Prenatal alpha-difluoromethylornithine treatment: effects on postnatal renal growth and function in the rat.

DFMO (alpha-difluoromethylornithine) is a specific irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, which in turn control macromolecule synthesis during cell proliferation. The current study was designed to investigate the effects of inhibition of ODC during discrete prenatal periods on renal growth and function. We administered 5 doses of 500 mg/kg DFMO or saline s.c. to timed pregnant Sprague-Dawley rats at 12 hr intervals beginning on gestation days (GD) 11, 14, or 17. Half the dams were killed on GD 20 for fetal morphological analyses and half were allowed to go to term. Renal function was assessed on postnatal days (PD) 3, 6, 10, and 14 by tests of basal renal clearance and urinary concentrating ability, and on PD 42-44 we measured serum chemistries. All three gestational treatment regimens resulted in postnatal deficits in general growth. Only in the GD 11-13 treatment group was there evidence of embryotoxicity and neonatal renal pathophysiology. Fetal weights and urogenital morphology were altered following GD 14-16 treatment and there were persistent deficits of renal growth. GD 17-19 treatment was associated only with transient postnatal deficits of renal growth. Thus, inhibition of ODC during critical prenatal periods induced distinct developmental effects. However, there were no associations between impaired renal growth and function. These data indicate that general tissue growth is not always a predictor of physiological development and support the necessity of multifaceted approaches to the understanding of adverse developmental effects.

Functional teratogens of the rat kidney. I. Colchicine, dinoseb, and methyl salicylate.

Substances known or suspected to cause subtle or transient anatomical alterations in renal development were administered prenatally or neonatally to rats in order to determine whether they are capable of altering renal functional development. Colchicine alters mitotic activity and cytoskeletal structure and is teratogenic in many species. Since the kidney of the newborn rat undergoes extensive cellular proliferation and nephron differentiation, it is possible that neonatal administration of colchicine may affect nephron development. Dinoseb and methyl salicylate have previously been reported to produce a high incidence of dilated renal pelvis in the term rat fetus. Colchicine was injected sc, at 75 micrograms/kg, to Postnatal Day (PD) 1 Sprague-Dawley rats. Dinoseb was administered ip to pregnant Sprague-Dawley rats on Gestation Days 10-12 at doses of 8 or 10.5 mg/kg/day, and methyl salicylate was administered ip at doses of 200, 250, or 300 mg/kg/day on Gestation Days 11-12. Renal function was examined in pups from immediately after birth through weaning. Maximal urine concentrating ability was measured after DDAVP (desmopressin acetate, a vasopressin analog) injection in suckling rats, and after 24 hr of water deprivation in weanlings. Proximal tubule transport was measured in renal cortical slices. Basal urinary parameters, including urine flow, osmolality, pH, and chloride content, were measured. Colchicine treatment had no effect on body weight or kidney weight. There was a significant decrease in maximal urine osmolality in PD 30 rats measured after 24 hr of water deprivation. The urine concentrating deficit detected in functionally mature PD 30 rats suggests that colchicine treatment during renal histogenesis causes a latent deficit in medullary function in the absence of any gross morphological effects. The 10.5 mg/kg/day dose of dinoseb caused a weight reduction in neonates which persisted after weaning. Urine volume after DDAVP challenge was increased over controls in both dose groups on PD 6, but maximal urine concentration was unaffected. On PD 14, maximal urine concentration after DDAVP injection was decreased in the 10.5 mg/kg/day group. By PD 30, urine concentrating ability was comparable to controls. Renal cortical slices from the 10.5 mg/kg/day dose group had an enhanced ability to accumulate organic anions on PD 3 and 31, but opposite effects were observed in the low-dose group. No other renal functional parameters were altered. Urine osmolality after DDAVP challenge was decreased over controls in the 250 mg/kg/day methyl salicylate group on PD 6, and urine volume was increased in this group after DDAVP injection on PD 14.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional teratogens of the rat kidney. II. Nitrofen and ethylenethiourea.

Nitrofen and ethylenethiourea (ETU), agents known to prenatally induce hydronephrosis in rats, were assessed for their effects on postnatal renal functional maturation. Both were given by gavage to pregnant Sprague-Dawley rats on Gestation Day 11. Nitrofen was given at concentrations of 50 or 100 mg/kg, and ETU at 20, 40, or 60 mg/kg. Renal function was examined in the offspring from birth until after weaning, the period of renal functional maturation in the rat. Maximal urine concentrating ability was measured after DDAVP (desmopressin acetate, a vasopressin analog) challenge or water deprivation. Proximal tubule transport was measured in renal cortical slices. Various urinary parameters were measured. Both prenatal nitrofen and ETU exposure caused a large number of neonatal deaths at the high dose, and hydronephrosis was observed. The severity of the lesion increased with age. Hydronephrotic animals were deficient in urine concentrating ability, which became more pronounced after weaning. A few other urinary parameters were altered, but cortical function appeared to be unaffected. Rats prenatally exposed to nitrofen, but with apparently normal kidneys, were significantly compromised in their ability to produce a concentrated urine in response to DDAVP challenge, on Postnatal Days (PDs) 6 and 14. By PD 30, they were not different from controls in urine concentrating response. Rats prenatally exposed to the higher doses of ETU, but with grossly normal kidneys, had significantly decreased plasma clearances of certain electrolytes early in life, but by PD 27, they were not different from controls. Proximal tubule transport of PAH was increased on PD 7 in ETU-exposed pups, but this effect did not persist.

