Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells.

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. METHODS: mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. RESULTS: Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. CONCLUSION: Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

Ferulic acid ameliorates lipopolysaccharide-induced tracheal injury via cGMP/PKGII signaling pathway.

BACKGROUND: Tracheal injury is a common clinical condition that still lacks an effective therapy at present. Stimulation of epithelial sodium channel (ENaC) increases Na+ transport, which is a driving force to keep tracheal mucosa free edema fluid during tracheal injury. Ferulic acid (FA) has been proved to be effective in many respiratory diseases through exerting anti-oxidant, anti-inflammatory, and anti-thrombotic effects. However, these studies rarely involve the level of ion transport, especially ENaC. METHODS: C57BL/J male mice were treated intraperitoneally with normal saline or FA (100 mg/kg) 12 h before, and 12 h after intratracheal administration of lipopolysaccharide (LPS, 5 mg/kg), respectively. The effects of FA on tracheal injury were not only assessed through HE staining, immunofluorescence assay, and protein/mRNA expressions of ENaC located on tracheas, but also evaluated by the function of ENaC in mouse tracheal epithelial cells (MTECs). Besides, to explore the detailed mechanism about FA involved in LPS-induced tracheal injury, the content of cyclic guanosine monophosphate (cGMP) was measured, and Rp-cGMP (cGMP inhibitor) or cGMP-dependent protein kinase II (PKGII)-siRNA (siPKGII) were applied in primary MTECs, respectively. RESULTS: Histological examination results demonstrated that tracheal injury was obviously attenuated by pretreatment of FA. Meanwhile, FA could reverse LPS-induced reduction of both protein/mRNA expressions and ENaC activity. ELISA assay verified cGMP content was increased by FA, and administration of Rp-cGMP or transfection of siPKGII could reverse the FA up-regulated ENaC protein expression in MTECs. CONCLUSIONS: Ferulic acid can attenuate LPS-induced tracheal injury through up-regulation of ENaC at least partially via the cGMP/PKGII pathway, which may provide a promising new direction for preventive and therapeutic strategy in tracheal injury.

DNA Methylation Patterns Correlate with the Expression of SCNN1A, SCNN1B, and SCNN1G (Epithelial Sodium Channel, ENaC) Genes.

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype-phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, ß and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5'-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.

Lipopolysaccharide Inhibits Alpha Epithelial Sodium Channel Expression via MiR-124-5p in Alveolar Type 2  Epithelial Cells.

Mesenchymal stem cells (MSCs) have been a potential strategy in the pretreatment of pulmonary diseases, while the mechanisms of MSCs-conditioned medium (MSCs-CM) involved with microRNAs on the regulation of lung ion transport are seldom reported. We investigated the role of miR-124-5p in lipopolysaccharide-involved epithelial sodium channel (ENaC) dysfunction and explored the potential target of miR-124-5p. We observed the lower expression of miR-124-5p after the administration of MSCs-CM, and the overexpression or inhibition of miR-124-5p regulated epithelial sodium channel α-subunit (α-ENaC) expression at protein levels in mouse alveolar type 2 epithelial (AT2) cells. We confirmed that α-ENaC is one of the target genes of miR-124-5p through dual luciferase assay and Ussing chamber assay revealed that miR-124-5p inhibited amiloride-sensitive currents associated with ENaC activity in intact H441 monolayers. Our results demonstrate that miR-124-5p can decrease the expression and function of α-ENaC in alveolar epithelial cells by targeting the 3'-UTR. The involvement of MSCs-CM in lipopolysaccharide-induced acute lung injury cell model could be related to the downregulation of miR-124-5p on α-ENaC, which may provide a new target for the treatment of acute lung injury.

Molecular Targets of Natriuretic Action of Prolactin in the Rat Model of Cholestasis of Pregnancy.

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.

Upregulation of SCNN1A Promotes Cell Proliferation, Migration, and Predicts Poor Prognosis in Ovarian Cancer Through Regulating Epithelial-Mesenchymal Transformation.

