sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.

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 full-length 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.     

Appearance of specific proteins in the apical plasma membrane of cultured renal collecting duct principal cell epithelium after chronic administration of aldosterone and arginine vasopressin

Principal cell epithelium of renal collecting duct from neonatal rabbit kidney was cultured in the presence of aldosterone (1 x 10(-6) M) and arginine vasopressin (AVP; 1 x 10(-6) M) for 10 days to investigate, by immunohistochemical methods using specific monoclonal antibodies, whether the hormones influence the expression and insertion of plasma membrane proteins. The experiments demonstrated that aldosterone alone or aldosterone plus AVP significantly increased the number of epithelial cells reacting at the luminal and lateral plasma membrane with the antibodies CD 2 and 3, specific for renal collecting duct, as we have shown in the kidney. In cultures treated with aldosterone and aldosterone plus AVP, nearly all epithelial cells were labelled by the antibodies, while controls or AVP treatment showed 41% and 24% unreactive cells, respectively. These findings were complemented with electrophysiological experiments, in which epithelia pretreated by aldosterone or aldosterone plus AVP showed significantly hyperpolarized transepithelial voltage (Vte) and higher resistance (Rte) than controls or AVP-treated specimens. The experiments demonstrated that chronic administration of aldosterone or of aldosterone plus AVP to increase Na+-transport was paralleled by the appearance of collecting-duct-specific proteins in the epithelium. Consequently, this result indicates that aldosterone influences the functional maturity of the cultured epithelium.     

Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart

RTP/Drg1/Cap43/rit42/TDD5/Ndr1/NDRG1 (referred to as NDRG1 hereafter) is a cytoplasmic protein involved in stress responses, hormone responses, cell growth, and differentiation. Recently, the mutation of this gene was reported to be causative for hereditary motor and sensory neuropathy-Lom. Here, we cloned two human cDNAs encoding NDRG3 and NDRG4, which are homologous to NDRG1. These two genes, together with NDRG1 and a previously deposited cDNA (designated NDRG2), constitute the NDRG gene family. The four members share 57-65% amino acid identity. NDRG4 was further characterized because its mRNA expression was quite specific in brain and heart, in contrast to the relatively ubiquitous expression of the other three members. NDRG4 mRNA consists of three isoforms, NDRG4-B, NDRG4-B(var), and NDRG4-H. Northern and Western blot analyses showed that NDRG4-B was expressed only in the brain, whereas NDRG4-H was expressed in both brain and heart. NDRG4-B(var) was a minor product. NDRG4 expression was more abundant in adult than fetal brain and heart and was markedly decreased in the Alzheimer's diseased brain. In situ hybridization showed that NDRG4 was localized in neurons of the brain and spinal cord. The NDRG4 gene contains 17 exons. mRNA expression of the three NDRG4 isoforms is regulated by alternative splicing and possibly by alternative promoter usage. The finely tuned expression of the NDRG gene family members suggests that they have different specific functions.     

Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.     

Methylation of cytosolic proteins may be a possible biochemical pathway of early aldosterone action in cultured renal collecting duct cells

The common model of aldosterone-dependent sodium transport is that the hormone increases sodium transport during the "early" and "late" response phases by inducing specific proteins (AIPs). However, in actual biochemical studies, AIPs were mostly detected 6-24 h after aldosterone application. Regarding the physiological early response phase, this implies temporal dissociation of the physiological and biochemical events. The discrepancy raises the question as to whether other biochemical events, such as protein modifications, may be involved in addition to the novo protein synthesis. Labelling of cultured renal collecting duct epithelia for 1-5 h with a radioactive methylgroup donor, S-adenosyl methionine (SAM), following tissue fractionation, resulted in progressive methylations of specific cytosolic proteins. Aldosterone-dependent methylations increased consistently with time, and accounted for a 60% increase in total cytosolic protein content as compared to controls after 5 h labelling. The different methylated proteins showed a molecular weight of 220, 97 and 75 kd and comprised groups of proteins with an isoelectric point of 5.1-5.7 and 6.0-7.5. Methylation of identical proteins was obtained by incubation of the epithelia with unlabelled SAM instead of aldosterone. SAM-induced as well as aldosterone-induced methylation of proteins with an isoelectric point of 6.0-7.5 could be inhibited by the methylation inhibitor S-adenosylhomocysteine. The results indicate that aldosterone may influence the SAM cycle in cultured collecting-duct epithelia during increase of the Na+-transport.     

14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels

Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density.     

E Prostanoid-1 receptor regulates renal medullary αENaC in rats infused with angiotensin II

E Prostanoid (EP) receptors play an important role in urinary Na⁺ excretion. In the kidney, the epithelial sodium channel (ENaC) is the rate-limiting-step for Na⁺ reabsorption. We hypothesized that activation of EP1/EP3 regulates the expression of ENaC in the face of renin-angiotensin-aldosterone-system (RAAS) activation. In primary cultures of inner medullary collecting duct (IMCD) cells, sulprostone (EP1 > EP3 agonist, 1 μM) and 17 Phenyl trinor (17 Pt, EP1 agonist, 10 μM) prevented the up-regulation of αENaC mRNA induced by aldosterone (10 nM). In Sprague-Dawley rats infused with angiotensin II (0.4 μg/kg/min), αENaC expression was up-regulated in renal cortex and medulla coincidently with high plasma aldosterone levels. Sulprostone and/or 17 Pt prevented this effect in renal medulla but not in cortex. Immunocytochemistry demonstrated that IMCD cells express EP1. Our results suggest that specific activation of EP1 receptor during RAAS activation antagonizes the action of aldosterone on αENaC expression in the renal medulla.     

Action of aldosterone on citrate synthase in cultured renal collecting duct cells

Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enzymatic activity of citrate synthase during increase in Na+ transport. Control epithelia showed citrate synthase activity of 71 +/- 3 mU/mg protein (n = 28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 +/- 6 mU/mg at 1 h (n = 5), to 88 +/- 6 mU/mg at 2 h (n = 6) and to 93 +/- 8 mU/mg protein at 3 h (n = 5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.     

Aldosterone regulates rapid trafficking of epithelial sodium channel subunits in renal cortical collecting duct cells via protein kinase D activation

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.     

The early non-genomic aldosterone-induced increase in sodium transport is a membrane-initiated event that requires protein carboxyl methylation in renal cells.

Effects of aldosterone on its target cells are generally considered to be mediated through the genomic pathway. However, recent studies have evidenced rapid effects of the hormone that involve a non-genomic mechanism. In this study, we show that, in the RCCD2 rat cortical collecting duct cell line, the early effect of the hormone on transepithelial sodium transport is neither antagonized by the mineralo- and glucocorticoid receptors antagonists RU26752 and RU486, nor blocked by mRNA and protein synthesis inhibitors. Interestingly, the plasma membranes of RCCD2 cells specifically bind 3H-aldosterone but not 3H-dexamethasone, a binding that is not displaced in the presence of RU26752 or RU486, suggesting the presence of an aldosterone membrane receptor. In addition, the early aldosterone-induced increase in sodium transport is blocked by the addition of a specific inhibitor of carboxyl methyl transferase. These results suggest that, in RCCD2 cells, the early aldosterone-induced increase in sodium transport is not mediated through the genomic pathway but through a membrane receptor-mediated signal and could involve a rapid carboxyl methylation process regulated by aldosterone.     

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.