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Background/Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium. [ABSTRACT FROM AUTHOR]

Epithelial sodium channel (ENaC) is responsible for regulating Na+ homeostasis. While its physiological functions have been investigated extensively in peripheral tissues, far fewer studies have explored its functions in the brain. Since our limited knowledge of ENaC's distribution in the brain impedes our understanding of its functions there, we decided to explore the whole-brain expression pattern of the Scnn1a gene, which encodes the core ENaC complex component ENaCα. To visualize Scnn1a expression in the brain, we crossed Scnn1a-Cre mice with Rosa26-lsl-tdTomato mice. Brain sections were subjected to immunofluorescence staining using antibodies against NeuN or Myelin Binding Protein (MBP), followed by the acquisition of confocal images. We observed robust tdTomato fluorescence not only in the soma of cortical layer 4, the thalamus, and a subset of amygdalar nuclei, but also in axonal projections in the hippocampus and striatum. We also observed expression in specific hypothalamic nuclei. Contrary to previous reports, however, we did not detect significant expression in the circumventricular organs, which are known for their role in regulating Na+ balance. Finally, we detected fluorescence in cells lining the ventricles and in the perivascular cells of the median eminence. Our comprehensive mapping of Scnn1a-expressing cells in the brain will provide a solid foundation for further investigations of the physiological roles ENaC plays within the central nervous system. [ABSTRACT FROM AUTHOR]

Epithelial sodium channel (ENaC) are integral to maintaining salt and water homeostasis in various biological tissues, including the kidney, lung, and colon. They enable the selective reabsorption of sodium ions, which is a process critical for controlling blood pressure, electrolyte balance, and overall fluid volume. ENaC activity is finely controlled through proteolytic activation, a process wherein specific enzymes, or proteases, cleave ENaC subunits, resulting in channel activation and increased sodium reabsorption. This regulatory mechanism plays a pivotal role in adapting sodium transport to different physiological conditions. In this review article, we provide an in-depth exploration of the role of proteolytic activation in regulating ENaC activity. We elucidate the involvement of various proteases, including furin-like convertases, cysteine, and serine proteases, and detail the precise cleavage sites and regulatory mechanisms underlying ENaC activation by these proteases. We also discuss the physiological implications of proteolytic ENaC activation, focusing on its involvement in blood pressure regulation, pulmonary function, and intestinal sodium absorption. Understanding the mechanisms and consequences of ENaC proteolytic activation provides valuable insights into the pathophysiology of various diseases, including hypertension, pulmonary disorders, and various gastrointestinal conditions. Moreover, we discuss the potential therapeutic avenues that emerge from understanding these mechanisms, offering new possibilities for managing diseases associated with ENaC dysfunction. In summary, this review provides a comprehensive discussion of the intricate interplay between proteases and ENaC, emphasizing the significance of proteolytic activation in maintaining sodium and fluid balance in both health and disease. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is a common lung disease with increasing morbidity and mortality rates due to the lack of specific drugs. Impaired alveolar fluid clearance (AFC) is a primary pathological feature of ALI. Epithelial sodium channel (ENaC) is a primary determinant in regulating the transport of Na+ and the clearance of alveolar edema fluid. Therefore, ENaC is an important target for the development of drugs for ALI therapy. However, the role of ENaC in the progression of ALI remains unclear. Inhibition of early growth response factor (EGR-1) expression has been reported to induce a protective effect on ALI; therefore, we evaluated whether EGR-1 participates in the progression of ALI by regulating ENaC-α in alveolar epithelium. We investigated the potential mechanism of EGR-1-mediated regulation of ENaC in ALI. We investigated whether EGR-1 aggravates the pulmonary edema response in ALI by regulating ENaC. ALI mouse models were established by intrabronchial injection of lipopolysaccharides (LPS). Lentiviruses with EGR-1 knockdown were transfected into LPS-stimulated A549 cells. We found that EGR-1 expression was upregulated in the lung tissues of ALI mice and in LPS-induced A549 cells, and was negatively correlated with ENaC-α expression. Knockdown of EGR-1 increased ENaC-α expression and relieved cellular edema in ALI. Moreover, EGR-1 regulated ENaC-α expression at the transcriptional level, and correspondingly promoted pulmonary edema and aggravated ALI symptoms. In conclusion, our study demonstrated that EGR-1 could promote pulmonary edema by downregulating ENaC-α at the transcriptional level in ALI. Our study provides a new potential therapeutic strategy for treatment of ALI. EGR-1 expression was increased in LPS-induced ALI mice and associated with aggravated pulmonary edema EGR-1 induced pulmonary edema relying on regulating the expression of ENaC-α at the transcriptional level by manipulating the promoter [ABSTRACT FROM AUTHOR]

