Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30--60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces alpha-ENaC subunit expression along the entire ASDN within 2 h, whereas beta- and gamma-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and alpha-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of alpha,beta,gamma-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.

The highly selective, amilorideblockable Na+ channel is a major target to the natriferic action of the mineralocorticoid aldosterone. This rat epithelial Na+ channel (rENaC) has been recently cloned from colon and is composed of three homologous subunits denoted alpha-, beta-, and gamma-rENaC (C. M. Canessa, L. Schild, G. Buell, B. Thorens, L. Gautschi, J.-D. Horisberger, and B. C. Rossier. Nature Lond. 367: 463-467, 1994). We have tested the effects of corticosteroids on the abundance of mRNA coding for each subunit in kidney cortex and distal colon. Chronic treatment of rats with aldosterone or dexamethasone evoked in kidney cortex a small induction of alpha-rENaC and no change in beta- and gamma-rENaC. In distal colon, however, beta- and gamma-rENaC were strongly induced by either aldosterone or dexamethasone, whereas alpha-rENaC was constitutively expressed. Most of the aldosterone-induced increase in beta- and gamma-rENaC mRNA took place during 3-24 h after plasma aldosterone was elevated. A similar differential induction of rENaC subunits in kidney and colon was also evoked by a Na(+)-free diet. The effects of salt deprivation were reversed by resalinating rats with a half time of < 2 h, suggesting a high turnover rate of at least beta- and gamma-rENaC. The data are consistent with the possibility that induction of channel subunits contributes to the chronic but not the acute response to aldosterone in the colon. Such a mechanism is not likely to play a major role in cortical collecting ducts.

Aldosterone is the major corticosteroid regulating Na(+) absorption in tight epithelia and acts primarily by activating the epithelial Na(+) channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na(+) current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na(+) channel activity. A point mutation in the beta-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

Phosphorylation of the epithelial Na؉ channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the ␤ and ␥ subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of ␤ and ␥ENaC, have been identified by an in vitro assay. One of these phosphorylates ␤Thr-613 and ␥Thr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of ␥Thr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating ␤Thr-613 and ␥Thr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions ␤613 or ␥623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on ␤Thr-613 and ␥Thr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERKmediated phosphorylation of ␤Thr-613 and ␥Thr-623 down-regulates the channel by facilitating its interaction with Nedd4.

FXYD5 (dysadherin or RIC) is a member of the FXYD family of single-span transmembrane proteins associated with the Na(+)-K(+)-ATPase. Several studies have demonstrated enhanced expression of FXYD5 during metastasis and effects on cell adhesion and motility. The current study examines effects of FXYD5 on the paracellular permeability in the mouse kidney collecting duct cell line M1. Expressing FXYD5 in these cells leads to a large decrease in amiloride-insensitive transepithelial electrical resistance as well as increased permeability to 4-kDa dextran. Impairment of cell-cell contact was also demonstrated by staining cells for the tight and adherence junction markers zonula occludens-1 and β-catenin, respectively. This is further supported by large expansions of the interstitial spaces, visualized in electron microscope images. Expressing FXYD5 in M1 cells resulted in a decrease in N-glycosylation of β1 Na(+)-K(+)-ATPase, while silencing it in H1299 cells had an opposite effect. This may provide a mechanism for the above effects, since normal glycosylation of β1 plays an important role in cell-cell contact formation (Vagin O, Tokhtaeva E, Sachs G. J Biol Chem 281: 39573-39587, 2006).