This project focuses on the molecular mechanisms through which aldosterone stimulates Na transport in target epithelia. During the last grant period we identified serum- and glucocorticoid-induced kinase-1 (SGK1) as an aldosterone-induced early response gene in the cortical collecting duct (CCD). Recent studies from our lab and by others suggest that SGK1 is essential for mediating corticosteroid effects on Na transport in cultured CCD cells. At the same time, SGK1 knockout mice have a milder Na-losing phenotype than mice lacking mineralocorticoid receptors (MRs) do. These observations suggest that SGK1, albeit important, is probably not the only gene that mediates aldosterone's effects on Na transport in vivo. Furthermore, the mechanism through which SGK1 alters Na homeostasis is still unknown. Thus the specific aims of this project are: (1) To determine the contribution of SGK1 to aldosterone's effect on Na homeostasis in vivo, in inducible, aldosterone target cell-specific SGK1 knockout mice. We will characterize the renal response to acute, aldosterone target cell-specific elimination of SGK1 by inducing Cre-activity with tamoxifen and determine the effects of aldosterone administration. We will also determine the role of SGK1 in aldosterone stimulated Na reabsorption in the colon in vivo and in vitro using colonic tissue originating from SGK1 knockout and wild type (WT) mice. (2) To determine the role of genes transcriptionally regulated by SGK1 in Na transport and in cell proliferation/survival in the CCD. Our recent DNA microarray studies identified significant transcriptional changes in SGKl-overexpressing vs. down-regulated M1 cells. Among the SGK1- regulated genes there are several categories with clear potential of contributing to SGK's effect on Na transport and cell survival/differentiation. We will determine the functional consequences of stably down regulating or overexpressing these genes in M1 cell lines. We will also determine the role of SGK1 in the maintenance of a well-differentiated CCD. Finally, in Aim 3, we will identify additional genes that are directly regulated by aldosterone in vivo in the CCD. By comparing gene expression profiles with or without aldosterone treatment of SGK1 knockout vs. WT mice, we will identify aldosterone-regulated genes that are not downstream of SGK1. CCD cells will be isolated by immuno-selection, and differentially expressed genes identified using DNA microarrays.
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