In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) is a heterotrimer and is widely distributed throughout the kidneys, blood vessels, lungs, colons, and many other organs. The basic role of the ENaC is to mediate the entry of Na+ into cells; the ENaC also has an important regulatory function in blood pressure, airway surface liquid (ASL), and endothelial cell function. Aldosterone, serum/glucocorticoid kinase 1 (SGK1), shear stress, and posttranslational modifications can regulate the activity of the ENaC; some ion channels also interact with the ENaC. In recent years, it has been found that the ENaC can lead to immune cell activation, endothelial cell dysfunction, aggravated inflammation involved in high salt-induced hypertension, cystic fibrosis, pseudohypoaldosteronism (PHA), and tumors; some inflammatory cytokines have been reported to have a regulatory role on the ENaC. The ENaC hyperfunction mediates the increase of intracellular Na+, and the elevated exchange of Na+ with Ca2+ leads to an intracellular calcium overload, which is an important mechanism for ENaC-related inflammation. Some of the research on the ENaC is controversial or unclear; we therefore reviewed the progress of studies on the role of ENaC-related inflammation in human diseases and their mechanisms. [ABSTRACT FROM AUTHOR]

Background: Non-voltage-gated sodium channel, also known as the epithelial sodium channel (ENaC), formed by heteromeric complexes consisting of SCNN1A, SCNN1B, and SCNN1G, is responsible for maintaining sodium ion and body fluid homeostasis in epithelial cells. However, no systematic study of SCNN1 family members has been conducted in renal clear cell carcinoma (ccRCC) to date. Objective: To investigate the abnormal expression of SCNN1 family in ccRCC and its potential correlation with clinical parameters. Methods: The transcription and protein expression levels of SCNN1 family members in ccRCC were analyzed based on the TCGA database, and were confirmed by quantitative RT-PCR and immunohistochemical staining assays, respectively. The area under curve (AUC) was used to evaluate the diagnostic value of SCNN1 family members for ccRCC patients. Results: The mRNA and protein expression of SCNN1 family members was significantly downregulated in ccRCC compared with normal kidney tissues, which might be due to DNA hypermethylation in the promoter region. It is worth noting that the AUC of SCNN1A, SCNN1B, and SCNN1G were 0.965, 0.979, and 0.988 based on the TCGA database (p < 0.0001), respectively. The diagnostic value was even higher when combing these three members together (AUC = 0.997, p < 0.0001). Intriguingly, the mRNA level of SCNN1A was significantly lower in females compared with males, while SCNN1B and SCNN1G were increased with the progression of ccRCC and remarkably associated with a worse outcome for patients. Conclusion: The aberrantly decrease of SCNN1 family members might serve as valuable biomarkers for the diagnosis of ccRCC. [ABSTRACT FROM AUTHOR]

Luteolin (Lut), a natural flavonoid compound existing in Perilla frutescens (L.) Britton, has been proven to play a protective role in the following biological aspects: inflammatory, viral, oxidant, and tumor-related. Lut can alleviate acute lung injury (ALI), manifested mainly by preventing the accumulation of inflammation-rich edematous fluid, while the protective actions of Lut on transepithelial ion transport in ALI were seldom researched. We found that Lut could improve the lung appearance/pathological structure in lipopolysaccharide (LPS)-induced mouse ALI models and reduce the wet/dry weight ratio, bronchoalveolar protein, and inflammatory cytokines. Meanwhile, Lut upregulated the expression level of the epithelial sodium channel (ENaC) in both the primary alveolar epithelial type 2 (AT2) cells and three-dimensional (3D) alveolar epithelial organoid model that recapitulated essential structural and functional aspects of the lung. Finally, by analyzing the 84 interaction genes between Lut and ALI/acute respiratory distress syndrome using GO and KEGG enrichment of network pharmacology, we found that the JAK/STAT signaling pathway might be involved in the network. Experimental data by knocking down STAT3 proved that Lut could reduce the phosphorylation of JAK/STAT and enhance the level of SOCS3, which abrogated the inhibition of ENaC expression induced by LPS accordingly. The evidence supported that Lut could attenuate inflammation-related ALI by enhancing transepithelial sodium transport, at least partially, via the JAK/STAT pathway, which may offer a promising therapeutic strategy for edematous lung diseases. [ABSTRACT FROM AUTHOR]

Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents. [ABSTRACT FROM AUTHOR]

