ENaC Expression Research Articles

Tempol Alters Urinary Extracellular Vesicle Lipid Content and Release While Reducing Blood Pressure during the Development of Salt-Sensitive Hypertension.

Salt-sensitive hypertension resulting from an increase in blood pressure after high dietary salt intake is associated with an increase in the production of reactive oxygen species (ROS). ROS are known to increase the activity of the epithelial sodium channel (ENaC), and therefore, they have an indirect effect on sodium retention and increasing blood pressure. Extracellular vesicles (EVs) carry various molecules including proteins, microRNAs, and lipids and play a role in intercellular communication and intracellular signaling in health and disease. We investigated changes in EV lipids, urinary electrolytes, osmolality, blood pressure, and expression of renal ENaC and its adaptor protein, MARCKS/MARCKS Like Protein 1 (MLP1) after administration of the antioxidant Tempol in salt-sensitive hypertensive 129Sv mice. Our results show Tempol infusion reduces systolic blood pressure and protein expression of the alpha subunit of ENaC and MARCKS in the kidney cortex of hypertensive 129Sv mice. Our lipidomic data show an enrichment of diacylglycerols and monoacylglycerols and reduction in ceramides, dihydroceramides, and triacylglycerols in urinary EVs from these mice after Tempol treatment. These data will provide insight into our understanding of mechanisms involving strategies aimed to inhibit ROS to alleviate salt-sensitive hypertension.

ANKRD36 Is Involved in Hypertension by Altering Expression of ENaC Genes.

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Conditioned Medium of Bone Marrow Mesenchymal Stem Cells Involved in Acute Lung Injury by Regulating Epithelial Sodium Channels via miR-34c

BackgroundOne of the characteristics of acute lung injury (ALI) is severe pulmonary edema, which is closely related to alveolar fluid clearance (AFC). Mesenchymal stem cells (MSCs) secrete a wide range of cytokines, growth factors, and microRNA (miRNAs) through paracrine action to participate in the mechanism of pulmonary inflammatory response, which increase the clearance of edema fluid and promote the repair process of ALI. The epithelial sodium channel (ENaC) is the rate-limiting step in the sodium–water transport and edema clearance in the alveolar cavity; the role of bone marrow-derived MSC-conditioned medium (BMSC-CM) in edema clearance and how miRNAs affect ENaC are still seldom known.MethodsCCK-8 cell proliferation assay was used to detect the effect of BMSC-CM on the survival of alveolar type 2 epithelial (AT2) cells. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of ENaC in AT2 cells. The effects of miR-34c on lung fluid absorption were observed in LPS-treated mice in vivo, and the transepithelial short-circuit currents in the monolayer of H441 cells were examined by the Ussing chamber setup. Dual luciferase reporter gene assay was used to detect the target gene of miR-34c.ResultsBMSC-CM could increase the viability of mouse AT2 cells. RT-PCR and western blot results showed that BMSC-CM significantly increased the expression of the γ-ENaC subunit in mouse AT2 cells. MiR-34c could restore the AFC and lung wet/dry weight ratio in the ALI animal model, and Ussing chamber assay revealed that miR-34c enhanced the amiloride-sensitive currents associated with ENaC activity in intact H441 cell monolayers. In addition, we observed a higher expression of miR-34c in mouse AT2 cells administrated with BMSC-CM, and the overexpression or inhibition of miR-34c could regulate the expression of ENaC protein and alter the function of ENaC. Finally, we detected that myristoylated alanine-rich C kinase substrate (MARCKS) may be one of the target genes of miR-34c.ConclusionOur results indicate that BMSC-CM may alleviate LPS-induced ALI through miR-34c targeting MARCKS and regulate ENaC indirectly, which further explores the benefit of paracrine effects of bone marrow-derived MSCs on edematous ALI.

