Adverse effects of hyperglycemia on kidney development in rats: in vivo and in vitro studies.

Congenital malformations occur more frequently in the offspring of diabetic mothers. These in vivo and in vitro studies investigate the potential adverse effects of hyperglycemia on kidney development in the rat. Female rats were made hyperglycemic throughout gestation with a single injection of streptozotocin (STZ) on day 0 of gestation, or for a short period encompassing the early stage of renal organogenesis by infusing glucose from gestational days 12-16. Kidney development in the pups was assessed by determining the total number of nephrons formed in the kidney. The number of nephrons was significantly reduced (10-35%) in the pups from STZ-treated dams, as a function of hyperglycemia. There were also fewer nephrons in pups from dams given glucose infusion whose hyperglycemia was transiently higher on day 13 of gestation. The in vitro experiments were done on metanephroi removed from 14-day-old fetuses and grown for 6 days in medium containing 0, 6.9, 13.8, or 27.5 mmol/l glucose. The development of explants grown in 0, 13.8, and 27.5 mmol/l glucose was impaired compared with that of explants grown in the 6.9 mmol/l control medium, showing that the glucose concentration must be closely controlled to ensure optimum in vitro metanephros development. Thus, exposure to hyperglycemia in utero can cause a nephron deficit, which in turn may have renal consequences later in life.

Altered nephrogenesis due to maternal diabetes is associated with increased expression of IGF-II/mannose-6-phosphate receptor in the fetal kidney.

We have recently demonstrated that the exposure to hyperglycemia in utero impairs nephrogenesis in rat fetuses (Amri K et al., Diabetes 48:2240-2245, 1999). Diabetic pregnancy is commonly associated with alterations in the IGF system in fetal tissues. It has also been shown that both IGF-I and IGF-II are produced within developing metanephros and promote renal organogenesis. Therefore, we investigated the effect of maternal diabetes on IGFs and their receptors in developing fetal rat kidney. Diabetes was induced in pregnant rats by a single injection of streptozotocin on day 0 of gestation. We measured the amounts of IGF and their receptors, both proteins and mRNAs, in the metanephroi of fetuses issued from diabetic subjects and in age-matched fetuses from control subjects (14-20 days of gestation). IGF-II was produced throughout fetal nephrogenesis, whereas IGF-I protein was not detected, suggesting a critical role of IGF-II in kidney development. Fetal exposure to maternal diabetes caused no change in IGF production in the early stages of nephrogenesis. Similarly, the amounts of IGF-I receptor and insulin receptor were not altered. By contrast, there was an increase in production of IGF-II/mannose-6-phosphate receptor throughout nephrogenesis. Because this receptor plays an essential role in regulating the action of IGF-II, the altered nephrogenesis in fetuses exposed to maternal diabetes may be linked to a decrease in IGF-II bioavailability.

Effect of 3,5,3'-triiodothyronine on maturation of rat renal phosphate transport: kinetic characteristics and phosphate transporter messenger ribonucleic acid and protein abundance.

The plasma concentration of T3 increases during the third postnatal week in rats when the young are weaned. Renal phosphate (Pi) transport matures during this period, with increases in the carrier affinity for Na-cotransported Pi(1/K(m)) and in the maximal transport rate (Vmax). As prevention of weaning prevents the normal increase in the plasma T3 concentration and blocks the maturation of Pi transport, this study examines the influence of T3 on Pi transport maturation. The brush border membranes from 21-day-old rats rendered hypothyroid with 6-n-propyl-2-thiouracil (PTU) or prevented from weaning (PW) were examined. These two treatments effectively prevented normal maturation, as the apparent K(m) was significantly higher in the PTU (0.243 +/- 0.04) and PW (0.159 +/- 0.008) rats than in controls [0.111 +/- 0.032 (n = 6; P < 0.05) and 0.104 +/- 0.005 (n = 6; P < 0.05)], and the Vmax was significantly lower [3192 +/- 170 vs. 4269 +/- 343 (n = 6; P < 0.01) and 3479 +/- 478 vs. 4184 +/- 368 (n = 6; P < 0.05)]. Administration of T3 to PTU and PW rats restored the apparent K(m) and V(max) to control values. The amounts of Na-Pi co-transporter messenger RNA and protein were also determined. The amounts of NaPi-2 messenger RNA and protein were decreased about 5-fold in PTU and PW rats, and T3 restored them to control levels. The effect of T3 on the maturation of Pi transport was independent of PTH, GH, or glucocorticoids. Thus, thyroid hormone helps control the maturation of Pi transport in the rat during weaning, and thyroid hormone may be a dominant triggering factor in this process.

