Epithelial Na(+) Channel Research Articles

Na restriction activates epithelial Na channels in rat kidney through two mechanisms and decreases distal Na+ delivery.

Dietary Na restriction, through the mineralocorticoid aldosterone, acts on epithelial Na channels via both fast (24h) and slow (5-7days) mechanisms in the kidney. The fast effect entails increased proteolytic processing and trafficking of channel protein to the apical membrane. It is rapidly reversible by the mineralocorticoid receptor antagonist eplerenone and is largely lost when tubules are studied ex vivo. The slow effect does not require increased processing or surface expression, is refractory to acute eplerenone treatment, and is preserved ex vivo. Both slow and fast effects contribute to Na retention in vivo. Increased Na+ reabsorption in the proximal tubule also promotes Na conservation under conditions of chronic dietary Na restriction, reducing Na+ delivery to the distal nephron. Changes in the activity of the epithelial Na channel (ENaC) help to conserve extracellular fluid volume. In rats fed a low-salt diet, proteolytic processing of ENaC increased within 1day, and was almost maximal after 3days. The rapid increase in the abundance of cleaved αENaC and γENaC correlated with decreased urinary Na+ excretion and with increased ENaC surface expression. By contrast, ENaC activity, measured ex vivo in isolated cortical collecting ducts, increased modestly after 3days and required 5days to reach maximal levels. The mineralocorticoid receptor antagonist eplerenone reversed the increase in cleaved γENaC and induced natriuresis after 1 or 3days but failed to alter either ENaC currents or Na+ excretion after 7days of Na restriction. We conclude that Na depletion, through aldosterone, stimulates ENaC via independent fast and slow mechanisms. In vivo, amiloride-induced natriuresis increased after 1day of Na depletion. By contrast, hydrochlorothiazide (HCTZ)-induced natriuresis decreased gradually over 7days, consistent with increased ability of ENaC activity to compensate for decreased Na+ reabsorption in the distal convoluted tubule. Administration of amiloride and HCTZ together increased Na+ excretion less in Na-depleted compared to control animals, indicating decreased delivery of Na+ to the distal nephron when dietary Na is restricted. Measurements of creatinine and Li+ clearances indicated that increased Na reabsorption by the proximal tubules is responsible for the decreased delivery. Thus, Na conservation during chronic dietary salt restriction entails enhanced transport by both proximal and distal nephron segments.

Urinary Plasmin(ogen) as a Prognostic Factor for Hypertension

IntroductionPlasmin and its precursor, plasminogen, are detectable in urine from patients with glomerular disease. Urinary plasmin(ogen) levels correlate with blood pressure (BP) and may contribute to renal Na+ retention by activating the epithelial Na+ channel (ENaC). In a longitudinal nested-cohort study, we asked whether urinary plasmin(ogen) levels predict subsequent increase in BP, incident hypertension, or mortality in subjects with type I diabetes, who often develop proteinuria.MethodsThe Pittsburgh Epidemiology of Diabetes Complications (EDC) study followed up type I diabetic subjects for 25 years. Urine specimens from 70 subjects with a spectrum of baseline urinary albumin levels were examined. Outcomes included increased BP after 2 years (≥1 SD over baseline systolic or diastolic BP, examined via logistic regression), 25-year incident hypertension (≥140/90 mm Hg or initiating BP-lowering medications), and all-cause or cardiovascular mortality, examined using Cox regression.ResultsSubjects experiencing a 2-year increase in BP had higher baseline urinary plasmin(ogen)/creatinine levels (uPl/Cr) than other subjects (P = 0.04); the difference in baseline urinary albumin/creatinine levels (uAlb/Cr) was similar (P = 0.07). Baseline uPl/Cr was associated with increased 25-year hypertension incidence (hazard ratio = 2.05, P = 0.001), all-cause mortality (HR = 2.05, P = 0.01) and cardiovascular mortality (HR = 3.30, P = 0.005), although not independent of uAlb/Cr.ConclusionThis is the first long-term prospective study addressing clinical outcomes associated with increased urinary plasmin(ogen). Findings are consistent with a role for plasmin(ogen) in promoting increased BP, but also demonstrate the difficulty in distinguishing effects due to plasmin(ogen) from those of albuminuria.