Fluoride effects on cell viability and ENaC expression in kidney epithelial cells

Fluoride (F) at micromolar (µM) concentrations induces apoptosis in several cell lines. Moreover, proteomic studies have shown major changes in the profile of proteins involved in signal transduction. These effects may negatively affect ion transport in the kidneys. The activity of epithelial sodium channels (ENaCs) is a limiting factor for sodium and water resorption in the kidneys, which is essential for the maintenance of the electrolyte balance and homeostasis of the body. Here we investigated the effects of F, at different concentrations (10, 40, 100, 200, and 400 μM), on the viability of renal epithelial cells (M-1), and ENaC expression. We showed that sodium fluoride (NaF) reduces cell viability in a concentration-dependent manner (p < 0.05) up to a 96-h time-point when compared to control. Sodium fluoride at moderate concentrations (100 and 200 μM), upregulated the ENaC subunit genes Scnn1a and Scnn1g, but not Scnn1b. Sodium fluoride downregulated all three ENaC subunit genes at a higher concentration of 400 μM (p < 0.05). Immunofluorescence analysis showed that Scnn1a and Scnn1g expression was decreased within 24 h of NaF treatment. After 48 h, NaF (400 μM) increased the expression of Scnn1a but not Scnn1g. However, NaF decreased the expression of Scnn1g at all studied concentrations. We conclude that F, at µM concentrations, modulates the expression of ENaC subunit genes, which is likely to significantly affect molecular signaling in kidney epithelial cells.

New generation ENaC inhibitors detach cystic fibrosis airway mucus bundles via sodium/hydrogen exchanger inhibition

Cystic fibrosis (CF) is a recessive inherited disease caused by mutations affecting anion transport by the epithelial ion channel cystic fibrosis transmembrane conductance regulator (CFTR). The disease is characterized by mucus accumulation in the airways and intestine, but the major cause of mortality in CF is airway mucus accumulation, leading to bacterial colonization, inflammation and respiratory failure. Several drug targets are under evaluation to alleviate airway mucus obstruction in CF and one of these targets is the epithelial sodium channel ENaC. To explore effects of ENaC inhibitors on mucus properties, we used two model systems to investigate mucus characteristics, mucus attachment in mouse ileum and mucus bundle transport in piglet airways. We quantified mucus attachment in explants from CFTR null (CF) mice and tracheobronchial explants from newborn CFTR null (CF) piglets to evaluate effects of ENaC or sodium/hydrogen exchanger (NHE) inhibitors on mucus attachment. ENaC inhibitors detached mucus in the CF mouse ileum, although the ileum lacks ENaC expression. This effect was mimicked by two NHE inhibitors. Airway mucus bundles were immobile in untreated newborn CF piglets but were detached by the therapeutic drug candidate AZD5634 (patent WO, 2015140527). These results suggest that the ENaC inhibitor AZD5634 causes detachment of CF mucus in the ileum and airway via NHE inhibition and that drug design should focus on NHE instead of ENaC inhibition.

G Protein‐Coupled Receptor Kinase 4 (GRK4) Variant 65L‐Mediated Impairment of Dopamine D1 Receptor Function causes Hypertension

