The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum-and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The fulllength sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88 -94% at the nucleotide level, and 96 -99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na ؉ channel results in a significantly enhanced Na ؉ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na ؉ transport.

The serum-and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloridesensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of ␤ and ␥ subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Highlights d SGK1 regulates autophagy in both C. elegans and mammalian cells d Elevated autophagy and mPTP opening shorten lifespan in sgk-1/mTORC2 mutant worms d SGK-1 phosphorylates mPTP component VDAC1 on Ser104, promoting its degradation d Loss of SGK function exaggerates mPTP-dependent hepatic ischemia/reperfusion injury

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an AGC kinase that regulates membrane sodium channel expression in renal tubular cells in an mTORC2-dependent manner. We hypothesized that SGK1 might represent a novel mTORC2-dependent regulator of T cell differentiation and function. Here we demonstrate that upon activation by mTORC2, SGK1 promoted TH2 differentiation by negatively regulating the NEDD4-2 E3 ligase-mediated destruction of transcription factor JunB. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling the expression of the long isoform of transcription factor TCF-1. Consistent with these findings, mice with a selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated robust amounts of IFN-γ in response to viral infections and more readily rejected tumors.

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green f luorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by f luorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclearto-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormoneactivated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.The mineralocorticoid receptor (MR) is a member of the steroid-thyroid receptor superfamily. These receptors are ligand-dependent transcription factors generally located in the nucleus even in the absence of the hormone (see ref. 1 for review). The glucocorticoid receptor (GR), however, is an exception, because it is mainly cytoplasmic in the absence of ligand, and nuclear translocation occurs only after hormone binding (1). The subcellular localization of the MR is still unclear. Because the MR shows significant sequence homology to the GR, one would expect it to also be cytoplasmic in the absence of hormone. However, previous data on its localization are controversial. On the one hand, earlier biochemical and immunocytochemical studies found the unliganded MR to be cytoplasmic (2, 3). Similar findings were reported for the recombinant MR overexpressed in Sf9 insect cells and mouse macrophages (4). On the other hand, immunohistochemistry of kidney sections from adrenalectomized animals revealed both nuclear and cytoplasmic staining (2, 5, 6). In addition, a study with enucleated GH3 cells localized MR entirely to the nucleus (7). All previous studies were done by using fixed and permeabilized cells and antibodies, and methodological differences might have contributed to the controversial results. Furthermore, these studies did not permit examination of the in vivo kinetics of receptor t...

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride-sensitive Na(+) channel activity was examined in the cortical collecting ducts of a mouse line (SGK1(-/-)) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na(+) channel (ENaC) protein. Neither SGK1(-/-) mice nor paired SGK1(+/+) animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current (I(Na)), under control conditions with standard chow. Administration of aldosterone (0.5 microg/h via osmotic minipump for 7 days) increased I(Na) to a similar extent in SGK1(+/+) (378 +/- 61 pA/cell at -100 mV) and in SGK1(-/-) (350 +/- 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of gamma-ENaC, was more pronounced in SGK(+/+) mice. The SGK1(-/-) animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, I(Na) was enhanced more in SGK1(-/-) (94 +/- 14 pA/cell) than in SGK(+/+) (23 +/- 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length gamma-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na(+)-Cl(-) cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.

WNK1 belongs to a unique family of Ser͞Thr kinases that have been implicated in the control of blood pressure. Intronic deletions in the WNK1 gene result in its overexpression and lead to pseudohypoaldosteronism type II, a disease with salt-sensitive hypertension and hyperkalemia. How overexpression of WNK1 leads to Na ؉ retention and hypertension is not entirely clear. Similarly, there is no information on the hormonal regulation of expression of WNK kinases. There are two main WNK1 transcripts expressed in the kidney: the originally described ''long'' WNK1 and a shorter transcript that is specifically expressed in the kidney (KS-WNK1). The goal of this study was to determine the effect of aldosterone, the main hormonal regulator of Na ؉ homeostasis, on the transcription of WNK1 isoforms in renal target cells, by using an unique mouse cortical collecting duct cell line that stably expresses functional mineralocorticoid receptors. Our results demonstrate that aldosterone, at physiological concentrations, rapidly induces the expression of the KS-WNK1 but not that of the long-WNK1 in these cells. Importantly, stable overexpression of KS-WNK1 significantly increases transepithelial Na ؉ transport in cortical collecting duct cells. Similarly, coexpression of KS-WNK1 and the epithelial Na ؉ channel in Fischer rat thyroid epithelial cells also stimulates Na ؉ current, suggesting that KS-WNK1 affects the subcellular location or activity but not the expression of epithelial Na ؉ channel. These observations suggest that stimulation of KS-WNK1 expression might be an important element of aldosteroneinduced Na ؉ retention and hypertension.hypertension ͉ collecting duct H igh blood pressure affects 20-25% of the adult population in industrialized countries; yet the etiology of the majority of cases is poorly understood. In recent years, however, the molecular mechanism of several monogenic forms of hypertension showing Mendelian inheritance has been identified. Interestingly, the mutations in these forms of hypertension converge on a common pathway, i.e., the aldosterone-regulated renal sodium reabsorption. For instance, Liddle's syndrome, a disease mimicking hyperaldosteronism, is caused by gain-of-function mutations in the epithelial sodium channel (ENaC), which leads to abnormally high renal Na ϩ reabsorption, low-renin hypertension, and hypokalemic alkalosis (1). In contrast, the features of another autosomal dominant form of familial hypertension, pseudohypoaldosteronism type II (PHAII) or Gordon's syndrome are more complex than Liddle's syndrome. In PHAII, the hypertension is associated with chloride-dependent sodium retention, hyperkalemia, and metabolic acidosis despite normal glomerular filtration rate (2). PHAII patients are markedly sensitive to thiazide diuretics (3). Despite the fact that these features indicate an overactive thiazide-sensitive Na-Cl cotransporter (NCC), no mutations in this transporter have been found in affected patients.WNKs (with no lysine [K]) are Ser͞Thr kinases that lack a key lysine in t...