Epithelial Sodium Channel Activity Research Articles

Activation of the Human Epithelial Sodium Channel (ENaC) by the Peptidomimetic S3969 Involves a Specific Binding Pocket in the Channel’s β-Subunit

The epithelial sodium channel (ENaC) mediates Na+ absorption in several epithelia. Its impaired function leads to severe disorders, like pseudohypoaldosteronism type 1 and respiratory distress. Interestingly, a small molecule ENaC activator, the peptidomimetic S3969, stimulates human but not mouse αβγ-ENaC (Lu et al. 2008, J Biol Chem). Our aim was to identify and characterize the S3969 binding site in human ENaC. ENaC was heterologously expressed in Xenopus laevis oocytes, amiloride-sensitive whole-cell currents (ΔIami) were measured using the two-electrode voltage clamp technique. A putative S3969 binding site was predicted by using molecular docking and molecular dynamics (MD) simulations based on a recently published ENaC structure (Noreng et al. 2020, Elife). In addition, the effect of S3969 on endogenously expressed ENaC was studied in human airway epithelial cells (H441) using transepithelial measurements in Ussing chambers. We confirmed that in oocytes S3969 activated human αβγ-ENaC by ~2-fold with an EC50 of ~0.3 μM (n=25). As reported, murine αβγ-ENaC was insensitive to S3969 in concentrations up to 10 μM (n=19). Using mouse-human chimeric ENaC, we found that a portion of the extracellular loop of the β-subunit (residues 256-404), which includes parts of the thumb, palm and β-ball domains, is critical for channel stimulation by S3969. Computer simulations predicted a putative S3969 binding pocket in this area, with βR388, βF391, and βY406 as key residues coordinating S3969. Mutating these residues strongly reduced (βR388A) or nearly abolished (βY406A; βF391G) the stimulatory effect of S3969 on ΔIami (n=12-18), without altering the effect of an alternative ENaC activator chymotrypsin (n=5). MD simulations also suggested that the binding of S3969 to ENaC increased the distance between the β-thumb and the γ-palm domain. Consistent with this, the stimulatory effect of S3969 was abolished when the β-thumb domain was covalently attached to the γ-palm domain following the introduction of two cysteine residues (βR437C, γS298C) to form a disulfide bridge (n=15). Importantly, reducing the disulfide bond with DTT partially rescued the effect of S3969 on ΔIami (relative stimulation 31±1%, n=15, p<0.001). We also showed that S3969 stimulated endogenously expressed human ENaC in H441 cells. In conclusion, we demonstrated that S3969 activates ENaC by interacting with a specific binding pocket in β-ENaC which probably increases the β-γ-intersubunit distance. These results may help to identify novel ENaC modulators with potential physiological or therapeutic implications. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 509149993, TRR 374 (subproject A4 to A.I. and C.K.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increased Epithelial Sodium Channel (ENaC) Activity Mediates Fructose-Induced Salt-Sensitive Hypertension

Increased Epithelial Sodium Channel (ENaC) Activity Mediates Fructose-Induced Salt-Sensitive Hypertension

The Antioxidative Effects of Flavones in Hypertensive Disease.

Hypertension is the leading remediable risk factor for cardiovascular morbidity and mortality in the United States. Excess dietary salt consumption, which is a catalyst of hypertension, initiates an inflammatory cascade via activation of antigen-presenting cells (APCs). This pro-inflammatory response is driven primarily by sodium ions (Na+) transporting into APCs by the epithelial sodium channel (ENaC) and subsequent NADPH oxidase activation, leading to high levels of oxidative stress. Oxidative stress, a well-known catalyst for hypertension-related illness development, disturbs redox homeostasis, which ultimately promotes lipid peroxidation, isolevuglandin production and an inflammatory response. Natural medicinal compounds derived from organic materials that are characterized by their anti-inflammatory, anti-oxidative, and anti-mutagenic properties have recently gained traction amongst the pharmacology community due to their therapeutic effects. Flavonoids, a natural phenolic compound, have these therapeutic benefits and can potentially serve as anti-hypertensives. Flavones are a type of flavonoid that have increased anti-inflammatory effects that may allow them to act as therapeutic agents for hypertension, including diosmetin, which is able to induce significant arterial vasodilation in several different animal models. This review will focus on the activity of flavones to illuminate potential preventative and potential therapeutic mechanisms against hypertension.