Salt sensitivity is associated with impaired dopamine receptor function, which is due, in part, to its desensitization by constitutively active GRK4γ variants. While GRK4 is essential for the normal desensitization and resensitization of dopamine receptors, certain GRK4 polymorphisms desensitize and internalize the dopamine D1 (D1R), thus impairing its natriuretic function. Activating variants of the human GRK4 (65R>L, 142A>V, and 486A>V) are involved in the desensitization of the D1R and are associated with hypertension in several ethnic groups. Furthermore, transgenic mice expressing the GRK4 65L are hypertensive when fed a high salt diet. We have reported that global transgenic mice expressing hGRK4 65L have salt-sensitive hypertension. To avoid the effect of protein overexpression in these transgenic mice, we generated mice globally expressing hGRK4 65L by crispr-cas9-mediated genome editing in C57Bl/6 mice (hGRK4 65L GE). Systolic blood pressure (SBP) in these mice under pentobarbital anesthesia was higher (126±2 vs 103±1 mmHg, n=3/group, P<0.05) than in their littermates not expressing the human gene (GE WT). Renal D1R expression was slightly but not significantly lower (100 ± 2 vs 85 ± 4%, n=3/group) but D1R phosphorylation was higher in hGRK4 65L GE than GE WT (112±2 vs 100±1%, n=3/group, P<0.05). Renal ENaC and NHE3 expressions were similar in both genotypes. To determine the role of the kidney in the salt sensitivity of these mice, we generated mice with kidney-specific (KS) expression of the GRK4 65L (KS hGRK4 65L) by the bilateral ureteral infusion of adeno-associated virus (AAV) vectors carrying hGRK4 65L in GRK4 knockout mice. Mice infused with hGRK4 wild type served as controls (KS hGRK4 WT). SBP (under anesthesia) before AAV was similar in both groups. SBP post AAV increased in KS hGRK4 65L (93±1 vs 117±4 mm Hg, P<0.05, n=4) but not in KS hGRK4 WT (96±2 vs 105±6, n=5). Renal cortical expression of D1R (63±1 vs 100±4 % n=5, P<0.01) was lower but D1R phosphorylation was higher (170 ± 10 vs 100 ±15%; n=5, P<0.02) in KS hGRK4 65L than KS hGRK4 WT. In addition, the renal expression of ENaC (1.4±0.14 vs 1.0±0.11, P<0.05) was increased KS hGRK4 65L but NHE3 was similar in both groups. We also studied human renal proximal tubule cells (hRPTCs) endogenously carrying GRK4 wild type or GRK4 65L. In hRPTCs with GRK4 65L, relative to GRK4 WT, D1R expression (599±80 vs 100±8 % n=6/group; P<0.05), as well as D1R phosphorylation (204 ± 22 vs 100±24%, n=6/group; P<0.05), were increased. Our results show across mouse models and hRPTCs that the presence of the GRK4 65L results in increased D1R phosphorylation, which impairs D1R function for normal renal sodium handling and increases blood pressure in GRK4 65L mice. These results show the important role of renal D1R in the regulation of blood pressure.

106 Maternal recognition of pregnancy (MRP) and mares: Is the epithelial sodium channel (ENaC) involved?

Maternal recognition of pregnancy (MRP) describes the prevention of luteolysis via the early conceptus. By prolonging the presence of the corpus luteum (CL), the conceptus ensures an environment capable of supporting pregnancy. In mares, MRP is enigmatic; the horse is one of few domesticated species wherein the MRP signal has yet to be discovered. Equine conceptus mobility throughout the uterine horns, termed migration, is known to be essential for pregnancy establishment. However, while the MRP signal in several other species is conceptus-derived, such a signal has yet to be observed from the equine embryo. We hypothesize that the migration of the equine conceptus itself activates MRP, potentially through epithelial sodium channel (ENaC). ENaC has been previously detected in multiple equine organs, observed in the uteri of various mammalian species, and also found to be upregulated during preimplantation in mice. However, neither the presence of ENaC within the mare uterus nor its potential involvement with the equine pregnancy has been researched. The aim of this study was 1) to determine if ENaC was present within the endometrium of the mare uterus and 2) to describe the location of ENaC if observed. Estrus synchronization was completed in mares (n = 14) during peak breeding season. Mares were subsequently blocked by age and breed and then placed into one of 2 groups: control mares (C; n = 7) and treatment mares (T; n = 7). Transrectal ultrasonography (US) was completed in all mares daily when one or more ovarian follicles reached 25 mm. When a mare had a 35 mm or larger follicle present, she received 2,000 international units of human chorionic gonadotropin intramuscularly to induce ovulation (OV) and US continued daily. Additionally, T mares were artificially inseminated (AI) with fresh, extended semen every day until the day after OV and were subsequently checked for pregnancy 12 d post-OV. C mares had the cervix manually manipulated the day after OV to replicate physical stimulation that occurs to the cervix when AI; however, no semen was deposited in C mare reproductive tracts. Endometrial biopsies were collected from all mares before OV, after OV, and during early pregnancy (when applicable) and samples were then processed for ENaC immunohistochemistry analysis. ENaC protein was immunodetected in all C and T mare endometrial samples and was localized to both uterine luminal epithelium and uterine glands. While further analysis will be conducted to quantify endometrial ENaC expression in C versus T (open versus pregnant) mares, the present histological data confirms that ENaC is present within the equine uterus.

