Epithelial Na(+) Channel Activity Research Articles

Gs‐DREADD Regulation of ENaC

Activity of the epithelial Na+ channel (ENaC) fine-tunes renal Na+ excretion and consequently, contributes to the normal control of blood pressure. ENaC is expressed in the apical membrane of principal cells. Importantly, ENaC is activated by the hormone vasopressin via the V2 receptor, which is coupled to the heterotrimeric Gs protein. Stimulation of the V2 receptor rapidly activates adenylyl cyclase to increase intracellular cAMP levels. We have recently developed a mouse model with targeted expression of the Gs designer receptors exclusively activated by designer drugs (DREADD) in renal principal cells (GsD). Moreover, this mouse line has a luciferase reporter downstream of a cAMP response element binding protein site. As such, it allows in vivo reporting of cAMP levels. DREADD receptors are activated by clozapine N-oxide (CNO), which normally is an inert drug with no biological activity in the absence of DREADD receptors. We test here if CNO affects the activity of ENaC to influence renal salt and water excretion in GsD mice similarly to vasopressin. In these mice, we longitudinally monitored CNO-stimulated cAMP levels in principal cells using an IVIS Lumina XR. In parallel, we quantified CNO-dependent ENaC activity and renal sodium excretion. We observed that CNO increased ENaC activity in principal cells in freshly isolated tubules from GsD mice compared to control mice. Metabolic cages experiments demonstrated that CNO treatment (0.1 mg/kg) increased cAMP levels concomitantly with reducing sodium excretion similarly to dDAVP (1 µg) in GsD mice. Finally, in COS-7 cells expressing murine ENaC tagged with YFP, CPT-cAMP increased translocation of ENaC to the plasma membrane. Here we demonstrated the utility of a new principal cell-specific Gs-DREADD mouse line. This new mouse model will be an important tool for investigating the effects of abnormal cAMP signaling in principal cells possibly informing understanding of the cellular mechanism underpinning conditions like polycystic kidney disease.

CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells.

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

Benzamil-mediated urine alkalization is caused by the inhibition of H+-K+-ATPases.

Epithelial Na+ channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H+ secretion via renal H+-K+-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K+ intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH4+ and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K+ diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K+ diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α1-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function.NEW & NOTEWORTHY Bolus application of epithelial Na+ channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H+-K+ pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.

LPS-induced epithelial barrier disruption via hyperactivation of CACC and ENaC.

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.

MST3 Involvement in Na+ and K+ Homeostasis with Increasing Dietary Potassium Intake.

K+ loading inhibits NKCC2 (Na-K-Cl cotransporter) and NCC (Na-Cl cotransporter) in the early distal tubules, resulting in Na+ delivery to the late distal convoluted tubules (DCTs). In the DCTs, Na+ entry through ENaC (epithelial Na channel) drives K+ secretion through ROMK (renal outer medullary potassium channel). WNK4 (with-no-lysine 4) regulates the NCC/NKCC2 through SAPK (Ste20-related proline-alanine-rich kinase)/OSR1 (oxidative stress responsive). K+ loading increases intracellular Cl−, which binds to the WNK4, thereby inhibiting autophosphorylation and downstream signals. Acute K+ loading-deactivated NCC was not observed in Cl−-insensitive WNK4 mice, indicating that WNK4 was involved in K+ loading-inhibited NCC activity. However, chronic K+ loading deactivated NCC in Cl−-insensitive WNK4 mice, indicating that other mechanisms may be involved. We previously reported that mammalian Ste20-like protein kinase 3 (MST3/STK24) was expressed mainly in the medullary TAL (thick ascending tubule) and at lower levels in the DCTs. MST3−/− mice exhibited higher ENaC activity, causing hypernatremia and hypertension. To investigate MST3 function in maintaining Na+/K+ homeostasis in kidneys, mice were fed diets containing various concentrations of Na+ and K+. The 2% KCl diets induced less MST3 expression in MST3−/− mice than that in wild-type (WT) mice. The MST3−/− mice had higher WNK4, NKCC2-S130 phosphorylation, and ENaC expression, resulting in lower urinary Na+ and K+ excretion than those of WT mice. Lower urinary Na+ excretion was associated with elevated plasma [Na+] and hypertension. These results suggest that MST3 maintains Na+/K+ homeostasis in response to K+ loading by regulation of WNK4 expression and NKCC2 and ENaC activity.

