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The epithelial NaWe explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests.We found that variants inOur results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal. Although there is no cure for this disease, specific genes that lead to different Bartter syndrome subtypes have been identified. Bartter syndrome type II specifically arises from mutations in theKCNJ1gene, which encodes the renal outer medullary potassium channel, ROMK. To date, over 40 Bartter syndrome-associated mutations inKCNJ1have been identified. Yet, their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carryingKCNJ1variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined ROMK genomic data in both the NIH TOPMed and ClinVar databases with the aid of a computational algorithm that predicts protein misfolding and disease severity. Subsequent phenotypic studies using a high throughput yeast screen to assess ROMK function—and analyses of ROMK biogenesis in yeast and human cells—identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced protein expression at the cell surface. Another ERAD-targeted ROMK mutant (L320P) was found in only one of the screens. In contrast, another mutation (T300R) was ERAD-resistant, but defects in ROMK activity were apparent after expression and two-electrode voltage clamp measurements inXenopusoocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies.Author SummaryBartter syndrome is a rare genetic disorder characterized by defective renal electrolyte handing, leading to debilitating symptoms and, in some patients, death in infancy. Currently, there is no cure for this disease. Bartter syndrome is divided into five types based on the causative gene. Bartter syndrome type II results from genetic variants in the gene encoding the ROMK protein, which is expressed in the kidney and assists in regulating sodium, potassium, and water homeostasis. Prior work established that some disease-associated ROMK mutants misfold and are destroyed soon after their synthesis in the endoplasmic reticulum (ER). Because a growing number of drugs have been identified that correct defective protein folding, we wished to identify an expanded cohort of similarly misshapen and unstable disease-associated ROMK variants. To this end, we developed a pipeline that employs computational analyses of human genome databases with genetic and biochemical assays. Next, we both confirmed the identity of known variants and uncovered previously uncharacterized ROMK variants associated with Bartter syndrome type II. Further analyses indicated that select mutants are targeted for ER-associated degradation, while another mutant compromises ROMK function. This work sets-the-stage for continued mining for ROMK loss of function alleles as well as other potassium channels, and positions select Bartter syndrome mutations for correction using emerging pharmaceuticals.

Significance: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for mortality and morbidity due to cardiovascular disease, and disproportionately affects blacks and women. Several mechanisms have been proposed, including exaggerated activation of sodium transporters in the kidney leading to salt retention and water. Recent Advances: Recent studies have found that in addition to the renal epithelium, myeloid immune cells can sense sodium via the epithelial Na(+) channel (ENaC), which leads to activation of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex, increased fatty acid oxidation, and production of isolevuglandins (IsoLGs). IsoLGs are immunogenic and contribute to salt-induced hypertension. In addition, aldosterone-mediated activation of ENaC has been attributed to the increased SSBP in women. The goal of this review is to highlight mechanisms contributing to SSBP in blacks and women, including, but not limited to increased activation of ENaC, fatty acid oxidation, and inflammation. Critical Issues: A critical barrier to progress in management of SSBP is that its diagnosis is not feasible in the clinic and is limited to expensive and laborious research protocols, which makes it difficult to investigate. Yet without understanding the underlying mechanisms, this important risk factor remains without treatment. Future Directions: Further studies are needed to understand the mechanisms that contribute to differential blood pressure responses to dietary salt and find feasible diagnostic tools. This is extremely important and may go a long way in mitigating the racial and sex disparities in cardiovascular outcomes. Antioxid. Redox Signal. 35, 1477–1493.

SUMMARYPolymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5 in cultured unpolarized cells. Using biotinylation of cell-surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+.