Background: There is little knowledge about the biological roles and clinical significance of SCNN1A in ovarian cancer. Thus, the objective of this study was to investigate the biological functions and prognosis value of SCNN1A in ovarian cancer to further seek a potential therapeutic target for patients with ovarian cancer. Materials and Methods: First, the expression level of SCNN1A in ovarian cancer samples obtained from ONCOMINE database was determined, and its expression in cell lines was also investigated. Moreover, correlation analysis was performed to determine the relationship between SCNN1A expression and prognosis in ovarian cancer patients according to the data obtained from GEPIA database and Kaplan-Meier plotter website. The biological roles of SCNN1A on cell growth, migration, and invasion were then examined by knockdown of SCNN1A in ovarian cancer cell line SK-OV-3. Ultimately, Western blotting analysis was carried out to investigate the expression of epithelial-mesenchymal transformation markers (including E-cadherin, N-cadherin, Vimentin, and Snail) after silencing SCNN1A. Results: Based on the ONCOMINE-related data and cell lines, SCNN1A was observed to be overexpressed in ovarian cancer samples and cell lines. Survival analysis showed that high expression of SCNN1A was associated with poor overall survival and progression-free survival in ovarian cancer patients. In addition, SCNN1A silence remarkably blocked SK-OV-3 cell growth ability, migration, and invasion potential. Western blotting results showed that SCNN1A silence led to an increase in E-cadherin, whereas a decrease in N-cadherin, Vimentin, and Snail in SK-OV-3 cells. Increased E-cadherin and decreased N-cadherin, Vimentin, as well as Snail inhibited cell invasion of ovarian cancer. Conclusions: SCNN1A might exert crucial roles in cell growth and invasion and migration in ovarian cancer, and might be a potential indicator for prognosis and a therapeutic target for ovarian cancer patients.

Post-Transcriptional Modulation of aENaC mRNA in Alveolar Epithelial Cells: Involvement of its 3' Untranslated Region.

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further. METHODS: Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro. RESULTS: First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR. CONCLUSION: Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells.

(Pro)Renin receptor mediates obesity-induced antinatriuresis and elevated blood pressure via upregulation of the renal epithelial sodium channel.

Recent studies have demonstrated that the renal (pro)renin receptor (PRR) regulates expression of the alpha subunit of the epithelial sodium channel (α-ENaC). In this study we hypothesized that the renal PRR mediates high fat diet (HFD)-induced sodium retention and elevated systolic blood pressure (SBP) by enhancing expression of the epithelial sodium channel (α-ENaC). In our study we used a recently developed inducible nephron specific PRR knockout mouse. Mice (n = 6 each group) were allocated to receive regular diet (RD, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 10 weeks. Body weight (BW), SBP, urine volume (UV) and urine sodium (UNaV), as well as renal interstitial Angiotensin II (Ang II), and renal medullary expression of PRR, p-SGK-1, α-ENaC were monitored in RD and HFD mice with or without PRR knockout. At baseline, there were no significant differences in BW, BP, UV or UNaV between different animal groups. At the end of the study, HFD mice had significant increases in SBP, BW, and significant reductions in UV and UNaV. Compared to RD, HFD significantly increased mRNA and protein expression of PRR, α-ENaC, p-SGK-1, and Ang II. Compared to HFD alone, PRR knockout mice on HFD had reduced mRNA and protein expression of PRR, p-SGK-1, and α-ENaC, as well as increased UV, UNaV and significantly reduced SBP. RIF Ang II was significantly increased by HFD and did not change in response to PRR knockout. We conclude that obesity induced sodium retention and elevated SBP are mediated by the PRR-SGK-1- α-ENaC pathway independent of Ang II.

Sodium Channel Subunit SCNN1B Suppresses Gastric Cancer Growth and Metastasis via GRP78 Degradation.