Regulation of renal Na transporters in response to dietary K.

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved γ-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by ~50% and ~80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle's loop and distal convoluted tubule along with Cl-, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis.

Cigarette smoke modifies and inactivates SPLUNC1, leading to airway dehydration.

Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Cigarette smoke (CS) exposure, a major cause of COPD, dysregulates airway epithelial ion transport and diminishes airway surface liquid (ASL) volume. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is secreted into the airway lumen where it maintains airway hydration via interactions with the epithelial Na+ channel (ENaC). Although ASL hydration is dysregulated in CS-exposed/COPD airways, effects of CS on SPLUNC1 have not been elucidated. We hypothesized that CS alters SPLUNC1 activity, therefore contributing to ASL dehydration. CS exposure caused irreversible SPLUNC1 aggregation and prevented SPLUNC1 from internalizing ENaC and maintaining ASL hydration. Proteomic analysis revealed αβ-unsaturated aldehyde modifications to SPLUNC1's cysteine residues. Removal of these cysteines prevented SPLUNC1 from regulating ENaC/ASL volume. In contrast, SPX-101, a peptide mimetic of natural SPLUNC1, that internalizes ENaC, but does not contain cysteines was unaffected by CS. SPX-101 increased ASL hydration and attenuated ENaC activity in airway cultures after CS exposure and prolonged survival in a chronic airway disease model. These findings suggest that the CS-induced defects in SPLUNC1 can be circumvented, thus making SPX-101 a novel candidate for the treatment of mucus dehydration in COPD. -Moore, P. J., Reidel, B., Ghosh, A., Sesma, J., Kesimer, M., Tarran, R. Cigarette smoke modifies and inactivates SPLUNC1, leading to airway dehydration.

β1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β1Pix knockdown, or overexpression of WT β1Pix or the β1Pix-Δ602-611 mutant. Moreover, we found that β1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.

Mechanistic target of rapamycin: integrating growth factor and nutrient signaling in the collecting duct.

The renal collecting duct and other postmacula densa sites are the primary tubular regions for fine-tuning of electrolyte homeostasis in the body. A role for the mechanistic target of rapamycin (mTOR), a serine-threonine kinase, has recently been appreciated in this regulation. mTOR exists in two distinct multiprotein functional complexes, i.e., mTORC1 and mTORC2. Upregulation of mTORC1, by growth factors and amino acids, is associated with cell cycle regulation and hypertrophic changes. In contrast, mTORC2 has been demonstrated to have a role in regulating Na+ and K+ reabsorptive processes, including those downstream of insulin and serum- and glucocorticoid-regulated kinase (SGK). In addition, mTORC2 can upregulate mTORC1. A number of elegant in vitro and in vivo studies using cell systems and genetically modified mice have revealed mechanisms underlying activation of the epithelial Na+ channel (ENaC) and the renal outer medullary K+ channel (ROMK) by mTORC2. Overall, mTOR in its systematic integration of phosphorylative signaling facilitates the delicate balance of whole body electrolyte homeostasis in the face of changes in metabolic status. Thus, inappropriate regulation of renal mTOR has the potential to result in electrolyte disturbances, such as acidosis/alkalosis, hyponatremia, and hypertension. The goal of this minireview is to highlight the physiological role of mTOR in its complexes in regulating electrolyte homeostasis in the aldosterone-sensitive distal nephron.