Background: Hypoxia 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. Methods: Excess 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. Results: Hypoxia (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. Conclusions: The expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Dyspnea and progressive hypoxemia are the main clinical features of patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary pathology shows diffuse alveolar damage with edema, hemorrhage, and the deposition of fibrinogens in the alveolar space, which are consistent with the Berlin Acute Respiratory Distress Syndrome Criteria. The epithelial sodium channel (ENaC) is a key channel protein in alveolar ion transport and the rate-limiting step for pulmonary edema fluid clearance, the dysregulation of which is associated with acute lung injury/acute respiratory distress syndrome. The main protein of the fibrinolysis system, plasmin, can bind to the furin site of γ-ENaC and induce it to an activation state, facilitating pulmonary fluid reabsorption. Intriguingly, the unique feature of SARS-CoV-2 from other β-coronaviruses is that the spike protein of the former has the same furin site (RRAR) with ENaC, suggesting that a potential competition exists between SARS-CoV-2 and ENaC for the cleavage by plasmin. Extensive pulmonary microthrombosis caused by disorders of the coagulation and fibrinolysis system has also been seen in COVID-19 patients. To some extent, high plasmin (ogen) is a common risk factor for SARS-CoV-2 infection since an increased cleavage by plasmin accelerates virus invasion. This review elaborates on the closely related relationship between SARS-CoV-2 and ENaC for fibrinolysis system-related proteins, aiming to clarify the regulation of ENaC under SARS-CoV-2 infection and provide a novel reference for the treatment of COVID-19 from the view of sodium transport regulation in the lung epithelium. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To assess changes in expression of renal epithelial sodium channel (ENaC) and NEDD4L, a ubiquitin ligase, in urinary extracellular vesicles (UEV) of pre-eclamptic women compared to normal pregnant controls. Urine was collected from pre-eclamptic women (PE, n = 20) or during normal pregnancy (NP, n = 20). UEV were separated by differential ultracentrifugation. NEDD4L, α-ENaC and γ-ENaC were identified by immunoblotting. There was no difference in the expression of NEDD4L (p = 0.17) and α-ENaC (p = 0.10). PE subjects showed increased expression of γ-ENaC by 6.9-fold compared to NP (p < 0.0001). ENaC expression is upregulated in UEV of pre-eclamptic subjects but was not associated with changes in NEDD4L. [ABSTRACT FROM AUTHOR]

Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease and furin. It has been reported that sPRR functions as an important regulator of intrarenal renin contributing to angiotensin II (ANG II)-induced hypertension. Relatively, less is known for the function of sPRR in ANG II-independent hypertension such as mineralocorticoid excess. In the present study, we used a novel mouse model with mutagenesis of the cleavage site in PRR (termed as PRRR279V/L282V or mutant) to examine the phenotype during aldosterone (Aldo)-salt treatment. The hypertensive response of mutant mice to Aldo-salt treatment was blunted in parallel with the attenuated response of plasma volume expansion and renal medullary a-epithelial Naþ channel expression. Moreover, Aldo-salt-induced hypertrophy in the heart and kidney as well as proteinuria were improved, accompanied by blunted polydipsia and polyuria. Together, these results represent strong evidence favoring endogenous sPRR as a mediator of Aldo-salt-induced hypertension and renal injury. [ABSTRACT FROM AUTHOR]

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a devastating respiratory disorder with high rates of mortality and morbidity, but the detailed underlying mechanisms of ALI/ARDS remain largely unknown. Mechanosensitive ion channels (MSCs), including epithelial sodium channel (ENaC), Piezo channels, transient receptor potential channels (TRPs), and two-pore domain potassium ion (K2P) channels, are highly expressed in lung tissues, and the activity of these MSCs can be modulated by mechanical forces (e.g., mechanical ventilation) and other stimuli (e.g., LPS, hyperoxia). Dysfunction of MSCs has been found in various types of ALI/ARDS, and MSCs play a key role in regulating alveolar fluid clearance, alveolar epithelial/endothelial barrier function, the inflammatory response and surfactant secretion in ALI/ARDS lungs. Targeting MSCs exerts therapeutic effects in the treatment of ALI/ARDS. In this review, we summarize the structure and functions of several well-recognized MSCs, the role of MSCs in the pathogenesis of ALI/ARDS and recent advances in the pharmacological and molecular modulation of MSCs in the treatment of ALI/ARDS. According to the current literature, targeting MSCs might be a very promising therapeutic approach against ALI/ARDS. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) causes significant morbidity and mortality in critically ill patients, which often presents with extensive accumulation of activated inflammatory cells and diffused alveolar damage accompanied by oxidative stress. Exosomes are nanovesicles, which have notable anti‐inflammatory and repair properties, thus alleviating the symptoms of ALI. Epithelial sodium channel (ENaC) is essential for the transepithelial absorption of Na+ and fluid from alveolar spaces. We studied the effects of bone marrow mesenchymal stem cell exosomes (BMSC‐exo) on the apoptosis and protein expression of ENaC in primary mouse alveolar epithelial type 2 cells (AT 2 cells). Moreover, the change of miR‐199a‐3p in AT 2 cells was detected by qRT‐PCR, and we studied the regulation of miR‐199a‐3p on ENaC protein expression. Our results demonstrated that BMSC‐exo could not only improve viability and reduce apoptosis in AT 2 cells, but also enhance the expression of ENaC protein and miR‐199a‐3p. Meanwhile, the upregulation of miR‐199a‐3p resulted in increased expression of ENaC protein. In summary, the BMSC‐exo could participate in the regulation of ENaC through miR‐199a‐3p originated from BMSC‐exo, thereby providing a new pharmacological tool for the treatment of ALI. [ABSTRACT FROM AUTHOR]

