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.

We conclude that vitamin A supply to the fetus is critical in determining the number of nephrons. Data available thus far on the frequency of mild VAD during pregnancy and on the long-term consequences of inborn nephron deficit highlight the clinical relevance of the present study.

SUMMARYIn humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

Vitamin A and its metabolic derivatives are known to be key signalling molecules in regulating morphogenetic events in vertebrate development. Here we investigated their possible involvement during mammalian kidney development using paired rat metanephros organ culture. Metanephroi were explanted from 14-day-old embryos and cultured for six days in a chemically defined medium containing a retinoid at a dose of 10(-11) to 10(-4) M. Retinol, all-trans and 9-cis retinoic acid were able to promote metanephros growth and differentiation in vitro. A significant increase in the number of nephrons was observed from 10(-8) M of retinol and 10(-10) M of all-trans retinoic acid, before any change in growth parameters. A threefold increase in the number of nephrons was obtained at a dose of 10(-6) M. At low retinoid concentrations, there was a modulating effect of triiodothyronine on retinoid-stimulated nephrogenesis since the absence of triiodothyronine in the medium enhanced the nephrogenic stimulation. Exposure of metanephroi from 13-day-old embryos to all-trans retinoic acid (10(-7) M) led to a sixfold increase of nephron formation. Finally, we analyzed the branching pattern of the ureteric bud and showed that within 48 hours of culture, it was significantly more developed upon retinoid exposure. In conclusion, this study demonstrates that retinoic acid is a key regulator of renal organogenesis in controlling nephrogenic induction processes and ureteric bud patterning, and that the younger the metanephros, the greater the effect.

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-α (GDNFR-α), and the receptor tyrosine kinase c-ret, known to play a major role in renal organogenesis. Expression of GDNF and GDNFR-α 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.

Development of the metanephric kidney during embryogenesis can be altered both in vivo and in vitro by exposure to gentamicin, which may lead to oligonephronia. To study the role of the ureteric bud in nephron deficit genesis, we used metanephros organ cultures exposed to gentamicin as a model of impaired nephrogenesis. Ultrastructural localization of the antibiotic showed that by eight hours it was already present within the epithelial cells of the ureteric bud and in its growing ends, and also trapped in the adjacent blastema. Using confocal microscopy and image analysis, we devised a quantitative approach to analyze the branching pattern of the ureteric bud, and showed that by 24 hours of culture, despite no change of explants growth, gentamicin had significantly decreased the number of branching points. This effect involved the early branching events and was limited to end buds that had no nephron anlagen nearby. Our findings indicate that impaired branching morphogenesis of the ureteric bud is the likely event of gentamicin-induced nephron deficit.

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Recent advances in developmental nephrology have provided new evidence that retinoids, vitamin A and its active metabolites such as all-trans retinoic acid, profoundly influence renal organogenesis. Retinoids are acknowledged as potent nephron mass regulators. They regulate embryonic kidney patterning through control of Ret expression in a dose-dependent manner, which modulates ureteric bud branching morphogenesis. Analysis of retinoid availability and utilization provides additional evidence of retinoid involvement at sites of epithelial-mesenchymal interactions, suggesting a tight control of vitamin A homeostasis for proper renal morphogenesis and differentiation. This provides a rationale for the search for and identification of retinoid target genes.