Regulation of the epithelial Na+ channel by Ras and Sgk

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DESCRIPTION (provided by applicant): Proper control of Na+-dependent fluid reabsorption at the distal nephron is critical to blood pressure homeostasis. The cellular mechanisms of aldosterone, the most important systemic modulator of discretionarily Na+ reabsorption, are poorly understood. The long-term goal of this (revised) first proposal of a newly appointed investigator is their elucidation. This research is relevant to the physiology of many organ systems including the urinary, cardiovascular and respiratory systems, as well as to the pathophysiology of hypertension and other diseases associated with fluid imbalance. Activity of the amiloride-sensitive, epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption. Aldosterone affects gene expression and then increases the activity of ENaC. However, the genes encoding ENaC are not themselves initially induced. Thus, aldosterone increases expression of intermediary signaling proteins that transduce information to ENaC. Genes encoding aldosterone-induced transcripts traditionally have been difficult to identify. With modem technology, two aldosterone-induced transcripts, relevant to signal transduction, recently have been identified: serum- and glucocorticoid-regulated kinase (Sgk), and the small G protein, K-RasA (K-rasA). Induction of these transcripts is a primary action of aldosterone in epithelia, and translates into an increase in Sgk and K-RasA protein levels. The relation of these proteins to Na+ reabsorption and ENaC remain poorly understood. The Specific Aims of the current proposal will directly determine the potential novel roles of aldosterone-induced Sgk and K-RasA signaling in regulating ENaC activity in epithelia. I hypothesize that aldosterone-activated KRasA and Sgk through signal transduction convergence stabilize ENaC in the open state and increase number of ENaC in the luminal membrane. The effect of Sgk and K-RasA signaling on ENaC will be investigated in the A6 cell model of distal nephron epithelia using a comprehensive and novel experimental approach. Biochemical assessment of the levels and activities of the protein constituents of Sgk and K-RasA signaling pathways in response to aldosterone will be one end-measurement. The other will be electrophysiological measurement of ENaC activity, kinetics and number. The effects of specific molecular and pharmacological modulators of Sgk and K-RasA signal transduction on these processes will be used to delineate in a systematic manner the cellular mechanisms of aldosterone action to increase Na+ reabsorption in native epithelia.