αENaC mRNA Research Articles

Three conserved domains in the 3’UTR region of αENaC mRNA modulates the stability of the transcript in alveolar epithelial cells (716.1)

The epithelial sodium channel (ENaC) plays an important role in the alveolar epithelium by mediating Na+ reabsorption, an essential process for resolution of pulmonary edema in acute respiratory distress syndrome (ARDS). Several proinflammatory conditions such as TNF‐α leads to a reduction of ENaC expression and activity that contributes to development of ARDS. To study how pro‐inflammatory conditions could modulate αENaC mRNA stability, we developped a Tet‐Off model that allows the specific inhibition of αENaC mRNA expression in presence of doxycycline. Using this model, we found that the half‐life (T1/2) for the transcript was about 1 hr. TNF‐α, a proinflammatory cytokine found in broncho‐alveolar lavage of ARDS patients, decreased T1/2 to 15 min, suggesting that destabilisation of αENaC transcript plays an important role in downregulation of ENaC expression in pro‐inflammatory conditions. The 3’ untranslated region (3’UTR) of a mRNA plays an important role in modulating mRNA stability. αENaC mRNA 3’UTR is very long (~30% of the transcript). Using a bioinformatic approach, we could define three domains (D1, D2, D3) able to form secondary structures that were highly conserved in different species. Alveolar epithelial cells were co‐transfected with 3’UTR deletion mutants inserted in pTRE‐tight vector along with the Tet‐Off vector. Deletion of the distal part of 3’UTR (D3) leaded to stabilization of the transcript with a T1/2 of 2.6 hrs. A longer deletion to remove also D2 showed a T1/2 of 4.6 hrs, while deletion of D1 decreased the half‐life to 0.5 hr. These results showed that the conserved domains have stabilizing (D1) and destabilizing (D2, D3) properties on αENaC mRNA expression. The hairpin structure of these domains could recruit RNA‐binding proteins responsible for modulation of αENaC mRNA stability. Altogether, our results suggest that modulation of αENaC mRNA stability via its 3’UTR could play an important role in regulation of ENaC expression during pro‐inflammatory conditions.Grant Funding Source: Supported by FRSQ, CFC and CIHR.

Renal epithelial sodium channel is critical for blood pressure maintenance and sodium balance in the normal late pregnant rat

Normal pregnancy is a state marked by avid sodium retention and plasma volume expansion. Insufficient plasma volume expansion results in the compromised maternal state of intrauterine growth restriction, which afflicts ∼5% of all human pregnancies. We have recently shown that renal epithelial sodium channel (ENaC) activity in vivo in the late pregnant (LP) rat is increased. To determine the importance of the renal versus extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy, we have chronically blocked the ENaC pharmacologically with daily subcutaneous injections of benzamil and genetically using intrarenal transfection of αENaC short hairpin RNA. Compared with untreated LP control animals, LP rats treated with benzamil retain less sodium and have reduced mean arterial blood pressure. Furthermore, LP rats treated with benzamil had lower maternal body weight gain. Intrarenal transfection of αENaC short hairpin RNA versus scrambled small RNA successfully decreased renal αENaC mRNA expression in LP rats. Intrarenal transfection of αENaC short hairpin RNA reduced maternal sodium retention, body weight gain and pup weight. Redundant physiological systems that protect blood pressure and sodium homeostasis were unable to compensate for the loss of ENaC activity in the pregnant rat. These findings demonstrate that the renal ENaC is necessary for maintaining pregnancy-mediated sodium retention, volume expansion and blood pressure regulation.

Epithelial sodium channel enhanced osteogenesis via cGMP/PKGII/ENaC signaling in rat osteoblast

The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na(+) regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.

Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line

The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)–Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2–Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.

Cycloheximide and lipopolysaccharide downregulate αENaC mRNA via different mechanisms in alveolar epithelial cells

Active Na(+) transport mediated by epithelial Na(+) channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.

Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increaseαENaCtranscription

Aldosterone is a major regulator of Na(+) absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na(+) channel (ENaC). MR(-/-) mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR(-/-) mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR(-/-) vs. MR(+/+) mice had significantly lower αENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR(-/-) mice.