The epithelial Na + channel (ENaC) plays a key role in regulating the extracellular fluid volume and blood pressure. Gain-of-function mutations in ENaC subunits are associated with hypertensive disorders including Liddle syndrome. We previously demonstrated that in male mice, dendritic cell ENaC mediates NADPH oxidase activation and increased accumulation of Isolevuglandins (IsoLGs), which act as neoantigens, promoting T cell activation and salt-induced hypertension. The mechanism by which dendritic cell ENaC is regulated, and the role of sex as a biological variable is still yet to be defined. One of the mechanisms by which ENaC is regulated is through sodium self-inhibition. We hypothesized that sodium self-inhibition plays a sex-specific role in regulating ENaC leading to salt-sensitive hypertension. To test this hypothesis, we generated mice with a gain-of-function mutation of a single nucleotide variant (W521R) in ENaC α-subunit using CRISPR/Cas9 in the wild-type (WT) salt-sensitive129/sv background. These mutant mice lack the sodium self-inhibition of ENaC. WT littermate male and female mice were used as controls. Using telemetry blood pressure monitoring, we found that WT males developed salt-induced hypertension, but WT females were protected (124.3 ± 5.15 vs. 95.37 ± 4.882 mmHg, p=0.0114). Moreover, W521R male mutants had elevated mean arterial pressure in response to 3 weeks of high salt-feeding (4% NaCl) compared to the salt-sensitive WT littermate males (169.3 ± 27.69 vs. 124.3 ± 5.15 mmHg, p= 0.0024). Similar to WT females, female W521R mice were protected from salt-induced blood pressure elevation (102.3± 5.23 mmHg). Using flow cytometry, we found that high Na + increased dendritic cell IsoLGs production in male W521R mice compared to littermate controls (3.97 ± 1.68% vs. 1.01 ± 0.08 % of dendritic cells, p=0.0386). Our findings suggest that W521R gain-of-function mutation contributes to inflammation and hypertension in male but not female mice.

Large-conductance K(+) (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K(+) secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K(+) diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K(+) diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K(+). We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K(+) diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K(+) (5% K(+), as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K(+) excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K(+) diet have higher blood K(+) concentration and reduced IC BK channel activity are consistent with impaired urinary K(+) secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K(+) diet. NEW & NOTEWORTHY When mice are placed on a high-K(+) diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K(+) channel, blunted large-conductance K(+) channel currents in intercalated cells, and increased blood K(+) concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K(+) diet.

Salt sensitivity of blood pressure is an independent risk factor for cardiovascular mortality not only in hypertensive but also in normotensive adults. The diagnosis of salt sensitivity of blood pressure is not feasible in the clinic due to lack of a simple diagnostic test, making it difficult to investigate therapeutic strategies. Most research efforts to understand the mechanisms of salt sensitivity of blood pressure have focused on renal regulation of sodium. However, salt retention or plasma volume expansion is not different between salt-sensitive and salt-resistant individuals. In addition, over 70% of extracellular fluid is interstitial and, therefore, not directly controlled by renal salt and water excretion. We discuss in this review how the seminal work by Harry Goldblatt paved the way for our attempts at understanding the mechanisms that underlie immune activation by salt in hypertension. We describe our findings that sodium, entering antigen-presenting cells via an epithelial sodium channel, triggers a PKC (protein kinase C)- and SGK1 (serum/glucocorticoid kinase 1)-stimulated activation of nicotinamide adenine dinucleotide phosphate oxidase, which, in turn, enhances lipid oxidation with generation of highly reactive isolevuglandins. Isolevuglandins adduct to proteins, with the potential to generate degraded peptide neoantigens. Activated antigen-presenting cells increase production of the TH17 polarizing cytokines, IL (interleukin)-6, IL-1β, and IL-23, which leads to differentiation and proliferation of IL-17A producing T cells. Our laboratory and others have shown that this cytokine contributes to hypertension. We also discuss where this sodium activation of antigen-presenting cells may occur in vivo and describe the multiple experiments, with pharmacological antagonists and knockout mice that we used to unravel this sequence of events in rodents. Finally, we describe experiments in mononuclear cells obtained from normotensive or hypertensive volunteers, which confirm that analogous processes of salt-induced immunity take place in humans.

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

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the γ subunit. Here, we investigated the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in Fisher rat thyroid cells by coexpressing the apical membrane-bound protease channel-activating protease 1 (CAP1; prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney, we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest COOH-terminal fragment of γENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in Fisher rat thyroid cells. Similar cleaved peptides were observed in total and cell surface fractions of the rat kidney. This outcome suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using nonreducing gels, in which the NH(2)- and COOH-terminal fragments of γENaC are linked by a disulfide bond. Under these conditions, the major cleaved form in the rat kidney had an apparent molecular mass of 56 kDa, ∼4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant γENaC species in the apical membrane of rat and mouse kidneys on a low-Na diet is the twice-cleaved, presumably activated form. NEW & NOTEWORTHY We have identified the major aldosterone-dependent cleaved form of the epithelial Na channel (ENaC) γ subunit in the kidney as a twice-cleaved peptide. This form appears to be identical in size with a subunit cleaved in vitro by the extracellular protease channel-activating protease 1 (prostasin). In the absence of reducing agents, it has an overall molecular mass less than that of the intact subunit, consistent with the excision of an inhibitory domain.