Extracellular vesicles from BALF of pediatric cystic fibrosis and asthma patients increase epithelial sodium channel activity in small airway epithelial cells

Extracellular Vesicles (EVs) are nanosized vesicles derived from all cell types. EV cargo allows for intercellular communication, intracellular signaling, and regulation of proteins in recipient cells. We tested the hypothesis that EVs isolated from the bronchoalveolar-lavage fluid (BALF) of pediatric cystic fibrosis (CF) or pediatric asthma patients increase epithelial sodium channel (ENaC) activity in normal human small airway epithelial cells (SAECs) and the mechanism involves specific EV lipids. We characterized EVs from BALF of pediatric CF and pediatric asthma patients by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. The CF and asthma pediatric groups were similar in BALF electrolytes concentration and cell count, except for neutrophils, which were higher in the CF group. Lipidomic analyses for each group of EVs were performed using targeted mass spectrometry. Phosphatidylethanolamine, sphingomyelins, and triacylglycerol were enriched in both groups, but phosphatidylcholine and phosphatidylinositol concentrations were greater in the CF group compared to the asthma group, and the opposite trend was found for phosphatidylserine. Endogenous ENaC activity, measured by the single-channel patch-clamp technique, increased in normal human SAECs after challenging SAEC with EVs from either the CF or asthma groups compared to control EVs. In conclusion, EVs isolated from BALF of pediatric patients with CF or asthma have unique lipid profiles. Despite the differences, both types of EVs increase ENaC activity in normal human SAECs compared to control EVs isolated from the conditioned media of these cells.

402-P: ENaC Regulation in Mouse Cortical Collecting Duct Cells by EVs Released during the Inactive and Active Cycles of Salt-Loaded Hypertensive Diabetic db/db Mice

The downregulation of renal epithelial sodium channel (ENaC) activity in response to salt-loading in the diabetic kidney is important to prevent the exacerbation of diabetic kidney disease. An inflammatory response in diabetic db/db mice is associated with constitutively high ENaC activity even after salt-loading. During salt-loading, diabetic db/db mice release extracellular vesicles (EVs) into the urine during the active and inactive cycles that are enriched in bioactive molecules. A vast majority of these EVs are released across the luminal membrane of renal epithelial cells from each segment of the nephron and have the potential to module ENaC activity in recipient collecting duct cells. Our hypothesis was the distinct cargo that is enriched in EVs released during the inactive and active cycles of salt-loaded hypertensive diabetic db/db mice is responsible for increased ENaC membrane protein expression and activity in recipient collecting duct cells. In this study, we measured blood pressure by telemetry during the light (inactive) or dark (active) cycles. In addition, osmolality, electrolytes, and creatinine were measured and EV were isolated from urine samples collected during both cycles. Western blotting and densitometric analysis showed greater ENaC alpha protein expression in membrane fractions of mouse cortical collecting duct (mpkCCD) cells while single-channel patch clamp studies show greater ENaC activity in mpkCCD cells treated with uEVs isolated from the active cycle compared to the inactive cycle of hypertensive diabetic db/db mice. Various proteins and metabolites were found to be enriched in each of the two groups of EVs. Taken together, these data suggest the packaged cargo within EVs released after salt-loading diabetic db/db mice changes over a 24 hour cycle, and various molecules enriched in active cycle EVs contribute to the upregulation of renal ENaC protein expression and activity. Disclosure L.Yu: None. M.F.Gholam: None. A.A.Alli: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK123078-01A1 to A.A.A.)