Aldosterone-dependenttrans-activation and epigenetic derepression of ENaC: where is the balance?

the critical role of the kidney collecting duct and the epithelial Na+ channel (ENaC) in the regulation of arterial blood pressure has been recognized for many years (5). However, the factors that alter renal function and the pathways involved remain to be determined. The mineralocorticoid hormone aldosterone increases tubular sodium absorption in large part by increasing α-ENaC transcription. The pioneering demonstration of epigenetic regulation of ENaC was published by the Kone's laboratory several years ago, when it was reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the α-ENaC promoter and that aldosterone and serum- and glucocorticoid-induced kinase-1 (Sgk1) act to disrupt this complex and its inhibitory effects (8, 9). Following studies indicated that either Af9 (in humans, encoded by MLLT3) or Sirt1 (encoded by SIRT1) directly binds to +78/+92 of α-ENaC and recruits Dot1a to repress basal and aldosterone-sensitive α-ENaC transcription (7, 10). Despite this epigenetic repression, basal α-ENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In an issue of the American Journal of Physiology-Renal Physiology, Kone and associates (6) provide evidence that begins to clarify this gap in knowledge and show that Sp1 is one of such positive regulators. Sp1, a promoter-specific factor required for transcription of the SV40 early and late promoters, was the first cloned mammalian transcription factor (2). Sp1 clearly has functional importance, since gene disruption in mice is lethal (3). Sp1, a candidate transcription factor, chosen based on sequence analysis of the α-ENaC promoter, binds to the α-ENaC promoter at a single, specific transcription factor binding site (a cis-element at +222/+229). Thus, Sp1 represents the first described constitutive driver of α-ENaC transcription, which also contributes to maximal aldosterone trans-activation of α-ENaC. Importantly, presented data complement the previous observations of this group. Additional experiments were done to study the role of Sp1 into the context of Dot1a-mediated repression of transcription or aldosterone-mediated induction of α-ENaC transcription. The authors reported that Sp1 acts independently from and overcomes the Dot1a-Af9 repressor complex (Fig. 1). Thus, the model and studies reflect the balance of genetic and epigenetic factors: Sp1 acting on the promoter DNA being the former, and Dot1 acting on histones being the latter. Surprisingly, the authors did not try to place in the global picture regulation of the transcription of ENaC genes by Af17, which competes with Af9 for binding to Dot1a to upregulate transcription of α-ENaC (4) and regulate ENaC-mediated sodium reabsorption and blood pressure (1). Fig. 1. Simplified model for the aldosterone-sensitive regulation of α-epithelial Na+ channel (ENaC) expression. Af9 directly binds +78/+92 of the α-ENaC promoter and recruits Dot1a to this position to basally repress α-ENaC transcription. ... This manuscript presents direct evidence of regulation of α-ENaC by Sp1 and highlights the need for further investigation of mechanisms that initiate or repress transcription of genes encoding ENaC subunits. A few questions are raised from the current study. First, this study is focused on Sp1, but it cannot exclude the possibility that other related factors also interact with the GAGGGCGT boxes of the α-ENaC promoter. Sp1 is part of a large family of proteins, including other Sp factors and the extensive set of Kruppel-like factors. Some of these factors are expressed and functionally important in the cardiorenal system. These factors might have a greater influence than Sp1, either as coactivators, or dominant-negative competitors with Sp1, like Af17, which competes with AF9 for binding to Dot1a (4). Similarly, it is not clear whether Sp1 plays a unique role in regulation of maximal aldosterone-dependent trans-activation of α-ENaC and how other factors like SGK1 modulate this regulation. Furthermore, current studies are performed in cultured mouse inner medullary collecting duct 3 cells in vitro. It would be nice if these findings will be further confirmed in vivo, similar to a recent study where the mice with connecting tubule/cortical collecting duct-specific inactivation of Dot1a revealed greater α-ENaC mRNA levels compared with control (10). Moreover, the functional correlation would improve the physiological significance of these molecular studies. Additional experiments are required to determine whether the Sp1 is functionally important in regulating ENaC or not by overexpressing or knocking down Sp1 and measuring a channel's activity. In conclusion, this study by Yu et al. (6) implicates a key role for the transcription factor Sp1 in regulation of ENaC. The authors propose a mechanism whereby endogenous Sp1 factor is functionally involved in conferring constitutive expression and aldosterone regulation of α-ENaC gene. Sp1 has the potential to be a critical point for understanding the physiological control of ENaC gene expression in the kidney. Importantly, this study is one of the first steps in understanding of genetic and epigenetic regulation of ENaC and emphasizes the need for further investigation of this important area of research.

Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

Modulation of αENaC mRNA expression under different stresses: Implication of its 3′UTR.

Active sodium transport by the epithelial Na+ channel (ENaC) is important for fluid movement across the alveolar epithelium in the lungs and for the resolution of pulmonary oedema in acute respiratory distress syndrome (ARDS). It was shown that cellular stress induced by lipopolysaccharides (LPS) or cycloheximide (Chx) downregulate αENaC mRNA via different molecular mechanisms. The purpose of this project was to investigate the importance of the αENaC 3′UTR in the mRNA modulation of ENaC. Alveolar epithelial cells were co‐transfected with 3′UTR mutants (V5/ENaC±3′UTR and Luc±3′UTR) inserted in pTRE‐tight vector along with the Tet‐Off vector. The cells were treated with Chx (1.0μM), LPS (15μg/mL) or Actinomycin D (5μg/mL) along with Dox (1μg/mL) to inhibit transcription. We found that 3′UTR deletion caused a ~60% decrease of luciferase activity and expression compared to the complete recombinant. The T1/2 of V5‐αENaC mRNA was 60 min and the deletion of 3′UTR reduced T1/2 to 37min. Chx reduced αENaC mRNA T1/2 to 33min whereas LPS had no effect. Actinomycin D, a transcription inhibitor, increased T1/2 >; 120min. These results suggest that the αENaC mRNA half‐life is shorter then previously estimated and that 3′UTR seems to be important for the stability of αENaC mRNA.Supported by FRSQ, CFC and CIHR.

Activation of the Heat Shock Response Attenuates the Interleukin 1β–Mediated Inhibition of the Amiloride-Sensitive Alveolar Epithelial Ion Transport

Acute lung injury (ALI) is a clinical syndrome characterized by hypoxia, which is caused by the breakdown of the alveolar capillary barrier. Interleukin 1β (IL-1β), a cytokine released within the airspace in ALI, downregulates the α subunit of the epithelial sodium channel (αENaC) transcription and protein expression via p38 MAP kinase-dependent signaling. Although induction of the heat shock response can restore alveolar fluid clearance compromised by IL-1β following the onset of severe hemorrhagic shock in rats, the mechanisms are not fully understood. In this study, we report that the induction of the heat shock response prevents IL-1β-dependent inhibition of αENaC mRNA expression and subsequent channel function. Heat shock results in IRAK1 detergent insolubility and a disruption of Hsp90 binding to IRAK1. Likewise, TAK1, another client protein of Hsp90 and signaling component of the IL-1β pathway, is also detergent insoluble after heat shock. Twenty-four hours after heat shock, both IRAK1 and TAK1 are again detergent soluble, which correlates with the IL-1β-dependent p38 activation. Remarkably, IL-1β-dependent p38 activation 24 h after heat shock did not result in an inhibition of αENaC mRNA expression and channel function. Further analysis demonstrates prolonged preservation of αENaC expression by the activation of the heat shock response that involves inducible Hsp70. Inhibition of Hsp70 at 24 h after heat shock results in p38-dependent IL-1β inhibition of αENaC mRNA expression, whereas overexpression of Hsp70 attenuates the p38-dependent IL-1β inhibition of αENaC mRNA expression. These studies demonstrate new mechanisms by which the induction of the heat shock response protects the barrier function of the alveolar epithelium in ALI.

Dexamethasone Regulates CFTR Expression in Calu-3 Cells with the Involvement of Chaperones HSP70 and HSP90

BackgroundDexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR). Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression.Methods and ResultsDose–response (1 nM to 10 µM) and time course (3 to 48 h) curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM) showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM) induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml) at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90) binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70) binding decreased 0.30 fold in an immunoprecipitation assay.ConclusionMature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene.

The epithelial Na(+) channel subunit-α (αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of αENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l(AC)) to determine the impact of Dot1l inactivation on renal αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.

