Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis.

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

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.

Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4-2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

Structural basis for excitatory neuropeptide signaling.

Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.

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

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

A mathematical model of ENaC and Slc26a6 regulation by CFTR in salivary gland ducts.

Cystic fibrosis (CF) is a genetic disease caused by the mutations of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis transmembrane conductance regulator gene. Cftr is a critical ion channel expressed in the apical membrane of mouse salivary gland striated duct cells. Although Cftr is primarily a Cl- channel, its knockout leads to higher salivary Cl- and Na+ concentrations and lower pH. Mouse experiments show that the activation of Cftr upregulates epithelial Na+ channel (ENaC) protein expression level and Slc26a6 (a 1Cl-:2[Formula: see text] exchanger of the solute carrier family) activity. Experimentally, it is difficult to predict how much the coregulation effects of CFTR contribute to the abnormal Na+, Cl-, and [Formula: see text] concentrations and pH in CF saliva. To address this question, we construct a wild-type mouse salivary gland model and simulate CFTR knockout by altering the expression levels of CFTR, ENaC, and Slc26a6. By reproducing the in vivo and ex vivo final saliva measurements from wild-type and CFTR knockout animals, we obtain computational evidence that ENaC and Slc26a6 activities are downregulated in CFTR knockout in salivary glands.NEW & NOTEWORTHY This paper describes a salivary gland mathematical model simulating the ion exchange between saliva and the salivary gland duct epithelium. The novelty lies in the implementation of CFTR regulating ENaC and Slc26a6 in a CFTR knockout gland. By reproducing the experimental saliva measurements in wild-type and CFTR knockout glands, the model shows that CFTR regulates ENaC and Slc26a6 anion exchanger in salivary glands. The method could be used to understand the various cystic fibrosis phenotypes.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Comprehensive mapping of Epithelial Na+ channel α expression in the mouse brain.

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.

Dendritic cell epithelial sodium channel induced inflammation and salt-sensitive hypertension.

PURPOSE OF REVIEW: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease. Epithelial sodium channel (ENaC) plays a critical role in renal electrolyte and volume regulation and has been implicated in the pathogenesis of SSBP. This review describes recent advances regarding the role of ENaC-dependent inflammation in the development of SSBP. RECENT FINDINGS: We recently found that sodium enters dendritic cells via ENaC, a process regulated by serum/glucocorticoid-regulated kinase 1 and epoxyeicosatrienoic acid 14,15. Sodium entry activates NADPH oxidase, leading to the production of isolevuglandins (IsoLGs). IsoLGs adduct self-proteins to form neoantigens in dendritic cells that activate T cells and result in the release of cytokines promoting sodium retention, kidney damage, and endothelial dysfunction in SSBP. Additionally, we described a novel mechanistic pathway involving ENaC and IsoLG-dependent NLRP3 inflammasome activation. These findings hold promise for the development of novel diagnostic biomarkers and therapeutic options for SSBP. SUMMARY: The exact mechanisms underlying SSBP remain elusive. Recent advances in understanding the extrarenal role of ENaC have opened a new perspective, and further research efforts should focus on understanding the link between ENaC, inflammation, and SSBP.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC ß- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Reverse Phenotypes of Patients with Genetically Confirmed Liddle Syndrome.

BACKGROUND: Liddle syndrome was initially characterized by hypertension, hypokalemia, metabolic alkalosis, and suppressed plasma renin and aldosterone, resulting from gain-of-function variants in the epithelial Na + channel (ENaC). Efficient treatment with ENaC inhibitors is available, but the phenotypic spectrum of genetically confirmed Liddle syndrome is unknown, and some patients may remain undiagnosed and at risk of inefficient treatment. In this study, we used a reverse phenotyping approach to investigate the Liddle syndrome phenotypic spectrum and genotype-phenotype correlations. METHODS: Pubmed, Embase, Scopus, and the Human Gene Mutation Database were searched for articles reporting Liddle syndrome variants. The genetic variants were systematically classified to identify patients with genetically confirmed Liddle syndrome. We identified 62 articles describing 45 unique variants within 86 Liddle syndrome families, and phenotypic data were pooled for 268 patients with confirmed Liddle syndrome. RESULTS: The Liddle syndrome variants localized to exon 13 of SCNN1B and SCNN1G , disrupting the PPPxY motif critical for downregulating ENaC activity. Hypertension sensitive to ENaC inhibition was present in 97% of adults carrying Liddle syndrome variants while hypokalemia, metabolic alkalosis, and plasma renin and aldosterone suppression showed incomplete penetrance. In addition, 95% and 55% of patients had a family history of hypertension or cerebrovascular events, respectively. The genotype had minor phenotypic effects; however, probands compared with relatives showed significant phenotypic discrepancies consistent with selection bias for initial genetic screening. CONCLUSIONS: Patients with genetically confirmed Liddle syndrome displayed a phenotypic spectrum, with ENaC-sensitive hypertension and family history of hypertension being the most common features. The phenotype seemed independent of the specific gene or variant type involved.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

