Alpha ENaC Research Articles

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the alpha-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type alpha beta gamma ENaC or mutant alpha(W493R)beta gamma ENaC in Xenopus oocytes. The alpha W493R mutation stimulated amiloride-sensitive whole-cell currents (Delta I(ami)) by approximately 4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P(o)) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the alpha W493R mutation abolished Na(+) self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the alpha W493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na(+) and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Amiloride‐sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.

Relationship between four single nucleotide polymorphisms of epithelial sodium channel alpha subunit gene and essential hypertension of Kazakhs in Xinjiang

To explore the relationship between G2139A,G3091A, T663A, and T3593C single nucleotide polymorphisms (SNPs), which are located at the promoter region,13th exon, and 2nd intron of epithelial sodium channel (ENaC) gene, and their haplotypes with essential hypertension (EH) in Kazakhs in Xinjiang. A case-control study was conducted including 252 EH patients (EH group) and 254 normotensive subjects (NT group) among Kazakhs in Xinjiang. The four genetic polymorphisms were identified by polymerase chain reaction - restriction fragment length polymorphism. The distribution of the genotypes and alleles in all subjects and the different frequency of these four SNPs between EH group and NT group were analyzed. The linkage disequilibrium and haplotypes of these four SNPs were analyzed. These four SNPs of alpha ENaC gene existed in Xinjiang Kazakhs. In all subjects, the distribution frequencies of genotypes AA, AG, and GG at G2139A were 26.2%, 52.3%, and 21.5%, respectively, and those of alleles (A, G) were 52.37% and 47.63%. The distribution frequencies of genotypes AA, AG, and GG at G3091A were 19.0%, 52.5%, and 28.5%, respectively, and those of and alleles (A, G) were 45.56% and 59.44%. The distribution frequencies of genotypes AA, AG, and GG at T663A were 15.6%, 49.9%, and 34.5%, respectively, and those of alleles (A, G) were 40.53% and 59.47%. The distribution frequencies of genotypes TT, TC, and CC at T3593C were 88.5%, 10.5%, and 1.0%, respectively, and those of alleles (T, C) were 93.77% and 6.23%. The distribution of genotypes at these four SNPs were all consistent with Hardy-Weinberg equilibrium in this population (P0.05). The distribution frequencies of genotypes and alleles about these four genetic polymorphisms were not significantly different between the EH group and NT group (P0.05). However, the frequencies of two haplotypes were found to be significantly different between these two groups (P0.05). The haplotype frequency which included 2139G, 3091A, 663G, and 3593T alleles was significantly increased in EH group (P0.01), while the haplotype frequency which included 2139A, 3091A, 663A, and 3593C alleles was significantly increased in NT group (P0.05). The haplotypes that are composed of G2139A, G3091A, T663A, and T3593C polymorphisms of alphaENaC gene may play an important role in the development of EH among Kazakhs in Xinjiang. The haplotypes that are composed of 2139G, 3091A, 663G, and 3593T alleles may aggravate the development of EH. The haplotypes that composed of 2139A, 3091A, 663A, and 3593C alleles may decrease the risk of EH among Kazakhs.

Combination ofENaCandCFTRmutations may predispose to cystic fibrosis-like disease: Table 1—

To the Editors: The epithelial sodium channel (ENaC) is part of the complex protein cystic fibrosis transmembrane conductance regulator (CFTR)-interactome that plays a key role in the composition of the airway surface liquid and the mucociliary clearance in the airways. We have recently screened ENaC beta and gamma genes in 55 patients with diffuse bronchiectasis and with only one or no CFTR mutation/variant and showed that eight (15%) patients carried at least one missense mutation in these ENaC genes 1. Azad et al. 2 have also investigated the frequency of ENaC mutations in ENaC alpha, beta and gamma genes in a cohort of 76 patients with cystic fibrosis (CF)-like disease and with only one or no mutation in the CFTR gene. They found a significant increase in missense mutations or variants in patients (15.3%) compared with controls (8.9%) 2. Moreover, a variant in …

