ENaC Function Research Articles

Transmembrane Serine Protease 2 and Proteolytic Activation of the Epithelial Sodium Channel in Mouse Kidney.

The renal epithelial sodium channel (ENaC) is essential for sodium balance and blood pressure control. ENaC undergoes complex proteolytic activation by not yet clearly identified tubular proteases. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). Murine ENaC and TMPRSS2 were (co-)expressed in Xenopus laevis oocytes. ENaC cleavage and function were studied in TMPRSS2-deficient murine cortical collecting duct (mCCDcl1) cells and TMPRSS2-knockout (Tmprss2-/-) mice. Short-circuit currents (ISC) were measured to assess ENaC-mediated transepithelial sodium transport of mCCDcl1 cells. The mCCDcl1 cell transcriptome was studied using RNA sequencing. The effect of low-sodium diet with or without high potassium were compared in Tmprss2-/- and wildtype mice using metabolic cages. ENaC-mediated whole-cell currents were recorded from microdissected tubules of Tmprss2-/- and wildtype mice. In oocytes, co-expression of murine TMPRSS2 and ENaC resulted in fully cleaved γ-ENaC and ∼2-fold stimulation of ENaC currents. High baseline expression of TMPRSS2 was detected in mCCDcl1 cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated ISC which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. Tmprss2-/- mice kept on standard diet exhibited no apparent phenotype, but renal γ-ENaC cleavage was altered. In response to a low-salt diet, particularly with high potassium intake, Tmprss2-/- mice increased plasma aldosterone significantly more than wildtype mice to achieve a similar reduction of renal sodium excretion. Importantly, the stimulatory effect of trypsin on renal tubular ENaC currents was much more pronounced in Tmprss2-/- mice than that in wildtype mice. This indicated the presence of incompletely cleaved and less active channels at the cell surface of TMPRSS2-deficient tubular epithelial cells. TMPRSS2 contributes to proteolytic ENaC activation in mouse kidney in vivo.

Revisiting voltage-coupled H+ secretion in the collecting duct.

Experimental studies have shown that V-type ATPase-driven H+ secretion is dependent on the transepithelial voltage. On this basis the "voltage hypothesis" of urinary acidification by the collecting duct was derived. Accordingly, it has been supposed that the lumen-negative potential created by the reabsorption of Na+ via the epithelial sodium channel (ENaC) enhances electrogenic H+ secretion via the V-type H+-ATPase. This concept continues to be widely used to explain acid/base disorders. Importantly, however, a solid proof-of-principle for the voltage hypothesis in physiologically relevant situations has not been reached. Rather, it has been challenged by recent in vivo functional studies. In this review we outline the arguments and experimental observations explaining why voltage-coupled H+ secretion in the collecting duct often appears poorly applicable to rationalize for changes of H+ secretion as a function of more or less ENaC function in the collecting duct.

Establishment of a conditionally reprogrammed primary eccrine sweat gland culture for evaluation of tissue-specific CFTR function

BackgroundSweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). MethodsFirst, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. ResultsESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. ConclusionsThis study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.

The small molecule activator S3969 stimulates the epithelial sodium channel by interacting with a specific binding pocket in the channel's β-subunit

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969 binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of β-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics (MD) simulations predicted critical residues in the thumb domain of β-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the β-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. MD simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (βR437C – γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

Da-Chuan-Xiong Decoction Ameliorates Sodium Sensitivity and Plasma Norepinephrine via Attenuation of Brain Oxidative Stress in the DOCA-Salt Hypertensive Rats.

Da-Chuan-Xiong Decoction (DCXD) is an aqueous extract from a classic Chinese herbal formula composed of dried rhizomes of Ligusticum chuanxiong Hort and Gastrodia elata Bl. in the mass ratio of 4 : 1. It has been long used to treat chronic cardiovascular disease caused by blood stasis and wind pathogen in the clinic. This experimental study aimed to investigate the blood pressure (BP)-lowering effect of DCXD treatment on hypertension and underlying mechanisms. Male Sprague-Dawley rats were used in the experiment, and the hypertensive models were created by administering deoxycorticosterone acetate (DOCA) in conjunction with a high salt intake in uninephrectomized rats. DCXD was administered to hypertensive rats by oral gavage daily at a dose of 5 g/kg or 2.5 g/kg bodyweight for 28 days. The brain sodium sensitivity, ENaC function, superoxide anion level, NADPH oxidase activity, and expression of ENaC, p67phox, p47phox, and Rac1 in the paraventricular nucleus were assessed by using the appropriate methods. The 28 days of DCXD (5 g/kg) treatment significantly reduced the increased BP effectively, inhibited the enhanced heart index, kidney index, and 24 h urinary protein, and improved the progressive pathological changes of heart and kidney, which was comparable to that of the positive control amlodipine. DCXD treatment also caused a marked reduction in plasma norepinephrine and induced a significant improvement in brain sodium sensitivity and ENaC function in DOCA-salt hypertensive rats. Rats in DCXD-treated groups also exhibited decreased superoxide anion levels and NADPH oxidase activity in the paraventricular nucleus. The level of ENaC, p67phox, and Rac1 protein expression in the paraventricular nucleus was significantly downregulated by DCXD treatment in DOCA-salt hypertensive rats. These findings indicate that the depressor action and sympathetic inhibition of DCXD on salt-sensitive hypertension may be by ameliorating brain sodium sensitivity, modulating ENaC function, and inhibiting the expression of ENaC and NADPH oxidase in the hypothalamic paraventricular nucleus.