The significance of the dilated renal pelvis in the nitrofen-exposed rat fetus: effects on morphology and function.

Previously (R. J. Kavlock, B. F. Rehnberg, and E. H. Rogers, 1987, Teratology 36, 51-58) we reported that gestational exposure of rats to adriamycin induced alterations in development of the fetal renal papilla that persisted postnatally. The morphological effect was associated with functional deficits in neonatal animals as seen by their performance during a test of renal concentrating ability in the second postnatal week. In the present study, we utilized an experimental approach similar to that in the adriamycin study to evaluate the fate of the dilated renal pelvis that is induced in fetal rats following prenatal exposure to nitrofen. Groups of Long-Evans rats were exposed to 0, 6.25, 12.5, or 25 mg/kg of nitrofen on gestation Days 7-16. Renal morphology of the offspring were determined on gestation Day 21 and postnatal Week 5. The postnatal cohort was tested in the second postnatal week for their ability to excrete an osmotically concentrated urine. As was the case with adriamycin, the renal concentrating ability in the neonate was reduced and poor performance in the function test was associated with permanence of the morphological effect. By utilizing a standardized semiquantitative procedure to describe the status of the kidneys and ureters during development in combination with physiological assessment of organ performance, we were able to assess the morphological and functional development of the kidney. In the absence of other anomalies we suggest that offspring be monitored during postnatal development when alterations of the fetal renal papilla are observed in standard teratology bioassays in order to determine whether the effect is transient or permanent.

Fate of adriamycin-induced dilated renal pelvis in the fetal rat: functional and morphological effects in the offspring.

Previously we reported that gestational exposure to Adriamycin, an anthracycline antibiotic used in the treatment of neoplasms, reduced renal function in the neonatal rat, and we suggested that alterations in the development of the renal papilla might be responsible for the dysfunction. In this study we exposed groups of Sprague-Dawley rats to 0, 1.0, 1.25, or 1.5 mg/kg of Adriamycin on gestation days 10-12, a period previously shown to be effective in altering postnatal renal function with this compound. Offspring were evaluated at several developmental periods in order to 1) precisely define the morphological status of the urogenital system in Adriamycin-treated offspring; 2) replicate the finding of a decreased renal concentrating ability in the neonates; 3) determine the transience/permanence of any morphological effect; and 4) correlate any permanent alterations in urogenital morphology with our indicator of neonatal functional competence. Maternal Adriamycin treatment induced alterations in the development of the renal papilla that persisted well into the postnatal life of the offspring. The first appearance of the morphological alteration occurred in the absence of other general indicators of developmental toxicity such as growth retardation. The determination of the ultimate fate and functional consequences of the structural alterations required postnatal evaluations of the renal system. Finally, a relatively simple test of renal function in the neonate proved to be predictive of the permanence of the morphological effect, and the absolute test result showed a strong correlation with the incidence of the morphological effect in the overall population.

Critical prenatal periods for chlorambucil-induced functional alterations of the rat kidney.

Previous studies have demonstrated that exposure of rats to chlorambucil during the period of metanephric differentiation results in morphological and functional alterations of the kidneys after birth. The present study describes the effects of chlorambucil treatment at various gestational ages on neonatal renal function to determine if critical periods other than day 11 exist for inducing functional developmental toxicity of the kidneys. Groups of pregnant Sprague-Dawley rats were exposed to 4.5 mg/kg of chlorambucil on gestation days 9, 11, 13 or 15 and the offspring were evaluated for neonatal growth and viability, gross malformations of the kidneys, and renal physiology. The results demonstrate that the critical period for the induction of specific renal defects and hypoplasia lies on day 11 of gestation, but functional alterations of the kidneys were observed after exposure on day 15 of gestation. In terms of practical application, a combination of the basal clearance test and the renal concentration test together provide an efficient means for detecting prenatally induced functional alterations of the kidneys. When renal malformations or anomalies are observed in standard teratology bioassays, studies using techniques similar to those described here may be extremely useful in determining the biological significance as well as permanence or transience of effects such as renal hypoplasia, dilated renal pelvis, and dilated ureter.

