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

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.

Luteolin Enhances Transepithelial Sodium Transport in the Lung Alveolar Model: Integrating Network Pharmacology and Mechanism Study.

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.

Ferulic Acid: A Review of Pharmacology, Toxicology, and Therapeutic Effects on Pulmonary Diseases.

Ferulic acid (FA), a prevalent dietary phytochemical, has many pharmacological effects, including anti-oxidation and anti-inflammation effects, and has been widely used in the pharmaceutical, food, and cosmetics industries. Many studies have shown that FA can significantly downregulate the expression of reactive oxygen species and activate nuclear factor erythroid-2-related factor-2/heme oxygenase-1 signaling, exerting anti-oxidative effects. The anti-inflammatory effect of FA is mainly related to the p38 mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways. FA has demonstrated potential clinical applications in the treatment of pulmonary diseases. The transforming growth factor-ß1/small mothers against decapentaplegic 3 signaling pathway can be blocked by FA, thereby alleviating pulmonary fibrosis. Moreover, in the context of asthma, the T helper cell 1/2 imbalance is restored by FA. Furthermore, FA ameliorates acute lung injury by inhibiting nuclear factor-kappaB and mitogen-activated protein kinase pathways via toll-like receptor 4, consequently decreasing the expression of downstream inflammatory mediators. Additionally, there is a moderate neuraminidase inhibitory activity showing a tendency to reduce the interleukin-8 level in response to influenza virus infections. Although the application of FA has broad prospects, more preclinical mechanism-based research should be carried out to test these applications in clinical settings. This review not only covers the literature on the pharmacological effects and mechanisms of FA, but also discusses the therapeutic role and toxicology of FA in several pulmonary diseases.

Competitive cleavage of SARS-CoV-2 spike protein and epithelial sodium channel by plasmin as a potential mechanism for COVID-19 infection.

Cleavage of the furin site in SARS-CoV-2 spike (S) protein accounts for increased transmissibility of COVID-19 by promoting the entry of virus into host cells through specific angiotensin-converting enzyme 2 (ACE2) receptors. Plasmin, a key serine protease of fibrinolysis system, cleaves the furin site of γ subunit of human epithelial sodium channels (ENaCs). Sharing the plasmin cleavage by viral S and host ENaC proteins may competitively inter-regulate SARS-CoV-2 transmissibility and edema resolution via the ENaC pathway. To address this possibility, we analyzed single-cell RNA sequence (scRNA-seq) data sets and found that PLAU (encoding urokinase plasminogen activator), SCNN1G (γENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in airway/alveolar epithelial cells. The expression levels of PLAU and FURIN were significantly higher compared with TMPRSS2 in healthy group. This difference was further amplified in both epithelial and immune cells in patients with moderate/severe COVID-19 and SARS-CoV-2 infected airway/alveolar epithelial cell lines. Of note, plasmin cleaved the S protein and facilitated the entry of pseudovirus in HEK293 cells. Conclusively, SARS-CoV-2 may expedite infusion by competing the fibrinolytic protease network with ENaC.

Effects of hypoxia on respiratory diseases: perspective view of epithelial ion transport.

The balance of gas exchange and lung ventilation is essential for the maintenance of body homeostasis. There are many ion channels and transporters in respiratory epithelial cells, including epithelial sodium channel, Na,K-ATPase, cystic fibrosis transmembrane conductance regulator, and some transporters. These ion channels/transporters maintain the capacity of liquid layer on the surface of respiratory epithelial cells and provide an immune barrier for the respiratory system to clear off foreign pathogens. However, in some harmful external environments and/or pathological conditions, the respiratory epithelium is prone to hypoxia, which would destroy the ion transport function of the epithelium and unbalance the homeostasis of internal environment, triggering a series of pathological reactions. Many respiratory diseases associated with hypoxia manifest an increased expression of hypoxia-inducible factor-1, which mediates the integrity of the epithelial barrier and affects epithelial ion transport function. It is important to study the relationship between hypoxia and ion transport function, whereas the mechanism of hypoxia-induced ion transport dysfunction in respiratory diseases is not clear. This review focuses on the relationship between hypoxia and respiratory diseases, as well as dysfunction of ion transport and tight junctions in respiratory epithelial cells under hypoxia.

Small Extracellular Vesicles Containing miR-34c Derived from Bone Marrow Mesenchymal Stem Cells Regulates Epithelial Sodium Channel via Targeting MARCKS.

