell so the measured tissue PAH con- centrations are representative of a free transport pool (Tune et al., 1969). The in vitro system is comparable to the intact kidney in that the active transport step is also accumulation of PAH in the tubular cell. Even though the lumen has collapsed, the proximal tubular cells are still intact (Fig. 1). There is also back diffusion or runout out of the slice into the medium and very slight diffusion still takes place into the collapsed lumen (Tune et al., 1969). A schematic representation of data obtained from the slice incubation system is shown in Figure 2. This shows the slice accumu- lation as measured by the S/M ratio for PAH plotted against time. It also shows that PAH is accumulated into the proximal tubular cell to a point of saturation. At that point, back diffusion equals active transport and the curve levels out. The system can be stimulated by adding acetate to the medium, or it can be inhibited by adding compounds like penicillin that compete with PAH fer the same transport system. 15 The nitrogen indicates practically no active transport taking place when slices are incubated under nitrogen instead of oxygen. The slice/media or S/M ratio is a measure of intrinsic transport capacity. It indicates the net intracellular concentration resulting from accumulation and runout. When the S/M ratio increases it is an indication that PAH secretion has increased (Fig. 2). When the S/M ratio decreases, PAH secretion decreases. The slice procedure has several advantages. The first is that the chemical composition of the medium can be rigidly controlled. Secondly, the influence of extrarenal factors such as renal blood flow which may alter tubular secretion in the intact animal are excluded. Thirdly, the technique permits measurement of effects of certain metabolic inhibitors which could not be tolerated in the intact animal. There are also several disadvantages of the slice technique. The secretory system in the slice preparation is incomplete. The technique doe not allow for a continuous filtration process. Furthermore, PAH, once it has accumulated intracellularly, is able to diffuse out of the cortical slices back into the medium. This is in contrast to the unidirectional movement of PAH in the intact kidney. Lastly, there is an inability to distinquish between the accumulation of substrate within the cells from that accumulated within the tubular lumen (Cross et al., 1950). Accumulation of PAH is used as a measure of tubular secretion of organic acids, and the accumulation of N-methylnicotinamide (NMN) is used to measure tubular secretion of organic bases. The pH for maximal uptake of PAH is about 8.0 and for NMN is 7.4 to 8.0 (Ross, 1968). The l6 rates of runout of both PAH and NMN are not significantly affected by pH changes from 3.8 to 8.0 (Ross, 1968). The ability to concentrate NMN is energy linked since a number of metabolic inhibitors and substrates can influence this concentrating mechanism. The amount of NMN taken up by renal slices is dependent on the concentration of NMN in the medium (Farah et al., 1958). Transport of PAH consists of four steps: 1) diffusion from medium to the interstitial fluid, 2) transfer from interstitial fluid into the cell, 3) intracellular concentration, and 4) diffusion into the lumen. Uptake involves steps one and two and runout involves steps three and four (Ross, 1968). Accumulation of PAH and NMN is dependent on the temperature as well as the pH. The rate of runout of PAH and NMN increases with temperature and pH, but at a much slower rate than uptake, until optimum is reached (Ross, 1968). Amino Acid Transport Free plasma amino acids are filtered by the glomeruli and then undergo differential active reabsorption in the proximal tubule. Amino acid transport may be associated with ATP production (Rea et al., 1972). a-aminoisobutyric acid (AIB) is a non-metabolizable amino acid which shows active transport of short chain polar amino acids. AIB is transported in the same way as naturally occurring metabolizable amino acids. Accumulation of AIB in renal cortical slices occurs against a chemical concentration gradient. The accumulation is dependent on aerobic metabolism, temperature and oxidative phosphorylation. It is also dependent on the initial concentration of amino acids under investigation (Rosenberg et al., 1961). l7 Immature humans tend to excrete larger amounts of amino acids than do mature individuals. The same is true for the rat (Webber et al., 1968). The immature rat appears to have the same sort of amino acid excretory pattern as the immature human. Young or regenerating tissues have a greater ability to concentrate amino acids than do mature tissues, at some stages of development (Webber et al., 1968). Renal slices from newborn rats showed a slower rate of accumulation, but attained higher tissue contents. Exit from the slices of AIB is also slower in the newborn rat. Even though accumulation occurs, renal tubular amino acid reabsorption is less Complete in the newborn than the adult rat (Christensen, 1973). Structural development of the kidney suggests that in the newborn the tubules account for less of the cortical mass while the glomeruli are proportionately larger. Morphological studies indicate that the brush border of the proximal tubule cells is much