Annexin II light chain p11 interacts with ENaC to increase functional activity at the membrane

The epithelial Na+ channel (ENaC) provides for Na+ absorption in various types of epithelia including the kidney, lung and colon where ENaC is localized to the apical membrane to enable Na+ entry into the cell. The degree of Na+ entry via ENaC largely depends on the number of active channels localized to the cell membrane. While regulation of ENaC endocytosis has been well studied, relatively little is understood of the proteins that govern ENaC exocytosis. We hypothesized that the annexin II light chain, p11 could participate in the transport of ENaC along the exocytic pathway. Our results demonstrate that all three ENaC channel subunits interacted with p11 in an in vitro binding assay. Quantitative mass spectrometry of affinity‐purified ENaC‐p11 complexes recovered several other trafficking proteins including HSP‐90, COMMD10 and annexin A6. We also found that p11 exhibits a robust protein expression in cortical collecting duct epithelial cells. However, the expression of p11 in these cells was not influenced by either short‐term or long‐term exposure to aldosterone. To determine whether the p11 interaction affected ENaC function, we measured amiloride sensitive Na+ currents in Xenopus oocytes or mammalian epithelia coexpressing ENaC and p11, or a siRNA to p11. Results from these experiments showed that p11 significantly augmented amiloride‐sensitive current, whereas knockdown of p11 decreased current. Further, knockdown of p11 reduced the ENaC cell surface population suggesting p11 promotes membrane insertion of ENaC. Overall, our findings reveal a novel protein‐protein interaction of p11 with ENaC that controls the number of ENaC channels inserted at the plasma membrane via the exocytic pathway.Support or Funding InformationThis work was supported by a University of Otago Research Grant, and an AIM grant from the Department of Physiology, University of Otago.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Epithelial Na+ Channel is Regulated by Biliary Components

Cirrhosis of the liver often leads to edema and increased circulating and urinary levels of bile acids and bilirubin conjugates. We hypothesized that ENaC activation by increased concentrations of biliary metabolites in renal tubular ultrafiltrate could contribute to renal Na+ retention and edema in cirrhosis of the liver. The epithelial Na+ channel (ENaC) plays a key role in regulating Na+ reabsorption in the kidney, and is regulated by an array of factors, including extracellular ligands (e.g. H+, Na+, Cl−) and post‐translational modifications (e.g. cleavage and palmitoylation). To begin testing this, we expressed wild type and mutant ENaCs in Xenopus oocytes and measured effects on channel current by two‐electrode voltage clamp. We found that specific bile acids and conjugated‐bilirubin activate ENaC in a dose‐dependent manner, with deoxycholic acid having the strongest stimulatory effect. Notably, deoxycholic acid stimulated both wild type channels and ‘near silent’ channels in which the furin cleavage sites were mutated. However, fully activating channels by trypsin abolished deoxycholic acid stimulation in both wild type channels and channels lacking furin cleavage sites, consistent with an effect on open probability. We hypothesized that bile acids, conjugated bilirubin and Cys‐palmitoylation (palmitate addition to specific intracellular cysteines on the β and γ subunits) regulate ENaC currents through a common mechanism: by increasing the membrane association of specific intracellular structures, driving conformation changes in the channel's pore. To begin testing our hypothesis, we determined whether mutation of all 4 palmitoylation sites in the β and γ subunits (βC43, βC557, γC33 and γC41) to alanine would affect activation by deoxycholic acid. We found that deoxycholic acid stimulated currents of ENaC lacking of all 4 palmitoylation site cysteines by 7‐fold over wild type ENaC. When we tested the effect of mutating individual sites, we found that removal of the N‐terminal palmitoylation site cysteines resulted in the strongest stimulatory effects. We also found that deoxycholic acid had a stronger stimulatory effect when we removed both sites from the γ subunit as compared to the β subunit. Our results demonstrate that ENaC can be activated by biliary metabolites, including bile acids and bilirubin, and that these metabolites may activate ENaC through a mechanism in common with ENaC activation by palmitoylation.Support or Funding InformationDK098204 to O.B.K. and DK110332 to E.C.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Loss‐of‐function Variants in the Epithelial Na+ Channel Extracellular Domain