The epithelial Na

The epithelial Na(+) channel (ENaC) promotes the absorption of Na(+) in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γ(mt)) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γ(mt/mt) mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γ(mt/mt) mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γ(mt/mt) mice and controls. γ(mt/mt) mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na(+) diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γ(mt/mt) mice relative to the controls. Lower blood pressures were variably observed in γ(mt/mt) mice on a high-salt diet compared with the controls. γ(mt/mt) also exhibited reduced diurnal blood pressure variation, a “nondipping” phenotype, on a high-Na(+) diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure. NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na(+) channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na(+) channel in regulating body fluid and blood pressure beyond classical transepithelial Na(+) transport mechanisms.

Acute kidney injury due to renal ischemia-reperfusion injury (IRI) may lead to chronic or end stage kidney disease. A greater understanding of the cellular mechanisms underlying IRI are required to develop therapeutic options aimed at limiting or reversing damage from IRI. Prior work has shown that deletion of the α subunit of the epithelial Na+ channel (ENaC) in endothelial cells protects from IRI by increasing the availability of nitric oxide. While canonical ENaCs consist of an α, β, and γ subunit, there is evidence of non-canonical ENaC expression in endothelial cells involving the α subunit. We therefore tested whether the deletion of the γ subunit of ENaC also protects mice from IRI to differentiate between these channel configurations. Mice with endothelial-specific deletion of the γ subunit and control littermates were subjected to unilateral renal artery occlusion followed by 48 h of reperfusion. No significant difference was noted in injury between the two groups as assessed by serum creatinine and blood urea nitrogen, levels of specific kidney injury markers, and histological examination. While deletion of the γ subunit did not alter infiltration of immune cells or cytokine message, it was associated with an increase in levels of total and phosphorylated endothelial nitric oxide synthase (eNOS) in the injured kidneys. Our studies demonstrate that even though deletion of the γ subunit of ENaC may allow for greater activation of eNOS, this is not sufficient to prevent IRI, suggesting the protective effects of α subunit deletion may be due, in part, to other mechanisms.

Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

Molecular chaperones are responsible for maintaining cellular homeostasis, and one such chaperone, GRP170, is an endoplasmic reticulum (ER) resident that oversees both protein biogenesis and quality control. We previously discovered that GRP170 regulates the degradation and assembly of the epithelial sodium channel (ENaC), which reabsorbs sodium in the distal nephron and thereby regulates salt-water homeostasis and blood pressure. To define the role of GRP170 - and, more generally, molecular chaperones in kidney physiology - we developed an inducible, nephron-specific GRP170-KO mouse. Here, we show that GRP170 deficiency causes a dramatic phenotype: profound hypovolemia, hyperaldosteronemia, and dysregulation of ion homeostasis, all of which are associated with the loss of ENaC. Additionally, the GRP170-KO mouse exhibits hallmarks of acute kidney injury (AKI). We further demonstrate that the unfolded protein response (UPR) is activated in the GRP170-deficient mouse. Notably, the UPR is also activated in AKI when originating from various other etiologies, including ischemia, sepsis, glomerulonephritis, nephrotic syndrome, and transplant rejection. Our work establishes the central role of GRP170 in kidney homeostasis and directly links molecular chaperone function to kidney injury.

Epithelial Na+ channel (ENaC)-mediated Na+ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K+]. Among the thousands of human ENaC variants, only a few exist whose functional consequences have been experimentally tested. Here, we used the Xenopus oocyte expression system to investigate the functional roles of four nonsynonymous human ENaC variants located within the β7-strand and its adjacent loop of the α-subunit extracellular β-ball domain. αR350Wβγ and αG355Rβγ channels exhibited 2.5- and 1.8-fold greater amiloride-sensitive currents than WT αβγ human ENaCs, respectively, whereas αV351Aβγ channels conducted significantly less current than WT. Currents in αH354Rβγ-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (αR350Wβγ, αV351Aβγ, and αG355Rβγ) were similar to that of WT. However, three mutant channels (αR350Wβγ, αH354Rβγ, and αG355Rβγ) exhibited a reduced Na+ self-inhibition response. Open probability of αR350Wβγ was significantly greater than that of WT. Moreover, other Arg-350 variants, including αR350G, αR350L, and αR350Q, also had significantly increased channel activity. A direct comparison of αR350W and two previously reported gain–of–function variants revealed that αR350W increases ENaC activity similarly to αW493R, but to a much greater degree than does αC479R. Our results indicate that αR350W along with αR350G, αR350L, and αR350Q, and αG355R are novel gain–of–function variants that function as gating modifiers. The location of these multiple functional variants suggests that the αENaC β-ball domain portion that interfaces with the palm domain of βENaC critically regulates ENaC gating.