less well developed in the newborn than in the adult. This may account for a slower rate of uptake. Newborn kidneys, however, concentrate amino acids better than adult kidneys (Webber et al., 1968). Several in- vestigators have attained much higher distribution ratios for AIB in the newborn than in the adult renal cortical slices (Webber et al., 1968). Reynolds et al. (1974) concluded that there is an adaptive control mechanism in newborn rat kidney cortex and that this mechanism can regulate the entry of certain amino acids into tubule cells. They suggested the presence of a time-dependent regulatory process for amino acid transport in cortical cells of newborn but not in adult rats. This time-dependent regulatory process is associated with protein synthesis and may be synthesis of new carrier proteins (Reynolds et al., 1974). 18 Glucose Transport Almost all glucose that is filtered at the glomerulus is reab- sorbed in the proximal tubule and returned to the venous circulation (Silverman et al., 1970). Reabsorption of glucose is restricted to the proximal segment of the nephron in most species. The tubular reabsorption of glucose is 99.95% of the filtered glucose (Wesson et al., 1969). Reabsorption of glucose across renal tubular epithelium is an active transport process. Glucose is reabsorbed and returned to the peritubular fluid against a concentration gradient (Taggart, 1958). The process of secretion across tubule cells does not involve breakdown and resynthesis of the glucose. The secretory process has its beginning at the barrier separating the tubule from the urine. The transport of glucose involves an intracellular glucose carrier complex and a site of formation at the cell-urine barrier (Chinard et al., 1959). Glucose transport involves two reactions: 1) a reversible combination with a cellular carrier and 2) dissociation of the carrier complex (Taggart, 1958). The two main barriers that glucose must cross from tubular fluid to blood are the brush border and the basal and lateral membranes. The brush border is the site of active transport of glucose (Silverman et al., 1970). Glucose penetrates the brush border of the tubule cell by diffusion and is converted into glucose-6-phosphate using ATP. Hydrolysis is the second reaction and occurs at a site within the cell furthest from the lumen (Taggart, 1958). Glucose exits primarily at the basal membrane (Rea et al., 1974). The capacity of the tubules to remove glucose from the lumen is limited. If glucose is filtered in excess of this limit, glucosuria occurs. Therefore, 19 there is a Tm for glucose (Chinard et al., 1959). Phlorizin inhibits reabsorption of xylose, glucose, fructose, and galactose. It does this by inhibiting the oxidative reactions of the citric acid cycle and inhibiting the generation of ATP (Taggart, 1958). The phlorizin receptor is identical to or part of the glucose transporter exposed at the lumen side of the brush border membrane of the proximal tubule. When phlorizin is given, glucose transport is inhibited 100%. These high affinity phlorizin sites have a well defined specificity of in- hibition by sugar substrates, particularly a-methyl-D-glucoside. a-methyl-D-glucoside (a-MG) has the highest affinity for glucose transport receptor sites, as high as glucose. There is a 100% inhibition of both glucose and a-MG when phlorizin is given (Silverman et al., 1975). Rea et al. (1974) compared the development of sugar uptake with o-MG using adult and newborn renal cortical slices. In newborn kidney slices they found that o-MG is weakly accumulated and is very low. The uptake of a-MG increased rapidly-from newborn to adult. This increase occurred at the same time that the microvilli on the brush border were increasing (Christensen, 1973). Gluconeogenesis Gluconeogenesis occurs in the kidney as well as in the liver. The content of glycogen and glucose is much less in the kidney than in the liver. The end product of gluconeogenesis in the kidney is glucose rather than glycogen (Krebs et al., 1963). Renal gluconeogenesis is stimulated by acidosis in vivo and in vitro. Glutamine is hydrolyzed to glutamate and ammonia by the catalytic action of glutaminase. During acidosis the amount of glutamine available for this reaction is increased. 20 Glutamate is then deaminated by glutamate dehydrogenase plus NAD+ to a-ketoglutarate (which enters the citric acid cycle), ammonia, and NADH. Renal glutaminase concentration and the response of renal glutamate to acidosis are both low during the first two weeks of age in the rat, but are near adult levels between the second and third weeks after birth. Renal glutamine concentration in rats nine days old is only 50% of adult concentrations and reaches adult concentrations by 12 days after birth. Renal glutaminase activity in nine day old rats is only 30% of adult activity and increases to 50% of adult activity by 14 days of age (Goldstein et al., 1973). Goldstein et al. (1973) found that the ability of kidney slices to convert glutamate to glucose was lower in ten day old rats than in adults but reached adult levels by 12 days after birth. They also found that the response of renal gluconeogenesis to acidosis was 50% lower in rats ten days old than in adult rats. It reached adult levels between 12-14 days of age. Renal gluconeogenic capacity matures to