Effects of prenatal and postnatal exposure to gentamicin on renal differentiation in the rat.

Pregnant rats were injected daily, from the 10th day of gestation to term, with 75 mg/kg of gentamicin. They gave birth about 15 h later than control pregnant rats injected with saline to pups with various degrees of growth retardation. In pups born of gentamicin-treated mothers, the number of nephrons present at birth, as well as the final number of nephrons, were reduced by at least 20%. Observation of the kidneys by light microscopy showed focal tubular lesions on the mature nephrons. The intrarenal concentration of gentamicin was higher in the severely growth retarded pups than in the others. In another series of experiments, rats were given 75 mg/kg of gentamicin daily from days 1 to 13 after birth. Although under these conditions the concentration of gentamicin reached in the postnatal kidney was higher than that reached after exposure in utero, no reduction of the final number of nephrons was observed. It is concluded that administration of gentamicin to pregnant rats caused focal tubular lesions in the developing kidney and a reduced rate of early nephrogenesis. The latter was probably due to growth retardation, though a more direct effect of gentamicin on early nephrogenesis may also have been involved.

Exposure in utero to maternal diabetes leads to glucose intolerance and high blood pressure with no major effects on lipid metabolism.

AIM: Recent evidence shows that adult metabolic disease may originate from an adverse fetal environment that can alter organ development and function in postnatal life. This study aimed to analyze the effect of exposure in utero to maternal diabetes on the development of the metabolic syndrome in the offspring. METHODS: Pregnant rats were made diabetic (blood glucose was 20mM) with a single streptozotocin injection on day 0 of gestation. Offspring from diabetic mothers (DMO) and control mothers (CMO) were followed from birth to 12 months of age. In these animals, metabolic parameters, such as glucose tolerance, insulin sensitivity and plasma lipid levels, as well as pancreatic insulin and morphology were studied. RESULTS: Compared with controls, DMO offspring had normal birth weights, but impaired postnatal growth that persisted throughout life. Metabolic tests revealed that DMO offspring also showed impaired glucose tolerance at six months associated with decreased insulin sensitivity and low insulin secretion. In older animals (12 months old), this phenotype persisted, but to a lesser extent. The DMO offspring also presented with high blood pressure and decreased levels of fasting plasma triglycerides, but normal plasma NEFA, and HDL and total cholesterol. CONCLUSION: Altogether, these results show that our model of exposure in utero to maternal diabetes led to normal birth weights, and induced transient glucose intolerance and increased blood pressure with no major effects on lipid metabolism. It also suggests that a hyperglycaemic fetal environment may be able to 'programme' hypertension and glucose intolerance, but not alter lipid metabolism.

Evaluation of the effect of autologous mesenchymal stem cell injection in a large-animal model of bilateral kidney ischaemia reperfusion injury.

OBJECTIVES: Adult mesenchymal stem cells (MSC) have been proven to be of benefit to the kidney in different experimental models of renal injuries. All studies have been performed in valuable rodent models, but the relevance of these results to large mammals and ultimately, to humans remains unknown. Therefore, the aim of this study was to investigate the effect of MSC transplantation in an alternative ovine large-animal model of bilateral kidney ischaemia reperfusion injury. MATERIAL AND METHODS: Sheep were divided into three groups: one sham-operated group and two groups submitted to renal bilateral ischaemia for 60 min. Animals with ischaemia reperfusion injury were treated with injection of autologous MSCs or with vehicle medium. RESULTS: The model sheep presented with renal histological manefestations that closely resembled lesions seen in patients. Transplanted MSCs were found in glomeruli but not in tubules and did not express glomerular cell markers (podocin, von Willebrand factor), but functional evaluation showed no beneficial effect of MSC infusion. Morphological and molecular analyses corroborated the functional results. MSCs did not repair kidney parenchyma and failed to modulate cell death and proliferation or cytokine release (tumour necrosis factor-alpha, vascular endothelial growth factor alpha (VEGF-alpha), Bcl-2, caspase). CONCLUSION: In this unique autologous large-animal model, MSCs did not exhibit reparative or paracrine protective properties.