The Influence of the Epithelial Sodium Channel in Breast Cancer Cells

Breast cancer is the most common cancer in women worldwide and leads to over half a million deaths annually. Most breast cancer deaths can be attributed to metastasis, in which the cancerous cells spread from the primary site in the breast tissue to other organs throughout the body. Thus, researching how breast cancer cells metastasise is vital. Ion channels, such as the epithelial sodium channel (ENaC) are an emerging area within cancer research due to their possible role in regulating cancer cell functions; such as epithelial- mesenchymal transition (EMT) which involves the cell undergoing phenotype changes before metastasising. Our research aims to investigate the influence of ENaC on breast cancer cell migration, EMT, invasion and metastasis. We hypothesise that high ENaC expression enables the cell to maintain a more epithelial and less migratory phenotype. We have shown mesenchymal cell lines have lower mRNA expression of alpha-ENaC and similarly less alpha-ENaC protein being expressed in mesenchymal cell lines compared to epithelial when examined using western blotting. Using Boyden chamber assays, an established model for cell migration, the influence of overexpressing ENaC subunits on the cells’ ability to migrate was investigated in two mesenchymal breast cancer cell lines, MDAMB231 and BT549. The overexpression of ENaC resulted in a reduction in the number of cells migrating through the Boyden chamber, suggesting a reduced migration ability compared to control cells. These results suggest ENaC has a role in the migration of breast cancer cells and highlights ENaC as a potential target in the future development of breast cancer therapies.

EVIDENCE HOW EPITHELIAL SODIUM CHANNEL PROMOTES STIFFNESS IN HUMAN AND MOUSE ENDOTHELIAL CELLS

Objective: The epithelial sodium channels (ENaCs) in the kidney maintain body salt/water balance and contributes to blood pressure regulation and hypertension. Recent finding suggests that ENaCs in endothelial cells are potential players mediating endothelial cell stiffness (ECS). Increased ECS is an important determinant of hypertension that coincides with altered endothelial nitric oxide (NO) production and endothelial dysfunction. Herein, we aim to uncover the molecular mechanism how ENaC influences ECS. I hypothesize that F-actin changes as a potential target through which ENaC mediates its effect. Design and method: ENaC subunit protein levels in Human Umbilical Vein Endothelial Cells (HUVECs) and Mouse Myocardial Endothelial Cells (MyEnd) cells were quantified by Western blotting. Aldosterone was used to increase ENaC expression. Changes in ECS assessed with Atomic Force Microscopy (AFM). If changes in F-actin cell cytoskeleton is correlated with ENaC expression and ECS, immunofluorescence microscopy was used and quantified. Results: Aldosterone was observed to significantly increase alpha-ENaC (both in human and mouse cells) and delta-ENaC (human specific) protein levels which were reversed by the additional incubation with amiloride (ENaC blocker). The elevated ENaC expression with aldosterone was associated with an increased Young's modulus that is indicative of elevated ECS observed in both human and mouse endothelial cells. The upregulated ENaC expression and increased stiffness was also translated into increased F-actin in response to aldosterone. Conclusions: Taken together, our study provides experimental evidence of increased ENaC protein level with increased F-actin cell cytoskeleton in endothelial cells that may promote ECS. This is a new mechanism through which ENaC mediate ECS and contributes to endothelial dysfunction and hypertension.

INCREASED EXPRESSION OF EPITHELIAL SODIUM CHANNEL AFFECTS MECHANICAL PROPERTIES OF ENDOTHELIAL CELLS BY REORGANISING THE F-ACTIN CYTOSKELETON