Mutations in epithelial Na+ channel (ENaC) subunits cause hypertensive or hypotensive human diseases. Several genetic variants in ENaC genes are associated with high blood pressure or salt sensitivity. Certain ENaC variants reportedly serve as modifiers to diseases such as cystic fibrosis. In this study, we sought to identify loss‐of‐function human ENaC variants in a heterologous expression system. A total of 48 variants in extracellular domains of α and γ ENaC subunits were selected. Mutant α subunits were co‐expressed with wild type β and γ subunits whereas mutant γ subunits were co‐expressed with wild type α and β subunits in Xenopus oocytes. Amiloride‐sensitive currents measured by two‐electrode voltage clamp in cells expressing wild type and mutant ENaCs were compared to identify functional variants. Among the tested 24 αENaC variants, four (αH255R, αT274M, αG454V and αΔF528) showed significantly less currents than wild type channels (p< 0.001, n=16~26). Four out of 24 γENaC variants (γS134F, γN282S, γE314K and γW486X) also had significantly less currents than wild type channels (p < 0.05, n=16~30). To explore the underlying mechanisms for the loss‐of function phenotype, we examined Na+ self‐inhibition responses of the mutant channels. All four α (H255R, T274M, G454V and ΔF528) and one γ (E314K) mutants displayed significantly greater Na+ self‐inhibition responses than wild type channels, corresponding to lower open probabilities. Our results identify eight novel loss‐of‐function ENaC variants in the extracellular domain and suggest that five variants are ENaC gating modifiers.Support or Funding InformationSupported by NIH DK079307, DK038470 and a DCI grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Collecting duct principal, but not intercalated, cell (pro)renin receptor modulates sodium and water transport

The collecting duct (CD) is the predominant site for prorenin receptor (PRR) expression within the nephron. We have previously demonstrated that the CD PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which CD cell type is involved is unclear. Hence, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR KO mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B‐1 subunit of the H+ ATPase promoters, respectively. In‐situ hybridization using RNAscope demonstrated abundant PRR RNA in IC with modest staining in PC in control mice with reduced cell‐specific PRR RNA in PC and IC KO mice. Compared to controls, PC KO, but not IC KO mice, had increased urinary volume (Control: 1.2 ± 0.4 vs PC KO: 2.0 ± 0.3, P < 0.05; IC KO: 1.4 ± 0.2 ml/day), reduced urine osmolality (Control: 3160 ± 279 vs PC KO: 2343 ± 247, P < 0.05; IC KO: 2748 ± 356 mosm/Kg H2O) and reduced expression of renal medullary AQP2 (50% of control). Further, PC KO mice had reduced renal medullary ENaC‐α expression with normal and low Na+ diet and lower Na+ balance (24‐hour intake – excretion) following low Na+ intake (controls: 60.5 ± 7.5 vs PC KO: 25.7 ± 10.6 μmol/day, P <0.05). In contrast, no differences in Na+ balance or ENaC expression was observed between control or IC KO mice. In acutely isolated cortical CD, under basal conditions, no significant differences in ENaC activity were observed between control, PC or IC specific PRR KO mice. Treatment with mouse prorenin (10 nM for 30 min) increased ENaC channel number and open probability by 2‐fold in control mice (basal: 0.15 ± 0.04; prorenin treatment: 0.74 ± 0.25). Addition of prorenin did not alter ENaC activity in PC specific PRR KO mice (basal: 0.28 ± 0.06; prorenin treatment: 0.33 ± 0.13) but increased ENaC activity in IC specific PRR KO mice (basal: 0.17 ± 0.08; prorenin treatment 1.29 ± 0.13). However, mean arterial pressure as measured by telemetry (Controls: 104 ± 1, PC KO: 106 ± 2, IC KO: 101 ± 4 mm Hg) was similar between controls and PC or IC KO mice on normal and low Na+ diets. Taken together, these findings indicate that under physiologic conditions, the PC, but not IC, PRR modulates ENaC expression and activity, urinary Na+ excretion and water transport. Further studies are needed to examine the role of PC and IC PRR in pathological states such as Ang‐II induced hypertension.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of basolateral ClC‐K2‐dependent Cl‐ reabsorption in collecting duct intercalated cells by dietary electrolyte intake and Ang II