Effects of weaning on phosphate transport maturation in the rat kidney. Clearance and brush border membrane studies.

Renal phosphate transport matures via an increase in the carrier affinity for phosphate during the 3rd post-natal week in the rat. This study examines whether weaning, which normally takes place during this period, plays a role in phosphate transport maturation. Early weaning (EW) and prevention of weaning (PW) both increased the fractional excretion of phosphate (EW 26.5 +/- 4.9, PW 26.7 +/- 2.2, controls 11.3 +/- 2.8, P < 0.001 and P < 0.05, n = 6 in each group). EW and PW also decreased the uptake of phosphate into brush border membrane vesicles (BBMV) isolated from the renal cortex of 21-day-old rats. Glucose transport in BBMV was not affected. The kinetics of phosphate uptake, measured in the presence of a sodium gradient, showed lower Vmax (4,112 +/- 362 pmol/mg protein per 10 s) in EW BBMV than in controls (6,030 +/- 200, n = 5, P < 0.001), but the affinity of the carrier for phosphate (1/Km) did not change. The decrease in Vmax may be due to the enhanced phosphate supply. The affinity of the carrier was lower in PW rats (Km = 0.31 +/- 0.04 mM) than in controls (0.18 +/- 0.04, n = 5, P < 0.01) but the Vmax remained unchanged. The low affinity may indicate that normal maturation of tubular transport, in which carrier affinity increases, is altered. The plasma concentrations of corticosterone, parathyroid hormone, insulin and triiodothyronine and their changes during EW and PW are also reported.(ABSTRACT TRUNCATED AT 250 WORDS)

The number of nephrons in the mammalian kidney: environmental influences play a determining role.

Several lines of evidence, mostly derived from animal studies, indicate that changes in the fetal environment may affect the renal development. Fetal growth retardation is associated with a nephron deficit in both humans and animals. Changes in the supply of vitamin A to the fetus may be responsible for the variations in the number of nephrons in the human kidney. In utero exposure to hyperglycemia or drugs may also cause a nephron deficit.

Otogeny of sugar transport in fetal rat kidney.

Concentration of alpha-methyl-D-glucopyranoside (alpha-MG) by kidney slices of rat fetuses increases during the studied period of gestation from day 18 until day 21 (last day of gestation). Glucoside transport is inhibited by phloridzin, D-glucose and D-galactose. Concentration dependence, over a substrate range from 0.01 to 0.2 mM, was studied on day 21: alpha-MG transport obeys a saturation kinetic with a low apparent Km (0.22 mM); D-glucose inhibition is of competitive nature (apparent Ki: 0.47 mM). Sodium dependence is demonstrated on day 21 of gestation: the phloridzine-sensitive transport, entirely inhibited in Na+-free medium, is enhanced when medium Na+ concentration increases from 0 to 143 mEq/l (Krebs-Ringer buffer concentration); further increase of Na+ concentration has no effect.

Effect of fetal exposure to gentamicin on phosphate transport in young rat kidney.

