Effect of puromycin on metanephric differentiation: morphological, autoradiographic and biochemical studies

Effect of aminonucleoside of puromycin (PAN) on metanephric development and proteoglycans (PGs) was investigated. Murine metanephric tissues, obtained on the thirteenth day of gestation, were exposed to PAN in a culture medium for one to seven days and processed for morphological, histochemical and immunofluorescent studies. For tissue autoradiographic and biochemical studies, kidneys were labelled with a precursor product of PGs, that is, [35S]-sulfate. A generalized decrease in the glomerular population along with swelling and deformation in the ureteric bud branches was observed. These changes were accompanied with a diminution in the total incorporated radioactivity and a reduction in the autoradiographic grains, especially over the tips of ureteric bud branches. Sepharose CL-4B chromatography revealed a major high molecular weight PG (Mr greater than 2.5 x 10(6], and a relative increase in the chondroitinase-ABC sensitive PGs. The media PGs were of relatively smaller size. Immunoprecipitation experiments with [35S]-methionine-labeled tissues and immunofluorescent studies revealed a significant decrease of PGs in metanephric tissues, while type IV collagen and laminin were relatively unaffected. Significant glomerular changes included failure in differentiation of the visceral epithelial foot processes, formation of villi and in maturation of glomerular basement membrane. The latter was seen as fragments of extracellular matrices interspersed among undifferentiated podocytes and had reduced staining with ruthenium red--a dye marker for the PGs. This deficiency of PGs was confirmed by electron microscopic autoradiography, where a reduction in the number of silver grains was observed. The fact that the PAN-induced cellular and extracellular alterations were associated with perturbances in biosynthesis of PGs, suggests that the morphogenetic regulators, that is, PGs play a vital role in various differentiation processes involved during metanephric development.