Role for ovarian hormones in purinoceptor-dependent natriuresis

BackgroundPremenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats.MethodsTo test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 μg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors.ResultsAcute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3.ConclusionThese data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Abstract P229: Site-1 Protease-derived Soluble (pro) Renin Receptor Mediates Aldosterone-induced Enac Activation In The Collecting Duct

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), generated by site-1 protease (S1P), a newly identified PRR cleavage protease, in ENaC regulation, and further tested its relevance to Aldo signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM for 24 h induced a significant increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique ( I eq : 7.5 ± 0.7 μA/cm 2 in sPRR group vs. 3.5 ± 0.5 μA/cm 2 in vehicle group, n = 6, p < 0.01) . In primary cultured rat IMCD cells, the same sPRR-His treatment induced a 1.7 fold increase in protein expression of the α-subunit but not β- or γ-subunit of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The upregulation of α-ENaC transcription depended on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Using chamber determination of short-circuit current showed that Aldo-induced ENaC activity was almost completely abolished by PF-429242 (PF), a S1P inhibitor, and the response was restored by supplement of sPRR-His ( I eq : 7.2 ± 0.7 μA/cm 2 in Aldo group vs. 5.0 ± 0.3 μA/cm 2 in Aldo/PF group vs. 6.8 ± 0.3 μA/cm 2 in Aldo/PF/sPRR-His group, n = 5, p < 0.05). Medium sPRR was elevated by Aldo and inhibited by PF. Male C57BL/6 mice were pretreated with PF (30 mg/kg/day) or vehicle via minipump, followed by 3 days of aldosterone (0.2 mg/kg/day via a second minipump). Amiloride-sensitive Na+ current in freshly isolated CCD as measured by using patch clamp lower in Aldo + PF group than in Aldo group. Together, these results support an essential role of S1P-derived sPRR in mediating Aldo-induced ENaC activation.

Abstract MP44: Proteolytic Activation Of ENaC Mediates Fructose-Induced Salt-Sensitive Hypertension

In nephrotic syndrome, glomerular injury leads to filtration of plasma proteases. Once in the urine, these enzymes cleave and activate the epithelial Na channel (ENaC) causing inappropriate Na retention and hypertension. Dietary fructose causes inappropriate Na retention and salt-sensitive hypertension but whether proteolytic activation of ENaC is involved, and the source of the proteases, is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt-sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high-salt diet without fructose (high salt) or one containing 4% NaCl plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 68±7% (p < 0.01). Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose) high salt/glucose) on blood pressure before and after oral amiloride. After 7 days the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but had no significant effect on the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose-induced salt-sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.

NOX4-dependent regulation of ENaC in hypertension and diabetic kidney disease.

NADPH oxidase 4 (NOX4) is the most abundant NOX isoform in the kidney; however, its importance for renal function has only recently emerged. The NOX4-dependent pathway regulates many factors essential for proper sodium handling in the distal nephron. However, the functional significance of this pathway in the control of sodium reabsorption during the initiation of chronic kidney disease is not established. The goal of this study was to test Nox4-dependent ENaC regulation in two models: SS hypertension and STZ-induced type 1 diabetes. First, we showed that genetic ablation of Nox4 in Dahl salt-sensitive (SS) rat attenuated a high-salt (HS)-induced increase in epithelial Na+ channel (ENaC) activity in the cortical collecting duct. We also found that H2 O2 upregulated ENaC activity, and H2 O2 production was reduced in both the renal cortex and medulla in SSNox4-/- rats fed an HS diet. Second, in the streptozotocin model of hyperglycemia-induced renal injury ENaC activity in hyperglycemic animals was elevated in SS but not SSNox4-/- rats. NaCl cotransporter (NCC) expression was increased compared to healthy controls, while expression values between SS and SSNox4-/- groups were similar. These data emphasize a critical contribution of the NOX4-mediated pathway in maladaptive upregulation of ENaC-mediated sodium reabsorption in the distal nephron in the conditions of HS- and hyperglycemia-induced kidney injury.