Probing the Ubiquitination-Mediated Function of Epithelial Sodium Channel in A549 Cells.

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in the tight epithelia of the lung is regulated by proteolytic activation and ubiquitination. Pathophysiology of lung diseases is directly related to changes in one or both of these mechanisms. In this study, we investigated the impact of ubiquitination and cathepsin-mediated proteolytic activation mechanisms on the functional regulation of ENaC in lung cancer A549 cells using the patch-clamp technique. Our findings suggest that inhibiting the proteasome (polyubiquitination) with MG132 improves ENaC activity, whereas altering the pH of the lysosome (monoubiquitination inhibition) with NH4Cl has no effect on ENaC activity. In A549 cells, inhibition of cathepsin B (CSTB) decreased the ENaC current, open probabilities (NPo and Po), and the number of active channels. These findings delineate novel modes of ENaC degradation and proteolytic activation of functional channels in A549 cells. Our findings indicate that both proteolytic activation and ubiquitination of ENaC significantly affect channel function and add new insights into the endogenous ENaC processing which might help to further understand the pathophysiology of the lung disease.

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia.

Localized and chronic hypoxia of airway mucosa is a common feature of progressive respiratory diseases, including cystic fibrosis (CF). However, the impact of prolonged hypoxia on airway stem cell function and differentiated epithelium is not well elucidated. Acute hypoxia alters the transcription and translation of many genes, including the CF transmembrane conductance regulator (CFTR). CFTR-targeted therapies (modulators) have not been investigated in vitro under chronic hypoxic conditions found in CF airways in vivo. Nasal epithelial cells (hNECs) derived from eight CF and three non-CF participants were expanded and differentiated at the air-liquid interface (26-30 days) at ambient and 2% oxygen tension (hypoxia). Morphology, global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility and ion transport) of basal stem cells and differentiated cultures were assessed. hNECs expanded at chronic hypoxia, demonstrating epithelial cobblestone morphology and a similar proliferation rate to hNECs expanded at normoxia. Hypoxia-inducible proteins and pathways in stem cells and differentiated cultures were identified. Despite the stem cells' plasticity and adaptation to chronic hypoxia, the differentiated epithelium was significantly thinner with reduced barrier integrity. Stem cell lineage commitment shifted to a more secretory epithelial phenotype. Motile cilia abundance, length, beat frequency and coordination were significantly negatively modulated. Chronic hypoxia reduces the activity of epithelial sodium and CFTR ion channels. CFTR modulator drug response was diminished. Our findings shed light on the molecular pathophysiology of hypoxia and its implications in CF. Targeting hypoxia can be a strategy to augment mucosal function and may provide a means to enhance the efficacy of CFTR modulators.

Triamterene Functions as an Effective Nonsense Suppression Agent for MPS I-H (Hurler Syndrome).

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene's diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC.

The serine protease plasmin plays detrimental roles in epithelial sodium channel activation and podocyte injury in Dahl salt-sensitive rats.

Salt-sensitive hypertension is associated with poor clinical outcomes. The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in sodium reabsorption and blood pressure regulation, in which its γ subunit is activated by extracellular serine proteases. In proteinuric nephropathies, plasmin filtered through injured glomeruli reportedly activates γENaC in the distal nephron and causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed hypertension and proteinuria along with γENaC activation and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remained unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as podocyte injury and the effects of plasmin inhibitors in DS rats. Five-week-old DS rats were divided into normal-salt diet, HS diet, and HS+plasmin inhibitor (either tranexamic acid [TA] or synthetic plasmin inhibitor YO-2) groups. After blood pressure measurement and 24 h urine collection over 5 weeks, rats were sacrificed for biochemical analyses. The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which was significantly attenuated by YO-2 but not TA. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by inhibiting apoptosis and inflammatory/profibrotic cytokines. YO-2 also suppressed upregulation of protease-activated receptor-1 and phosphorylated ERK1/2. These results indicate an important role of plasmin in the development of salt-sensitive hypertension and related podocyte injury, suggesting plasmin inhibition as a potential therapeutic strategy.

Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

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

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

Mineralocorticoid Receptor Antagonists Cause Natriuresis in the Absence of Aldosterone.

MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. We studied AS (aldosterone synthase)-deficient (AS-/-) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR-/-) mice. KS-MR-/- mice were used to compare MR expression and ENaC localization and cleavage with AS-/- mice. MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR-/- mice but were fully preserved along DCT2 and were partially reduced along CCD in AS-/- mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS-/- mice and were not increased along CCD after low salt. AS-/- mice exhibited transient Na+ wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS-/- mice led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR-/- mice. Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS-/- mice.

Urine Di-(2-ethylhexyl) Phthalate Metabolites Are Independently Related to Body Fluid Status in Adults: Results from a U.S. Nationally Representative Survey

Purpose: Di-(2-ethylhexyl) phthalate (DEHP) has been utilized in many daily products for decades. Previous studies have reported that DEHP exposure could induce renin–angiotensin–aldosterone system activation and increase epithelial sodium channel (ENaC) activity, which contributes to extracellular fluid (ECF) volume expansion. However, there is also no previous study to evaluate the association between DEHP exposure and body fluid status. Methods: We selected 1678 subjects (aged ≥18 years) from a National Health and Nutrition Examination Survey (NHANES) in 2003–2004 to determine the relationship between urine DEHP metabolites and body composition (body measures, bioelectrical impedance analysis (BIA)). Results: After weighing the sampling strategy in multiple linear regression analysis, we report that higher levels of DEHP metabolites are correlated with increases in body measures (body weight, body mass index (BMI), waist circumference), BIA parameters (estimated fat mass, percent body fat, ECF, and ECF/intracellular fluid (ICF) ratio) in multiple linear regression analysis. The relationship between DEHP metabolites and the ECF/ICF ratio was more evident in subjects of younger age (20–39 years old), women, non-Hispanic white ethnicity, and subjects who were not active smokers. Conclusion: In addition to being positively correlated with body measures and body fat, we found that urine DEHP metabolites were positively correlated with ECF and the ECF/ICF ratio in the US general adult population. The finding implies that DEHP exposures might increase ECF volume and the ECF/ICF ratio, which may have adverse health outcomes on the cardiovascular system. Further research is needed to clarify the causal relationship.

PATHOGENICITY AND LONG-TERM OUTCOMES OF LIDDLE SYNDROME CAUSED BY A NONSENSE MUTATION OF SCNN1G IN A CHINESE FAMILY

Objective: Liddle syndrome (LS) is a common monogenic hypertension with continuous activation of epithelial sodium channels (ENaCs) encoded by SCNN1A, SCNN1B, and SCNN1G. This study aimed to identify the pathogenicity of a nonsense mutation in SCNN1G in a Chinese family with LS and the outcomes of tailored treatment with amiloride. Design and method: Clinical data were collected from six family members and this mutation site was reported by our team at 2015. To explore the pathogenicity of candidate variants, we constructed mutant and wild-type models in vitro and measured amiloride-sensitive current in Chinese Hamster Ovary cells using patch clamp technique. Participants were followed up for 6 years to monitor their target organ damage after tailored treatment with amiloride. Results: Four family members presented with severe hypertension and hypokalemia, and two had stroke. This nonsense variant resulted in a termination codon at codon 572, truncating the Pro-Pro-Pro-X-Tyr motif. The mutant epithelial sodium channels displayed higher amiloride-sensitive currents than the wild-type channels (P < 0.05). Tailored treatment with amiloride achieved ideal blood pressure control in the patients, and no adverse events occurred during follow-up. Conclusions: We found the pathogenicity of a nonsense SCNN1G mutation (p.Glu571*) with enhanced amiloride-sensitive currents in a LS family. Tailored treatment with amiloride may be an effective strategy for the long-term control of blood pressure and the prevention of cardiovascular events in these patients.