Objective: Epithelial sodium channel (ENaC) in the kidney maintain body salt/water balance and regulate blood pressure. Recent finding suggests that endothelial cells express ENaC. Here elevated ENaC contributes to increased endothelial cell stiffness (ECS) that associates with endothelial dysfunction and hypertension. dysfunction and hypertension. However, the mechanism how ENaC influence ECS remains unknown. This study aims to investigate the potential role of ENaC subunits in mediating ECS by cytoskeletal changes. I hypothesize ENaC mediated ECS via an increased expression of F-actin. To challenge our hypothesis, ENaC protein expression was quantified by Western blot in human umbilical vein endothelial cells (HUVECs) treated with aldosterone under static condition (0 dyn/cm2) and under laminar shear stress (LSS, 10 dyn/cm2, 24 h). Atomic Force Microscopy (AFM) was used to measure cell stiffness and changes of F-actin were assessed by immunofluorescence microscopy. Under static condition, aldosterone significantly increased ?-, and ?-ENaC protein levels. On the other hand, under LSS conditions aldosterone increased ?-, and ?-ENaC protein levels. Also under static condition, a significantly increased Young's modulus was determined in both fixed and live HUVEC cells treated with aldosterone. Furthermore, the increased stiffness in aldosterone treated cells revealed increased F-actin levels. Overall, these findings indicate that aldosterone has different effects on ENaC subunit expression in HUVECs grown under static and LSS conditions. The increased ENaC expression does increase ECS. This seems to involve an increased F-actin expression as a major determinant of ECS. Therefore, this study may provide a new mechanism for ENaC-mediated ECS and blood pressure regulation.

Effects of SGLT2 inhibitor ipragliflozin alone and combined with pioglitazone on fluid retention in type 2 diabetic mice with NASH

Several antidiabetic agents, including thiazolidinediones and sodium-glucose cotransporter (SGLT) 2 inhibitors, attenuate the symptoms of nonalcoholic steatohepatitis (NASH). However, thiazolidinediones have serious side effects such as fluid retention and increased risk of congestive heart failure. We examined the effects of SGLT2 inhibitor ipragliflozin, pioglitazone, and ipragliflozin + pioglitazone on fluid retention in type 2 diabetic mice with NASH. Four-week repeated administration of pioglitazone caused significant increases in heart weight (31% increase in 30 mg/kg pioglitazone-treated group compared to vehicle-treated group) concomitant with fluid retention, as estimated by a decrease in plasma osmolality and increase in water intake/urine volume ratio. In addition, pioglitazone significantly increased (by 1.5 to 2-fold) mRNA expression of α, β, and γ subtypes of ENaC and AQP2 and 3 subtypes in the renal medulla. Thus, pioglitazone-induced fluid retention may arise from enhanced reabsorption of sodium and water associated with increased expression of these channels in the kidney. In contrast, ipragliflozin alone did not induce these symptoms and did not affect ENaC or AQP expression. Combination treatment with ipragliflozin + pioglitazone attenuated these symptoms by ipragliflozin-induced osmotic diuresis. These findings demonstrate that treatment with ipragliflozin monotherapy or coadministered with pioglitazone may be a potential therapeutic option for the treatment of type 2 diabetes with NASH without fluid retention as a side effect.

Relationship between Phenotypic Characteristics from the Donors, Predictive Biomarkers from the Donor Grafts and the Development of Primary Graft Dysfunction in Lung Transplant Recipients

Purpose Lung transplantation is the only therapeutic option for patients with a terminal lung condition. Unfortunately, survival rates after transplantation remains too low. Primary graft dysfunction (PGD) is the first cause of death in the perioperative period and is associated with acute respiratory distress syndrome (ARDS), higher risk of lung infections, chronic rejection, and lower survival rates. We hypothesized that the alveolar epithelial dysfunction in donor grafts and then in the recipient, is a critical component of PGD pathophysiology. Our goal was to identify markers of alveolar damage and dysfunction in the donor grafts associated with PGD development within recipients. Methods We collected human samples and the clinical data from 174 donors and their recipients at different time points (before, during and after lung transplantation, in the ICU and follow-ups at the transplantation clinic). We also isolated alveolar epithelial cells from donor lung biopsies to assess the expression of integrity (ZO-1) and functionality biomarkers (such as ENaC and CFTR channels, which play a key role in the resorption of lung edema, a key feature of ARDS/PGD). Results Among our cohort of 174 lung transplants, suffering from various lung diseases (i.e. CF, PF/IPF, COPD, PAH), 26% developed a PGD. Several phenotypic factors from donors and intervention/procedures during transplantations were analysed. Among them, we found that although below the defined acceptable limit (6h), the mean duration of cold ischemia of the donor grafts among recipients subsequently developing a PGD was significantly longer (266 min) than the one from non-PGD recipients (233 min). We also demonstrated that alveolar cells, isolated from donor grafts within recipients developing a PGD, featured a severe decrease in ZO-1, ENaC and CFTR expression. Moreover, longer cold ischemia duration was associated with the observed ENaC and CFTR downregulation. Conclusion This study provides novel insights on epithelial alteration within donor graft, associated with subsequent PGD development. Moreover, our data pave the way for the identification of novel biomarkers and therapeutic targets for the restoration of alveolar integrity and functionality, with the ultimate goal to prevent PGD in lung transplants.