Characterization of the epithelial sodium channel α subunit coding and non-coding transcripts and their corresponding mRNA expression levels in Dahl R versus S rat kidney cortex on normal and high salt diet

Aims/hypothesisThe α subunit of the amiloride-sensitive epithelial sodium channel (α ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of α ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to examine the presence and consequently investigate the mRNA expression levels of alternatively spliced forms of α ENaC in kidney cortex of Dahl salt-resistant rats (R) versus Dahl salt-sensitive rats (S) on high salt and normal diets.MethodsUsing quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported α ENaC-a and -b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to wildtype α ENaC mRNA levels. We identified 2 novel non-coding C-terminus spliced forms and examined their mRNA expression in Dahl R versus S rat kidney cortex. We also tested the presence of five previously reported lung-specific α ENaC spliced forms in Dahl rat kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980).ResultsPreviously reported α ENaC-a and -b alternatively spliced forms are present in Dahl rat kidney cortex and are significantly higher in Dahl R versus S rats (P < 0.05). Four-week high salt diet significantly increases α ENaC-b (P < 0.05), but not α ENaC-a transcript abundance in Dahl R, but not S rats. Two non-coding α ENaC spliced forms -c and -d are newly identified in the present study, whose levels are comparable in Dahl R and S rats. Compared to α ENaC-wt, α ENaC-a, -c and -d are low abundance transcripts (4 +/- 2, 110 +/- 20, and 10 +/- 2 fold less respectively), in contrast to α ENaC-b abundance that exceeds α ENaC-wt by 32 +/- 3 fold. We could not identify any of the five previously reported lung-specific α ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rat kidney cortex.Conclusion/interpretationα ENaC alternative splicing might regulate α ENaC by the formation of coding RNA species (α ENaC-a and -b) and non-coding RNA species (α ENaC-c and -d). α ENaC-a and -b mRNA levels are significantly higher in Dahl R versus S rats. Additionally, α ENaC-b is a salt-sensitive transcript whose levels are significantly higher 4-weeks post high salt diet compared to normal salt diet in Dahl R rats. Among the four α ENaC transcripts (-a, -b, -c and -d), α ENaC-b is a predominant transcript that exceeds α ENaC-wt abundance by ~32 fold. α ENaC-a and -b spliced forms, particularly, α ENaC-b, might potentially act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. On the other hand, non-coding α ENaC-c and -d might assist alternative splicing, facilitate RNA processing, or regulate α ENaC as well as each other.

Preferential Assembly of Epithelial Sodium Channel (ENaC) Subunits in Xenopus Oocytes: ROLE OF FURIN-MEDIATED ENDOGENOUS PROTEOLYSIS

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.

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.

Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle's syndrome

Liddle's syndrome is an autosomal dominant form of human hypertension, caused by gain-of-function mutations of the epithelial sodium channel (ENaC) which is expressed in aldosterone target tissues including the distal colon. We used a mouse model for Liddle's syndrome to investigate ENaC-mediated Na+ transport in late distal colon by measuring the amiloride-sensitive transepithelial short circuit current (Delta I SC-Ami) ex vivo. In Liddle mice maintained on a standard salt diet, Delta I SC-Ami was only slightly increased but plasma aldosterone (P Aldo) was severely suppressed. Liddle mice responded to a low or a high salt diet by increasing or decreasing, respectively, their P Aldo and Delta I SC-Ami. However, less aldosterone was required in Liddle animals to achieve similar or even higher Na+ transport rates than wild-type animals. Indeed, the ability of aldosterone to stimulate Delta I SC-Ami was about threefold higher in Liddle animals than in the wild-type controls. Application of aldosterone to colon tissue in vitro confirmed that ENaC stimulation by aldosterone was not only preserved but enhanced in Liddle mice. Aldosterone-induced transcriptional up-regulation of the channel's beta- and gamma-subunit (beta ENaC and gamma ENaC) and of the serum- and glucocorticoid-inducible kinase 1 (SGK1) was similar in colon tissue from Liddle and wild-type animals, while aldosterone had no transcriptional effect on the alpha-subunit (alpha ENaC). Moreover, Na+ feedback regulation was largely preserved in colon tissue of Liddle animals. In conclusion, we have demonstrated that in the colon of Liddle mice, ENaC-mediated Na+ transport is enhanced with an increased responsiveness to aldosterone. This may be pathophysiologically relevant in patients with Liddle's syndrome, in particular on a high salt diet, when suppression of P Aldo is likely to be insufficient to reduce Na+ absorption to an appropriate level.

