This is a proposal to examine the regulation of development of intercalated cells in the embryonic rat kidney. Immunocytochemical studies from our laboratory indicated that intercalated cells differentiate and exhibit amplified expression of the vacuolar H+ATPase in the fetal kidney. Our laboratory recently discovered that an isoform of one of the subunits of the H+ATPase has amplified expression in vitro in the embryonic kidney explant model.
The specific aims of this proposal are: 1.To define the time course and sequence of cellular events leading to the development of intercalated cells in vitro in the embryonic kidney explant model. Embryonic kidneys will be stained at different time points with antibodies to the intercalated cell-specific isoform, and with antibodies to other collecting duct transport proteins, to define the extent and time course of differentiation in vitro. 2.To investigate the intercellular signals that induce the differentiation of intercalated cells in the embryonic kidney. Ureteric bud from day 12.5- 13 rat embryos will be removed from the metanephric blastema, and the conditions required to restore differentiation, assessed by expression of the intercalated cell-specific isoform, will be examined. 3.To define the promoter elements on the kidney isoform of the kidney vacuolar H+ATPase that confer the capacity for amplification of expression during development. The control elements in the 5' flanking region of the intercalated cell-specific isoform will be mapped by placing different deletion constructs into enhancerless retroviral vectors with a reporter gene, and by other methods. These experiments will have a major impact on our understanding of the cell interactions and cellular mechanisms of development of the collecting duct, and may provide new insights into the causes of developmental abnormalities of the collecting system.
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