Dexmedetomidine alleviates pulmonary edema through the epithelial sodium channel (ENaC) via the PI3K/Akt/Nedd4-2 pathway in LPS-induced acute lung injury

Dexmedetomidine (Dex), a highly selective α2-adrenergic receptor (α2AR) agonist, has an anti-inflammatory property and can alleviate pulmonary edema in lipopolysaccharide (LPS)-induced acute lung injury (ALI), but the mechanism is still unclear. In this study, we attempted to investigate the effect of Dex on alveolar epithelial sodium channel (ENaC) in the modulation of alveolar fluid clearance (AFC) and the underlying mechanism. Lipopolysaccharide (LPS) was used to induce acute lung injury (ALI) in rats and alveolar epithelial cell injury in A549 cells. In vivo, Dex markedly reduced pulmonary edema induced by LPS through promoting AFC, prevented LPS-induced downregulation of α-, β-, and γ-ENaC expression, attenuated inflammatory cell infiltration in lung tissue, reduced the concentrations of TNF-α, IL-1β, and IL-6, and increased concentrations of IL-10 in bronchoalveolar lavage fluid (BALF). In A549 cells stimulated with LPS, Dex attenuated LPS-mediated cell injury and the downregulation of α-, β-, and γ-ENaC expression. However, all of these effects were blocked by the PI3K inhibitor LY294002, suggesting that the protective role of Dex is PI3K-dependent. Additionally, Dex increased the expression of phosphorylated Akt and reduced the expression of Nedd4-2, while LY294002 reversed the effect of Dex in vivo and in vitro. Furthermore, insulin-like growth factor (IGF)-1, a PI3K agonists, promoted the expression of phosphorylated Akt and reduced the expression of Nedd4-2 in LPS-stimulated A549 cells, indicating that Dex worked through PI3K, and Akt and Nedd4-2 are downstream of PI3K. In conclusion, Dex alleviates pulmonary edema by suppressing inflammatory response in LPS-induced ALI, and the mechanism is partly related to the upregulation of ENaC expression via the PI3K/Akt/Nedd4-2 signaling pathway.

Impact of ENaC downregulation in transgenic mice on the outcomes of acute lung injury induced by bleomycin.

What is the central question of this study? How does the downregulation of ENaC, the major driving force for alveolar fluid clearance, impact acute lung injury outcomes induced by bleomycin, featuring alveolar damage, as observed during ARDS exudative phase? What is the main finding and its importance? ENaC downregulation in αENaC(-/-)Tg+ mice did not elicit a substantial worsening impact on the main bleomycin outcomes. In ARDS patients, both ENaC alteration and alveolar damage are observed. Thus, novel therapeutic avenues, favouring alveolar integrity restauration, in addition to lung oedema resolution capacity, mainly driven by ENaC, would be essential. The exudative phase of acute respiratory distress syndrome (ARDS) is characterized by extended alveolar damage, resulting in accumulation of protein-rich inflammatory oedematous fluid in the alveolar space. Na+ reabsorption through ENaC channels is a major driving force for alveolar fluid clearance (AFC) in physiological and pathological conditions. It has previously been shown that partial αENaC impairment in transgenic (αENaC(-/-)Tg+) mice results in reduced AFC in basal conditions and increased wet/dry ratio after thiourea-induced lung oedema, a model in which the integrity of the alveolar epithelium is preserved. The goal of this study was to further investigate the impact of αENaC downregulation in αENaC(-/-)Tg+ mice using an experimental model of acute lung injury induced by bleomycin. A non-significant trend in enhanced weight loss and mortality rates was observed after the bleomycin challenge in αENaC(-/-)Tg+ compared to wild-type (WT) mice. Bronchoalveolar lavage analyses revealed increased TNFα levels and protein concentrations, as indexes of lung inflammation and alveolar damage, in αENaC(-/-)Tg+ mice, compared to WT, at day 3 post-bleomycin, although a statistical difference was no longer measured at day 7. Differential immune cell counts were similar in WT and αENaC(-/-)Tg+ mice challenged with bleomycin. Moreover, lung weight measurements indicated similar oedema levels in WT mice and in transgenic mice with impaired ENaC channels. Altogether, our data indicated that change in ENaC expression does not elicit a significant impact on lung oedema level/resolution in the bleomycin model, featuring alveolar damage.