The renal phosphate (Pi)-transporting capacity normally increases, due to increased carrier system affinity, during the third postnatal week in rats. However, the tubular Pi reabsorption of rat pups born from gentamicin-treated mothers does not increase during this period. This study determines whether exposure to gentamicin in utero selectively alters the postnatal maturation of the carrier affinity for Pi. Pi and glucose transports by proximal tubule brush-border membrane (BBM) were studied. The maximal rate of uptake (Vmax) of Na-Pi cotransport was significantly lower (536 +/- 169 pmol.mg protein-1.10 s-1; n = 6, P < 0.01) in gentamicin-exposed rats than in controls (1,021 +/- 167 pmol.mg protein-1.10 s-1, n = 6), whereas the Michaelis constant (Km) values were the same. Gentamicin exposure had no effect on plasma parathyroid hormone concentration or on BBM glucose transport activity. The total phospholipid content of BBM, their phospholipid composition, cholesterol content, and cholesterol-to-total phospholipid mole ratio were unaltered, suggesting that membrane fluidity was unchanged. The Vmax of BBM alkaline phosphatase was lower in gentamicin-exposed rats than in controls.

Maturation of rat renal phosphate transport: effect of triiodothyronine.

1. Prevention of weaning prevents the increase in the carrier affinity for Na(+)-cotransported phosphate (Pi) and the normal elevation of plasma 3,5,3'-triiodothyronine (T3) which occur between postnatal days 14 and 21. 2. This study examines the possible role of T3 in the control of the maturation process of Pi transport. Clearance experiments and brush-border membrane studies were performed on 14-day-old rats given T3. 3. The fractional excretion of Pi of T3-treated rats was 33% lower compared with controls (P < 0.01). After Pi perfusion, it remained at a lower level, and the amount of Pi reabsorbed per minute, corrected for the glomerular filtration rate (RPi/GFR), was higher. 4. The membrane vesicles from 14-day-old rats given T3 showed a 30% increase in carrier affinity for Na(+)-cotransported Pi. In addition to this maturational effect of T3, a 46% increase in Vmax was also observed. 5. The amount of immunoreactive Pi transporter, detected using anti-(NaPi-2) antiserum, was increased in T3-treated rats. 6. Glucose transport, another Na(+)-dependent transport system, was not altered by T3. 7. It is concluded that exogenous T3 given before the third postnatal week specifically induced precocious maturation of renal Pi transport in 14-day-old rats, suggesting that thyroid hormone is normally involved in this maturation.

Long-term effects of mild oligonephronia induced in utero by gentamicin in the rat.

Renal clearance studies and morphologic observations were performed in rats aged 14, 21, and 28 d and 3, 6, 9, and 12 mo born with a 20% reduction in nephron number after administration of 75 mg/kg/d of gentamicin to their mothers during the second half of gestation. Gentamicin was still present in urine 3 mo after birth. Morphologic damage characteristic of gentamicin accumulation was observed in the kidney on d 14 and 21. Adequate compensatory adaptation to oligonephronia occurred for glomerular function within 14 d of birth, but tubular phosphate reabsorption was significantly low on d 21. On d 28, no evidence of histologic or functional damage to the kidney was observed. At 3 mo, mesangial lesions were observed in rats of the gentamicin group, whereas they were rarely present in 6-mo-old control rats. Furthermore, glomerular sclerotic lesions were already evident in about 5% of the juxtamedullary nephrons. The same percentage of injured nephrons was not observed before 12 mo in controls. Complementary morphologic data obtained in 24-mo-old rats showed that glomerulosclerosis involved 40% of the juxtamedullary nephron population at this age in animals of the gentamicin group versus 21% in controls. It is concluded that in the young rats born with oligonephronia of gentamicin-treated mothers neither the gentamicin remaining in the kidney cells nor the injuries it caused them prevented compensatory adaptation of the kidney to a reduced number of nephrons. However, although this oligonephronia was mild, it might have been sufficient to cause early development of glomerular sclerosis in the adults.

Immediate and long-term renal effects of fetal exposure to gentamicin.