Functional consequences of prenatal methylmercury exposure: effects on renal and hepatic responses to trophic stimuli and on renal excretory mechanisms.

The effects of prenatal exposure to methylmercury on the functional development of renal and hepatic response systems was examined in the developing rat. Methylmercury produced an elevation of basal activity of renal ornithine decarboxylase (ODC, an enzyme involved in regulation of cellular maturation) and an eventual relative hypertrophy; liver ODC was reduced and hypertrophy was not evident. In contrast, the reactivity of liver ODC to trophic stimulants (vasopressin, isoproterenol) was markedly enhanced by prenatal methylmercury exposure, whereas renal ODC responses were much less affected (vasopressin) or actually reduced (isoproterenol). Targeted actions of methylmercury on renal excretory function were also prominent, with increased fractional excretions of urea and electrolytes and an eventual reduction in glomerular filtration as assessed by creatinine clearance. In addition, the reactivity of the kidney to challenge with desmopressin was altered coincidentally with the appearance of the effects on basal clearance mechanisms. These studies show that doses of methylmercury ordinarily associated with selective actions on development of neurobehavioral patterns also influence the functional ontogeny of other organ systems; furthermore, the fact that the target tissues are different for prenatal vs. postnatal methylmercury exposure, indicates that the functional teratology of methylmercury exhibits critical periods of sensitivity.

Chlorambucil induced congenital renal hypoplasia: effects on basal renal function in the developing rat.

Administration of chlorambucil to pregnant rats on day 11 of gestation induced dose-related alterations in renal growth and function in the postnatal offspring. These effects occurred above and beyond the reductions in body growth and were evident in animals that displayed no overt malformation of the urogenital tract. Reductions in overall growth amounted to 0, 6 and 15% in the 3,4.5 and 6 mg/kg groups, respectively, while kidney weights were reduced by 7, 15 and 23%. The weights of the kidneys relative to the body were reduced 5, 9 and 10% with increasing dose. Although basal renal function was not affected by the degree of hypoplasia seen in the low dose group, reduced glomerular and tubular function were evident following a basal clearance test in the 2 highest dose groups. The data indicate that chlorambucil induced renal hypoplasia results in reductions in renal function that persist for at least the first 3 weeks after birth in the rat and that physiological assessment of developmental toxicity can provide an extremely useful addendum to the more classical morphological criteria.

Toxicity of mercuric chloride to the developing rat kidney. III. Distribution and elimination of mercury during postnatal maturation.

Mercuric chloride is a potent nephrotoxicant in the adult rat, but has little effect on newborns. Nephrotoxicity increases with postnatal maturation. This study assesses the changes in tissue distribution and excretion of Hg during postnatal development. Sprague-Dawley rats were injected sc with 5 mg/kg 203HgCl2 on postnatal Day 1, 8, 15, 22, or 29. Hg concentration was measured in the whole body, renal cortex, medulla and papilla, liver, and subcellular fractions of liver and kidney. Binding to cytosolic metallothionein was assessed. Whole-body elimination of Hg was slow at the three younger age groups, as only 20% of the initial load was eliminated by 5 days after injection. Excretion was much more rapid in the two older groups, which eliminated about half of the initial load within 5 days. Concentration of Hg was highest in renal cortex (the principal site of Hg toxicity), and there was an age-related increase in cortical Hg concentration. This may explain the increased toxicity of Hg with age. There was an age-related decrease in hepatic Hg concentration. The high levels of metallothionein present in perinatal rat liver may protect the renal cortex from receiving a toxic dose of Hg; however, the increased concentration of hepatic Hg in newborns is insufficient to account for all of the cortical decrease. It is probable that Hg was distributed to other tissues. In liver and kidney cells of neonates, Hg concentration was highest in the cytosol, decreasing in an age-related manner. This was accompanied by an age-related increase of Hg in the nuclear/mitochondrial fraction. Hg in the cytosol was largely bound to metallothionein, although there were substantial amounts associated with very low-molecular-weight molecules and high-molecular-weight proteins. There are significant maturational changes in the organ, cellular and subcellular distribution of Hg in the rat during the first 4 weeks after birth. These probably explain the increasing sensitivity with maturity to Hg nephrotoxicity.

Renal functional teratogenesis resulting from adriamycin exposure.

