The long-term objective of these studies is to understand the molecular mechanisms through which aldosterone stimulates Na transport and thereby regulates blood pressure. The foundation for the proposed studies is the recent identification of serum- and glucocorticoid-induced kinase (sgk) as an aldosterone-induced early response gene in the cortical collecting duct (CCD). We also demonstrated that in Xenopus oocytes expressing the epithelial Na channel (ENaC) sgk stimulates amiloride-sensitive Na current. These observations make sgk a strong candidate for mediating the early actions of aldosterone on Na transport. We established that in oocytes sgk increases the number of ENaC subunits in the cell membrane and has no effect on open the probability of ENaCs or their rate of their endocytosis, suggesting that in this system sgk increases ENaC exocytosis or recycling. We propose the following specific aims.
Aim 1 will test the hypothesis that sgk increases Na absorption in mammalian collecting duct cells by increasing exocytosis or recycling of ENaC, similarly as it does in Xenopus oocytes. The effects of up- or down-regulating sgk in CCD cells on ENaC will be determined by measuring amiloride-sensitive short-circuit current, and analyzing its fluctuations by noise analysis, by determining the amount of ENaC in the apical membrane using biochemical approaches, and by determining the rate of removal of ENaC from the apical membrane.
Aim 2 is to elucidate the mechanism by which sgk increases surface expression of ENaC. We will determine if the effect of sgk is mediated by post-translational or transcriptional events by monitoring ENaC current in oocytes following injection of sgk protein and/or RNA synthesis inhibitors. The subcellular localization of sgk will be determined using GFP-sgk chimeras or antibodies against sgk. Finally, in this aim we will identify the downstream targets of sgk action by testing the effects of candidate proteins and by phosphopeptide mapping.
Aim 3 is to determine the role of sgk in AVP-stimulated Na transport in CCD cells. The effects of AVP and PI3K inhibitors on the activity and phosphorylation of sgk in mammalian CCD cells will be determined, and these effects will be correlated with changes in amiloride-sensitive current, in normal CCD cells and cells in which endogenous sgk levels are down- regulated. Since the ultimate target of sgk action seems to be a step in ENaC trafficking, identification of the downstream effectors of sgk could lead to insights into the mechanism of ENaC trafficking, which is poorly understood. Unraveling the molecular steps leading to hormone-stimulated Na transport in the CCD could also lead to the identification of genes that might be mutated in some forms of human hypertension, and may eventually lead to therapeutic measures that interrupt this pathway.
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|Martel, Jessica A; Michael, Donna; Fejes-Toth, Geza et al. (2007) Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells. Am J Physiol Renal Physiol 293:F904-13|
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