We studied the content of aquaporin-5 (AQP5) and epithelial sodium channel (ENaC) in rat lungs during the development of toxic pulmonary edema (TPE) caused by intoxication with phosgene and perfluoroisobutylene (1.5 LC50). The lung body weight index (LBI) was calculated and histological examination of the lung tissues was performed. Localization and expression of AQP5 and ENaC were determined by immunohistochemistry. Intoxication led to a significant (p<0.05) increase in LBI and histological changes typical of TPE 1 and 3 h after the exposure. In 1 and 3 h after phosgene intoxication, the AQP5 and ENaC content significantly (p<0.05) increased in comparison with the control. Similar changes in the AQP5 and ENaC content were observed 1 and 3 h after exposure to perfluoroisobutylene. It was hypothesized that AQP5 plays an important role in the formation of TPE caused by intoxication with acylating pulmonotoxicants. An increase in the content of ENaC can be considered as a compensatory reaction of the body aimed at clearance of the alveolar fluid. [ABSTRACT FROM AUTHOR]

Objective(s): Previous studies have shown that ulinastatin (UTI) alleviates pulmonary edema in acute lung injury (ALI) caused by lipopolysaccharide (LPS), although the mechanism behind this action is uncertain. This research aimed to identify the fundamental mechanism by which UTI alleviates pulmonary edema. Materials and Methods: We established a model of acute lung injury (ALI) in rats by using LPS as the inciting agent.The control, LPS, and LPS+UTI groups were each comprised of a specific number of randomly selected Wistar rats. We evaluated lung injury and determined pulmonary edema. The concentrations of TNF-α, IL-1β and IL-6 in BALF and the expression levels of α1Na, k-ATPase, β1Na, K-AtPase, α-ENaC, β-ENaC, γ-ENaC, Zonula occludens (ZO)-1, Occludin, Caludin-5, PI3K, Akt, TLR4, MyD88 and NF-κBwere identified in lung tissues. Results: The presence of UTI was associated with a reduction in lung pathological alterations, lung injury scores, the lung W/D ratio, and MPO activity, in addition to the improved gas exchange (P<0.01). Furthermore, UTI alleviated EB leakage and stimulated AFC (P<0.01). Importantly, UTI increased the expression of ZO-1, Occludin, Caludin-5, α1Na, K-ATPase, β1Na, K-AtPase, α-ENaC, β-ENaC, and γ-ENaC (P<0.01). Furthermore, UTI inhibited the inflammatory response, enhanced the expression of PI3K and Akt and hindered TLR4, MyD88, and NF-κB expression (P<0.01) in lung tissues. Conclusion: Our results demonstrated that UTI attenuated pulmonary edema by reducing pulmonary permeability and promoting AFC through inhibiting the inflammatory response, and the mechanism is related to promoting PI3K/Akt signaling pathways and suppressing TLR4/MyD88/NF-κB signaling pathways. [ABSTRACT FROM AUTHOR]

The ischemia–reperfusion injury (IRI) of rat kidneys is used as a model of acute kidney injury. Salt-sensitive hypertension occurs in rats after IRI, and the distal nephrons play important roles in the development of this condition. We investigated the role of the mineralocorticoid receptor (MR) in the progression of IRI-induced salt-sensitive hypertension in rats. Fourteen days after right-side nephrectomy, IRI was induced by clamping the left renal artery, with sham surgery performed as a control. IRI rats were provided with normal water or water with 1.0% NaCl (IRI/NaCl), or they were implanted with an osmotic mini-pump to infuse vehicle or aldosterone (IRI/Aldo). Esaxerenone, a non-steroidal MR blocker (MRB), was administered to IRI/NaCl and IRI/Aldo rats for 6 weeks. MR expression increased by day 7 post-IRI. Blood pressure and urinary protein excretion increased in IRI/NaCl and IRI/Aldo rats over the 6-week period, but these effects were negated by MRB administration. The MRB attenuated the expression of the gamma-epithelial sodium channel (ENaC) and renal damage. The ENaC inhibitor, amiloride, ameliorated hypertension and renal damage in IRI/NaCl and IRI/Aldo rats. Our findings thus showed that MR upregulation may play a pivotal role in ENaC-mediated sodium uptake in rats after IRI, resulting in the development of salt-sensitive hypertension in response to salt overload or the activation of the renin–angiotensin–aldosterone system. [ABSTRACT FROM AUTHOR]

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria. [ABSTRACT FROM AUTHOR]

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells' suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC—amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist. [ABSTRACT FROM AUTHOR]