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, ß 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.

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

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

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

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.

Ion transport mechanisms for smoke inhalation-injured airway epithelial barrier.

Smoke inhalation injury is the leading cause of death in firefighters and victims. Inhaled hot air and toxic smoke are the predominant hazards to the respiratory epithelium. We aimed to analyze the effects of thermal stress and smoke aldehyde on the permeability of the airway epithelial barrier. Transepithelial resistance (RTE) and short-circuit current (ISC) of mouse tracheal epithelial monolayers were digitized by an Ussing chamber setup. Zonula occludens-1 tight junctions were visualized under confocal microscopy. A cell viability test and fluorescein isothiocyanate-dextran assay were performed. Thermal stress (40 °C) decreased RTE in a two-phase manner. Meanwhile, thermal stress increased ISC followed by its decline. Na+ depletion, amiloride (an inhibitor for epithelial Na+ channels [ENaCs]), ouabain (a blocker for Na+/K+-ATPase), and CFTRinh-172 (a blocker of cystic fibrosis transmembrane regulator [CFTR]) altered the responses of RTE and ISC to thermal stress. Steady-state 40 °C increased activity of ENaCs, Na+/K+-ATPase, and CFTR. Acrolein, one of the main oxidative unsaturated aldehydes in fire smoke, eliminated RTE and ISC. Na+ depletion, amiloride, ouabain, and CFTRinh-172 suppressed acrolein-sensitive ISC, but showed activating effects on acrolein-sensitive RTE. Thermal stress or acrolein disrupted zonula occludens-1 tight junctions, increased fluorescein isothiocyanate-dextran permeability but did not cause cell death or detachment. The synergistic effects of thermal stress and acrolein exacerbated the damage to monolayers. In conclusion, the paracellular pathway mediated by the tight junctions and the transcellular pathway mediated by active and passive ion transport pathways contribute to impairment of the airway epithelial barrier caused by thermal stress and acrolein. Graphical abstract Thermal stress and acrolein are two essential determinants for smoke inhalation injury, impairing airway epithelial barrier. Transcellular ion transport pathways via the ENaC, CFTR, and Na/K-ATPase are interrupted by both thermal stress and acrolein, one of the most potent smoke toxins. Heat and acrolein damage the integrity of the airway epithelium through suppressing and relocating the tight junctions.

The Sorting Nexin 3 Retromer Pathway Regulates the Cell Surface Localization and Activity of a Wnt-Activated Polycystin Channel Complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by inactivating mutations in PKD1 (85%) or PKD2 (15%). The ADPKD proteins encoded by these genes, polycystin-1 (PC1) and polycystin-2 (PC2), form a plasma membrane receptor-ion channel complex. However, the mechanisms controlling the subcellular localization of PC1 and PC2 are poorly understood. Here, we investigated the involvement of the retromer complex, an ancient protein module initially discovered in yeast that regulates the retrieval, sorting, and retrograde transport of membrane receptors. Using yeast two-hybrid, biochemical, and cellular assays, we determined that PC2 binds two isoforms of the retromer-associated protein sorting nexin 3 (SNX3), including a novel isoform that binds PC2 in a direct manner. Knockdown of SNX3 or the core retromer protein VPS35 increased the surface expression of endogenous PC1 and PC2 in vitro and in vivo and increased Wnt-activated PC2-dependent whole-cell currents. These findings indicate that an SNX3-retromer complex regulates the surface expression and function of PC1 and PC2. Molecular targeting of proteins involved in the endosomal sorting of PC1 and PC2 could lead to new therapeutic approaches in ADPKD.

Transepithelial Fluid and Salt Re-Absorption Regulated by cGK2 Signals.

Transepithelial fluid and salt re-absorption in epithelial tissues play an important role in fluid and salt homeostasis. In absorptive epithelium, fluid and salt flux is controlled by machinery mainly composed of epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), Na⁺/H⁺ exchanger (NHE), aquaporin, and sodium potassium adenosine triphosphatase (Na⁺/K⁺-ATPase). Dysregulation of fluid and salt transport across epithelium contributes to the pathogenesis of many diseases, such as pulmonary edema and cystic fibrosis. Intracellular and extracellular signals, i.e., hormones and protein kinases, regulate fluid and salt turnover and resolution. Increasing evidence demonstrates that transepithelial fluid transport is regulated by cyclic guanosine monophosphate-dependent protein kinase (cGK) signals. cGK2 was originally identified and cloned from intestinal specimens, the presence of which has also been confirmed in the kidney and the lung. cGK2 regulates fluid and salt through ENaC, CFTR and NHE. Deficient cGK2 regulation of transepithelial ion transport was seen in acute lung injury, and cGK2 could be a novel druggable target to restore edematous disorder in epithelial tissues.