There remains a paucity of functional biomarkers in gastric cancer. Here, we report the identification of the sodium channel subunit SCNN1B as a candidate biomarker in gastric cancer. SCNN1B mRNA expression was silenced commonly by promoter hypermethylation in gastric cancer cell lines and primary tumor tissues. Tissue microarray analysis revealed that high expression of SCNN1B was an independent prognostic factor for longer survival in gastric cancer patients, especially those with late-stage disease. Functional studies demonstrated that SCNN1B overexpression was sufficient to suppress multiple features of cancer cell pathophysiology in vitro and in vivo Mechanistic investigations revealed that SCNN1B interacted with the endoplasmic reticulum chaperone, GRP78, and induced its degradation via polyubiquitination, triggering the unfolded protein response (UPR) via activation of PERK, ATF4, XBP1s, and C/EBP homologous protein and leading in turn to caspase-dependent apoptosis. Accordingly, SCNN1B sensitized gastric cancer cells to the UPR-inducing drug tunicamycin. GRP78 overexpression abolished the inhibitory effect of SCNN1B on cell growth and migration, whereas GRP78 silencing aggravated growth inhibition by SCNN1B. In summary, our results identify SCNN1B as a tumor-suppressive function that triggers UPR in gastric cancer cells, with implications for its potential clinical applications as a survival biomarker in gastric cancer patients. Cancer Res; 77(8); 1968-82. ©2017 AACR.

Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of ß-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

[Expression of Catechol-O-Methyltransferase (Comt), Mineralocorticoid Receptor (Mlr), and Epithelial Sodium Channel (ENaC) Genes in Kidneys of Hypertensive ISIAH Rats at Rest and during Response to Stress].

Emotional stress plays a significant role in the processes of the development of arterial hypertension, especially in the presence of genetic predisposition. The origin and maintenance of hypertensive status during stress development can be activated by the sympathetic nervous system. An increase in sympathetic stimulation can, in turn, result in a change in the functions of kidneys, which provide fluid and electrolyte balance of the organism. A comparative study of the mRNA expression level of catechol-o-methyltransferase (Comt), mineralocorticoid receptor (Mlr), and ß-subunit of epithelial sodium channel (ß-ENaC) genes was conducted on the kidneys of hypertensive ISIAH rats and normotensive WAG rats at rest and after the effect of emotional stress. The discovered changes in the expression level of the selected genes confirm their involvement in increased sympathetic stimulation of the kidney, along with changes in the function of kidney regulation of fluid and electrolyte balance, which is an important factor of the development of sustained hypertension in the ISIAH rats strain.

1,25-Dihydroxyvitamin D Enhances Alveolar Fluid Clearance by Upregulating the Expression of Epithelial Sodium Channels.

Vitamin D is implicated in the pathogenesis of asthma, acute lung injury, and other respiratory diseases. 1,25-Dihydroxyvitamin D (1,25(OH)2D3), the hormonal form of vitamin D, has been shown to reduce vascular permeability and ameliorate lung edema. Therefore, we speculate that 1,25(OH)2D3 may regulate alveolar Na(+) transport via targeting epithelial Na(+) channels (ENaC), a crucial pathway for alveolar fluid clearance. In vivo total alveolar fluid clearance was 39.4 ± 3.8% in 1,25(OH)2D3-treated mice, significantly greater than vehicle-treated controls (24.7 ± 1.9 %, n = 10, p < 0.05). 1,25(OH)2D3 increased amiloride-sensitive short-circuit currents in H441 monolayers, and whole-cell patch-clamp data confirmed that ENaC currents in single H441 cell were enhanced in 1,25(OH)2D3-treated cells. Western blot showed that the expression of α-ENaC was significantly elevated in 1,25(OH)2D3-treated mouse lungs and 1,25(OH)2D3-treated H441 cells. These observations suggest that vitamin D augments transalveolar fluid clearance, and vitamin D therapy may potentially be used to ameliorate pulmonary edema.

Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.

The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αßγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.

Reducing αENaC expression in the kidney connecting tubule induces pseudohypoaldosteronism type 1 symptoms during K+ loading.