adult capacity by 12 days after birth (Goldstein et al., 1973). The purpose of this study was to determine whether increasing nutrient supply above normal in utero, and thereby producing larger offspring, will affect the functional capacity of the kidneys. This may be of clinical relevance in treating infants of diabetic mothers who are also larger than normal infants at birth. METHODS Animals Female Sprague-Dawley rats weighing 250-350 grams were obtained from a local supplier (Spartan Research Animals, Inc., Haslett, Mi.). These were divided into three groups: Control non-operated, sham operated, and operated with one uterine horn ligated. Males for breeding were obtained from the same supplier. Males and females were fed laboratory chow (Lab Blox, Allied Mills, Inc.) throughout the experiment except when females were maintained on a grain ration (Campbell et al., 1966) from the time of conception until subsequent litters were used. Tap water and food were available ad libitum. Animals were housed in a temperature controlled room with a 12-12 hour light-dark cycle. Uterine Ligation Procedure Non-pregnant female rats were anesthetized with pentobarbital sodium (30 mg/kg i.p.), shaved, and a laparotomy performed. One uterine horn was identified and two silk ligatures were tied securely around it near the caudal end. The horn was out between the two ligatures. Care was taken to avoid unnecessary injury to tissue and blood supply. Sham-operated controls were manipulated similarly except that the ligatures were not tied and the horn was left intact. The abdominal musculature was closed with interrupted stitches and the skin closed with wound clips. Clean, but not aspectic, technique was used. 21 22 The females were allowed to recover from surgery for one week prior to breeding. Timed Pregnancy Female rats were placed in cages with male breeder rats nightly before the beginning of the dark cycle. Early the next morning, males were removed and females examined for the presence of sperm. Several drops of distilled water were placed into the vagina with a medicine dr0pper, gently withdrawn, placed on a glass slide and examined microscopically for the presence of sperm. As soon as an animal had a positive vaginal smear, she was housed individually and fed the grain ration (Campbell et al., 1966). At 20 days gestation pups were taken by cesarean section or allowed to reach full-term and used at one day of age. Cesarean Section Female rats at 20 days gestation were anesthetized with ether, shaved, and laparotomized. Uterine horns containing the fetuses were exposed and a small incision was made in each horn. Each fetus and the attached placenta was removed through the opening, cleaned of mucus, and separated from the placenta. After all fetuses were removed, the uterine horns were sutured closed. Abdominal musculature was closed with interrupted stitches. The skin was closed with wound clips, and the surgical area cleaned with zephrin HCl (Benzalkonium HCl, Pierce Co.). Pups were kept warm on a heating pad and stimulated to breath by intermittent touching until used within a 30 minute time period. Preparation of Renal Cortical Slices Animals were weighed and killed by decapitation. The kidneys were immediately removed, weighed, and placed in cold saline. 23 Renal cortical slices 0.3-0.4 mm thick were prepared freehand and pooled slices weighing about 50 mg were kept in cold saline until incubated. Accumulation of Organic Ions by Renal Cortical Slices Slices of renal cortex were incubated in 2.7 ml of Cross and Taggart (1950) phosphate medium. The medium, adjusted to pH 7.4, contained 7.4 x 10‘5 M p-aminohippuric acid (PAH) (Eastman Kodak Co.) and 6.0 x 10"5 M (4.6 mc/mm) 14C-N-methylnicotinamide (NMN) (New England Nuclear). Incubations were carried out in a Dubnoff Metabolic Shaker at 25°C under a gas phase of 100% oxygen for 90 minutes. After incuba- tion the slices were quickly removed from the beakers, blotted, weighed, and placed in tissue grinders. A two ml aliquot of medium was taken from each beaker. Three ml of 10% trichloracetic acid (TCA) were added to tissue grinders containing tissue or medium. Tissue was macerated and the final volume adjusted to 10 ml with distilled water. Samples were centrifuged at 1400 rpm for 15 minutes. After centrifuging, 2.0 ml of the supernatant were used to determine PAH spectrophoto- metrically as described by Smith et a1. (1945). In addition, one ml of slice or medium homogenate was placed in scintillation vials and 10 ml of modified Bray's solution (6 g of 2,5-diphenyloxazole and 100 g of napthalene per liter of dioxane) added. Radioactivity was determined using a Beckman LS-250 liquid scintillation spectro- meter, employing internal standardization. Samples were counted to an accuracy of 1 2.00%. Transport was expressed as the slice to medium (S/M) ratio which is equal to the concentration of PAH per gram of tissue (wet weight) divided by the concentration of PAH per ml of 24 medium or in the case of NMN-14C disintegrations per minute per gram of tissue (wet weight) divided by the disintegrations per minute per ml of medium. Transport of Amino Acids and Glucose by Renal Cortical Slices Renal cortical slices were pre-incubated in 2.0 ml of Krebs- Ringer bicarbonate buffer (Goldstein et al., 1973) in 25 ml incuba- tion