Cisplatin Decreases ENaC Activity Contributing to Renal Salt Wasting Syndrome.

Cisplatin (CDDP) is an important anticancer drug. A common side effect of CDDP is renal salt and water-wasting syndrome (RSWS). The origin of RSWS is obscure. Emerging evidence, though, suggests that broad inhibition of sodium transport proteins by CDDP may result in decreases in tubular reabsorption, causing increases in sodium and water excretion. In this sense, CDDP would be acting like a diuretic. The effect of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter fine-tuning renal Na+ excretion, is unknown. We test here whether CDDP affects ENaC to promote renal salt and water excretion. The effects of CDDP and benzamil (BZM), a blocker of ENaC, on excretion of a sodium load were quantified. Similar to BZM, CDDP facilitated renal Na+ excretion. To directly quantify the effects on ENaC, principal cells in split-open tubules were patch clamped. CDDP, at doses comparable to those used for chemotherapy (1.5 µM), significantly decreased ENaC activity in native tubules. To further elaborate on this mechanism, the dose-dependent effects of CDDP on mouse ENaC (mENaC) heterologously expressed in Chinese Hamster Ovary (CHO) cells were tested using patch clamping. As in native tubules, CDDP significantly decreased the activity of mENaC expressed in CHO cells. Dose–response curves and competition with amiloride identified CDDP as a weak inhibitor of ENaC (apparent IC50 = 1 µM) that competes with amiloride for inhibition of the channel, weakening the inhibitory actions of the latter. Such observations are consistent with CDDP being a partial modulator of ENaC, which possibly has a binding site that overlaps with that of amiloride. These findings are consistent with inhibition of ENaC by CDDP contributing to the RSWS caused by this important chemotherapy drug.

ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR modulator therapies.

ENaC inhibition with BI 1265162 is a promising strategy to optimise outcomes in patients with CF either eligible, or ineligible, for CFTR modulator therapy. Phase II clinical trials of BI 1265162 must now show this translates into clinical benefit. https://bit.ly/2OQ1IUI

Aldosterone-dependent and -independent regulation of Na+ and K+ excretion and ENaC in mouse kidneys.

We investigated the regulation of Na+ and K+ excretion and the epithelial Na+ channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods to assess excretion and electrophysiology and Western blot analysis to test for ENaC activity and processing. After 1 day of dietary Na+ restriction, AS-/- mice lost more Na+ in the urine than AS+/+ mice did. After 1 wk on this diet, both genotypes strongly reduced urinary Na+ excretion, but creatinine clearance decreased only in AS-/- mice. Only AS+/+ animals exhibited increased ENaC function, assessed as amiloride-sensitive whole cell currents in collecting ducts or cleavage of αENaC and γENaC in Western blots. To assess the role of aldosterone in the excretion of a K+ load, animals were fasted overnight and refed with high-K+ or low-K+ diets for 5 h. Both AS+/+ and AS-/- mice excreted a large amount of K+ during this period. In both phenotypes the excretion was benzamil sensitive, indicating increased K+ secretion coupled to ENaC-dependent Na+ reabsorption. However, the increase in plasma K+ under these conditions was much larger in AS-/- animals than in AS+/+ animals. In both groups, cleavage of αENaC and γENaC increased. However, Na+ current measured ex vivo in connecting tubules was enhanced only in AS+/+ mice. We conclude that in the absence of aldosterone, mice can conserve Na+ without ENaC activation but at the expense of diminished glomerular filtration rate. Excretion of a K+ load can be accomplished through aldosterone-independent upregulation of ENaC, but aldosterone is required to excrete the excess K+ without hyperkalemia.