Short Term Exposure of Sheep Tracheal Epithelium to Cigarette Smoke Extract Reduces ENaC Current: A Pilot Study.

Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 μA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 μA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.

Select amino acids recover cytokine-altered ENaC function in human bronchial epithelial cells.

The airway epithelium plays a pivotal role in regulating mucosal immunity and inflammation. Epithelial barrier function, homeostasis of luminal fluid, and mucociliary clearance are major components of mucosal defense mechanisms. The epithelial sodium channel (ENaC) is one of the key players in controlling airway fluid volume and composition, and characteristic cytokines cause ENaC and barrier dysfunctions following pulmonary infections or allergic reactions. Given the limited understanding of the requisite duration and magnitude of cytokines to affect ENaC and barrier function, available treatment options for restoring normal ENaC activity are limited. Previous studies have demonstrated that distinct amino acids can modulate epithelial ion channel activities and barrier function in intestines and airways. Here, we have investigated the time- and concentration-dependent effect of representative cytokines for Th1- (IFN-γ and TNF-α), Th2- (IL-4 and IL-13), and Treg-mediated (TGF-β1) immune responses on ENaC activity and barrier function in human bronchial epithelial cells. When cells were exposed to Th1 and Treg cytokines, ENaC activity decreased gradually while barrier function remained largely unaffected. In contrast, Th2 cytokines had an immediate and profound inhibitory effect on ENaC activity that was subsequently followed by epithelial barrier disruption. These functional changes were associated with decreased membrane protein expression of α-, β-, and γ-ENaC, and decreased mRNA levels of β- and γ-ENaC. A proprietary blend of amino acids was developed based on their ability to prevent Th2 cytokine-induced ENaC dysfunction. Exposure to the select amino acids reversed the inhibitory effect of IL-13 on ENaC activity by increasing mRNA levels of β- and γ-ENaC, and protein expression of γ-ENaC. This study indicates the beneficial effect of select amino acids on ENaC activity in an in vitro setting of Th2-mediated inflammation suggesting these amino acids as a novel therapeutic approach for correcting this condition.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Direct endothelial ENaC activation mitigates vasculopathy induced by SARS-CoV2 spike protein.

Although both COVID-19 and non-COVID-19 ARDS can be accompanied by significantly increased levels of circulating cytokines, the former significantly differs from the latter by its higher vasculopathy, characterized by increased oxidative stress and coagulopathy in lung capillaries. This points towards the existence of SARS-CoV2-specific factors and mechanisms that can sensitize the endothelium towards becoming dysfunctional. Although the virus is rarely detected within endothelial cells or in the circulation, the S1 subunit of its spike protein, which contains the receptor binding domain (RBD) for human ACE2 (hACE2), can be detected in plasma from COVID-19 patients and its levels correlate with disease severity. It remains obscure how the SARS-CoV2 RBD exerts its deleterious actions in lung endothelium and whether there are mechanisms to mitigate this. In this study, we use a combination of in vitro studies in RBD-treated human lung microvascular endothelial cells (HL-MVEC), including electrophysiology, barrier function, oxidative stress and human ACE2 (hACE2) surface protein expression measurements with in vivo studies in transgenic mice globally expressing human ACE2 and injected with RBD. We show that SARS-CoV2 RBD impairs endothelial ENaC activity, reduces surface hACE2 expression and increases reactive oxygen species (ROS) and tissue factor (TF) generation in monolayers of HL-MVEC, as such promoting barrier dysfunction and coagulopathy. The TNF-derived TIP peptide (a.k.a. solnatide, AP301) -which directly activates ENaC upon binding to its a subunit- can override RBD-induced impairment of ENaC function and hACE2 expression, mitigates ROS and TF generation and restores barrier function in HL-MVEC monolayers. In correlation with the increased mortality observed in COVID-19 patients co-infected with S. pneumoniae, compared to subjects solely infected with SARS-CoV2, we observe that prior intraperitoneal RBD treatment in transgenic mice globally expressing hACE2 significantly increases fibrin deposition and capillary leak upon intratracheal instillation of S. pneumoniae and that this is mitigated by TIP peptide treatment.