Epithelial Sodium Channel and Salt-Sensitive Hypertension.

The development of high blood pressure is influenced by genetic and environmental factors, with high salt intake being a known environmental contributor. Humans display a spectrum of sodium-sensitivity, with some individuals displaying a significant blood pressure rise in response to increased sodium intake while others experience almost no change. These differences are, in part, attributable to genetic variation in pathways involved in sodium handling and excretion. ENaC (epithelial sodium channel) is one of the key transporters responsible for the reabsorption of sodium in the distal nephron. This channel has an important role in the regulation of extracellular fluid volume and consequently blood pressure. Herein, we review the role of ENaC in the development of salt-sensitive hypertension, and present mechanistic insights into the regulation of ENaC activity and how it may accelerate sodium-induced damage and dysfunction. We discuss the traditional role of ENaC in renal sodium reabsorption and review work addressing ENaC expression and function in the brain, vasculature, and immune cells, and how this has expanded the implications for its role in the initiation and progression of salt-sensitive hypertension.

ENaC expression correlates with the acute furosemide-induced K+ excretion.

BackgroundIn the aldosterone‐sensitive distal nephron (ASDN), epithelial sodium channel (ENaC)‐mediated Na+ absorption drives K+ excretion. K+ excretion depends on the delivery of Na+ to the ASDN and molecularly activated ENaC. Furosemide is known as a K+ wasting diuretic as it greatly enhances Na+ delivery to the ASDN. Here, we studied the magnitude of acute furosemide‐induced kaliuresis under various states of basal molecular ENaC activity.MethodsC57/Bl6J mice were subjected to different dietary regimens that regulate molecular ENaC expression and activity levels. The animals were anesthetized and bladder‐catheterized. Diuresis was continuously measured before and after administration of furosemide (2 µg/g BW) or benzamil (0.2 µg/g BW). Flame photometry was used to measure urinary [Na+] and [K+]. The kidneys were harvested and, subsequently, ENaC expression and cleavage activation were determined by semiquantitative western blotting.ResultsA low K+ and a high Na+ diet markedly suppressed ENaC protein expression, cleavage activation, and furosemide‐induced kaliuresis. In contrast, furosemide‐induced kaliuresis was greatly enhanced in animals fed a high K+ or low Na+ diet, conditions with increased ENaC expression. The furosemide‐induced diuresis was similar in all dietary groups.ConclusionAcute furosemide‐induced kaliuresis differs greatly and depends on the a priori molecular expression level of ENaC. Remarkably, it can be even absent in animals fed a high Na+ diet, despite a marked increase of tubular flow and urinary Na+ excretion. This study provides auxiliary evidence that acute ENaC‐dependent K+ excretion requires both Na+ as substrate and molecular activation of ENaC.