Reactive species generated by heme impair alveolar epithelial sodium channel function in acute respiratory distress syndrome

We previously reported that the highly reactive cell-free heme (CFH) is increased in the plasma of patients with chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time that CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl2, with plasmalogens) are increased in the plasma of patients exposed to Cl2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloride-sensitive epithelial Na+ channel (ENaC) and cation sodium (Na+) channels in mouse alveolar cells and trans-epithelial Na+ transport across human airway cells with EC50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86–1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na+ transport across the channel. A single intramuscular injection of the heme-scavenging protein, hemopexin (4 μg/kg body weight), one hour post halogen gas exposure, decreased plasma CFH and improved lung ENaC activity in mice. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.

Upregulation of Renal ENaC Activity in Spontaneously Hypertensive Rats Is Independent of Aldosterone

Spontaneously hypertensive rat (SHR) has been widely used as a model to interrogate the contribution of the kidney to the pathogenesis of essential hypertension in humans. Elevation of blood pressure in SHR has been associated with altered renal function, since predisposition to hypertension can be transferred with a transplanted kidney into a normotensive Wistar‐Kyoto (WKY) strain. Renal defects result in excessive water and sodium retention leading to increased circulating volume and blood pressure. Abnormally elevated Na+ reabsorption in SHR is associated with the overactive renin‐angiotensin‐aldosterone system (RAAS) and with the increased abundance of the renal epithelial Na+ channel (ENaC) responsible for final regulation of sodium reuptake in the kidney and pivotal for long‐term blood pressure control. The existing data on regulation of ENaC by RAAS components in SHR is inconsistent and molecular determinants of excessive ENaC activation remain largely obscure. In the present study, we tested the efficacy of aldosterone antagonism to inhibit ENaC activity, decrease renal Na+ reabsorption and reduce blood pressure in SHR of both sexes. Where necessary, age‐matched (12–15 week‐old) WKY rats were used as controls. Consistent with previously published data, enzyme immunoassay analysis revealed elevated circulating aldosterone levels and semi‐quantitative immunoblotting – higher expression of renal ENaC protein in SHR when compared to WKY controls. Patch‐clamp experiments in split‐opened collecting ducts showed that ENaC activity was moderately increased in SHR when compared to WKY. Systemic administration of aldosterone antagonist, spironolactone (30 mg/kgBW·day) did not affect renal ENaC activity or protein abundance in SHR. Aldosterone antagonism did not alter the expression of sodium‐chloride co‐transporter (NCC) in the kidneys of SHR. Tail‐cuff plethysmography showed that treatment with spironolactone at doses 30–200 mg/kgBW·day for 2 weeks failed to decrease blood pressure in SHR and did not result in any detectable changes in urinary sodium excretion. We did not observe any sex‐specific differences pertaining to ENaC activity or responsiveness to spironolactone in SHR males and females. Thus, we conclude that aldosterone is not a primary driver of excessive ENaC‐dependent renal Na+ reabsorption in SHR.