Hypertension is the major clinically relevant problem, which commonly stems from excessive renal salt retention. Both basic and clinical studies show that elevated dietary Na+ can promote volume expansion and increased blood pressure only when accompanied with high Cl−. The collecting duct (CD) is the only tubular segment with capacity to separately reabsorb Na+ and Cl−. Here, principal cells perform electrogenic Na+ reabsorption via the epithelial Na+ channel (ENaC), whereas activity of the basolateral ClC‐K2 chloride channel mediates trans‐cellular Clmovement in electrically uncoupled intercalated cells. In contrast to ENaC, little is known about dietary and hormonal means controlling ClC‐K2 activity in the CD. Using patch clamp electrophysiology at both single channel and macroscopic levels in freshly isolated CDs, we found that ClC‐K2 single channel activity and macroscopic Cl− conductance in intercalated cells are inversely related to dietary Cl−, but not K+ intake. Furthermore, stimulation of mineralocorticoid receptors with deoxycorticosterone acetate (DOCA) failed to affect ClC‐K2 activity arguing against a role of aldosterone in regulation of ClC‐K2 by dietary Cl−. In contrast, Angiotensin II (Ang II) exerts a set of stimulatory actions by acutely increasing ClC‐K2 single channel open probability, ClC‐K2/b‐dependent Cl− conductance in ICs and on a longer timescale promotes ClC‐K2/b trafficking to the basolateral membrane of ICs in freshly isolated CDs implicating its potential role during variations in dietary Cl− intake. Inhibition of AT1 receptors (AT1R) with losartan precluded regulation of ClC‐K2 by Ang II. Moreover, ClC‐K2 abundance was significantly reduced in mice lacking AT1R. Overall, our results point to a critical role of dietary Cl− in controlling ClC‐K2 activity in the CD likely in an Ang II‐AT1R dependent manner. We propose that this mechanism could be instrumental for the discrete CD responses to hypovolemia (when Ang II is high) and hyperkalemia (Ang II is low).Support or Funding InformationThis research was supported by NIH‐NIDDK DK095029 (to O. P.), AHA 17GRNT33660488 (to O. P.), and ASN Ben J. Lipps Research Fellowship (to V. T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Mechanism for Regulation of Blood Pressure by the Circadian Clock Protein PER1

Work from our lab suggests that the clock protein PER1 is critical for maintaining the circadian rhythm of blood pressure (BP). BP is highest during the active phase of the day whereas during the rest period BP dips at least 10%. Individuals that do not experience the dip are termed “non‐dippers.” Non‐dipping hypertension is associated with an increased risk of cardiovascular disease. The mechanisms underlying non‐dipping hypertension are not understood. Male C57BL/6J Per1 KO mice develop non‐dipping hypertension on a high‐salt diet plus mineralocorticoid treatment (HS/DOCP). Sodium (Na) handling by the kidney has large effects on blood volume and BP. Excretion of Na also demonstrates a circadian pattern with a peak of excretion during active periods. We hypothesized that Per1 contributes to circadian regulation of BP via a renal Na handling‐dependent mechanism. To test this hypothesis, we treated WT and Per1 KO mice housed in metabolic cages with a normal salt diet or HS/DOCP. We measured changes in timed urinary Na excretion using flame photometry. The night:day ratio of Na in the urine of Per1 KO mice was decreased compared to WT (4x vs. 7x, P<0.001, n=6 per group). ELISA was used to measure the fold increase of urinary endothelin‐1 (ET‐1) in response to HS/DOCP treatment. Per1 KO mice exhibited a higher fold increase of ET‐1 compared to WT with HS/DOCP (5.9x vs 3.6x, P<0.01, n=9 WT, 11 KO). Using real‐time quantitative PCR, we determined that the expression of Na handling genes was altered in the KO mice on HS/DOCP. ET‐1 was upregulated in the cortex of Per1 KO mice on HS/DOCP treatment (P<0.01, n=6 per group). This upregulation was not present in WT mice on HS/DOCP. Nitric oxide synthase 1 (NOS1), a negative regulator of Na reabsorption, was significantly downregulated in Per1 KO medulla on HS/DOCP (P<0.05, n=6 per group). Preliminary data from the first distal‐nephron specific Per1 KO mice also shows an inappropriate increase in expression of the epithelial Na channel α subunit (αENaC) and the Na chloride cotransporter (NCC) relative to control mice. These results support our hypothesis that Per1 mediates control of circadian BP rhythms via the regulation of renal Na transport genes, specifically in the distal nephron of the kidney. These findings have implications for understanding the etiology of non‐dipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.Support or Funding InformationR01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of FloridaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conserved cysteines in the finger domain of the epithelial Na+ channel α and γ subunits are proximal to the dynamic finger–thumb domain interface