Effects of simvastatin on lipopolysaccharide induced α-subunit epithelial sodium channel mRNA in rat lung alveolar type II epithelial cells

To study the impact of simvastatin on α-subunit epithelial sodium channel (α-ENaC) mRNA expression in primary culture alveolar typeII (ATII) epithelial cell of rats induced by lipopolysaccharide (LPS) in vitro. ATII of primary generation were isolated from adult Sprague-Dawley (SD) rats. The cells were randomly divided into five groups: blank control group, LPS injured group (final concentration of LPS 1 mg/L), simvastatin low and high concentration groups (final concentration of simvastatin 20 μmol/L, 30 μmol/L, respectively), solution control group. Then, after being intervened for 1, 12 and 24 hours, the level of human tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were monitored by enzyme-linked immunosorbent assay (ELISA), and α-ENaC mRNA expression was tested by reverse transcription-polymerase chain reaction (RT-PCR). After being intervened for 1, 12 and 24 hours, expressions of TNF-α and IL-1β in LPS injured group were obviously higher than those in blank control group. Expressions of TNF-α and IL-1β at 1, 12 and 24 hours in simvastatin low concentration group were significantly decreased compared with those in LPS injured group (TNF-α 1 hour: 1178.80±127.43 ng/L vs. 2336.00±170.04 ng/L, 12 hours: 1003.60±59.61 ng/L vs. 2479.80±210.41 ng/L, 24 hours: 695.80±25.24 ng/L vs. 1167.60±132.72 ng/L; IL-β 1 hour: 285.00±42.60 ng/L vs. 429.60±27.39 ng/L, 12 hours: 238.60±24.12 ng/L vs. 822.20±12.74 ng/L, 24 hours: 213.40±17.87 ng/L vs. 637.60±22.96 ng/L, all P<0.05). Expressions of TNF-α and IL-1β in high concentration group were decreased more obviously than those in low concentration group (TNF-α 1 hour: 965.60±24.45 ng/L vs. 1178.80±127.43 ng/L, 12 hours: 522.80±16.89 ng/L vs. 1003.60±59.61 ng/L, 24 hours: 252.40±17.64 ng/L vs. 695.80±25.24 ng/L; IL-1β 1 hour: 225.60±34.44 ng/L vs. 285.00±42.60 ng/L, 12 hours: 190.60±17.64 ng/L vs. 238.60±24.12 ng/L, 24 hours: 152.80±14.70 ng/L vs. 213.40±17.87 ng/L, all P<0.05), but increased compared with those in blank control group. After being intervened for 1 hour, no evident changes were observed in expression of α-ENaC mRNA in all groups. After being intervened for 12 hours and 24 hours, evident decrease in expression of α-ENaC mRNA (A value) was observed in LPS injured group compared with blank control group (12 hours: 0.211±0.021 vs. 0.496±0.027, 24 hours: 0.253±0.030 vs. 0.482±0.030, both P<0.05). Expressions of α-ENaC mRNA in simvastatin low concentration group evidently increased compared with those in LPS injured group (12 hours: 0.363±0.030 vs. 0.211±0.021, 24 hours: 0.309±0.024 vs. 0.253±0.030, both P<0.05). Expressions of α-ENaC mRNA in simvastatin high concentration group increased more obviously compared with those in low concentration group (12 hours: 0.413±0.034 vs. 0.363±0.030, 24 hours: 0.346±0.024 vs. 0.309±0.024, both P<0.05), but decreased compared with blank control group. No evident difference in expressions of all indexes in solution control group was observed compared with those in blank control group. High dose simvastatin could improve α-ENaC mRNA expression in primary culture ATII epithelial cells of rats. This may act by modulation the level of TNF-α and IL-1β.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1δ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC), which contributes to blood pressure regulation. Casein kinases 1δ and 1ε (CK1δ/ε) are critical regulators of clock proteins. CK1δ/ε must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1δ/ε inhibitor, PF670462, to test the effect of CK1δ/ε blockade and inhibited Per1 nuclear entry on αENaC in a model of the renal cortical collecting duct (mpkCCD(c14) cells). CK1δ/ε blockade prevented Per1 and Clock from interacting with an E-box from the αENaC promoter. CK1δ/ε inhibition reduced αENaC mRNA levels by <60%. A similar decrease in αENaC mRNA was observed following siRNA-mediated CK1δ/ε knock-down. Inhibition of CK1δ/ε effectively prevented the transcriptional response of αENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of αENaC. CK1δ/ε inhibition significantly reduced αENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1δ/ε inhibition. The present study shows for the first time that CK1δ/ε inhibition and impaired Per1 nuclear entry results in decreased αENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock‐regulatory kinases CKIδ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function. We previously showed that the circadian clock protein Per1 positively regulates the expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Casein kinase I is a critical regulator of clock proteins. In order to enter the nucleus, Per1 must be phosphorylated by casein kinase Iε (CKIε). Renal cortical collecting duct (mpkCCDc14) cells were treated with the CKIδ/ε inhibitor PF670462. DNA affinity purification assays showed that CKIδ/ε inhibition prevented Per1 from interacting with a target E‐box from the αENaC promoter. Steady state αENaC mRNA levels were reduced more than 60% following CKIδ/ε inhibition. CKIδ/ε inhibition significantly reduced αENaC protein levels. ENaC activity is tightly controlled by aldosterone and αENaC is a well‐known transcriptional target of aldosterone. Inhibition of CKIδ/ε effectively prevented the aldosterone response of αENaC, suggesting an interaction between Per1 and aldosterone‐mediated regulation of this gene. Importantly, single channel analysis in the amphibian renal cell line 2F3 demonstrated that CKIδ/ε inhibition resulted in decreased ENaC NPo. The present study shows for the first time that CKIδ/ε inhibition represses αENaC expression and ENaC activity, providing further evidence for direct control of ENaC by the circadian clock.