PURPOSE OF REVIEW: This review describes recent findings regarding the epithelial Na(+) channel (ENaC) and its roles in physiologic and pathophysiologic states. We discuss new insights regarding ENaC’s structure, its regulation by various factors, its potential role in hypertension and nephrotic syndrome, and its roles in the immune system and vasculature. RECENT FINDINGS: A recently resolved structure of ENaC provides clues regarding mechanisms of ENaC activation by proteases. The use of amiloride in nephrotic syndrome, and associated complications are discussed. ENaC is expressed in dendritic cells and contributes to immune system activation and increases in blood pressure in response to NaCl. ENaC is expressed in endothelial ENaC and has a role in regulating vascular tone. SUMMARY: New findings have emerged regarding ENaC and its role in the kidney, immune system and vasculature.

Elevated cardiovascular risk including stroke, heart failure, and heart attack is present even after normalization of blood pressure in patients with hypertension. Underlying immune cell activation is a likely culprit. Although immune cells are important for protection against invading pathogens, their chronic overactivation may lead to tissue damage and high blood pressure. Triggers that may initiate immune activation include viral infections, autoimmunity, and lifestyle factors such as excess dietary salt. These conditions activate the immune system either directly or through their impact on the gut microbiome, which ultimately produces chronic inflammation and hypertension. T cells are central to the immune responses contributing to hypertension. They are activated in part by binding specific antigens that are presented in major histocompatibility complex molecules on professional antigen-presenting cells, and they generate repertoires of rearranged T-cell receptors. Activated T cells infiltrate tissues and produce cytokines including interleukin 17A, which promote renal and vascular dysfunction and end-organ damage leading to hypertension. In this comprehensive review, we highlight environmental, genetic, and microbial associated mechanisms contributing to both innate and adaptive immune cell activation leading to hypertension. Targeting the underlying chronic immune cell activation in hypertension has the potential to mitigate the excess cardiovascular risk associated with this common and deadly disease.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding ENaC’s subunits results in early post-natal mortality. We present initial characterization of a mouse with dramatically suppressed expression of the γ subunit. We use this hypomorphic (γmt) allele to explore the importance of ENaC’s γ subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ subunit expression in γmt/mt mice is markedly suppressed in kidney and lung, while electrolytes resemble those of littermate controls. Challenge with a high K+ diet does not cause significant differences in blood K+, but provokes higher aldosterone in γmt/mt mice than controls. Quantitative magnetic resonance (QMR) measurement of body composition reveals similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. Surprisingly, euvolemia is sustained without significant changes in aldosterone or atrial natriuretic peptide. γmt/mt mice exhibit a more rapid decline in body water and lean tissue mass in response to a low Na+ diet than controls. Replacement of drinking water with 2% saline induces dramatic increases in body fat in both genotypes, and a selective transient increase in body water and lean tissue mass in γmt/mt mice. While ENaC in renal tubules and colon work to prevent extracellular fluid volume depletion, our observations suggest that ENaC in non-epithelial tissues may have a role in preventing extracellular fluid volume overload.

Salt-sensitivity is a major risk factor for hypertension and cardiovascular disease (CVD). Reducing dietary Na + decreases blood pressure and CVD risk. However, the precise mechanisms of how Na + leads to hypertension are still not well defined. Recently, we found that dendritic cells (DCs) in response to increases in extracellular [Na + ] exhibit an amiloride-sensitive epithelial Na + channel (ENaC)-dependent activation of NADPH-oxidase, superoxide production, reactive isolevuglandin (IsoLG)-protein adduct formation, and cytokine secretion which promote hypertension. We hypothesized that the NLRP3 inflammasome in antigen-presenting cells (APCs) mediates salt-sensitive hypertension through an ENaC-dependent mechanism. To test this hypothesis, we cultured mouse splenocytes in normal-salt or high-salt (HS) media with or without co-treatment with the ENaC inhibitor, amiloride (20 μM). Using flow cytometry, we found that HS increased monocyte and DC IL-1β production, which was confirmed through an ELISA assay detecting IL-1β release (2.131 ± 0.733 vs 12.75 ± 1.108 pg/mL, pin vitro data, we treated salt-sensitive mice on a 129-SvJ background with a HS diet (4% NaCl) for 28 days with or without amiloride (1mg/kg/day in drinking water) or NLRP3 inflammasome inhibitor MCC950 (10mg/kg i.p.). Amiloride or MCC950 treated mice developed blunted hypertension in response to HS (120.4 ± 2.99; 101.0 ± 3.74) compared to vehicle controls (140.5 ± 3.98). Amiloride treated mice also exhibited less expression of NLRP3, pro-IL1β, and IsoLGs in DCs and monocytes compared to controls. Interestingly, MCC950 treated mice exhibited decreased pro-IL1β but not NLRP3 expression or IsoLG production. Using the DOCA-salt model, we found similar increases in NLRP3, pro-IL1β, and IsoLGs expression in DCs and monocytes, which was abolished after treatment with IsoLG scavenger 2-HOBA (1g/L). Our findings suggest a role for ENaC-dependent NLRP3 inflammasome activation in APCs in response to a HS diet, which may represent a promising treatment approach to salt-induced hypertension.