The epithelial Na+ channel (ENaC) is a member of the ENaC/degenerin family of ion channels. In the structure of a related family member, the "thumb" domain's base interacts with the pore, and its tip interacts with the divergent "finger" domain. Between the base and tip, the thumb domain is characterized by a conserved five-rung disulfide ladder holding together two anti-parallel α helices. The ENaC α and γ subunits' finger domains harbor autoinhibitory tracts that can be proteolytically liberated to activate the channel and also host an ENaC-specific pair of cysteines. Using a crosslinking approach, we show that one of the finger domain cysteines in the α subunit (αCys-263) and both of the finger domain cysteines in the γ subunit (γCys-213 and γCys-220) lie near the dynamic finger-thumb domain interface. Our data suggest that the αCys-256/αCys-263 pair is not disulfide-bonded. In contrast, we found that the γCys-213/γCys-220 pair is disulfide-bonded. Our data also suggest that the γ subunit lacks the terminal rung in the thumb domain disulfide ladder, suggesting asymmetry between the subunits. We also observed functional asymmetry between the α and γ subunit finger-thumb domain interfaces; crosslinks bridging the α subunit finger-thumb interface only inhibited ENaC currents, whereas crosslinks bridging the γ subunit finger-thumb interface activated or inhibited currents dependent on the length of the crosslinker. Our data suggest that reactive cysteines lie at the dynamic finger-thumb interfaces of the α and γ subunits and may play a yet undefined role in channel regulation.

The phosphorylation site T613 in the β-subunit of rat epithelial Na+ channel (ENaC) modulates channel inhibition by Nedd4-2.

The epithelial Na+ channel (ENaC) is a heteromeric channel composed of three subunits (α, β, γ). At the C-terminus of each subunit, a PY-motif allows binding of the ubiquitin ligase Nedd4-2 which plays a key role in promoting ENaC retrieval from the plasma membrane. Phosphorylation of Nedd4-2 by the serum and glucocorticoid-inducible kinase 1 (Sgk1) reduces Nedd4-2 binding to the PY-motifs. In β and γENaC, threonine residues (βT613, γT623) belong to an extracellular signal-regulated kinase (ERK) motif and directly precede the PY-motifs. Thus, phosphorylation of these residues may modulate the interaction of their adjacent PY-motifs with Nedd4-2. In this study, a phosphospecific antibody was used to demonstrate phosphorylation of βT613 in Xenopus laevis oocytes heterologously expressing rat αβγENaC. Treating the oocytes with progesterone to stimulate ERK increased phosphorylation of βT613. Inactivation of the putative phosphorylation sites by mutating both threonine residues to alanine (βT613A/γT623A) increased ENaC-mediated amiloride-sensitive whole-cell currents (ΔIami) and expression of βENaC at the cell surface. Co-expression of Nedd4-2 largely reduced ΔIami in oocytes expressing αβγENaC or channels with mutated PY-motifs in α and γENaC or in α and βENaC. Importantly, the inhibitory effect of co-expressed Nedd4-2 was largely reduced in channels with mutated PY-motifs in α and γENaC when combined with the βT613A mutation but conserved in channels with mutated PY-motifs in α and βENaC combined with the γT623A mutation. These results suggest that phosphorylation and dephosphorylation of βT613 play a prominent role in regulating Nedd4-2-mediated ENaC retrieval from the plasma membrane.