Epithelial sodium channel (ENaC) is a pivotal regulator of alveolar fluid clearance in the airway epithelium and plays a key role in the treatment of acute lung injury (ALI), which is mainly composed of the three homologous subunits (α, β and γ). The mechanisms of microRNAs in small extracellular vesicles (sEVs) derived from mesenchymal stem cell (MSC-sEVs) on the regulation of lung ion transport are seldom reported. In this study, we aimed at investigating whether miR-34c had an effect on ENaC dysfunction induced by lipopolysaccharide and explored the underlying mechanism in this process. Primarily, the effect of miR-34c on lung edema and histopathology changes in an ALI mouse model was investigated. Then the uptake of PKH26-labeled sEVs was observed in recipient cells, and we observed that the overexpression of miR-34c in MSC-sEVs could upregulate the LPS-inhibited γ-ENaC expression. The dual luciferase reporter gene assay demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS) was one of target genes of miR-34c, the protein expression of which was negatively correlated with miR-34c. Subsequently, either upregulating miR-34c or knocking down MARCKS could increase the protein expression of phospho-phosphatidylinositol 3-kinase (p-PI3K) and phospho-protein kinase B (p-AKT), implying a downstream regulation pathway was involved. All of the above suggest that miR-34c in MSC-sEVs can attenuate edematous lung injury via enhancing γ-ENaC expression, at least partially, through targeting MARCKS and activating the PI3K/AKT signaling pathway subsequently. [ABSTRACT FROM AUTHOR]

Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis. [ABSTRACT FROM AUTHOR]

Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF's anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, β, γ, δ), forming heterotrimeric αβγ- or δβγ-ENaCs. Although the physiology of αβγ-ENaC is well understood, for decades the field has stalled with respect to δβγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δβγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αβγ- and δβγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC. [ABSTRACT FROM AUTHOR]

Systemic pseudohypoaldosteronism (PHA) is a rare, salt-wasting syndrome that is caused by inactivating variants in genes encoding epithelial sodium channel subunits. Hyponatremia, hyperkalemia, metabolic acidosis, increased aldosterone and renin levels are expected findings in PHA. Clinical management is challenging due to high dose oral replacement therapy. Furthermore, patients with systemic PHA require life-long therapy. Here we report a patient with systemic PHA due to SCNN1B variant whose hyponatremia and hyperkalemia was detected at the 24th hour of life. Hyperkalemia did not improve with conventional treatments and dialysis was required. He also developed myocarditis and hypertension in follow-up. Challenges for diagnosis and treatment in this patient are discussed herein. In addition, published evidence concerning common features of patients with SCNN1B variant are reviewed. [ABSTRACT FROM AUTHOR]

Aims: The occurrence of vascular permeability pulmonary edema in acute lung injury (ALI) is related to the imbalance of alveolar fluid transport. Regulating the active transport of alveolar fluid by aquaporins (AQPs), epithelial sodium channels (ENaCs), and Na+-K+-ATPase can effectively reduce the edema fluid in the alveolar cavity and protect against ALI. We evaluated the therapeutic effects of total flavonoids, extracted from Nervilia fordii (TFENF), and investigated its potential mechanisms of alveolar fluid transport in a rat ALI model. Materials and methods: A model of lipopolysaccharide (LPS, 5 mg/kg)-induced ALI was established in Sprague-Dawley (SD) rats through the arteriae dorsalis penis. SD rats were divided into six groups, including the vehicle, LPS model, TFENF (6 mg/kg, 12 mg/kg, 24 mg/kg), and dexamethasone group (DEX group, 5 mg/kg). The wet-to-dry (W/D) lung weight ratio, oxygenation index, and histopathological observation were used to evaluate the therapeutic effect of TFENF. The mRNA expression of AQPs, ENaCs , and pro-inflammatory cytokines was determined using real-time polymerase chain reaction, whereas protein expression was determined using immunohistochemistry. The Na + -K + -ATPase activity was assessed using enzyme-linked immunosorbent assay. Results: LPS significantly stimulated the production of inflammatory mediators including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and disrupted the water transport balance in the alveolar cavity by inhibiting AQPs/ENaCs/Na + -K + -ATPase. Pretreatment with TFENF reduced the pathological damage and W/D ratio of the lungs and ameliorated the arterial blood oxygen partial pressure (PaO2) and oxygenation index. TFENF further decreased the mRNA level of TNF-α and IL-1β ; increased the expression of AQP-1, AQP-5, αENaC, and βENaC ; and increased Na + -K + -ATPase activity. Moreover, the regulation of AQPs, βENaC, and Na + -K + -ATPase and the inhibition of TNF-α and IL-1β by TFENF were found to be dose dependent. Conclusion: TFENF protects against LPS-induced ALI, at least in part, through the suppression of inflammatory cytokines and regulation of the active transport capacity of AQPs/ENaCs/Na + -K + -ATPase. These findings suggest the therapeutic potential of TFENF as phytomedicine to treat inflammation and pulmonary edema in ALI. [ABSTRACT FROM AUTHOR]

