The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulating the cell-surface expression and function of the epithelial sodium channel (ENaC) in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of serum-and glucocorticoid-regulated kinase 1 (Sgk1), a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. In vivo early aldosterone-regulated mRNA now has been identified in microselected mouse distal nephron by microarray. From 22 mRNA that displayed a two-fold or more change, 13 were downregulated and nine were upregulated. Besides Sgk1, the induced mRNA include Grem2 (protein related to DAN and cerebrus [PRDC]), activating transcription factor 3, cAMP responsive element modulator, and the ubiquitin-specific protease Usp2-45. The induction of this last enzyme isoform was verified in mouse distal nephron tubule at the protein level. With the use of Hek293 cells, Xenopus oocytes, and mpkCCD c14 cells as expression systems, it was shown that Usp2-45 deubiquitylates ENaC and stimulates ENaC-mediated sodium transport, an effect that is not additive to that of Sgk1. A deubiquitylating enzyme that targets ENaC in vitro and thus may play a role in sodium transport regulation was identified within a series of new in vivo early aldosterone-regulated gene products.

Aldosterone increases sodium reabsorption across kidney target tubules already before it increases the number of transport proteins, indicating that the early functional response to aldosterone depends on the activation of preexisting channels and pumps. A central mediator of this action is the early aldosterone-induced kinase Sgk1 that de-represses the surface expression and activity of the epithelial sodium channel (ENaC). A main mechanism by which Sgk1 exerts this de-repression is the phosphorylation of the ubiquitin ligase Nedd4-2 that is thereby prevented from ubiquitylating ENaC. Among a series of new early aldosterone-induced gene products recently identified in kidney target tubules, an additional regulator of ENaC ubiquitylation, the deubiquitylating enzyme Usp2-45, was identified. Coexpression of Usp2-45 was shown to increase ENaC surface expression and activity, and to decrease its ubiquitylation in expression systems, whereas other Usps such as the splice variant Usp2-69 had no effect. Since both Sgk1 and Usp2-45 are similarly induced in distal colon as well, in contrast to other gene products strongly induced in kidney that are not regulated in colon, we suggest that (de)ubiquitylation is the major ENaC regulatory mechanism targeted by aldosterone in the short-term via transcriptional regulation.

In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl- secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl-]i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP(V163S). Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl-]i (0-100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 microM) and forskolin (2 microM) increased [Cl-]i by 9.6+/-1.5 mM (n=35). Similarly, ATP (100 microM) increased [Cl-]i transiently by 9.5+/-2.2 mM (n=17). The increase in [Cl-]i was reduced by the Na+/K+/2 Cl- -cortransporter-1 (NKCC1) blocker azosemide (100 microM), the CFTR blocker SP-303 (50 microM), the blocker of Ca2+-activated Cl- channels DIDS (100 microM) or the ENaC blocker amiloride (10 microM). Changes in YFP(V163S) fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl-]i following stimulation with secretagogues, which could participate in the inhibition of ENaC.

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