BACKGROUND: The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. METHODS: Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. RESULTS: Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. CONCLUSIONS: Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

BACKGROUND: Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. METHODS: We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. RESULTS: Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and ßENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. CONCLUSIONS: AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

MicroRNA-19 is regulated by aldosterone in a sex-specific manner to alter kidney sodium transport.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.

Molecular mechanisms of dexamethasone actions in COVID-19: Ion channels and airway surface liquid dynamics.

The COVID-19 pandemic has been a global health crisis of unprecedented magnitude. In the battle against the SARS-CoV-2 coronavirus, dexamethasone, a widely used corticosteroid with potent anti-inflammatory properties, has emerged as a promising therapy in the fight against severe COVID-19. Dexamethasone is a synthetic glucocorticoid that exerts its therapeutic effects by suppressing the immune system and reducing inflammation. In the context of COVID-19, the severe form of the disease is often characterized by a hyperactive immune response, known as a cytokine storm. Dexamethasone anti-inflammatory properties make it a potent tool in modulating this exaggerated immune response. Lung inflammation may lead to excessive fluid accumulation in the airways which can reduce gas exchange and mucociliary clearance. Pulmonary oedema and flooding of the airways are hallmarks of severe COVID-19 lung disease. The volume of airway surface liquid is determined by a delicate balance of salt and water secretion and absorption across the airway epithelium. In addition to its anti-inflammatory actions, dexamethasone modulates the activity of ion channels which regulate electrolyte and water transport across the airway epithelium. The observations of dexamethasone activation of sodium ion absorption via ENaC Na+ channels and inhibition of chloride ion secretion via CFTR Cl- channels to decrease airway surface liquid volume indicate a novel therapeutic action of the glucocorticoid to reverse airway flooding. This brief review delves into the early non-genomic and late genomic signaling mechanisms of dexamethasone regulation of ion channels and airway surface liquid dynamics, shedding light on the molecular mechanisms underpinning the action of the glucocorticoid in managing COVID-19.

A frameshift mutation in the SCNN1B gene in a family with Liddle syndrome: A case report and systematic review.

Liddle syndrome is an autosomal dominant form of monogenic hypertension that is caused by mutations in SCNN1A, SCNN1B or SCNN1G, which respectively encode the α, ß and γ subunits of the epithelial sodium channel. In the present study, DNA was extracted from leukocytes in peripheral blood obtained from all members of a family with Liddle syndrome. Whole­exome sequencing and Sanger sequencing were performed to assess the candidate variant and a co­segregation analysis was conducted. A frameshift mutation in SCNN1B (NM_ 000336: c.1806dupG, p.Pro603Alafs*5) in the family was identified, characterized by early­onset hypertension and hypokalemia. The mutation led to the truncation of the ß subunit of the epithelial sodium channel and a lack of the conservative PY motif. Furthermore, a systematic review of follow­up data from patients with Liddle syndrome with SCNN1B mutations was performed. The follow­up data of 108 patients with pathogenic SCNN1B mutations from 47 families were summarized. Phenotypic heterogeneity was evident in patients with Liddle syndrome and early­onset hypertension was the most frequent symptom. Patients responded well to targeted amiloride therapy with significant improvements in blood pressure and serum potassium concentration. The present study demonstrates that confirmatory genetic testing and targeted therapy can prevent premature onset of clinical endpoint events in patients with Liddle syndrome.

Discovery and development of BI 1265162, an ENaC inhibitor for the treatment of cystic fibrosis.

Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and development of BI 1265162, the first ENaC inhibitor devoid of the amiloride structural motif that entered clinical trials. The design of BI 1265162 focused on its suitability for inhalation via the Respimat® Soft Mist™ Inhaler and a long duration of action. A convergent and scalable route for the synthesis of BI 1265162 as dihydrogen phosphate salt is presented, that was applied to support clinical trials. A phase 2 study with BI 1265162 did not provide a clear sign of clinical benefit. Whether ENaC inhibition will be able to hold its promise for CF patients remains an open question.