Pregnant Sprague-Dawley rats were exposed to adriamycin and offspring were evaluated for renal functional competence. Exposure consisted of 0, 1.0 or 1.5 mg/kg/day by intraperitoneal injection on either gestational days 7-10 or 10-12. The exposed offspring were evaluated for 1) growth and viability; 2) serum concentrations and renal clearances of creatinine, urea, glucose, sodium, potassium, chloride, and total osmotic particles; 3) the ability to excrete an osmotically concentrated urine following fluid deprivation; 4) the ability to excrete an osmotically dilute urine following isotonic volume expansion; and 5) the ability to secrete hydrogen ions following administration of a fixed acid. Exposure during days 7-10 of gestation produced greater evidence of overt developmental toxicity than did exposure during days 10-12 of gestation. The reverse was true, however, for the effects of adriamycin on renal function, as the majority of effects on these measures were found in the high-dose pups exposed during days 10-12 of gestation. The application of the renal function tests did not lower the observed effect level for adriamycin-induced developmental toxicity, but it did provide confirmatory information on the nature of the effect, on the magnitude of the effect in the exposed population, and on the possible morphological site of observed functional lesion. For reasons discussed in the text, a combination of the basal clearance test and the renal concentrating test appears to provide the most efficient means for detecting the presence of prenatally induced functional alterations of the kidneys.

Amphotericin B- and folic acid-induced nephropathies in developing rats.

The kidneys of newborn rats, which are both morphologically and physiologically immature, have been shown to be relatively insensitive to the nephrotoxic effects of several chemicals. To examine the specificity of these age-related differences, pups received sc injections of either 20 mg/kg of amphotericin B or 250 mg/kg folic acid, two known nephrotoxins in adult animals, on postnatal Day 1, 8, or 15. Renal function was examined by a basal clearance test and a hydropenia challenge at 1, 2, or 5 (6 in the case of amphotericin B) days after treatment. We observed no difference in degree of renal toxicity with age, but repair of renal damage tended to proceed slower at the youngest age. Amphotericin treatment produced uremia, increased fractional excretion of water and sodium, a decreased fractional excretion of urea, and a diminished hydropenia response but no change in creatinine clearance and no renal pathology. The observed pattern of renal toxicity may be attributed to an inability to maintain urea gradients in the distal segment of the nephron. Folic acid treatment resulted in greatly increased kidney weights with marked pathology, uremia with decreased creatinine clearance, increased fractional excretion of water, and a decreased hydropenia response. Unlike the renal toxicity observed following amphotericin treatment, renal toxicity from folic acid appears to be a nonspecific response to cell injury within the renal tubules. The data indicate that, in general, neonates do not possess a relative insensitivity to nephrotoxins and that renal physiological measurements which can be performed in neonatal rats are useful in evaluating and interpreting alterations in renal function.

Toxicity of sodium fluoride to the postnatally developing rat kidney.

The adult rat kidney is an important target organ for sodium fluoride; however, the toxicity of fluoride to the developing kidney is unknown. This study examined renal function following NaF exposure during the first 4 weeks after birth. Sprague-Dawley rats received a single ip injection of 0, 30, or 48 mg/kg NaF on postnatal Day 1, 8, 15, or 29. Alterations in renal function, histology, and morphology were determined 24, 48, and 120 hr after exposure. Measurements of renal function included urine volume, osmolality, the ability to concentrate urine during water deprivation, urinary pH, and chloride content. Rats were then sacrificed and their kidneys processed for observation by light microscopy. Some minor alterations in renal function were observed in the three youngest age groups after NaF exposure. These changes included decreased body weight after treatment with 30 or 48 mg/kg NaF but increased kidney-body weight ratio in the 48 mg/kg group on Day 1; decreased urinary pH in both dose groups after treatment on Day 1 or 8; increased urinary volume 120 hr after treatment on Day 8; and decreased chloride excretion in the 48 mg/kg group 24 hr after injection on Day 1. None of these effects was severe. In contrast to these results, marked renal toxicity was observed in postweaning rats treated on Day 29. The NaF exposure resulted in increased kidney weight and kidney/body weight ratio, profound diuresis, decreased urinary osmolality, and decreased ability to concentrate urine during water deprivation. Urinary chloride excretion was decreased for the first 2 days after NaF exposure, then increased in water-deprived rats 120 hr after treatment. Glucosuria and hematuria were present for 2 days after treatment with 48 mg/kg. Histological lesions were apparent in the proximal tubules of the treated Day 29 rats. Thus, the kidney of the suckling rat is largely unresponsive to NaF toxicity. Renal sensitivity increases abruptly after weaning in the Day 29 rat.

Biological effects of long-term exposure of rats to 970-MHz radiofrequency radiation.

Rats (N = 16) exposed individually in circularly polarized waveguides to 970-MHz electromagnetic radiation (SAR = 2.5 mW/g, 22 h daily for 70 consecutive days) had significantly higher serum levels of triglycerides, albumin, and total protein compared with sham-irradiated controls. No difference was observed in the weights, hematologic profile, or in vitro lymphocyte responses to mitogens between these two groups. The higher serum levels of triglycerides in radiofrequency-radiation-exposed rats suggest a non-specific stress reaction.