A mild and transient form of autosomal recessive pseudohypoaldosteronism type 1 caused by a novel mutation in the SCNN1A gene.

We investigate the genetic etiology in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of a mild and transient form of pseudohypoaldosteronism type 1 (PHA1). Twelve patients with PHA1 from four different families with clinical and biochemical data were analyzed. The coding regions of NR3C2 and SCNN1A genes were sequenced. Human α-epithelial sodium channel (ENaC) wild-type (wt), αPhe226Cys and αPhe226Ser ENaC variants were expressed in Xenopus laevis oocytes to evaluate ENaC activity. The protein expression of α-ENaC wt and mutants was determined by Western blot. All patients were homozygotes for the p.Phe226Cys mutation of the α subunit of ENaC. In functional studies in X. laevis oocytes, p.Phe226Cys caused a significant reduction of ENaC activity (83% reduction), reduced the number of active ENαC mutant channels, and reduced the basal open probability compared with wt. Quantitative Western blot analysis revealed that the reduced activity of ENαC mutant channels was due to a reduced ENaC protein expression for the αPhe226Cys compared with wt. We present 12 patients from four different families with a mild and transient autosomal recessive PHA1 due to a novel homozygous missense mutation in the SCNN1A gene. Functional studies showed that the p.Phe226Cys substitution mutation in ENaC leads to a partial loss of function resulting mainly from both a decrease in the intrinsic ENaC activity and a reduction in channel expression at the protein level. The partial loss of ENaC function could explain the mild phenotype, variable expressivity, and the transient course of the disorder in these patients.NEW & NOTEWORTHY This paper demonstrates that mild autosomal recessive pseudohypoaldosteronism type 1 (PHA1) due to p.Phe226Cys missense mutation in the extracellular domain of ENαC α subunit can be transient, with phenotypic variability even with the normal sweat test, and incomplete penetrance. Functional studies explain the phenotype and denote the importance of the location on the extracellular domain of the SCNN1A p.Phe226Cys mutation for the intrinsic ENaC activity and the channel expression at the protein level.

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Submersion and hypoxia inhibit alveolar epithelial Na+ transport through ERK/NF-κB signaling pathway

BackgroundHypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which forms oxygen delivery barriers and is responsible for the disruption of ion transport. Epithelial sodium channel (ENaC) on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na+ and water reabsorption, thus becomes the key point for edema fluid removal under hypoxia. Here we sought to explore the effects of hypoxia on ENaC expression and the further mechanism related, which may provide a possible treatment strategy in edema related pulmonary diseases.MethodsExcess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema, supported by the evidence of increased hypoxia-inducible factor-1 expression. The protein/mRNA expressions of ENaC were detected, and extracellular signal-regulated kinase (ERK)/nuclear factor κB (NF-κB) inhibitor was applied to explore the detailed mechanism about the effects of hypoxia on epithelial ion transport in AEC. Meanwhile, mice were placed in chambers with normoxic or hypoxic (8%) condition for 24 h, respectively. The effects of hypoxia and NF-κB were assessed through alveolar fluid clearance and ENaC function by Ussing chamber assay.ResultsHypoxia (submersion culture mode) induced the reduction of protein/mRNA expression of ENaC, whereas increased the activation of ERK/NF-κB signaling pathway in parallel experiments using human A549 and mouse alveolar type 2 cells, respectively. Moreover, the inhibition of ERK (PD98059, 10 µM) alleviated the phosphorylation of IκB and p65, implying NF-κB as a downstream pathway involved with ERK regulation. Intriguingly, the expression of α-ENaC could be reversed by either ERK or NF-κB inhibitor (QNZ, 100 nM) under hypoxia. The alleviation of pulmonary edema was evidenced by the administration of NF-κB inhibitor, and enhancement of ENaC function was supported by recording amiloride-sensitive short-circuit currents.ConclusionsThe expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway.