Aminoglycoside antibiotics, like gentamicin, given to pregnant females cross the placenta and accumulate in the fetal kidney, which, like the adult kidney, was found to be the major site of deposition. In young guinea-pigs whose mothers were given gentamicin during the week following nephrogenesis in the fetus, nephron growth was found to be retarded temporarily. In rats whose mothers were given gentamicin during the period of fetal nephrogenesis, the final number of nephrons was reduced by about 20%. In both cases, renal development was impaired, although the concentration of gentamicin in the fetal kidney was lower than that measured in the kidney of human fetuses whose mothers had received a single injection of aminoglycoside. In rats exposed to gentamicin in utero, cellular damage of the undifferentiated and differentiating renal tissues was observed. It is, there are likely that the oligonephronia observed in animals born of gentamicin-treated mothers resulted from a direct effect of the drug at early stages of nephrogenesis. When gentamicin administration to the mother was prolonged, part of the oligonephronia observed at birth might have also resulted from fetal growth retardation, secondary to adverse effects of the drug on the mother. Providing it was not associated with fetal growth retardation, the presence of high gentamicin concentrations in the fetal kidney at late stages of nephrogenesis did not affect nephron differentiation. Long-term studies of rats born with gentamicin-induced oligonephronia showed that neither the antibiotic still present in kidney several weeks after birth, nor the injuries it caused, prevented renal growth and morphological adaptation of the nephrons to their reduced number.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an early weaning on phosphate transport maturation in the rat kidney: influence of the phosphate content of the diet.

The renal phosphate (Pi) transport system matures during the 3rd postnatal wk in the rat by an increase in the carrier affinity for sodium-cotransported phosphate. This study examines the ability of pups to adapt their renal Pi transport to the dietary phosphorus content during this period of carrier affinity maturation, corresponding to the weaning period in the rat. Clearance experiments and brush border membrane studies were performed on 21-d-old rats weaned early on d 16 onto a low phosphate diet (LPD, 0.19 g/100 g), a normal phosphate diet (control, 0.78 g/100 g), or a high phosphate diet (HPD, 1.5 g/100 g). In LPD rats, the Pi fractional excretion (0.3 +/- 0.1%) was lower than in controls (18 +/- 3%, p < 0.001). It remained very low (0.21 +/- 0.05% in LPD rats versus 40.5 +/- 6.3% in controls, p < 0.001) after Pi perfusion (1.5 mumol.min-1.100 g-1) and the reabsorbed Pi per min, corrected for the glomerular filtration rate, was higher than in the two other groups. The calcium fractional excretion (12.6 +/- 1.02%) in the LPD rats was much higher than in the controls (0.42 +/- 0.2%, p < 0.001). In contrast, HPD rats had an elevated Pi fractional excretion (41 +/- 4%, p < 0.001), whereas reabsorbed phosphate per min corrected for the glomerular filtration rate was not increased by a Pi load.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrauterine growth retardation leads to a permanent nephron deficit in the rat.

Intrauterine growth retardation (IUGR) was induced in Sprague-Dawley rats by partial artery ligation of one uterine horn in the mother on day 17 of gestation or by feeding the mother a 5% protein diet from day 8 of gestation. The controls were pups of the contralateral uterine horn or pups born to mothers fed a normal (22%) protein diet. The number of nephrons present at birth and the final number of nephrons in 2-week-old rats were counted throughout the entire kidney. The number of nephrons present at birth and the final number of nephrons were significantly correlated with birth weight for growth-retarded rats of both groups and their corresponding controls (P < 0.02 for the poorest correlation). Clearance experiments and morphometric studies of 2-week-old rats born to mothers with uterine artery ligation indicated that, despite a large compensatory hypertrophy of the nephrons in those animals born with a nephron deficit of about 30%, the overall renal function was impaired. We conclude that IUGR is accompanied by a nephron deficit which may not be fully compensated for within the first weeks after birth.

Role of retinoids in renal development: pathophysiological implication.

Vitamin A closely modulates nephron endowment at birth. It is also required for the development of renal vasculature. Fetal vitamin A status may thus be responsible for most of the variations in nephron number found in the general population, and may play a major role in the intrauterine programming of chronic renal disease and hypertension.

Regulation of c-ret expression by retinoic acid in rat metanephros: implication in nephron mass control.