ENaCs as Both Effectors and Regulators of MiRNAs in Lung Epithelial Development and Regeneration.

Epithelial sodium channels (ENaC) play an important role in re-absorbing excessive luminal fluid by building up an osmotic Na+ gradient across the tight epithelium in the airway, the lung, the kidney, and the colon. The ENaC is a major pathway for retention of salt in kidney too. MicroRNAs (miRs), a group of non-coding RNAs that regulate gene expression at the post-transcriptional level, have emerged as a novel class of regulators for ENaC. Given the ENaC pathway is crucial for maintaining fluid homeostasis in the lung and the kidney and other cavities, we summarized the cross-talk between ENaC and miRs and recapitulated the underlying regulatory factors, including aldosterone, transforming growth factor-ß1, and vascular endothelial growth factor-A in the lung and other epithelial tissues/organs. We have compared the profiling of miRs between normal and injured mice and human lungs, which showed a significant alteration in numerous miRs in mouse models of LPS and ventilator induced ARDS. In addition, we reiterated the potential regulation of the ENaC by miRs in stem/ progenitor cell-based re-epithelialization, and identified a promising pharmaceutic target of ENaC for removing edema fluid in ARDS by mesenchymal stem cells-released paracrine. In conclusion, it seems that the interactions between miRs and scnn1s/ENaCs are critical for lung development, epithelial cell turnover in adult lungs, and re-epithelialization for repair.

Docetaxel modulates the delayed rectifier potassium current (IK) and ATP-sensitive potassium current (IKATP) in human breast cancer cells.

Ion channel expression and activity may be affected during tumor development and cancer growth. Activation of potassium (K(+)) channels in human breast cancer cells is reported to be involved in cell cycle progression. In this study, we investigated the effects of docetaxel on the delayed rectifier potassium current (I K) and the ATP-sensitive potassium current (I KATP) in two human breast cancer cell lines, MCF-7 and MDA-MB-435S, using the whole-cell patch-clamp technique. Our results show that docetaxel inhibited the I K and I KATP in both cell lines in a dose-dependent manner. Compared with the control at a potential of +60 mV, treatment with docetaxel at doses of 0.1, 1, 5, and 10 µM significantly decreased the I K in MCF-7 cells by 16.1 ± 3.5, 30.2 ± 5.2, 42.5 ± 4.3, and 46.4 ± 9% (n = 5, P < 0.05), respectively and also decreased the I KATP at +50 mV. Similar results were observed in MDA-MB-435S cells. The G-V curves showed no significant changes after treatment of either MCF-7 or MDA-MB-435S cells with 10 µM docetaxel. The datas indicate that the possible mechanisms of I K and I KATP inhibition by docetaxel may be responsible for its effect on the proliferation of human breast cancer cells.

Network pharmacology-based research into the mechanism of ferulic acid on acute lung injury through enhancing transepithelial sodium transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferulic acid (FA) has shown potential therapeutic applications in treating lung diseases. However, the underlying mechanisms by which FA ameliorates acute lung injury (ALI) have not been distinctly elucidated. AIM OF THE STUDY: The project aims to observe the therapeutic effects of FA on lipopolysaccharide-induced ALI and to elucidate its specific mechanisms in regulating epithelial sodium channel (ENaC), which majors in alveolar fluid clearance during ALI. MATERIALS AND METHODS: In this study, the possible pathways of FA were determined through network pharmacology analyses. The mechanisms of FA in ALI were verified by in vivo mouse model and in vitro studies, including primary alveolar epithelial type 2 cells and three-dimensional alveolar organoid models. RESULTS: FA ameliorated ALI by improving lung pathological changes, reducing pulmonary edema, and upregulating the α/γ-ENaC expression in C57BL/J male mice. Simultaneously, FA was observed to augment ENaC levels in both three-dimensional alveolar organoid and alveolar epithelial type 2 cells models. Network pharmacology techniques and experimental data from inhibition or knockdown of IkappaB kinase ß (IKKß) proved that FA reduced the phosphorylation of IKKß/nuclear factor-kappaB (NF-κB) and eliminated the lipopolysaccharide-inhibited expression of ENaC, which could be regulated by nuclear protein NF-κB p65 directly. CONCLUSIONS: FA could enhance the expression of ENaC at least in part by inhibiting the IKKß/NF-κB signaling pathway, which may potentially pave the way for promising treatment of ALI.