Function and regulation of epithelial sodium transporters in the kidney of a salt-sensitive hypertensive rat model

To determine the function and regulation of thiazide-sensitive NaCl co-transporters (NCC), NaK2Cl co-transporters (NKCC2), and epithelial sodium channels (ENaC) in the kidneys of a salt-sensitive hypertensive model. Neonatal Wistar rats were treated with capsaicin or vehicle. Seven-week-old male rats were treated for 2 weeks with: vehicle plus a normal (Con-NS) or high (Con-HS) sodium diet, and capsaicin pretreatment plus a normal (Cap-NS) or high (Cap-HS) sodium diet. Mean arterial pressure (MAP), renal excretory function, and protein expression determined by western blot were performed. MAP was increased in Cap-HS compared with other groups. Trichlormethiazide increased urine sodium excretion (UNaV) and urine flow rate (UFR) and decreased MAP in Cap-HS rats only. Furosemide increased UNaV and UFR in Cap-NS, Con-HS and Cap-HS, and decreased MAP in Cap-HS rats only. Amiloride had no effect on UNaV, UFR and MAP in any group. Renal NCC contents were increased in Cap-HS compared with Con-NS, Con-HS and Cap-NS rats, and NKCC2 expression was increased in Cap-NS, Con-HS and Cap-HS compared with Con-NS rats. No change was found in ENaC alpha subunit expression. The capsaicin-induced release of calcitonin gene-related peptide from renal tissues was decreased in Cap-HS and Cap-NS compared with Con-HS and Con-NS rats. NCC and possibly NKCC2, but not ENaC, were functionally upregulated in the kidneys of rats subjected to sensory nerve degeneration plus high salt intake, suggesting that sensory neurotransmitters may regulate the expression of the former but not the latter, which may underlie the development of salt-sensitive hypertension in this model.

Cloning and functional expression of a new epithelial sodium channel δ subunit isoform differentially expressed in neurons of the human and monkey telencephalon

Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them.

Thrombin Impairs Alveolar Fluid Clearance by Promoting Endocytosis of Na+,K+-ATPase

Coagulation is an emerging area of interest in the pathogenesis and treatment of acute lung injury. Concentrations of the edemagenic coagulation protease thrombin are elevated in plasma and lavage fluids from afflicted patients. We explored the impact of thrombin on the formation and resolution of alveolar edema. Intravascularly applied thrombin inhibited active transepithelial 22Na transport in intact rabbit lungs, suppressing alveolar fluid clearance. Epithelial permeability was unaffected, whereas endothelial permeability was increased. In A549 human lung epithelial cells and in mouse primary alveolar type II cells, thrombin blocked ouabain-sensitive Na+,K+-ATPase-mediated 86Rb+ uptake, without altering amiloride-sensitive sodium currents. Furthermore, thrombin downregulated cell-surface expression of Na+,K+-ATPase, but not ENaC alpha and beta subunits. The endocytosis inhibitor phalloidin oleate blocked all thrombin-induced effects on sodium transport activity. Similarly, diphenyleneiodonium chloride, an inhibitor of reactive oxygen radical production, as well as a protein kinase C-zeta inhibitor, prevented these thrombin-induced effects. Thus, thrombin signaling via reactive oxygen species and protein kinase C-zeta promotes Na+,K+-ATPase endocytosis, resulting in loss of function. We propose here a dual role for thrombin in mediating disturbances to fluid balance in the lung: thrombin concomitantly provokes edema formation by increasing endothelial permeability, and inhibits alveolar edema resolution by blocking Na+,K+-ATPase function.