Epithelial Na+ channel differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice.

A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.

Renal Na-handling defect associated with PER1-dependent nondipping hypertension in male mice

Many physiological functions have a circadian rhythm, including blood pressure (BP). BP is highest during the active phase, whereas during the rest period, BP dips 10-20%. Patients that do not experience this dip at night are termed "nondippers." Nondipping hypertension is associated with increased risk of cardiovascular disease. The mechanisms underlying nondipping hypertension are not understood. Without the circadian clock gene Per1, C57BL/6J mice develop nondipping hypertension on a high-salt diet plus mineralocorticoid treatment (HS/DOCP). Our laboratory has shown that PER1 regulates expression of several genes related to sodium (Na) transport in the kidney, including epithelial Na channel (ENaC) and Na chloride cotransporter (NCC). Urinary Na excretion also demonstrates a circadian pattern with a peak during active periods. We hypothesized that PER1 contributes to circadian regulation of BP via a renal Na-handling-dependent mechanism. Na-handling genes from the distal nephron were inappropriately regulated in KO mice on HS/DOCP. Additionally, the night/day ratio of Na urinary excretion by Per1 KO mice is decreased compared with WT (4 × vs. 7×, P < 0.001, n = 6 per group). Distal nephron-specific Per1 KO mice also show an inappropriate increase in expression of Na transporter genes αENaC and NCC. These results support the hypothesis that PER1 mediates control of circadian BP rhythms via the regulation of distal nephron Na transport genes. These findings have implications for the understanding of the etiology of nondipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.

SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Cystic fibrosis (CF) is a common genetic disease with significantly increased mortality. CF airways exhibit ion transport abnormalities, including hyperactivity of the epithelial Na+ channel (ENaC). Short-palate lung and nasal epithelial clone 1 (SPLUNC1) is a multifunctional innate defense protein that is secreted into the airway lumen. We have previously demonstrated that SPLUNC1 binds to and inhibits ENaC to maintain fluid homeostasis in airway epithelia and that this process fails in CF airways. Despite this, how SPLUNC1 actually regulates ENaC is unknown. Here, we found that SPLUNC1 caused αγ-ENaC to internalize, whereas SPLUNC1 and β-ENaC remained at the plasma membrane. Additional studies revealed that SPLUNC1 increased neural precursor cell-expressed developmentally down-regulated protein 4-2-dependent ubiquitination of α- but not β- or γ-ENaC. We also labeled intracellular ENaC termini with green fluorescent protein and mCherry, and found that extracellular SPLUNC1 altered intracellular ENaC Forster resonance energy transfer. Taken together, our data indicate that SPLUNC1 is an allosteric regulator of ENaC that dissociates αβγ-ENaC to generate a new SPLUNC1-β-ENaC complex. These data indicate a novel mode for regulating ENaC at the plasma membrane.-Kim, C. S., Ahmad, S., Wu, T., Walton, W. G., Redinbo, M. R., Tarran, R. SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Epithelial Na+ Channel（ENaC）Trafficking Process between the Intracellular Space and the Apical Membrane using Mathematical Analysis with a Four–state Model

Epithelial Na+ Channel（ENaC）Trafficking Process between the Intracellular Space and the Apical Membrane using Mathematical Analysis with a Four–state Model