Regulation of Epithelial Sodium Transport by SARS-CoV-2 Is Closely Related with Fibrinolytic System-Associated Proteins.

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 ß-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.

Fibrinolytic system and COVID-19: From an innovative view of epithelial ion transport.

Lifeways of worldwide people have changed dramatically amid the coronavirus disease 2019 (COVID-19) pandemic, and public health is at stake currently. In the early stage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, fibrinolytic system is mostly inhibited, which is responsible for the development of hypofibrinolysis, promoting disseminated intravascular coagulation, hyaline membrane formation, and pulmonary edema. Whereas the common feature and risk factor at advanced stage is a large amount of fibrin degradation products, including D-dimer, the characteristic of hyperfibrinolysis. Plasmin can cleave both SARS-CoV-2 spike protein and γ subunit of epithelial sodium channel (ENaC), a critical element to edematous fluid clearance. In this review, we aim to sort out the role of fibrinolytic system in the pathogenesis of COVID-19, as well as provide the possible guidance in current treating methods. In addition, the abnormal regulation of ENaC in the occurrence of SARS-CoV-2 mediated hypofibrinolysis and hyperfibrinolysis are summarized, with the view of proposing an innovative view of epithelial ion transport in preventing the dysfunction of fibrinolytic system during the progress of COVID-19.

Mesenchymal stem cell conditioned medium alleviates acute lung injury through KGF-mediated regulation of epithelial sodium channels.

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression.

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.

Characterization of a novel splice variant of δ ENaC subunit in human lungs.

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. δ ENaC (termed δ1 in this article) has been detected in human lung epithelial cells in addition to α, ß, and γ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: 8233-8241, 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: 2663-2676, 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the δ1 ENaC, namely, δ2 ENaC in human bronchoalveolar epithelial cells (16HBEo). δ2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1 ENaC. δ2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of δ1ßγ, δ2ßγ, and αßγ channels. Oocytes injected with δ2ßγ cRNAs exhibited whole cell currents significantly greater than those expressing δ1ßγ and αßγ channels. Single channel activity, unitary conductance, and open probability of δ2ßγ channels were significantly greater compared with δ1ßγ and αßγ channels. In addition, δ2ßγ and δ1ßγ channels displayed significant differences in apparent Na(+) affinity, dissociation constant for amiloride (K(i)(amil)), the EC(50) for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na(+) channels.

Expression and activity of epithelial sodium channel in hyperoxia-induced bronchopulmonary dysplasia in neonatal rats.

BACKGROUND: The aim of the present study was to investigate the expression and activity of epithelial sodium channel (ENaC) in hyperoxia-induced bronchopulmonary dysplasia (BPD) in neonatal rats. METHODS: Neonatal rats were exposed to hyperoxia to establish BPD models (control group was exposed to air), lung water was measured and Western blot was applied to detect the expression of three homologous subunits: α-, ß- and γ-ENaC in the lung tissues. Furthermore, ATII cells were isolated from neonatal rats, and primarily cultured under normoxic or hyperoxic conditions. The ENaC expression was also examined in these cells. In addition, the amiloride-sensitive Na(+) currents induced by hyperoxia were recorded using the whole-cell patch clamp technique. RESULTS: The α-ENaC expression was increased after 5 days of hyperoxia in rat lung tissues, whereas not after 1, 3 and 7 days. ATII cells showed α-ENaC expression was reduced after 1 and 2 days' hyperoxia, but no change after 3 days. In contrast, ß- and γ-ENaC expression was increased after hyperoxia in both in vivo and in vitro experiments. The amiloride-sensitive Na(+) currents in hyperoxia-exposed ATII cells were also increased, which was consistent with the upregulated expression of ß- and γ-ENaC. CONCLUSION: Hyperoxia upregulates the expression of ENaC, especially ß- and γ-ENaC subunits, in both neonatal rat lung tissues and ATII cells. Hyperoxia also enhanced the activity of ENaC in neonatal rat ATII cells. Dysfunctional transport of Na(+) may not be a key factor involving pulmonary edema at the early stage of BPD.