In the small intestine, the presence of transitional cells or cells intermediate between Paneth cells and goblet cells has been reported frequently for 100 years. Light microscopy and, more recently, fine structural studies have indicated that secretory granules observed in intermediate cells share some morphologic characteristics with those of granular goblet cells and of Paneth cells. In order to verify if intermediate cells in the jejunum and ileum of the adult mouse have functional similarities with either granular goblet or Paneth cells, we have studied the incorporation of sulfur-35 by radioautography and the localization of lysozyme by immunocytochemistry. After radioautography, goblet cells and, to a lesser extent, granular goblet cells had incorporated sulfur-35, whereas Paneth cells and intermediate cells were completely negative. Immunolocalization of lysozyme was done by using rabbit anti-rat lysozyme and protein A-peroxidase. After demonstration of peroxidase activity, only Paneth cells were stained and intermediate cells were negative. Therefore, intermediate cells do not contain sulfomucin or lysozyme, and they are functionally different from goblet and Paneth cells. Their function remains unknown.

We have investigated the individual and combined actions of epidermal growth factor (EGF), transferrin and hydrocortisone on the maturation of whole fetal mouse metanephroi maintained in serum-free conditions for up to 5 days. The presence of EGF (100 ng/ml) resulted in elevated levels of [3H]-thymidine incorporation when compared to controls; autoradiograms showed that the proliferation of mesenchymal cells in the nephrogenic zone is particularly enhanced as verified by cell counting. Brush border hydrolase activities (alkaline phosphatase and gamma-glutamyltransferase), on the other hand, were significantly diminished. Transferrin (5 micrograms/ml) slightly stimulated DNA synthesis and potentiated EGF mitogenic action. The activation of DNA replication by the growth factor seems to be mediated through the protein kinase C pathway. When added alone, hydrocortisone (10(-6) M) strongly inhibited DNA synthesis, stimulated hydrolase activities and exerted a positive effect on brush border differentiation. When combined with EGF or to EGF + transferrin, hydrocortisone counteracted the effects of these latter peptides on DNA synthesis and enzyme activities. Considering the earlier observation of a reciprocal relation between proliferation and differentiation during the neotubulogenic phase of kidney development, the results described in the current study suggest that synergistic and synarchic actions of these heterologous factors are involved in the regulation of tubulogenesis.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

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