flasks. “The medium adjusted to pH 7.4 by bubbling with 95% oxygen-5% CO2 contained 0.154 M KH P04, 0.154 M NaCl, 0.154 M KC1, 2 0.11 M CaClz, 0.154 M MgSO4, and 0.154 M NaHCO3. Pre-incubations were carried out in a Dubnoff Metabolic Shaker. The slices were gassed for 1-2 minutes with 95% 02-5% C02, then stoppered and in- cubated for 30 minutes at 37°C. After pre-incubation, slices were transferred to a clean flask containing 2.7 ml Krebs-Ringer bicarbo- nate buffer with either 0.10 mM/L 14C-a-aminoisobutyric acid (9.0 mc/mm) (New England Nuclear) or 0.125 M 14C-a-methyl-D-glucoside (177.0 mc/mm) (New England Nuclear). The flasks were stoppered and incubated for one hour at 37°C. After incubation slices were removed, blotted, and weighed. A 2.0 ml aliquot of medium was removed from each flask. Slice and medium were analyzed for AIB or a-MG as reported for NMN., Gluconeogenesis Two flasks of Krebs-Ringer bicarbonate buffer media, one with water and one with 10 mM L-monosodium glutamate as substrate, were made as described above for AIB pre-incubations. The media was bubbled for 15 minutes at 37°C with 95% 02-5% C02. While the media was bubbling, renal cortical slices were prepared as described 25 previously. Slices were rinsed 3 times in saline to remove exogenous glucose. Prior to incubation, slices were placed in 25 ml flasks containing the medium adjusted to a pH of 7.4. These were gassed with 95% 02-5% CO for 1-2 minutes, the flasks stoppered, and then 2 incubated for 90 minutes at 37°C in a Dubnoff Metabolic Shaker. After incubation slices were removed, blotted, placed in crucibles dried to constant weight, and dried in a dessicator for 24 hours. After 24 hours the dry tissues were weighed. The medium from slices was prepared according to Goldstein et a1. (1973). Glucose concen- tration was analyzed using a glucose oxidase reagent containing glucose oxidase peroxidase and afchromagen (Worthington Glucostat Reagent, Worthington Biochemical Corp., New Jersey). Glucose produced was expressed as umoles of glucose/hour/gram tissue (dry weight). Protein and Water Composition of Renal Cortical Slices Slices were prepared as described previously for accumulation studies and kept in iced saline until blotted, weighed, and placed in a dessicator for 24 hours. Total water content of cortical slices was determined as the differences between the wet and dry weights and tissue water was expressed as a percent of the wet weight. After drying, the slices were dissolved in 3 N KOH and diluted with distilled water. A one ml aliquot was used for protein analysis (Lowry et al., 1951). Statistical Analysis All data obtained was subjected to statistical analysis using analysis of variance, complete random design or random complete block. Certain data were analyzed using the Least Significant Difference 26 (LSD) test (Sokal and Rohlf, 1969). The 0.05 level of probability was used as the criterion of significance in all statistical tests. RESULTS Food Intake During Pregnancy The food intake of pregnant rats from days 1-4 of gestation was the same for control, sham operated and ligated operated dams (Table l). Rats from all three groups consumed more food on days 18-21 of gestation than on days 1—4 (Table 1). The food intake during late gestation was the same for all three groups. Length of Gestation The length of gestation was measured as the time from a vaginal smear positive for sperm to birth of a litter. The gestational period for all three groups of animals was 22 days (Table II). Body Weight Pups from dams with one ligated uterine horn were heavier at 20 days gestation (5.95 t 0.15 g) than those from control dams (4.66 E 0.18 g) or sham operated dams (5.22 i 0.48 3) (Fig. 3). Animals from all groups were heavier at one day of age than at 20 days gestation (Fig. 3). Pups from ligated dams, however, were signifi- cantly heavier (8.03 i 0.32 g) than those from sham operated (7.11 i 0.12 g) or control (7.33 i 0.27 g) dams at day one (Fig. 3). Kidney Weight and Kidney Weight/Body Weight Ratio (KW/8W) Kidneys from pups of ligated dams were significantly heavier (0.047 t 0.003 g) at 20 days gestation than those from pups of sham operated (0.035 t 0.004 g) or control (0.035 t 0.003 g) dams (Fig. 4). 27 28 At day one, there was also a significant increase in kidney weights of pups from ligated dams (0.073 t 0.007 g) when compared with those of pups from sham (0.060 t 0.003 g) or control (0.063 t 0.002 g) dams (Fig. 4). The KW/BW ratio was significantly greater in pups from ligated dams (0.777 t 0.038) than in pups from sham dams (0.685 1 0.050) (Fig. 5) at 20 days gestation. At one day of age there was also an increase in the KW/BW ratio for pups from ligated dams (1.061 t 0.125) when com- pared with pups from shams (0.840 t 0.028) or controls (0.871 t 0.022) (Fig. 5). Protein Protein concentration was expressed as percent protein per mg wet weight. In 20 day gestation pups, there were no significant differences in the protein content of the renal cortical slices in the three groups of pups (Fig. 6). There were also no differences in protein content of renal cortical slices from the three groups of pups at one day of age (Fig. 6). 