Cisplatin Decreases ENaC Activity Contributing to Renal Sodium Wasting

Cisplatin (CDDP) is a platinum‐containing compound that has antineoplastic effects due to its ability to bind DNA and inhibit replication. While an important chemotherapy drug, CDDP results in a renal salt and water‐wasting syndrome and is nephrotoxic. The origin of the renal salt and water‐wasting caused by CDDP is obscure; though, some evidence suggests that broad inhibition of the Na+/K+‐ATPase by CDDP results in generalized decreases in tubular reabsorption causing increases in sodium and water excretion. The effects of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter of renal Na+ excretion, are unknown. We test here if CDDP affects the activity of ENaC to influence renal salt and water excretion. These studies focus on understanding the cellular mechanism by which CDDP may affect ENaC. For these studies, C57Bl/6 male mice were randomly divided into 3 groups, as follow: 1) Saline, 2) CDDP and 3) Benzamil (BZM) treated. For metabolic cages experiments, C57Bl/6 male mice received a Na+ load (100 μL of a 0.9% NaCl solution) and were treated with CDDP (25 mg/kg, i.p.) or BZM (1.4 mg/kg, i.p.). Then, water consumption, Na+ intake, urine volume, urinary Na+ and creatine excretion were evaluated every 2 h over a 10 h period. To directly quantify ENaC activity in these animals, principal cells were patch‐clamped in split‐open tubules using the cell‐attached configuration. After recording baseline ENaC activity for 5 min in these tubules, they were treated with CDDP (0.5 to 1.5 μM) with ENaC activity quantified in the presence of this drug over the next 5 min. Preliminary results show that CDDP increases excretion of a Na+ load, increases fractional excretion of sodium (FeNa) and decreases ENaC activity in isolated tubules. Importantly, our studies show that, unlike ouabain, CDDP does not affect channel conductance in cell‐attached patches. Such observations are most consistent with CDDP not affecting the electrochemical forces driving Na+ transport across principal cells, which would change in response to changes in pump activity, but rather decreasing ENaC activity through direct effects on the channel or a channel regulator. In conclusion, these observations are consistent with CDDP actions on ENaC, independent of effects on the pump, contributing to the renal salt and water wasting caused by this chemotherapy drug.Support or Funding InformationThis research was supported by NIH/NIDDK grants R01DK113816 and R01DK117909.

Contribution of Kir4.1/Kir5.1 Channels to the Control of ENaC‐Mediated Apical Sodium Transport in the Cortical Collecting Duct

Recent studies using genetically modified animal models reveal that inwardly rectifying potassium channels Kir4.1 and Kir5.1, encoded by Kcnj10 and Kcnj16 genes, play an essential role in determining plasma potassium level and regulating blood pressure. Moreover, we have revealed recently that the deletion of Kcnj16 gene in Dahl salt‐sensitive (SS) rats has the profound effect on the blood pressure and renal damage. These data uncover a new potential target for the treatment of salt‐induced hypertension. Protein products of Kcnj10 and Kcnj16 are localized to the basolateral membrane of distal nephron segments forming functional homomeric (Kir4.1) and heteromeric (Kir4.1/Kir5.1) potassium channels that play an essential role in establishing a negative basolateral membrane potential and recycling K+ ions moved into the cytosol by the Na+/K+‐ATPase. In the present study we examined the effect of pharmacological inhibition of Kir4.1 and Kir4.1/Kir5.1 channels on sodium transport through the epithelial Na+ channel (ENaC) in the cortical collecting duct (CCD) cells. We have reported recently that Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM) inhibited whole‐cell potassium currents recorded from native principal CCD cells. First we examined whether fluoxetine and nortriptyline have different selectivity in targeting Kir4.1 and Kir4.1/Kir5.1 expressed in Chinese hamster ovary (CHO) cells. Single channel analysis revealed that application of 100 μM of amitriptyline produces a significant decrease in NPo of Kir4.1/Kir5.1 but does not affect Kir4.1 current activity, whereas fluoxetine selectively inhibits Kir4.1. Next we examined whether selective inhibition of Kir4.1 and Kir4.1/Kir5.1 affects an apical sodium transport via ENaC. We showed that amitriptyline, but not fluoxetine reduced (IC50=15.0 μM) the amiloride‐sensitive short‐circuit current (ISC) measured in polarized epithelial mCCDcl1 cells. In control experiments, application of amitriptyline did not directly affect ENaC activity overexpressed in CHO cells. Using different concentrations of recently developed Kir4.1 inhibitor, VU0134992, and patch‐clamp studies of single‐channel activity on mCCD cell line and native rodent and human CCD tubules, we confirmed that inhibition of Kir4.1/Kir5.1 channel but not Kir4.1 produces suppressive effect on ENaC activity. In summary, inhibition of Kir4.1/Kir5.1 channels in CCD cells indirectly reduces apical sodium conductance through ENaC, demonstrating the importance of basolateral potassium channels in the control of sodium reabsorption in distal nephron.Support or Funding InformationNational Institutes of Health grants R01 DK120821 (to J.D. and A.S.), R56 DK121750 (to O.P.), and R35 HL135749 (to A.S.)