Genetic inactivation of the epithelial Na(+) channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na(+) and K(+) homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) symptoms in subjects already on a standard diet. In the present study, we further examined the importance of αENaC in the CNT. Knockout mice with αENaC deleted primarily in a part of the CNT (CNT-KO) were generated using Scnn1a(lox/lox) mice and Atp6v1b1::Cre mice. With a standard diet, plasma Na(+) concentration ([Na(+)]) and [K(+)], and urine Na(+) and K(+) output were unaffected. Seven days of Na(+) restriction (0.01% Na(+)) led to a higher urine Na(+) output only on days 3-5, and after 7 days plasma [Na(+)] and [K(+)] were unaffected. In contrast, the CNT-KO mice were highly susceptible to a 2-day 5% K(+) diet and showed lower food intake and relative body weight, lower plasma [Na(+)], higher fractional excretion (FE) of Na(+), higher plasma [K(+)], and lower FE of K(+). The higher FE of Na(+) coincided with lower abundance and phosphorylation of the Na(+)-Cl(-) cotransporter. In conclusion, reducing ENaC expression in the CNT induces clear PHA-1 symptoms during high dietary K(+) loading.

Chronic Hypoxemia in Children With Congenital Heart Defect Impairs Airway Epithelial Sodium Transport.

OBJECTIVE: Ambient hypoxia impairs the airway epithelial Na transport, which is crucial in lung edema reabsorption. Whether chronic systemic hypoxemia affects airway Na transport has remained largely unknown. We have therefore investigated whether chronic systemic hypoxemia in children with congenital heart defect affects airway epithelial Na transport, Na transporter-gene expression, and short-term lung edema accumulation. DESIGN: Prospective, observational study. SETTING: Tertiary care medical center responsible for nationwide pediatric cardiac surgery. PATIENTS: Ninety-nine children with congenital heart defect or acquired heart disease (age range, 6 d to 14.8 yr) were divided into three groups based on their level of preoperative systemic hypoxemia: 1) normoxemic patients (SpO2% ≥ 95%; n = 44), 2) patients with cyanotic congenital heart defect and moderate hypoxemia (SpO2 86-94%; n = 16), and 3) patients with cyanotic congenital heart defect and profound systemic hypoxemia (SpO2 ≤ 85%; n = 39). MEASUREMENTS AND MAIN RESULTS: Nasal transepithelial potential difference served as a surrogate measure for epithelial Na transport of the respiratory tract. Profoundly hypoxemic patients had 29% lower basal nasal transepithelial potential difference (p = 0.02) and 55% lower amiloride-sensitive nasal transepithelial potential difference (p = 0.0003) than normoxemic patients. In profoundly hypoxemic patients, nasal epithelial messenger RNA expressions of two airway Na transporters (amiloride-sensitive epithelial Na channel and ß1- Na-K-ATPase) were not attenuated, but instead α1-Na-K-ATPase messenger RNA levels were higher (p = 0.03) than in the normoxemic patients, indicating that posttranscriptional factors may impair airway Na transport. The chest radiograph lung edema score increased after congenital cardiac surgery in profoundly hypoxemic patients (p = 0.0004) but not in patients with normoxemia or moderate hypoxemia. CONCLUSIONS: The impaired airway epithelial amiloride-sensitive Na transport activity in profoundly hypoxemic children with cyanotic congenital heart defect may hinder defense against lung edema after cardiac surgery.

Intracellular Na+ regulates epithelial Na+ channel maturation.