Sodium retention in rats with liver cirrhosis is associated with increased renal abundance of NaCl cotransporter (NCC)

Liver cirrhosis is associated with enhanced renal tubular sodium retention, the mechanism of which is still debated. We hypothesized that liver cirrhosis is associated with increased expression of renal epithelial sodium transporter(s). Liver cirrhosis was induced by bile duct ligation (BDL) in rats. Steady state mRNA of ENaC subunits alpha, beta, gamma serum and glucocorticoid inducible kinase (Sgk1) were measured by TaqMan PCR in kidney homogenates at week 1, 2, 3 and 4 after BDL. Renal protein content of ENaC subunits, ubiquitin-protein-ligase Nedd4-2 and NaCl cotransporter (NCC) were assessed by western blot. Subcellular localization of ENaC subunits and NCC were analysed by immunohistochemistry. Steady state mRNA of ENaC alpha, beta and gamma were unchanged during the 4 weeks investigated, while ENaC protein decreased most prominently at week 2 (control vs BDL; alpha, -46%; beta, -81%; and gamma, -63%; n = 6). Subcellular localization of ENaC subunits was not altered at week 2. Sgk1 mRNA did not change, whereas Nedd4-2 protein was reduced by >50% 2-4 weeks after BDL. NCC protein significantly increased at week 1 (control vs BDL: +66%, n = 6, P<0.05) and decreased at week 3 (control vs BDL: -85%, n = 6, P<0.0005). Enhanced abundance of NCC was observed in the initial stage after BDL, followed by a marked decrease. ENaC transcription, translation or cell surface abundance was not increased after BDL.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

BackgroundThe intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.MethodsPseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.ResultsThe infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.ConclusionsThese results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.

New insights into the role of serum- and glucocorticoid-inducible kinase SGK1 in the regulation of renal function and blood pressure

The serum and glucocorticoid inducible kinase 1 (SGK1) is induced in the aldosterone sensitive distal nephron (ASDN) where it may stimulate Na reabsorption, partly by inhibiting ubiquitin ligase Nedd4-2-mediated retrieval of epithelial Na+ channel ENaC from the luminal membrane. We describe recent advances in our understanding of SGK1 function in the regulation of renal function and blood pressure. Thiazolidinediones, i.e. activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), upregulate SGK1 and ENaC mRNA expression and increase cell-surface expression of ENaC alpha in a human cortical-collecting-duct cell line. cAMP/protein kinase A can induce phosphorylation and inhibition of Nedd4-2-independent of SGK1. Part of ENaC stimulation by SGK1 appears dependent on a SGK1 consensus motif in ENaC alpha and independent of Nedd4-2. SGK1-dependent upregulation of Na+ reabsorption in ASDN contributes to upregulation of renal K+ excretion. In oocytes, SGK1 activates various renal transport proteins including Na+/glucose cotransporter SGLT1, Na+-coupled dicarboxylate transporter NaDC-1, epithelial Ca+ channel TRPV5, renal outer medullary K+ channel ROMK and voltage gated K+ channels KCNE1/KCNQ1 and Kv1.3. A variant of the SGK1 gene associates with increased blood pressure and body mass index. PPAR gamma activators may increase renal Na reabsorption by stimulating SGK1 and ENaC. Nedd4-2 integrates influences of cAMP/protein kinase A and SGK1. SGK1 can activate ENaC in part directly and independent of Nedd4-2. K+ homeostasis requires SGK1-dependent Na+ reabsorption in ASDN. SGK1 may affect renal transport mechanisms beyond Na+ reabsorption and K+ secretion in ASDN. Polymorphisms of SGK1 may be relevant to the pathophysiology of hypertension and other diseases.