MTOR Modulates Sodium and Potassium Conduction in Heterologous Expression System

The role of mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 in ion transport in kidney tubule has been an area of increasing interest in epithelial research. We and others have shown both in cultured cells and in vivo that mTORC2 is the hydrophobic motif kinase for serum and glucocorticoid kinase 1 (SGK1) and that its activity is required for epithelial Na+ channel (ENaC)‐dependent sodium reabsorption in the aldosterone‐sensitive distal nephron (ASDN). However, recent evidence has shown that mTORC2 can also modulate the renal outer medullary K+ channel (ROMK) activity directly. Electrogenic sodium reabsorption through ENaC generates the main driving force in ASDN for potassium secretion through ROMK, and is a key locus of acute regulation of K+ secretion. We suspected that differences observed in various studies may be due to acute vs. chronic regulation. We therefore compared the effects on ENaC and ROMK activity of chronic (through gene‐knockout) and acute (through a pharmacological approach) mTOR inhibition. We used CRISPR‐Cas9 to generate Sin1 (an essential component of mTORC2)‐deficient HEK‐293T cells, which were compared with wild type cells. The cells were transiently transfected with ENaC (alpha, beta gamma subunits) and ROMK1 channels. After recovery, the cells were treated with either vehicle or the mTOR inhibitor AZD8055 for 30 min and subjected to whole cell patch clamp to assess ENaC and ROMK currents. We found that in Sin1 KO cells (chronic loss of mTORC2) ENaC current was unaffected, while ROMK was markedly reduced. In contrast, acute mTOR inhibition with AZD8055 inhibited ENaC but not ROMK whole cell currents. These data provide initial support for the idea that acute pharmacologic inhibition of mTORC2 and chronic selective inhibition of mTORC2 have opposite effects on ENaC and ROMK activities. However, the results are also consistent with the possibility that global inhibition of mTOR (blocking mTORC1 and mTORC2) has distinct effects from selective inhibition of mTORC2. These observations may provide further insight into such diverse processes as aldosterone escape and the distinct plasma K+ concentrations seen in various types of hypertension.Support or Funding InformationThis work was supported by Relypsa Nephrolgy and Cardiology fellowship 2019–2020 to WS and NIH RO1‐DK 56695 to DP

The Terrestrial Migration of Vertebrates Drove the Evolution of ENaC Activation by Cleavage α

The epithelial Na+ channel (ENaC) is critical to regulating extracellular fluids through its activity in several epithelia, including the aldosterone‐sensitive distal nephron and colon, and the airway and alveoli. Human ENaC subunits are subject to activating proteolysis. The ENaC α and γ subunits may each be cleaved twice at polybasic tracts in their respective extracellular GRIP domains. This liberates imbedded inhibitory tracts, which activate the channel. Activation through either subunit requires double cleavage. To examine the evolution of ENaC cleavage sites, we generated a phylogenetic tree of ENaC and ENaC‐related subunits to identify key branch points in the evolution of ENaC subunits. The four ENaC subunits arose from three gene duplication events. First, a single gene diverged to an α/δ precursor and a β/γ precursor. Second, the β/γ precursor diverged to β and γ subunits. These two events occurred before the emergence of jawless fishes, as lampreys have α, β, and γ subunits. After the emergence of jawed fishes, α and δ diverged from the α/δ precursor; the lobe‐finned coelacanth is earliest rooted species to have all four subunits. We observed that the activating cleavage sites in the α and γ subunits appeared around the transition to terrestrial life. Among the fishes, only the Australian lungfish γ subunit had two cleavage sites, and all tetrapod α and γ subunits had two apparent cleavage sites. We ran nested likelihood models in BayesTraits and found a significant relationship between terrestrial status and having two cleavage sites in the α and γ subunits. Both sites showed a statistically significant coevolutionary pattern with the terrestrial state (proximal site, p = 0.004; distal site, p = 0.006). The sites also showed a coevolutionary pattern with each other (p = 0.0003). In contrast, the C‐terminal PY motif, which is important for aldosterone regulation through Sgk1/Nedd4‐2, did not show a dependence on terrestrial status (p = 0.58). We also performed ancestral reconstructions to determine whether these protein sequences arose through divergent or convergent evolution. Competing evolutionary models, where common ancestors were constrained to states reflecting each model, were compared using marginal likelihoods from Markov chain Monte Carlo simulations of each model. Our analysis strongly supports a convergent evolutionary model for the cleavage sites, and a divergent evolutionary model for the PY motifs. Our data suggest that aldosterone signaling adopted both long existing and newly evolved mechanisms to regulate ENaC function.

