The main objective of this proposal is to determine the ontogeny, localization and regulation of expression by glucocorticoids for the ENaC subunits in the developing kidney, uroepithelium, lung and colon. The recent evidence that hypertension associated with Liddle~s syndrome is due to mutations in the beta and gamma ENaC subunits and the finding that young prehypertensive Dahl salt-sensitive rates have increase Na+ absorption via apical membrane amiloride-sensitive sodium channels int he IMCD epithelia provide the rationale for the further study of EnaC as a potentially important molecule in the pathogenesis of hypertension. there are 2 specific aims. The first specific aim is to determine the ontogeny of expression and cell specific localization of the ENaC subunits in normal and Dahl S & R rats. ENaC plays important functional roles in the developing kidney, lung and colon and may be very important in the transition from intrauterine to extrauterine life. the expression of functional Na+ channels is dependent on the synthesis of at least 3 proteins, thus the regulation of each may be important for the appropriate cell- and organ-specific function. Since ontogeny is organ specific, learning the pattern of mRNA expression is a critical first step in understanding how each subunit is regulated. Knowing the pattern of expression will permit the generation of hypotheses regarding factors influencing regulation. We hypothesize that differences in the ontogeny or level of expression of ENaC subunits in the kidney plan an important role in the development of hypertension in Dahl rats. To test this hypothesis we will compare the ontogeny of expression and localization of ENaC subunit mRNA and protein in Na+ transporting tissues of normal and Dahl S and R rates using ribonuclease protection assays, in situ hybridization, and immunochemistry. The second specific aim is to determine the role of glucocorticoids (GC) in the regulation of ENaC expression in development. In adult animals corticosteroids influence ENaC mRNA and protein expression in kidney, lung and colon. We hypothesize that ENaC expression during development is regulated in part by GC hormones. To test this hypothesis we will use an in vivo model to assess the effects of GC's on expression of ENaC subunit mRNAs and proteins. The corticicotropin releasing hormone (CRH) knockout (KO) mouse, which has a severely impaired ability to secrete GC will serve as a model to study the role of GCs in the regulation of ENaC expression. This will allow us to selectively determine the effect of GCs on ENaC subunit expression in the developing kidney and lung. The ontogeny and localization of ENaC subunit mRNA expression will be determined in wild type mice. CRH KO mice and CRH Ko in which the mothers receive GC replacement. These studies should conclusively determine the importance of glucocorticoids in the regulation of ENaC expression during development.
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