The dual GLP-1 and GLP-2 receptor agonist dapiglutide promotes barrier function in murine short bowel.

Short bowel syndrome can occur after extensive intestinal resection, causing intestinal insufficiency or intestinal failure, which requires long-term parenteral nutrition. Glucagon-like peptide-2 (GLP-2) pharmacotherapy is now clinically used to reduce the disease burden of intestinal failure. However, many patients still cannot be weaned off from parenteral nutrition completely. The novel dual GLP-1 and GLP-2 receptor agonist dapiglutide has previously been shown to be highly effective in a preclinical murine short bowel model. Here, we studied the effects of dapiglutide on intestinal epithelial barrier function. In the jejunum, dapiglutide increased claudin-7 expression and tightened the paracellular tight junction leak pathway. At the same time, dapiglutide promoted paracellular tight junction cation size selectivity in the jejunum. This was paralleled by extension of the cation selective tight junction proteins claudin-2 and claudin-10b and preserved claudin-15 expression and localization along the crypt-villus axis in the jejunum. In the colon, no barrier effects from dapiglutide were observed. In the colon, dapiglutide attenuated the short bowel-associated, compensatorily increased epithelial sodium channel activity, likely secondary, by improved volume status. Future studies are needed to address the intestinal adaptation of the colon.

Tubular IL-1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages.

Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. Thirty-four-week-old db/db mice display significantly higher levels of IL-1β in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1β in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1β when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1β promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1β in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. Renal tubular epithelial cell-derived IL-1β polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1β synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

Two adjacent phosphorylation sites in the C-terminus of the channel’s α-subunit have opposing effects on epithelial sodium channel (ENaC) activity

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.

Sphingomyelin‐enriched extracellular vesicles from hypertensive diabetic mice regulate calcium mobilization and amiloride‐sensitive transepithelial current in mouse cortical collecting duct cells

Uncontrolled hypertension (HTN) and diabetes mellitus (DM) are two major public health burdens contributing to increased hospitalization, readmission costs, and adverse clinical outcomes. Moreover, DM is a leading cause of death and long‐term morbidity. Overexpression and activity of epithelial sodium channels (ENaC) in the kidney results in hypertension, but the mechanism for its regulation in the diabetic kidney is not entirely understood. Cholesterol is known to increase ENaC activity in cultured kidney cells in a calcium and PI(4,5)P2‐dependent mechanism. Presumably ENaC is regulated by other lipids in pathophysiology. Extracellular vesicles (EVs) are nano‐sized vesicles that play an important role in intercellular communication and intracellular signaling. Here, we investigated whether EVs isolated from the urine of hypertensive diabetic db/db mice or diabetic db/db mice augment ENaC activity by inhibiting calcium mobilization in mpkCCD cells. Adult diabetic db/db mice were salt loaded (4% NaCl) for 7 days to induce hypertension. Blood pressure was measured by tail‐cuff, and urine from db/db mice with systolic blood pressure of greater than 140mmHg or from normotensive db/db mice were used to isolate EVs. Nanoparticle tracking analysis was performed to determine EV concentration. EVs from hypertensive diabetic db/db mice (N=5) compared to diabetic db/db mice (N=5) suppressed ionomycin‐induced calcium mobilization in mpkCCD cells cultured on glass bottom 35 mm dishes (N=3 experiments, p<0.05) which was measured by confocal microscopy and augmented amiloride‐sensitive transepithelial current in mpKCCD cells cultured in 12mm diameter permeable supports (N=6 permeable supports per group) measured by an epithelial volt ohm meter (EVOM). Sphingomyelin were found to be enriched in urinary EVs from hypertensive diabetic db/db mice compared to diabetic only db/db mice. Exogenous application of sphingomyelin to mpkCCD cells mimicked the suppression of calcium mobilization and increase in amiloride‐sensitive transepithelial current in these cells. Taken together, these data suggest differences in the packaged cargo of urinary EVs between HTN and DM mice that presumably plays a role in the regulation of ENaC activity.