Hypertension is a major risk factor for development of cardiovascular disease. Excess dietary salt contributes to inflammation and the genesis of hypertension. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and salt-sensitive hypertension. We hypothesized that Jak2 expression, specifically in CD11c + myeloid cells, regulates expression of ENaCγ and promotes salt-sensitive hypertension. Our results indicate that deletion of Jak2 in CD11c + myeloid cells reduced the salt-induced expression of ENaCγ in CD11c+ cells. Moreover, mice lacking Jak2 in CD11c+ cells developed a blunted hypertensive response (123.8±4.7) during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L- NAME)/high salt model of salt-sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt-feeding when compared to the wildtype litter mate controls (57.89±9.5). These results indicate that Jak2 expression in CD11c + myeloid cells plays a role in salt- sensitive hypertension through an ENaC-dependent mechanism.

Purpose of Review The main goal of this article is to discuss the role of the epithelial sodium channel (ENaC) in extracellular fluid and blood pressure regulation. Recent Findings Besides its role in sodium handling in the kidney, recent studies have found that ENaC expressed in other cells including immune cells can influence blood pressure via extra-renal mechanisms. Dendritic cells (DCs) are activated and contribute to salt-sensitive hypertension in an ENaC-dependent manner. We discuss recent studies on how ENaC is regulated in both the kidney and other sites including the vascular smooth muscles, endothelial cells, and immune cells. We also discuss how this extra-renal ENaC can play a role in salt-sensitive hypertension and its promise as a novel therapeutic target. Summary The role of ENaC in blood pressure regulation in the kidney has been well studied. Recent human gene sequencing efforts have identified thousands of variants among the genes encoding ENaC, and research efforts to determine if these variants and their expression in extra-renal tissue play a role in hypertension will advance our understanding of the pathogenesis of ENaC-mediated cardiovascular disease and lead to novel therapeutic targets.

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The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum–resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na(+) channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na(+) transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na(+) currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate–induced activation of αβ(S518C)γ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

The Caenorhabditis elegans MEC-4/MEC-10 channel mediates the worm's response to gentle body touch and is activated by laminar shear stress (LSS) when expressed in Xenopus oocytes. Substitutions at multiple sites within the second transmembrane domain (TM2) of MEC-4 or MEC-10 abolish the gentle touch response in worms, but the roles of these residues in mechanosensing are unclear. The present study therefore examined the role of specific MEC-4 and MEC-10 TM2 residues in the channel's response to LSS. We found that introducing mutations within the TM2 of MEC-4 or MEC-10 not only altered channel activity, but also affected the channel's response to LSS. This response was enhanced by Cys substitutions at selected MEC-4 sites (Phe(715), Gly(716), Gln(718), and Leu(719)) between the degenerin and the putative amiloride-binding sites in this subunit. In contrast, the LSS response was largely blunted in MEC-10 variants bearing single Cys substitutions in the regions preceding and following the amiloride-binding site (Gly(677)–Leu(681)), as well as with four MEC-10 touch-deficient mutations that introduced charged residues into the TM2 domain. An enhanced response to LSS was observed with a MEC-10 mutation in the putative selectivity filter. Overall, MEC-4 or MEC-10 mutants that altered the channel's LSS response are primarily clustered between the degenerin site and the selectivity filter, a region that probably forms the narrowest portion of the channel pore. Our results suggest that pore-lining residues of MEC-4 and MEC-10 have important yet different roles in tuning the channel's response to mechanical forces.