Loss of renal Nedd4‐2 in adult mice leads to PHAII compensated by ENaC down‐regulation and ROMK up‐regulation

Constitutive Nedd4‐2 knockout (KO) mice show Na+ retention and salt‐sensitive hypertension. To determine the role of renal Nedd4‐2 in adult mice, we generated inducible renal tubule‐specific Nedd4‐2 KO mice (Nedd4‐2Pax8/LC1) using a combination of the Tet‐On and Cre‐loxP systems. Under standard and high‐Na+ diets, KO displayed decreased plasma aldosterone but normal Na+/K+ balance. Under high Na+ diet, KO mice showed hypercalciuria and hypertension, both treated by thiazides. NCC expression and phosphorylation were increased in the KO. Interestingly, KO showed increased intracellular β‐ and γENaC abundance, but decreased α‐ and γENaC proteolytic cleavage and decreased αENaC mRNA and protein expression, suggesting down‐regulated ENaC. In contrast, ROMK protein expression and cell surface localization were increased in the distal convoluted tubules and connecting tubules of KO, in keeping with the absence of hyperkalemia. Thus, in adult mice, loss of renal Nedd4‐2 causes a pseudohypoaldosteronism type II‐like syndrome with NCC up‐regulation leading to salt‐sensitive hypertension and hypercalciuria, suggesting that Nedd4‐2 primarily targets NCC. The accompanying hypoaldosteronism with down‐regulation of ENaC‐mediated Na+ reabsorption together with an up‐regulation of K+ secretion via ROMK likely contributes to the compensated phenotype of the KO mice and explains the maintained Na+/K+ balance.

Investigation on Genetic Heterogeneity of α-Subunit of the Epithelial Sodium Channel in Rat Renal Cortex

Genotypic variability of αENaC mRNA in adult rat kidney caused by deletion at the 3'-end of α-subunit DNA was studied by real-time PCR using specific probes with a fluorescent dye (TaqMan). It was found that mRNA deletion forms (a and b), products of alternative splicing of aENaC gene, are absent in the cortex of adult rat kidney.

Effect of angiotensin II on expression of alveolar epithelial sodium channel in rat