11122. The water content of renal cortical slices was determined in pups from ligated, sham, and control dams at 20 days gestation and one day. There were no significant differences in the three groups of pups at either age (Fig. 7). £§H_ The accumulation of PAH by renal cortical slices was determined at 20 days gestation and one day of age in pups from ligated dams, shams, and controls._ At 20 days gestation there were no differences 29 in PAH transport in the three groups of pups. Similarly, at one day of age there were no differences in the S/M ratio fbr the three groups of pups (Fig. 8). Renal cortical slices from pups at 20 days gesta- tion accumulated more PAH irregardless of group than those from pups one day old (Fig. 8). NMN_ NMN accumulation was measured in kidney slices from pups from ligated dams, shams, and control dams at 20 days gestation and day one. NMN accumulation was the same in all three groups at both 20 days and one day of age. There was an increase in NMN transport from 20 days gestation to one day (Fig. 9). ALB Amino acid transport, as measured by AIB, was less at 20 days gestation in pups from ligated dams (5.49 t 0.21) than pups from sham (9.58 t 1.09) or control (7.54 i 1.12) dams. At one day of age kidneys from animals in all three groups accumulated AIB the same (Fig. 10). a-Methylglucoside Accumulation of a-MG by renal cortical slices was less in pups from ligated dams (1.02 i 0) than in pups from shams (1.42 i 0.07) or controls (1.24 t 0.03) at 20 days gestation. However, there were no differences in a-MG accumulation at one day of age in the three groups of pups (Fig. 11). Glucoside transport did not increase with age from 20 days gestation to one day old (Fig. 11). 30 Gluconeogenesis Glucose synthesis with or without L-glutamate as substrate was the same for renal cortical slices from pups of ligated, sham, or control dams at 20 days gestation and day one (Fig. 12, 13). The capacity to synthesize glucose at one day was greater than at 20 days gestation, with and without the addition of substrate to the medium (Fig. 12, 13). DISCUSSION Altering litter size, by reducing the number of pups per litter after birth, will produce overnutrition. Overnutrition is described as an increase in the amount of nutrients-protein, carbohydrate, fats, vitamins, minerals, oxygen, and water above the nutrient requirement for normal growth. Rats that were suckled in small groups grow more rapidly and attain a larger final size than controls (Winick et al., 1967). Litter size can be altered prenatally to increase the availability of nutrients to the fetus. Van Marthens et al. (1969) showed that ligation of one uterine horn in the rat caused a decrease in litter size with a subsequent increase in body weight. Johnson et al. (1974) showed that accumulation of PAH and NMN by renal cortical slices was increased in pups from ligated dams when compared to pups from sham operated dams. Since other investigators have demonstrated that inadequate nutrition in utero has an effect on delaying renal development (Allen et al., 1973) it was of interest to determine whether overnutrition in utero would also alter renal development. The purpose of this study was to determine whether ligation of one uterine horn prior to breeding, and thereby increasing nutrient supply to the fetuses, would alter renal function pre and postnatally. Pups born of dams with one uterine horn ligated were heavier (Fig. 3) at both 20 days gestation and day one than those of control 31 32 or sham operated dams. Johnson et al. (1974) showed that rats from ligated dams were larger at birth, at five, and ten days of age than pups from sham operated dams. Food intake measured in early and late pregnancy was not different for the three groups of rats (Table I); therefore, increased body weight of the pups could not be attributed to an increase in food consumption by the ligated dams. Body weight increases in the pups from ligated dams could also not be due to a prolonged gestational period since gestational periods were the same fbr all three groups of rats (Table II). Pups from ligated dams had heavier kidneys than pups from sham or control dams (Fig. 4). KW/BW ratios (Fig. 5) were increased in pups from ligated dams. In 15 day old rats, however, Solomon et al. (1973) showed that for animals from reduced litters the KW/BW ratios were lower than animals from intact litters. Van Marthens et al. (1969) also showed that in rabbits, uterine ligation produced offspring which had an increase in cerebral weight. The effect of uterine ligation prior- to breeding on the development of other organ systems has not been determined. The protein and water content of the kidneys of pups from ligated dams, shams, and controls did not differ at either age (Fig. 6, 7). This suggests the increased kidney weight in the pups from ligated dams was not due to an increase in one specific constituent. Previously, it has been shown that the ability of renal cortical slices from newborn rats to accumulate PAH demonstrates a pattern of functional development (Kim et al., 1972). Rennick et al. (1961) 33 observed a correlation between histological development of the renal cortex and the ability of the tissue to accumulate PAH. In contrast to this, in this series of experiments the PAH S/M ratio decreased from 20 days gestation to one day old in kidneys from rats in all three groups (Fig. 8). This could be caused by an increase in plasma free fatty acids