The activity of the Epithelial Na+ Channel (ENaC) in renal principal cells (PC) fine-tunes sodium excretion and consequently, affects blood pressure. The Gs-adenylyl cyclase-cAMP signal transduction pathway is believed to play a central role in the normal control of ENaC activity in PCs. The current study quantifies the importance of this signaling pathway to the regulation of ENaC activity in vivo using a knock-in mouse that has conditional expression of Gs-DREADD (designer receptors exclusively activated by designer drugs; GsD) in renal PCs. The GsD mouse also contains a cAMP response element-luciferase reporter transgene for non-invasive bioluminescence monitoring of cAMP signaling. Clozapine N-oxide (CNO) was used to selectively and temporally stimulate GsD. Treatment with CNO significantly increased luciferase bioluminescence in the kidneys of PC-specific GsD but not control mice. CNO also significantly increased the activity of ENaC in principal cells in PC-specific GsD mice compared to untreated knock-in mice and CNO treated littermate controls. The cell permeable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate, significantly increased the activity and expression in the plasma membrane of recombinant ENaC expressed in CHO and COS-7 cells, respectively. Treatment of PC-specific GsD mice with CNO rapidly and significantly decreased urinary Na+ excretion compared to untreated PC-specific GsD mice and treated littermate controls. This decrease in Na+ excretion in response to CNO in PC-specific GsD mice was similar in magnitude and timing as that induced by the selective vasopressin receptor 2 agonist, desmopressin, in wild type mice. These findings demonstrate for the first time that targeted activation of Gs signaling exclusively in PCs is sufficient to increase ENaC activity and decrease dependent urinary Na+ excretion in live animals. [ABSTRACT FROM AUTHOR]

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FIO2 )-induced lung injury. Exposure to high FIO2 from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG (400 mM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 mM) or amiloride (1 mM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10 mM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high FIO2 exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the antiproteinuric and hyperkalemic mechanisms activated by dual renin-angiotensin system (RAS) blockade in renovascular hypertensive rats (2-kidney 1-clip model [2K-1C]). Six weeks after clipping the left renal artery or sham operation (2K), rats were treated with losartan, enalapril, or both drugs for two weeks. We found that 2K-1C rats displayed higher tail-cuff blood pressure (BP), increased non-clipped kidney Ang II concentration, and more pronounced urinary albumin excretion than 2K. BP was decreased by the treatment with either enalapril or losartan, and the combination of both drugs promoted an additional antihypertensive effect in 2K-1C rats. Renal Ang II content and albuminuria were reduced by either enalapril or losartan in monotherapy and restored to control levels by dual RAS blockade. Albuminuria in 2K-1C rats was accompanied by downregulation of the glomerular slit protein podocin, reduction of the endocytic receptors megalin and cubilin, and a marked decrease in the expression of the ClC-5 chloride channel, compared to 2K animals. Treatment with losartan and enalapril in monotherapy or combination increased the expression of podocin, cubilin, and ClC-5. However, only the combined therapy normalized podocin, cubilin, and ClC-5 protein abundance in the non-clipped kidney of 2K-1C rats. Renovascular hypertensive 2K-1C rats had a lower concentration of plasma potassium compared to 2K rats. Single RAS blockade normalized potassium plasma concentration, whereas 2K-1C rats treated with dual RAS blockade exhibited hyperkalemia. Hypokalemia in 2K-1C rats was accompanied by an increase in the cleaved activated forms of α-ENaC and γ-ENaC and the expression of β-ENaC. Combined RAS blockade but not monotherapy significantly reduced the expression of these ENaC subunits in 2K-1C rats. Indeed, double RAS blockade reduced the abundance of cleaved-α-ENaC to levels lower than those of 2K rats. Collectively, these results demonstrate that the antiproteinuric effect of dual RAS blockade in 2K-1C rats is associated with the restored abundance of podocin and cubilin, and ClC-5. Moreover, double RAS blockade-induced hyperkalemia may be due, at least partially, to an exaggerated downregulation of cleaved α-ENaC in the non-clipped kidney of renovascular hypertensive rats. [ABSTRACT FROM AUTHOR]

Aim: The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co‐expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo. Methods: We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8‐S238A) or a mutation leading to a zymogen‐locked state (Prss8‐R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8‐S238A and Prss8‐R44Q was studied in vitro using the Xenopus laevis oocyte expression system. Results: Co‐expression of murine ENaC with Prss8‐wt or Prss8‐S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co‐expressing Prss8‐R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8‐R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8‐S238A and Prss8‐R44Q mice but with higher plasma aldosterone concentrations in Prss8‐R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8‐wt and Prss8‐S238A mice, whereas Prss8‐R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure. Conclusion: Unlike proteolytically inactive Prss8‐S238A, zymogen‐locked Prss8‐R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity. [ABSTRACT FROM AUTHOR]

Vascular epithelial sodium channels (ENaCs) made up of canonical α, β, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, μ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, μ-ENaC is the least understood subunit. However, the recent discovery of μ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the d subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of μ-ENaC in the vasculature to identify whether μ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of μ-ENaC and implications for vascular physiology and pathophysiology in humans. [ABSTRACT FROM AUTHOR]

Vascular epithelial sodium channels (ENaCs) made up of canonical α, β, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans. [ABSTRACT FROM AUTHOR]

Vascular epithelial sodium channels (ENaCs) made up of canonical α, β, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans. [ABSTRACT FROM AUTHOR]