To research the effect of exogenous angiotensin II (AngII) on alveolar fluid clearance (AFC) and alveolar epithelial sodium channel (ENaC) expression in rats. Fifteen healthy Sprague-Dawley (SD) rats were randomly divided into control group, AngII group and AngII type 1 (AT1) receptor blocker ZD7155 pretreatment group, with 5 rats in each group. Exogenous AngII 10 μg× kg(-1)×min(-1)was administered by micro pump via catheter in left jugular vein in AngII group and ZD7155 pretreatment group, whereas control group rats received only normal saline. ZD 7155 10 mg/kg was injected intraperitoneal 30 minutes before administration of exogenous AngII in ZD7155 pretreatment group. The pathological changes in lung were observed after 6 hours. AFC was estimated by Evans-blue labeled 5% albumin. The mRNA and protein expression of ENaC were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. AFC in AngII group was significantly lower than that of control group [(6.16 ± 3.01)% vs. (16.10 ± 3.46)%, P<0.01], and AFC in ZD7155 pretreatment group was significantly higher than that of AngII group [(10.60 ± 2.05)% vs. (6.16 ± 3.01)%, P<0.05]. α-ENaC mRNA expression was significantly increased in AngII group compared with control group (0.663 ± 0.068 vs. 0.236 ± 0.030, P<0.01), but significantly decreased in ZD7155 pretreatment group when compared with AngII group (0.386 ± 0.061 vs. 0.663 ± 0.068, P<0.01). There was no significant difference in β-ENaC and γ-ENaC mRNA expression among three groups. Compared with control group, α-ENaC protein was significantly increased in AngII group (0.343 ± 0.053 vs. 0.145 ± 0.030, P<0.01), but β-ENaC and γ-ENaC proteins were significantly decreased (β-ENaC: 0.286 ± 0.038 vs. 0.512 ± 0.055, γ-ENaC : 0.144 ± 0.040 vs. 0.460 ± 0.066, both P<0.01). Compared with AngII group, α-ENaC protein was significantly lower(0.228 ± 0.045 vs.0.343 ± 0.053, P<0.01), whereas β-ENaC and γ-ENaC proteins were significantly higher (β-ENaC: 0.358 ± 0.043 vs. 0.286 ± 0.038, γ-ENaC: 0.220 ± 0.033 vs. 0.144 ± 0.040, both P<0.05) in ZD7155 pretreatment group. Exogenous AngII modulates ENaC expression of gene and protein by AT1 receptor pathway, attenuates AFC, and aggravates lung edema.

Localization of ENaC subunit mRNAs in adult bullfrog skin

Adult amphibian skin has served as a model for the investigation of Na+-transporting epithelia, such as mammalian renal tubules. The amiloride-blockable epithelial Na+ channel (ENaC), which is located in the apical membrane of the outer living cell layer, regulates Na+ transport across the epithelium. ENaC is thought to develop during the terminal differentiation of epidermal cells, but the details are unclear. Here, we used in situ hybridization to examine the localization of the ENaC subunit mRNAs in skin of adult bullfrogs, to clarify the development of ENaC. We found that α-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while β-ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of γ-ENaC mRNA, possibly for technical reasons. α- and β-ENaC mRNAs, at least, were present in the sub-apical cells, in which ENaC protein is not necessary for amphibian skin to possess its Na+-transport function. Our results may mean that the sub-apical cells are already producing the ENaC subunit mRNAs prior to the final step in their differentiation.

Lung fluid absorption is induced in preterm guinea pigs ventilated with low tidal volumes

ABSTRACTThe objective of this study was to determine if low tidal volume (Vt) ventilation was beneficial when ventilating preterm fetuses. The authors ventilated preterm guinea pig fetuses at gestation day (GD) 67, 3 days before birth, newborn, and 10-day-old (PD10) guinea pigs with low Vt (6 mL/kg body weight [bw]) and compared them to age-matched fetuses/animals ventilated with higher potentially injurious Vt (12 mL/kg bw). Lung fluid absorption was measured after intratracheal instillation of 5% albumin in 0.9% NaCl. Low Vt ventilation stimulated lung fluid absorption when compared to higher Vt in all groups. The increased lung fluid absorption in low Vt–ventilated fetuses was associated with increased α epithelial Na channel (αEnaC) mRNA. However, αENaC and βENaC protein was unchanged over the 1-hour study. Because stretch induces mitogen-activated protein (MAP) kinase expression and MAP kinases may affect lung fluid absorption, the authors investigated if MAP kinase (MAPK) expression was affected by Vt. Extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK) were phosphorylated in the higher Vt–ventilated guinea pig fetuses. This suggested that a reduced activation of MAP kinases might explain the increased lung fluid absorption in the low Vt–ventilated fetuses. Thus these data suggest that low Vt ventilation increases fetal lung fluid absorption and thus may be preferential to use clinically.