or some other inhibitor from the mothers milk which would compete for transport of PAH. The decreased ability to accumulate PAH from in utero to immediate postnatal period could also be because the kidney is assuming excretory functions that were previously handled by the placenta in utero. Hirsch et al. (1970) reported that kidneys from rabbits just prior to birth accumulated PAH to a greater extent than kidneys from rabbits at day one. In kidneys of pups from ligated dams, PAH transport is not in- creased above sham pups or control pups (Fig. 8). However, Johnson et al. (1974) demonstrated that kidneys from pups of ligated dams accumulated significantly more PAH at 5 days of age than those from non-ligated dams. This would suggest that more profound development of the organic acid transport system occurs between 1 and 5 days of age in the rat than prior to day one. The pattern of development of organic base uptake in renal cortical slices was different than that of organic acid. There is a progressive increase in the ability to accumulate TEA by renal cortical slices in the puppy (Rennick et al., 1961). Similarly, accumulation of NMN shows an increase from 20 days gestation to one day of age (Fig. 9). The pattern of development of NMN accumulation by rat renal cortex appears to be more closely associated with organ growth than 34 that of PAH within the first few days of life (Fig. 8). Johnson et al. (1974) previously demonstrated a significant increase in NMN transport in kidneys from rats 5 to 10 days of age. However, in these experiments, there were no differences seen in the ability of larger kidneys of pups from ligated dams to accumulate NMN (Fig. 9) suggesting the critical developmental period for organic base transport is after 5 days of age in the rat. Similarly, the renal transport of amino acids is not the same at all ages. Reynolds et al. (1974) reported that the distribution ratio for AIB is higher in kidneys from one day old rats than in adult kidneys. Enhanced accumulation of amino acids into tubular cells from newborn kidneys may be due to the fact that these are required for protein synthesis. Protein synthesis at the transcription and trans- lational levels has been demonstrated to occur in the proximal tubular cells of newborn rat kidney cortex (Reynolds et al., 1974). The AIB S/M ratio obtained in these experiments for kidneys from control and sham newborn rats were similar to the distribution ratios fer new— born rat kidney cortex previously reported (Reynolds et al., 1974). Amino acid transport as measured by AIB accumulation into renal cortical slices was significantly decreased in pups from ligated dams at 20 days gestation (Fig. 10) suggesting faster maturity of these kidneys. At one day of age, however, there were no differences in the three groups of animals (Fig. 10). This ability of renal cortical slices from pups of ligated dams appeared to increase from the twentieth day of gestation to day one. The significance of this, if it is a real effect, is not known. 35 Sugar transport was measured by a-MG. The S/M ratio for a-MG was very low in kidneys from 20 day gestation and one day old animals (Fig. 11). At 20 days gestation it is even lower for kidneys of pups from ligated than sham or control dams. Reynolds et al. (1974) measured glucose transport with a-MG in one day old animals and re- ported distribution ratios similar to the S/M ratios obtained at one day of age (Fig. 11). The larger kidneys of pups from ligated dams showed no increase in the ability to transport glucoside compared to kidneys of pups from control or sham dams. This indicates that deve- lopment of this system probably occurs at a later age than day one. Since an S/M of one is obtained in this system when transport occurs mainly by diffusion, these S/M ratios are indicative of very little active glucoside transport. Furthermore, glucoside transport did not increase from 20 days gestation to day one (Fig. 11). This may be because tubular reabsorption matures by increasing the number of functional units or by maturity of cellular reabsorptive mechanisms, and this probably has not occurred at day one in the rat. Gluconeogenic enzyme activity rapidly increases in rat kidney cortex during the first two postnatal weeks (Zorzoli et al., 1969). Renal gluconeogenesis is stimulated by acidosis. Renal glutaminase concentration and the response of renal glutamate to acidosis are low during the first two weeks of life in the rat (Goldstein et al., 1973). There could be a relationship between the patterns of biochemical and physiological development in the rat kidney. There were no significant differences in the gluconeogenic capacity of animals from ligated dams than from shams or controls at 20 days gestation 36 with or without substrate added to the media (Fig. 12). These experiments showed glucose production with L-glutamate at 20 days gestation in the rat to be similar to the values obtained by Zorzoli et al. (1969) at 21 days gestation. The capacity to synthesize glucose at one day is greater than at 20 days gestation (Fig. 12, 13). This is due to gluconeogenic enzyme development. Wacher et a1. (1961) demonstrated that at birth the rat kidney glutaminase was low compared to adult rat kidneys. These experiments showed that uterine ligation does not alter the