Vascular epithelial sodium channels (ENaCs) made up of canonical α, β, and c subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, ƍ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, d-ENaC is the least understood subunit. However, the recent discovery of ƍ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the d subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of ƍ-ENaC in the vasculature to identify whether ƍ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of ƍ-ENaC and implications for vascular physiology and pathophysiology in humans. [ABSTRACT FROM AUTHOR]

Objective: The study was performed to investigate the association of hypertension in pregnancy with prostasin gene polymorphism in Pakistani females. Methods: This case-control study was performed at University of Karachi, Pakistan from April 2018 to May 2019. A total of 160 females, including 90 hypertensives and 70 healthy pregnant females, were recruited by purposive sampling after obtaining informed written consent. Genotyping was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: The frequencies of the TC and CC genotypes were higher in hypertensive pregnant females compared to healthy controls. A significant difference was evident for CC (P=0.012) genotype; however, no significant difference was observed for TC (P=0.49) and TT genotypes (P=0.06) between control and hypertensive groups. The adjusted odds ratio for CC genotype was 6.2 (P=0.025) and 1.48 (P=0.44) for TC genotype compared to the TT genotype. There was a significantly higher prevalence of the C allele of the prostasin gene at rs12597511 in the hypertensive group, suggesting that this allele is a risk factor for hypertension and cardiovascular diseases. Conclusion: C allele at rs12597511 of prostasin gene demonstrate as a risk factor for having hypertension in pregnancy. [ABSTRACT FROM AUTHOR]

Aim: Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in NS. However, a proof of the essential role of plasmin in experimental NS is lacking. Methods: We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6‐Nphs2tm3.1Antc*Tg(Nphs1‐rtTA*3G)8Jhm*Tg(tetO‐cre)1Jaw or nphs2Δipod). These mice were crossed with plasminogen deficient mice (Bl6‐Plgtm1Jld or plg−/−) to generate double knockout mice (nphs2Δipod*plg−/−). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days. Results: Uninduced nphs2Δipod*plg−/− mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2Δipod*plg+/+ and nphs2Δipod*plg−/− mice. Western blot revealed the urinary excretion of plasminogen and plasmin in nphs2Δipod*plg+/+ mice which were absent in nphs2Δipod*plg−/− mice. After the onset of proteinuria, amiloride‐sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes. Conclusions: This study shows that mice lacking urinary plasminogen are not protected from ENaC‐mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen. [ABSTRACT FROM AUTHOR]

Aims: Acute 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.Methods: We 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.Results: Our 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.Conclusion: In 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. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSCs) were shown to harbor therapeutic potential in models of respiratory diseases, such as bronchopulmonary dysplasia (BPD), the most common sequel of preterm birth. In these studies, cells or animals were challenged with hyperoxia or other injury-inducing agents. However, little is known about the effect of MSCs on immature fetal lungs and whether MSCs are able to improve lung maturity, which may alleviate lung developmental arrest in BPD. Methods: We aimed to determine if the conditioned medium (CM) of MSCs stimulates functional and structural lung maturation. As a measure of functional maturation, Na+ transport in primary fetal distal lung epithelial cells (FDLE) was studied in Ussing chambers. Na+ transporter and surfactant protein mRNA expression was determined by qRT-PCR. Structural maturation was assessed by microscopy in fetal rat lung explants. Results: MSC-CM strongly increased the activity of the epithelial Na+ channel (ENaC) and the Na,K-ATPase as well as their mRNA expression. Branching and growth of fetal lung explants and surfactant protein mRNA expression were enhanced by MSC-CM. Epithelial integrity and metabolic activity of FDLE cells were not influenced by MSC-CM. Since MSC's actions are mainly attributed to paracrine signaling, prominent lung growth factors were blocked. None of the tested growth factors (VEGF, BMP, PDGF, EGF, TGF-β, FGF, HGF) contributed to the MSC-induced increase of Na+ transport. In contrast, inhibition of PI3-K/AKT and Rac1 signaling reduced MSC-CM efficacy, suggesting an involvement of these pathways in the MSC-CM-induced Na+ transport. Conclusion: The results demonstrate that MSC-CM strongly stimulated functional and structural maturation of the fetal lungs. These effects were at least partially mediated by the PI3-K/AKT and Rac1 signaling pathway. Thus, MSCs not only repair a deleterious tissue environment, but also target lung cellular immaturity itself. [ABSTRACT FROM AUTHOR]