PIP2 Binds Multiple Cationic Sites of ENaC

Phosphatidylinositol 4,5‐bisphosphate (PIP2) regulates the activity and gating of many different ion channels to include the epithelial Na+ channel (ENaC). The negatively charged phosphoryl groups of the PIP2 headgroup are expected to interact with cationic residues of the cytoplasmic tails of ENaC. To date, understanding of the biophysical interaction between ENaC and PIP2 has been limited to indirect functional assays. Consequently, the precise binding sites and their affinities have not been defined. We began this study using secondary structure prediction tools to show that helical conformation that may cluster the cationic residues of ENaC cytoplasmic tails. Such clustering would form an electropositive region attractive to the PIP2 headgroup. We used a CIBN/CRY2‐5‐ptase optogenetic dimerization system in HEK cells to show that the C5 phosphoryl group of PIP2 plays a role in regulating ENaC activity. To more precisely define the PIP2 binding sites of ENaC, we designed short peptides corresponding to the cationic clusters of ENaC. Steady state intrinsic fluorescence ligand binding data showed that PIP2 interacted with the extreme amino terminus of the β‐ENaC subunit (βN1 peptide) and the amino and carboxy termini of γ‐ENaC nearest the transmembrane domains (γN2 and γC peptides, respectively), but no significant changes were observed with the other peptides or mutant controls. Microscale thermophoresis data revealed that the βN1 and γN2 sites has the strongest interaction with the PIP2 headgroup with Kd ~5.2 and 15 μM, respectively, whereas the γC site interacts with much weaker affinity. Again, no interaction was observed between PIP2 and the other peptides and mutant controls. These results are consistent with PIP2 binding ENaC to stabilize the open conformation of channel via moderate and weak electrostatic interactions within the cytoplasmic termini.Support or Funding InformationThis work is supported by NIH/HHLBI T32 HL07446 to CRA and JDS, and NIH/NIDDK R01 DK987460 and DK117909 to JDS.

Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force.

Canonical epithelial sodium channels (ENaCs) are heterotrimers formed by α, β, and γ ENaC subunits in vertebrates and belong to the Degenerin/ENaC family of proteins. Proteins from this family form mechanosensitive channels throughout the animal kingdom. Activity of canonical ENaC is regulated by shear force (SF) mediating Na+ absorption in the kidney and vascular tone of arteries. Expression analysis suggests that non-canonical ENaC, formed by single or only two subunits, exist in certain tissues, but it is unknown if these channels respond to SF. α, β, γ, and δ ENaC subunits were expressed either alone or in combinations of two subunits in Xenopus oocytes. Amiloride-sensitive currents and the responses to SF were assessed using two-electrode voltage clamp recordings. With the exception of γ ENaC, all homomeric channels provided amiloride-sensitive currents and responded to SF applied via a fluid stream directed onto the oocytes. Channels containing two subunits were also activated by SF. Here, the presence of the γ ENaC subunit when co-expressed with α or δ augmented the SF response in comparison to the αβγ/δβγ ENaC. Overall, we provide evidence that non-canonical ENaC can form channels that respond to SF. This supports a potential function of non-canonical ENaC as mechanosensors in epithelial, vascular, and sensory cells.