renal functions examined at 20 days gestation or day one in the rat. However, previous work in 5 and 10 day old rats indicates altered renal function in PAH and NMN transport in kidneys of pups frOm ligated dams (Johnson et al., 1974). This suggests that alterations in kidney function can occur at a later stage of development in the rat. Apparently renal development begins after birth. Clinically, over- nutrition in utero may have an effect on kidney development at a later stage of development but poSsibly has no effect on the newborn within the first days of life. These data are among the first available for a variety of renal functions in the rat at day 20 of gestation and day one. All organs do not develop at the same time. Van Marthens et al. (1969) showed that overnutrition had an effect on newborn rat cerebrum. However, these data suggest that the kidney is not effected by over- nutrition at birth or 20 days gestation. Therefore, the kidney probably develops later than the brain and possibly develops later than other organs. {[1 l SUMMARY Rats with one uterine horn ligated were used to determine alterations in renal development from overnutrition in 20 day gestation and one day old pups. Ligation of one uterine horn produced heavier offspring than sham or control dams. The off- spring also had heavier kidneys than pups from sham or control dams. Protein and water contents of all kidneys from the three groups of animals were the same. This suggests that uterine ligation does produce overnutrition in utero. Accumulation of organic ions was determined in kidneys of pups at 20 days gestation and one day. There were no differences in PAH transport in the kidneys of pups from ligated, sham, or control dams. A developmental pattern for PAH was established for these two ages. At 20 days gestation PAH transport is high and then falls to a lower level at one day of age. NMN accumulation was also determined and there were no differences in the S/M ratios for kidneys of pups from ligated, sham, or control dams at either age. Accumulation of NMN increased in kidneys from pups at 20 days gestation to higher S/M ratios in kidneys of pups at one day of age. These data suggest that overnutrition has no effect on PAH or NMN transport systems at 20 days gestation or one day. Transport of amino acids was determined using AIB. There were no differences in amino acid transport in kidneys of pups from 37 38 ligated, sham, or control dams at one day of age. However, there was a significant decrease in AIB transport in kidneys of pups from ligated dams at 20 days gestation. This may suggest a more mature kidney in pups from ligated dams since the S/M ratio for AIB decreases from newborn kidneys to adult kidneys. There were no differences in AIB accumulation between the two ages. Glucose transport was also measured using a-MG. There was a decrease in glucoside transport in kidneys of pups from ligated dams when compared to kidneys of pups from sham or control dams. There were no differences at one day of age in the three groups of pups suggesting overnutrition had no effect on glucose transport in the kidney at this age. Glucose transport development must occur later than one day of age since no increases in the S/M ratio for a-MG occurred between 20 days gestation and day one. There is some development of gluconeogenic enzymes in the kidneys of pups 20 days gestation to day one. There were increases in the amount of glucose produced in kidneys from 20 day gestation animals to one day old animals. However, there were no changes in glucose production in kidneys of pups from ligated, sham, or control dams at either age. This suggests that overnutrition had no effect on the gluconeogenic capacity of the kidneys of pups from ligated dams at 20 days gestation or one day of age. These data show that uterine ligation does not alter renal function at 20 days gestation or one day of age in the rat. However, these data do suggest that renal development takes place at a later date than one day after birth. 39 Table I. Food Intake of Rats During Gestationa Group' Period Control Sham ‘ Ligated Days 1-4 . of Pregnancy 24.63 i 1.62b 24.70 i 1.40 20.82 1 1.51 Days 18-21 of Pregnancy 30.67 i 1.64 39.25 i 9.71 35.27 i 3.45 aValues represent the means i S. E. obtained from 3 females in each group from days l-4 of gestation and from 3 sham, 6 controls, and 4 ligated females from days 18-21 of gestation. bGrams of food consumed per day. Table II. Group Control Sham Ligated 40 Gestational Period of Rats Days 22 0.13a H- 22 i 0.00 22 i 0.00 aValues represent the means i S. E. obtained from six females in each group. 41 Figure 1. Schematic diagram of the in vivo system described by Tune et al. (1969) and the in vitro system. The diagram compares the two systems and shows that in both the active transport step is accumulation of PAH. 42 , active organic " acid trans on poo passive flux passive flux blood lumen cell In Viva In Vitro Figure l 43 Figure 2. Schematic diagram of the p-aminohippurate (PAH) slice incubation system under several conditions. The PAH S/M ratio is equal to the concentration of PAH in the slice to that in the medium (Johnson, 1972). PAH S/M RATIO I2 IO 44 30 60 90 TIME(minutes) Figure 2 I20 STIMULATION CONTROL INHIBITION NITROGEN Figure 3. 