Vitamin A and its derivatives have been shown to promote kidney development in vitro in a dose-dependent fashion. To address the molecular mechanisms by which all-trans-retinoic acid (RA) may regulate the nephron mass, rat kidneys were removed on embryonic day 14 (E14) and grown in organ culture under standard or RA-stimulated conditions. By using RT-PCR, we studied the expression of the glial cell line-derived neurotrophic factor (GDNF), its cell surface receptor-alpha (GDNFR-alpha), and the receptor tyrosine kinase c-ret, known to play a major role in renal organogenesis. Expression of GDNF and GDNFR-alpha transcripts was high at the time of explantation and remained unaffected in culture with or without RA. In contrast, c-ret mRNA level, which was low in E14 metanephros and dropped rapidly in vitro, was increased by RA in a dose-dependent manner. The same is true at the protein level. Exogenous GDNF barely promotes additional nephron formation in vitro. Thus the present data establish c-ret as a key target of retinoids during kidney organogenesis.

Kinetics of gentamicin in plasma of nonpregnant, pregnant, and fetal guinea pigs and its distribution in fetal tissues.

Nonpregnant and pregnant guinea pigs in the last third of gestation were injected intramuscularly with 4 mg of gentamicin per kg, and drug concentrations in plasma were determined by radioimmunoassay at several intervals after injection. The maximum gentamicin concentration was lower in pregnant than in nonpregnant animals (14.6 +/- 0.7 micrograms/ml versus 21.6 +/- 0.7 micrograms/ml), and the peak time occurred significantly later (0.57 +/- 0.12 h versus 0.13 +/- 0.02 h). Four hours after gentamicin injection, drug concentrations in plasma were 2.1 +/- 0.8 and 0.3 +/- 0.1 micrograms/ml in pregnant and nonpregnant animals, respectively. Pregnant animals therefore eliminated the drug from their plasma more slowly. These data provide good evidence that the kinetics of plasma gentamicin varies in pregnant females because its volume of distribution was larger in pregnant than in nonpregnant animals. Detectable but small amounts of gentamicin (less than or equal to 0.50 microgram/ml) were found in the plasma of 46 of 57 fetuses. However, no net variations in these concentrations were observed during the period between 15 min and 6 h after injection to the mother. Gentamicin concentrations were also determined in the kidneys, liver, lungs, heart, and brain of fetal guinea pigs after administration to their mothers of one daily injection of 4 mg/kg for 7 days. Gentamicin was present in all these fetal organs; however, as in the adult organs, the kidneys contained far more than any of the others. Gentamicin concentrations were not significantly different in the kidney cortex and medulla (1.79 +/- 0.16 versus 1.48 +/- 0.92 micrograms/g), indicating that, contrary to what is observed for adults, renal accumulation of gentamicin in the fetus does not occur preferentially in the cortex.

Effect of fetal exposure to gentamicin on kidneys of young guinea pigs.

Clearance experiments were performed with young pups born of guinea pigs given a daily injection of 4 mg of gentamicin per kg (body weight) from days 48 to 54 of gestation (term, 68 days). For 3-day-old animals, the glomerular filtration rate was similar to that measured in control guinea pigs of the same age whose mothers were given saline during the same period of gestation. The same applied to fractional excretion of water, urea, total solutes, Na, K, Ca, and Mg but not to fractional phosphate excretion, which increased significantly in the gentamicin group when compared with the controls (mean +/- standard error, 21.7 +/- 4.9 versus 7.3 +/- 1.8%; P less than 0.05; n = 6 for both). The glomerular volume of the juxtamedullary nephrons diminished by about 40%, and their proximal tubule length decreased by about 20%. The glomerular volume of the superficial nephrons also diminished, by about 30%, but their proximal tubule length did not change. The gentamicin concentration was higher in the renal cortex than in the medulla (13.1 +/- 2.6 versus 5.7 +/- 2.2 micrograms/g [dry wt]; P less than 0.01; n = 6 for each). It decreased significantly from days 3 to 20 in both tissues. No functional impairment of the kidney was found in 10-day-old animals, and normal or even supranormal morphometry of the nephrons was observed in the 20-day-old animals. It is concluded that fetal exposure to gentamicin impairs proximal tubular function in the developing animal and might also adversely affect glomerular and tubular growth. However, both the functional and morphometric impairments of nephrons are transitory.