Pendrin abundance, subcellular distribution, and function are unaffected by either αENaC gene ablation or by increasing ENaC channel activity

The intercalated cell Cl−/HCO3− exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Effect of irinotecan administration on amiloride-sensitive sodium taste responses in mice.

Taste alteration is a frequently reported side effect in patients receiving the chemotherapeutic agent, irinotecan. However, the way in which irinotecan causes taste disturbance and the type of taste impairment that is affected remain elusive. Here, we used the two-bottle preference test to characterize behavioral taste responses and employed immunohistochemical analyses to clarify the types and mechanisms of taste alteration induced, in mice, by irinotecan administration. Irinotecan administration resulted in a reduced intake of sodium taste solution but had no effect on sweet taste responses, as determined in the two-bottle preference test. In the presence of amiloride, which inhibits the function of the epithelial sodium channel (ENaC) in the periphery, the intake of sodium taste solution was comparable between the irinotecan-treated and control groups. Immunohistochemical analyses revealed that α-ENaC immunoreactivity detected in taste bud cells decreased slowly after irinotecan administration, and that administration of irinotecan had little effect on the number of cells expressing the cellular proliferation marker, Ki67, within or around taste buds. Our results imply that irinotecan administration may be responsible for altered behavioral sodium taste responses originating from ENaC function in the periphery, while being accompanied by the reduction of α-ENaC expression at the apical membrane of taste receptor cells without disturbing taste cell renewal.

Upregulation of α-ENaC induces pancreatic β-cell dysfunction, ER stress, and SIRT2 degradation.

Islet beta cells (β-cells) produce insulin in response to high blood glucose levels, which is essential for preserving glucose homeostasis. Voltage-gated ion channels in β-cells, including Na +, K +, and Ca 2+ channels, aid in the release of insulin. The epithelial sodium channel alpha subunit (α-ENaC), a voltage-independent sodium ion channel, is also expressed in human pancreatic endocrine cells. However, there is no reported study on the function of ENaC in the β-cells. In the current study, we found that α-ENaC was expressed in human pancreatic glandule and pancreatic islet β-cells. In the pancreas of db/db mice and high-fat diet-induced mice, and in mouse islet β-cells (MIN6 cells) treated with palmitate, α-ENaC expression was increased. When α-ENaC was overexpressed in MIN6 cells, insulin content and glucose-induced insulin secretion were significantly reduced. On the other hand, palmitate injured islet β-cells and suppressed insulin synthesis and secretion, but increased α-ENaC expression in MIN6 cells. However, α-ENaC knockout ( Scnn1a -/-) in MIN6 cells attenuated β-cell disorder induced by palmitate. Furthermore, α-ENaC regulated the ubiquitylation and degradation of sirtuin 2 in β-cells. α-ENaC also modulated β-cell function in correlation with the inositol-requiring enzyme 1 alpha/X-box binding protein 1 (IRE1α/XBP1) and protein kinase RNA-like endoplasmic reticulum kinase/C/EBP homologous protein (PERK/CHOP) endoplasmic reticulum stress pathways. These results suggest that α-ENaC may play a novel role in insulin synthesis and secretion in the β-cells, and the upregulation of α-ENaC promotes islet β-cell dysfunction. In conclusion, α-ENaC may be a key regulator involved in islet β-cell damage and a potential therapeutic target for type 2 diabetes mellitus.

Effect of kokumi taste-active γ-glutamyl peptides on amiloride-sensitive epithelial Na+ channels in rat fungiform taste cells

Kokumi taste-active compounds enhance salty taste perception. In animal models, sodium (salt) detection is mediated by the amiloride-sensitive epithelial sodium channel, ENaC. This ion channel works as a sodium receptor in the so-called sodium-taste cells. It is not known whether kokumi taste substances are able to affect the activity of functional ENaCs in these cells. Here, we use the patch-clamp technique to study the effect of kokumi-active tripeptides, glutathione (GSH) and γ-glutamyl-valyl-glycine (EVG), on the ENaC-mediated membrane current in rat fungiform sodium-taste cells. GSH and EVG reduced slightly this current and the effect disappeared in the presence of amiloride, a specific ENaC blocker. No effect on membrane current was detected in other taste cells (Type II and Type III cells) that do not express functional ENaC. Our findings suggest that the enhancing effect of kokumi taste-active γ-glutamyl peptides on salt reception is not explained by an increase in the activity of ENaC.

Lessons learned about epithelial sodium channels from transgenic mouse models.