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Intra-amniotic exposure to proinflammatory cytokines such as interleukin-1 (IL-1) correlates with a decreased incidence of respiratory distress syndrome (RDS) in infants following premature birth. At birth, inadequate absorption of fluid from the fetal lung contributes to the onset RDS. Lung fluid clearance is coupled to Na+ transport via epithelial sodium channels (ENaC). In this study, we assessed the effects of IL-1 on the expression of ENaC, particularly the α-subunit which is critical for fetal lung fluid clearance at birth. Cultured mouse lung epithelial (MLE-12) cells were treated with either IL-1α or IL-1β to determine their effects on α-ENaC expression. Changes in IL-1-induced α-ENaC levels in the presence of IL-1 receptor antagonist (IL-1ra), cycloheximide, NF-κB inhibitor, and MAP kinase inhibitors were investigated. IL-1α and IL-1β independently induced a significant increase of α-ENaC mRNA and protein after 24 h compared to untreated cells. IL-1-dependent increases in α-ENaC protein were mitigated by IL-1ra and cycloheximide. IL-1 exposure induced NF-κB binding activity. Attenuation of IL-1-induced NF-κB activation by its inhibitor SN50 decreased α-ENaC protein abundance. Inhibition of ERK 1,2 MAPK significantly decreased both IL-1α and β-induced α-ENaC protein expression whereas inhibition of p38 MAPK only blocked IL-1β-induced α-ENaC protein levels. In contrast, IL-1-induced α-ENaC protein levels were unaffected by a c-Jun N-terminal kinase (JNK) inhibitor. Our results suggest that in MLE-12 cells, IL-1-induced elevation of α-ENaC is mediated via NF-κB activation and in part involves stimulation of the ERK 1,2 and p38 MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

Background: In COVID 19 related lung disease, which is a leading cause of death from this disease, cytokines like tumor necrosis factor-alpha (TNF alpha) may be pivotal in the pathogenesis. TNF alpha reduces fluid absorption due to impairment of sodium and chloride transport required for building an osmotic gradient across epithelial cells, which in the airways maintains airway surface liquid helping to keep airways open and enabling bacterial clearance and aids water absorption from the alveolar spaces. TNF alpha can, through Rho-kinase, disintegrate the endothelial and epithelial cytoskeleton, and thus break up intercellular tight junctional proteins, breaching the intercellular barrier, which prevents flooding of the interstitial and alveolar spaces with fluid. Hypotheses: (1) Preservation and restoration of airway and alveolar epithelial sodium and chloride transport and the cytoskeleton dependent integrity of the cell barriers within the lung can prevent and treat COVID 19 lung disease. (2) TNF alpha is the key mediator of pulmonary edema in COVID 19 lung disease. Confirmation of hypothesis and implications: The role of a reduction in the function of epithelial sodium and chloride transport could with regards to chloride transport be tested by analysis of chloride levels in exhaled breath condensate and levels correlated with TNF alpha concentrations. Reduced levels would indicate a reduction of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and a correlation with TNF alpha levels indicative of its involvement. Anti-TNF alpha treatment with antibodies is already available and needs to be tested in randomized controlled trials of COVID 19 lung disease. TNF alpha levels could also be reduced by statins, aspirin, and curcumin. Chloride transport could be facilitated by CFTR activators, including curcumin and phosphodiesterase-5 inhibitors. Sodium and chloride transport could be further regulated to prevent accumulation of alveolar fluid by use of Na(+)/K(+)/2Cl(−) cotransporter type 1 inhibitors, which have been associated with improved outcome in adults ventilated for acute respiratory distress syndrome (ARDS) in randomized controlled trials. Primary prevention of coronavirus infection and TNF alpha release in response to it could be improved by induction of antimicrobial peptides LL-37 and human beta defensin-2 and reduction of TNF alpha production by vitamin D prophylaxis for the population as a whole. [ABSTRACT FROM AUTHOR]

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, β-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and β-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, β-, and γ-subunits and ask for a careful investigation of the channel composition. [ABSTRACT FROM AUTHOR]

BACKGROUND "Neuronal precursor cell expressed developmentally down-regulated 4-like" (NEDD4L) is considered a candidate gene for hypertension—both functionally and genetically—through the regulation of the ubiquitination of the epithelial sodium channel (ENaC). This study explores the relationship between genetic variation in NEDD4L and hypertension with chronic kidney disease (CKD) in the southeastern Han Chinese population. METHODS We recruited 623 CKD patients and measured ambulatory blood pressure monitoring (ABPM), and the rs4149601 and rs2288774 polymorphisms in NEDD4L were genotyped using quantitative polymerase chain reaction. RESULTS For rs4149601, significant differences in genotype frequencies in an additive model (GG vs. GA vs. AA) were observed between normotensive patients and hypertensive patients when hypertension was classified into ambulatory hypertension, clinical hypertension, and ambulatory systolic hypertension (P = 0.038, 0.005, and 0.006, respectively). In a recessive model (GG + GA vs. AA), the frequency of the AA genotype of rs4149601 in the hypertension groups was all higher than that in the normotensive groups. The genotype distribution of rs2288774 did not differ significantly between the normotensive and hypertensive patients. In both the full cohort and the propensity score matching (PSM) cohort, the AA genotype of rs4149601 (compared with the GG + GA genotype group) was independently correlated with ambulatory hypertension, clinical hypertension, and ambulatory systolic hypertension by multivariate logistic regression analysis. CONCLUSIONS The present study indicates that the AA genotype of rs4149601 associates with hypertension in CKD. Consequently, the rs4149601 A allele might be a risk factor for hypertension with CKD. [ABSTRACT FROM AUTHOR]