Dissociation of hypertension and renal damage after cessation of high salt diet in Dahl rats

BackgroundEvery year, thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. Most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying the self‐sustaining character of hypertension. We hypothesize that chronic hypertension leads to sustained renal damage and excessive sodium reabsorption, staying even after removing the trigger, caused the initial blood pressure raise.MethodsWe used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4%NaCl diet to induce hypertension, and then the diet was switched back to normal (0.4%NaCl).Patch‐clamp analysis was performed in freshly isolated, split‐open cortical collecting ducts to characterize activity of epithelial sodium channel (ENaC), responsible for sodium reabsorption in aldosterone‐sensitive distal nephron. Flow cytometry, trichrome staining and FITC‐inulin clearance estimated immune cell infiltration, renal damage and renal function.ResultsWe found that blood pressure increase is reversible if induced by a short 3‐day long feeding with 4%NaCl diet. A 3‐week long high salt diet develops a sustaining hypertension (MAP ~150 mmHg): switching back to 0.4%NaCl diet slightly reduces blood pressure (to 145 mmHg) next day, but later hypertension progresses, reaching ~158 mmHg. Notably, the self‐sustaining phase of hypertension was sensitive to benzamil treatment, which lowered BP to ~136 mmHg.Patch clamp analysis revealed that development of hypertension was accompanied with elevated ENaC activity which also stayed high, despite withdrawal of 4%NaCl diet.A separate subset of experiments demonstrated that dietary salt reduction in hypertensive animals decreases the number of total CD45+CD3+CD4+ and CD45+CD3+CD8+ cells in renal tissues.Also, we found a reduced area of protein casts abundance in the post‐salt hypertensive group in comparison with age‐matched rats kept on a high salt diet, and improving a glomerular filtration rate in the post‐salt period.ConclusionBased on our earlier publications and current data, we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge. However, dietary salt restriction reduces renal inflammation and damage in hypertensive animals.

Eplerenone‐induced natriuresis and hyperkalemia in mice lacking aldosterone results from aldosterone‐independent mineralocorticoid receptor occupancy

BackgroundMineralocorticoid receptor (MR) antagonists are recommended for patients with resistant hypertension independent of circulating aldosterone levels. Aldosterone, secreted in response to salt depletion or hyperkalemia, activates MR to increase epithelial sodium channel (ENaC) activity. While glucocorticoids can also activate MR, they are metabolized by 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), protecting MR from illicit glucocorticoid occupation. 11β‐HSD2 is expressed at increasing levels from the DCT through the collecting duct. Here, we hypothesized that glucocorticoid occupies MR to stimulate ENaC in the DCT2 and early connecting tubule.MethodsWe studied aldosterone synthase knockout (AS−/−) mice provided by Dr. JM Luther. Kidney–specific MR knockout (MR−/−) mice were used for validation of Western blot for ENaC and immunofluorescence for MR. We measured plasma and urinary electrolytes at baseline, and following dietary salt restriction with or without administration of the MR antagonist eplerenone.ResultsAS−/− mice displayed higher plasma K+ levels than AS+/+ mice but not salt wasting under baseline. Under low salt (LS) diet, AS−/− mice exhibited higher urine Na+ excretion, but preserved body weight. Cleaved αENaC and γENaC abundances were lower in AS−/− than AS+/+ mice, and were not increased in AS−/− mice after LS diet. Notably, we found nuclear localization of MR was preserved in DCT2 of AS−/− mice. Administration of the MR blocker eplerenone to AS−/− mice fed LS diet led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice.ConclusionsOur results provide evidence that MR is occupied in the absence of aldosterone, suggesting glucocorticoid‐binding to MR preserves sodium homeostasis in the DCT2 of AS−/− mice. This likely explains the milder phenotype of AS−/− mice compared with MR−/− mice.