This review provides an up-to-date understanding about the regulation of epithelial sodium channel (ENaC) expression and function. In particular, we will focus on its implication in renal Na+ and K+ handling and control of blood pressure using transgenic animal models. In kidney, the highly amiloride-sensitive ENaC maintains whole body Na+ homeostasis by modulating Na+ transport via epithelia. This classical role is mostly confirmed using genetically engineered animal models. Recently identified key signaling pathways that regulate ENaC expression and function unveiled some nonclassical and unexpected channel regulatory processes. If aberrant, these dysregulated mechanisms may also result in the development of salt-dependent hypertension.The purpose of this review is to highlight the most recent findings in renal ENaC regulation and function, in considering data obtained from animal models. Increased ENaC-mediated Na+ transport is a prerequisite for salt-dependent forms of hypertension. To treat salt-sensitive hypertension it is crucial to fully understand the function and regulation of ENaC.

Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension.

Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. For the first time, expression of α, β, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αβγ- and δβγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.

COVID-19 and Liquid Homeostasis in the Lung-A Perspective through the Epithelial Sodium Channel (ENaC) Lens.

Infections with a new corona virus in 2019 lead to the definition of a new disease known as Corona Virus Disease 2019 (COVID-19). The sever cases of COVID-19 and the main cause of death due to virus infection are attributed to respiratory distress. This is associated with the formation of pulmonary oedema that impairs blood oxygenation and hypoxemia as main symptoms of respiratory distress. An important player for the maintenance of a defined liquid environment in lungs needed for normal lung function is the epithelial sodium channel (ENaC). The present article reviews the implications of SARS-CoV-2 infections from the perspective of impaired function of ENaC. The rationale for this perspective is derived from the recognition that viral spike protein and ENaC share a common proteolytic cleavage site. This cleavage site is utilized by the protease furin, that is essential for ENaC activity. Furin cleavage of spike ‘activates’ the virus protein to enable binding to host cell membrane receptors and initiate cell infection. Based on the importance of proteolytic cleavage for ENaC function and activation of spike, it seems feasible to assume that virus infections are associated with impaired ENaC activity. This is further supported by symptoms of COVID-19 that are reminiscent of impaired ENaC function in the respiratory tract.

MO068: Paraoxonase 2 Protects the Kidney Tissue from Aldosterone-Promoted Oxidative Stress in Milan Normotensive Rats Strain

Abstract BACKGROUND AND AIMS Kidney plays a pivotal role on the maintenance of blood pressure by the regulation of sodium reabsorption. Aldosterone sensitive distal nephron finely tunes salt reabsorption through several transporters, including the epithelial sodium channel (ENaC). Alteration in ENaC function leads to clinical relevant conditions associated with high or low blood pressure when gain or loss of function mutations occur, respectively. Recently, accumulating evidence suggests the putative role of renal Paraoxonase 2 (PON2) as regulator of ENaC function. PON2 is a membrane-associated enzyme showing hydrolytic activity against 3-oxo-C12 homoserine lactone, a signaling molecule produced by bacteria and mainly Pseudomonas aeruginosa. PON2 also exhibits antioxidant property. In the distal nephron, PON2 is expressed in principal cells, and in vitro it physically interacts with ENaC subunits and inhibits ENaC channel activity in Xenopus oocytes likely by modulating channel assembly in the endoplasmic reticulum. However, the role of PON2 in the kidney is still not known. In the present study, we investigate whether PON2 is regulated by changes in aldosterone state and its role in aldosterone-induced oxidative stress in the kidneys. METHOD We addressed these issues utilizing both in vivo and in vitro approaches, such as RESULTS We first demonstrated that in Milan normotensive rats strain, PON2 is upregulated by low-salt state according with α-ENaC activation. This regulation is aldosterone sensitive since pretreatment with mineralocorticoid receptor antagonist spironolactone prevented the stimulatory effects of sodium restricted diet. Furthermore, low-sodium diet inducing PON2 upregulation was associated with reduced ROS levels, more specifically superoxide levels, but treatment with spironolactone prevented this beneficial effect, by increasing ROS level in kidney cortex. Since antihypertensive drugs spironolactone and canrenone contain a lactone residue in their molecular structure, we tested whether they could directly interact with PON2. We demonstrated in vitro that spironolactone and canrenone were able to inhibit PON2 lactonase activity and so to directly interfere with PON2 function. Finally, Milan hypertensive rats, carrying a mutation of α-adducin as determinant of their hypertensive phenotype, presented with higher level of ROS while PON2 was upregulated. CONCLUSION These results demonstrate PON2 as a novel target of steroid hormone aldosterone. PON2 may serve as a potential ROS scavenger mechanism protecting the kidneys from aldosterone-promoted oxidative stress.