Epithelial Na(+) channel (ENaC) function is regulated by the intracellular Na(+) concentration ([Na(+)]i) through a process known as Na(+) feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na(+)]i alters ENaC cleavage. We show here that [Na(+)]i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na(+)]i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na(+)]i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na(+)]i. The hypothesis that [Na(+)]i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na(+)]i. Therefore, increased [Na(+)]i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Electroporation-mediated gene delivery of Na+,K+ -ATPase, and ENaC subunits to the lung attenuates acute respiratory distress syndrome in a two-hit porcine model.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a common cause of organ failure with an associated mortality rate of 40%. The initiating event is disruption of alveolar-capillary interface causing leakage of edema into alveoli. HYPOTHESIS: Electroporation-mediated gene delivery of epithelial sodium channel (ENaC) and Na+,K+ -ATPase into alveolar cells would improve alveolar clearance of edema and attenuate ARDS. METHODS: Pigs were anesthetized and instrumented, and the superior mesenteric artery was clamped to cause gut ischemia/reperfusion injury and peritoneal sepsis by fecal clot implantation. Animals were ventilated according to ARDSnet protocol. Four hours after injury, animals were randomized into groups: (i) treatment: Na+,K+ -ATPase/ENaC plasmid (n = 5) and (ii) control: empty plasmid (n = 5). Plasmids were delivered to the lung using bronchoscope. Electroporation was delivered using eight-square-wave electric pulses across the chest. Following electroporation, pigs were monitored 48 h. RESULTS: The Pao2/Fio2 ratio and lung compliance were higher in the treatment group. Lung wet/dry ratio was lower in the treatment group. Relative expression of the Na+,K+ -ATPase transgene was higher throughout lungs receiving treatment plasmids. Quantitative histopathology revealed a reduction in intra-alveolar fibrin in the treatment group. Bronchoalveolar lavage showed increased surfactant protein B in the treatment group. Survival was improved in the treatment group. CONCLUSIONS: Electroporation-mediated transfer of Na+,K+ -ATPase/ENaC plasmids improved lung function, reduced fibrin deposits, decreased lung edema, and improved survival in a translational porcine model of ARDS. Gene therapy can attenuate ARDS pathophysiology in a high-fidelity animal model, suggesting a potential new therapy for patients.

Collecting duct-specific knockout of renin attenuates angiotensin II-induced hypertension.

The physiological and pathophysiological significance of collecting duct (CD)-derived renin, particularly as it relates to blood pressure (BP) regulation, is unknown. To address this question, we generated CD-specific renin knockout (KO) mice and examined BP and renal salt and water excretion. Mice containing loxP-flanked exon 1 of the renin gene were crossed with mice transgenic for aquaporin-2-Cre recombinase to achieve CD-specific renin KO. Compared with controls, CD renin KO mice had 70% lower medullary renin mRNA and 90% lower renin mRNA in microdissected cortical CD. Urinary renin levels were significantly lower in KO mice (45% of control levels) while plasma renin concentration was significantly higher in KO mice (63% higher than controls) during normal-Na intake. While no observable differences were noted in BP between the two groups with varying Na intake, infusion of angiotensin II at 400 ng·kg(-1)·min(-1) resulted in an attenuated hypertensive response in the KO mice (mean arterial pressure 111 ± 4 mmHg in KO vs. 128 ± 3 mmHg in controls). Urinary renin excretion and epithelial Na(+) channel (ENaC) remained significantly lower in the KO mice following ANG II infusion compared with controls. Furthermore, membrane-associated ENaC protein levels were significantly lower in KO mice following ANG II infusion. These findings suggest that CD renin modulates BP in ANG II-infused hypertension and these effects are associated with changes in ENaC expression.

Sodium transport is modulated by p38 kinase-dependent cross-talk between ENaC and Na,K-ATPase in collecting duct principal cells.

In relation to dietary Na(+) intake and aldosterone levels, collecting duct principal cells are exposed to large variations in Na(+) transport. In these cells, Na(+) crosses the apical membrane via epithelial Na(+) channels (ENaC) and is extruded into the interstitium by Na,K-ATPase. The activity of ENaC and Na,K-ATPase must be highly coordinated to accommodate variations in Na(+) transport and minimize fluctuations in intracellular Na(+) concentration. We hypothesized that, independent of hormonal stimulus, cross-talk between ENaC and Na,K-ATPase coordinates Na(+) transport across apical and basolateral membranes. By varying Na(+) intake in aldosterone-clamped rats and overexpressing γ-ENaC or modulating apical Na(+) availability in cultured mouse collecting duct cells, enhanced apical Na(+) entry invariably led to increased basolateral Na,K-ATPase expression and activity. In cultured collecting duct cells, enhanced apical Na(+) entry increased the basolateral cell surface expression of Na,K-ATPase by inhibiting p38 kinase-mediated endocytosis of Na,K-ATPase. Our results reveal a new role for p38 kinase in mediating cross-talk between apical Na(+) entry via ENaC and its basolateral exit via Na,K-ATPase, which may allow principal cells to maintain intracellular Na(+) concentrations within narrow limits.