Effect of spironolactone on K+ homeostasis and ENaC expression in lymphocytes from chronic hemodialysis patients

Cardiac disease is the major cause of death in hemodialysis patients (HD). It is now clear that aldosterone has deleterious effects in the cardiovascular system. In the present study, we evaluated the effects of an aldosterone-antagonist, spironolactone, on the extrarenal regulation of potassium in HD patients. Furthermore, to validate the effectiveness of the spironolactone dose-design, we measured the expression of Na(+)-channel (ENaC alpha subunit) in peripheral blood mononuclear cells (PBMC), before and after a two-week course of spironolactone. The study design included a two-week baseline period, followed by spironolactone treatment (50 mg three times weekly for 15 days), and by a two-week washout period and then a two-week placebo period. An oral K(+) load (0.3 mEq/K(+) kg body weight plus carbohydrates) was administered at the end of each period, and time-course of plasma potassium was evaluated. ENaC expression in PBMC was assessed before and after spironolactone. The maximal increase in plasma potassium after the K(+) carbohydrate load was: control 5.33 +/- 0.88 mEq K(+)/L; spironolactone 5.23 +/- 0.68 mEq K(+)/L; placebo 5.38 +/- 0.61 mEq K(+)/L (N= 9). No patients developed hyperkalemia during the spironolactone treatment period. ENaC expression was significantly higher in all six HD patients studied, compared to control subjects (P < 0.05). Treatment with spironolactone in HD patients reduced alpha subunit mRNA expression to values similar to those of normal subjects. Spironolactone may be considered for the treatment of selected chronic HD patients. The effect of the drug on a known target of aldosterone, the ENaC, demonstrates the effectiveness of the drug to block aldosterone effects in nonepithelial tissues.

Cystic Fibrosis Transmembrane Conductance Regulator Differentially Regulates Human and Mouse Epithelial Sodium Channels in Xenopus Oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with alphabetagamma mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.

Cystic fibrosis transmembrane conductance regulator and Na+ channel subunits mRNA transcripts, and Cl- efflux, show a different distribution in rat duodenum and colon.

We compared the distribution and putative association of Cl- channel transport, CFTR mRNA transcripts, and Na+ channel (ENaC) alpha- and beta-subunit mRNA transcripts in villus and crypt epithelial cells of duodenum, with corresponding surface and crypt cells of colon from sodium-depleted rats. Cells were loaded with 36Cl- and forskolin-stimulated efflux was determined. RT-PCR was performed for CFTR mRNA transcripts and ENaC alpha- and beta-subunit mRNA. Duodenal epithelial cell response to VIP was assessed by measuring intracellular cAMP. Forskolin-stimulated Cl- efflux occurred with decreasing magnitude in duodenal crypt, duodenal villus, colonic crypt and colonic surface cells in Na(+)-depleted animals. CFTR expression was correlated directly with Cl- efflux (r=0.91, P<0.01). Na+ channel alpha-subunit was expressed in colon and duodenum in animals fed diets with a high or low sodium content. While the beta-subunit mRNA was detected in the colon of sodium-restricted rats, it was absent in the duodenum under control conditions and after Na+ restriction. There was an inverse correlation between mRNA transcripts for CFTR and the ENaC alpha-subunit (r=-0.93, P<0.003) and beta-subunit (r=-0.91, P<0.004) in colon. VIP-stimulated cAMP in duodenal epithelial cells was greater in crypt than villus (P<0.05). Cl- efflux, CFTR transcription and forskolin-stimulated cAMP activity occur in both crypt and villus epithelial cells in duodenum. Possible interaction between CFTR and Na+ channels is apparently limited to parts of the colonic crypt. Lack of duodenal beta-subunit expression makes ENaC activity unlikely.

Short-term aldosterone action on Na,K-ATPase surface expression: role of aldosterone-induced SGK1?