A Functionalized Screen-Printed Carbon with CeO &lt;sub&gt;2&lt;/sub&gt;/AuNP-Streptavidin-Biotinylated Aptamer as an Electrochemical Biosensor for Detection of Epithelial Sodium Channel (ENaC)

Epithelial sodium channel proteins (ENaC) are highly Na+ selective channels expressed in epithelia which are independently capable of producing sustained severe hypertension. ENaC expression is proportional to sodium intake and is clinically associated with hypertension. The higher the sodium concentration, the higher the amount of ENaC protein in the cell. Electrochemical aptasensors are highly sensitive, robust, and providing a rapid response to a variety of targets, as well as having the potential for miniaturization. This study developed an aptasensor for the detection of ENaC as a biomarker of hypertension via a gold nanoparticle (AuNP)-Aptamer bioconjugate formation that was attached to a screen-printed carbon electrode modified with cerium oxide. The concentration of ENaC was monitored using differential pulse voltammetry of [Fe(CN)6]4−/3- redox system. The limit of detection of ENaC protein with this aptasensor was obtained of 0.110 ng mL-1, in the linear range of concentrations 0.05–3.0 ng mL-1. The developed aptasensor is simple and can be miniaturized to determine the ENaC concentration in urine for the realization of a point-of-care device for the early detection of hypertension.

Inhibition of Pre-B Cell Colony Enhancing Factor Reduces Lung Injury in Rats Receiving Cardiopulmonary Bypass.

ObjectivePre-B cell colony enhancing factor (PBEF) is an important proinflammatory cytokine involved in acute lung injury. However, whether PBEF participates in lung injury caused by cardiopulmonary bypass (CPB) is still unknown. This study aimed to investigate the effects of silencing PBEF on lung injury and the sodium and water transport system in rats receiving CPB.MethodsMorphological changes in lung tissues were evaluated using hematoxylin and eosin (H&E) staining. PBEF was detected using immunohistochemistry. The sodium and water transport system-related proteins and cellular signaling pathways were detected by Western blotting.ResultsRats receiving CPB (model group) had more severe alveolar wall damage and higher expression of PBEF in free form than the control rats. Western blotting showed that the expression of PBEF, surfactant protein D (SP), aquaporin (AQP) 1, AQP5, and epithelial sodium channel (ENaC) was significantly higher in the lung tissue of CPB rats than control rats. By contrast, adenovirus-encoding sh-PBEF significantly reduced the expression of PBEF, SP, AQP1, AQP5, and ENaC in the lung tissues of rats treated with CPB. The phosphorylation levels of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (AKT), and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the lung tissue of rats that received CPB, and were downregulated by adenovirus-encoding sh-PBEF.ConclusionAdenovirus-encoding sh-PBEF could reduce lung injury and repair the sodium–water transport system in rats receiving CPB, likely through reducing MAPK, ERK1/2, and Akt signaling pathways.

Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis

Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency.

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN

AimsAcute lung injury (ALI) is a clinical syndrome with high morbidity and mortality, and severe pulmonary edema is one of the characteristics. Epithelial sodium channel (ENaC) located on the apical side of alveolar type 2 epithelial (AT2) cells is the primary rate limiting segment in alveolar fluid clearance. Many preclinical studies have revealed that mesenchymal stem cells (MSCs) based therapy has great therapeutic potential for ALI, while the role of ENaC in this process is rarely known.MethodsWe studied the effects of bone marrow-derived MSCs (BMSCs) on the protein/mRNA expression and activity of ENaC in primary mouse AT2 and human H441 cells by co-culture with them, respectively. Moreover, the changes of miRNA-130b in AT2 cells were detected by qRT-PCR, and we studied the involvement of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and the downstream PI3K/AKT pathway in the miRNA-130b regulation of ENaC.ResultsOur results demonstrated that BMSCs could increase ENaC protein expression and function, as well as the expression level of miRNA-130b. The dual luciferase target gene assay verified that PTEN was one of the target genes of miR-130b, which showed adverse effects on the protein expression of α/γ-ENaC and PTEN in AT2 cells. Upregulating miR-130b and/or knocking down PTEN resulted in the increase of α/γ-ENaC protein level, and the protein expression of p-AKT/AKT was enhanced by miR-130b. Both α and γ-ENaC protein expressions were increased after AT2 cells were transfected with siPTEN, which could be reversed by the co-administration of PI3K/AKT inhibitor LY294002.ConclusionIn summary, miRNA-130b in BMSCs can enhance ENaC at least partially by targeting PTEN and activating PI3K/AKT pathway, which may provide a promising new direction for therapeutic strategy in ALI.