Epithelial sodium channel regulated by aldosterone-induced protein sgk

Abstract

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na ؉ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na ؉ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially.Rather, the open probability and͞or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na ؉ absorption and thus in the control of extracellular f luid volume, blood pressure, and sodium homeostasis.

Targeted Disruption of the Protein Kinase SGK3/CISK Impairs Postnatal Hair Follicle Development

“…Western blot analysis was performed as described previously (Chen et al, 1999). Protein extracts from WT and Sgk3 KO mice were used for Western blot analysis by using an SGK antibody (gift from Dr. G. Firestone, University of California, Berkeley, CA) that cross-reacts with SGK1, SGK2, and SGK3.…”

SGK1 regulation by miR-466g in cortical collecting duct cells

“…In particular, aldosterone stimulates expression of SGK1 in the late distal convoluted tubule, connecting segment, and collecting duct (7,8,18,25,27), which together constitute the aldosterone-sensitive distal nephron. In turn, SGK1 stimulates ENaC activity through several mechanisms, including an increase in the number and residency time of ENaC in the apical membrane (3,10,21,25,32) and an increase in open probability of ENaC (2,11,34,37).…”

“…SGK1 mRNA expression increases in CCD cells as early as 15 min after mineralocorticoid or glucocorticoid stimulation (8,27). Since miRNAs decrease gene expression by binding to sites in the 3=-UTR of target mRNAs, aldosterone might increase SGK1 mRNA expression in part by decreasing miR466g expression.…”

“…Human embryonic kidney-293 cells (passages [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] were transfected in 24-well cell culture plates with 37.5 ng of luciferase reporter plasmid, 25 ng ␤-galactosidase control plasmid, and 5-100 ϫ 10 Ϫ9 M of scrambled or precursor miR-466g (Applied Biosystems). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used as a transfection reagent in all experiments.…”

“…In the early phase of induction, aldosterone activates preexisting ENaC channels through transcriptional regulation of proteins that stimulate ENaC activity. Serum and glucocorticoid regulated kinase 1 (SGK1) is now recognized as a prototypic early-response gene and a key mediator of the stimulatory effects of aldosterone on ENaC activity in CCD cells (1,8,17,27).…”