45 Body weight (g) of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean t (S. E.) obtained from 10 litters for each of the three groups of pups at day 20 of gestation and 12 litters for each of the three groups of pups at day one. The value obtained for body weight for pups from ligated dams is significantly greater (i) than far pups from sham or control dams at both ages. (P<0.05) u7////////// i a///////// mm.y///////////// a .5052, Soc SHAM IIGATED CONTROL Figure 4. 47 Kidney weight (g) of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained from 10 litters for each of the three groups of pups at day 20 of gestation and 12 litters for each of the three groups at day one. The value obtained for kidneys of pups from ligated dams is significantly greater (1") than for pups from sham or control dams at both ages. (P<0.05) 8 0. .|.r/////////// 1.7////////// ”m .7///////////// 7 O 5 4. 3 o o. o. o. o. a .5032, $296. 2 0. m. SHAM LIGATED CONTROL Figure 5. 49 Kidney weight/body weight ratio (x 100) of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained from 10 litters for each of the three groups of pups at day 20 of gestation and 12 litters for each of the three groups of pups at day one. The value obtained for KW/BW ratio for pups from ligated dams is significantly greater ( in than for pups from sham or control dams at both ages. (P <0.05) KW/BW x IOO L2 1.1 1.0 - Day 20 of gestation 50 ”4 DayI ? CONTROL \\\\‘- SHAM Figure 5 * LIGATED Figure 6. 51 Protein content (% protein/mg wet weight) of renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained for 3 litters at 20 days gestation and 2 litters at day one. 'A single value was obtained for each litter by pooling the results for individual beakers. 20 I5 IO % PROTEIN/mg wet wt - Day 20 of gestation 52 W‘Dayl CONTROL / / / / / / / / / / / / / é / SHAM Figure 6 LIGATED 53 Figure 7. Water content (%) of renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean t (S. E.) obtained for 3 litters at both ages. A single value was obtained for each litter by pooling the results for individual beakers. WATER, % J; C) 54 I 00 - Day 20 of gestation 90 7/1 Day I 80 70 (F C) Ln C) 00 C3 20 IO / CONTROL SHAM LIGATED / Figure 7 Figure 8. 55 Accumulation of PAH (S/M ratio) in renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained for 3 litters at both ages. A single value Was obtained for each litter by pooling the results of individual beakers. PAH S/M RATIO 56 - Day 20 of gestation 7’}. Day] CONTROL SHAM Figure 8 LIGATED Figure 9. 57 Accumulation of NMN (S/M ratio) by renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained far 3 litters in each group at both ages. A single value was obtained for each litter by pooling the results of individual beakers. NMN S/M RATIO CONTROL SHAM LIGATED Figure 10. 59 Accumulation of AIB (S/M ratio) by renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean i (S. E.) obtained for 3 litters in each group at both ages. A single value was obtained for each litter by pooling the results for individual beakers. The value obtained for kidneys of pups from ligated dams at 20 days gestation is significantly less ('fir) than that of pups from sham or control dams. (P<0.05) AIB S/M RATIO I2 II IO 60 - Day 20 of gestation V/A Day I CONTROL SHAM Figure 10 LIGATED Figure 11. 61 Accumulation of a-MG (S/M ratio) by renal cortical slices from kidneys of pups from ligated, sham, and control dams at day 20 of gestation and day one. Each bar represents the mean t (S. E.) obtained for 3 litters in each group at both ages. A single value was obtained for each litter by pooling the results for individual beakers. The value obtained for kidneys of pups from ligated dams at 20 days gestation is significantly less (i) than that for kidneys of pups from sham or control dams. (P<0.05) «It-MG S/M RATIO 1.8 1.6 1.4 1.2 1.0 62 - Day 20 of gestation _ VJ Dav I ' J A CONTROL SHAM Figure 11 LlGATED Figure 12. 63 Gluconeogenic capacity (umoles of glucose/hour/g dry weight) with and without 10 mM L—glutamate added as substrate in kidneys from pups from ligated, sham, and control dams at day 20 of gestation. Each bar represents the mean i (S. E.) obtained for 3 litters in each group. A single value was obtained for each litter by pooling the results of indivi- dual beakers. .—I __. N N C) en (3 h" GLUCONEOGENESIS, pm glucose/hr/g dry wt u: 64 DAY 20 OF GESTATION - With substrate 'I/A Without substrate CONTROL SHAM Figure 12 LIGATED Figure 13. 65 Gluconeogenic capacity (umoles of glucose/hour/g dry weight) with and without 10 mM L-glutamate added as substrate in renal cortical slices of kidneys from pups of ligated, sham, and control dams at day one. Bath bar represents the mean i (S. E.) obtained for 3 litters in each group. A single value was obtained for each litter by pooling the results of individual beakers. 50 45 CD (0 O GLUCONEOGENESIS, pm glucose/hr/g dry wt no 0 O - With substrate 7" Without substrate CONTROL 66 DAY 1 SHAM Figure 13 LIGATED BIBLIOGRAPHY BIBLIOGRAPHY Allen, L. H. and Zeman, F. J.: Kidney function in the progeny of protein-deficient rats. J. Nutr. 103:1467-1477, 1973. Arant, B., C. Edelmann, and Nash, M.: The renal reabsorption of glucose in the developing canine kidney: A study of glomerulotubular balance. Pediat. Res. 83638-646, 1974. 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