Aldosterone controls extracellular volume and blood pressure by regulating Na(+) reabsorption across epithelial cells of the aldosterone-sensitive distal nephron (ASDN). This effect is mediated by a coordinate action on the luminal channel ENaC (generally rate limiting) and the basolateral Na,K-ATPase. Long-term effects of aldosterone (starting within 3 to 6 hours and increasing over days) are mediated by the direct and indirect induction of stable elements of the Na(+) transport machinery (e.g., Na,K-ATPase alpha subunit), whereas short-term effects appear to be mediated by the upregulation of short-lived elements of the machinery (e.g., ENaC alpha subunit) and of regulatory proteins, such as the serum- and glucocorticoid-regulated kinase SGK1. We have recently shown that in cortical collecting duct (CCD) from adrenalectomized (ADX) rats, the increase in Na,K-ATPase activity (approximately threefold in 3 h), induced by a single aldosterone injection, can be fully accounted for by the increase in Na,K-ATPase cell-surface expression. Using the model cell line mpkCCD(cl4), we showed that the parallel increase in Na,K-ATPase function [assessed by Na(+) pump current (I(p)) measurements] and cell-surface expression depends on transcription and translation, and that it is not secondary to a change in apical Na(+) influx. As a first approach to address the question whether the aldosterone-induced regulatory protein SGK1 might play a role in mediating Na,K-ATPase translocation, we have used the Xenopus laevis expression system. SGK1 coexpression indeed increased both the Na(+) pump current and the surface expression of pumps containing the rat alpha1 subunits. In summary, aldosterone controls Na(+) reabsorption in the short term not only by regulating the apical cell-surface expression of ENaC but also by coordinately acting on the basolateral cell-surface expression of the Na,K-ATPase. Results obtained in the Xenopus oocyte expression system suggest the possibility that this effect could be mediated in part by the aldosterone-induced kinase SGK1.

The serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na+ channel alpha,beta,gamma-ENaC.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been identified as a signalling molecule up-regulated by aldosterone, which stimulates the renal epithelial Na(+) channel ENaC. It is therefore thought to participate in the antinatriuretic action of this hormone. More recently, two isoforms, SGK2 and SGK3, have been cloned. The present study was performed to establish whether SGK2 and SGK3 influence ENaC activity similarly to SGK1. Dual-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing alpha,ss,gamma-ENaC with or without SGK1, SGK2 or SGK3 revealed a stimulatory effect of all three kinases on the amiloride-sensitive current (I(Na)). To establish whether the SGK isoforms exert their effects through direct phosphorylation, we replaced the serine at the SGK consensus site of alphaENaC (alpha(S622A)ENaC) by site-directed mutagenesis. alpha(S622A),beta,gamma-ENaC was up-regulated similar to wild-type ENaC, suggesting that SGK isoforms do not act via direct phosphorylation of the transport proteins. In conclusion, SGK2 and SGK3 mimic the function of SGK1 and are likely to participate in the regulation of ENaC activity.

Endogenously Expressed Epithelial Sodium Channel Is Present in Lipid Rafts in A6 Cells

The epithelial sodium channel (ENaC) present in the kidney collecting duct, distal colon, and the lung is responsible for salt reabsorption and whole body volume regulation. It is composed of three homologous subunits, alpha, beta, and gamma, and mutations to these subunits can lead to the salt wasting disease pseudohypoaldosteronism type I, associated with decreased channel density at the plasma membrane or to the hypertensive disorder, Liddle's syndrome, in which channel residency time at the plasma membrane is enhanced. Regulation of ENaC trafficking and turnover is therefore critical to sodium homeostasis. In this study we examined whether ENaC is present in the cholesterol-enriched microdomains commonly called lipid rafts, in the endogenously expressing A6 cell line. We demonstrate that a fraction of alpha, beta, and gamma ENaC is present in detergent-insoluble membranes, that subunits exist in membranes that float on discontinuous sucrose density gradients, and that methyl-beta-cyclodextrin treatment causes a redistribution of ENaC subunits to higher density membranes. Furthermore, chronic aldosterone stimulation results in a shift in the membrane density of all three subunits. Biotinylation of apical membrane proteins revealed that ENaC is present in lipid rafts on the plasma membrane. In conclusion, these results show that ENaC is present in lipid rafts both intracellularly and on the cell surface. Raft association may be important for trafficking and/or function of the channel.