ENaC Activity Research Articles

Transmembrane Serine Protease 2 and Proteolytic Activation of the Epithelial Sodium Channel in Mouse Kidney.

The renal epithelial sodium channel (ENaC) is essential for sodium balance and blood pressure control. ENaC undergoes complex proteolytic activation by not yet clearly identified tubular proteases. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). Murine ENaC and TMPRSS2 were (co-)expressed in Xenopus laevis oocytes. ENaC cleavage and function were studied in TMPRSS2-deficient murine cortical collecting duct (mCCDcl1) cells and TMPRSS2-knockout (Tmprss2-/-) mice. Short-circuit currents (ISC) were measured to assess ENaC-mediated transepithelial sodium transport of mCCDcl1 cells. The mCCDcl1 cell transcriptome was studied using RNA sequencing. The effect of low-sodium diet with or without high potassium were compared in Tmprss2-/- and wildtype mice using metabolic cages. ENaC-mediated whole-cell currents were recorded from microdissected tubules of Tmprss2-/- and wildtype mice. In oocytes, co-expression of murine TMPRSS2 and ENaC resulted in fully cleaved γ-ENaC and ∼2-fold stimulation of ENaC currents. High baseline expression of TMPRSS2 was detected in mCCDcl1 cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated ISC which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. Tmprss2-/- mice kept on standard diet exhibited no apparent phenotype, but renal γ-ENaC cleavage was altered. In response to a low-salt diet, particularly with high potassium intake, Tmprss2-/- mice increased plasma aldosterone significantly more than wildtype mice to achieve a similar reduction of renal sodium excretion. Importantly, the stimulatory effect of trypsin on renal tubular ENaC currents was much more pronounced in Tmprss2-/- mice than that in wildtype mice. This indicated the presence of incompletely cleaved and less active channels at the cell surface of TMPRSS2-deficient tubular epithelial cells. TMPRSS2 contributes to proteolytic ENaC activation in mouse kidney in vivo.

Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury.

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NADPH oxidase 2 (NOX2), involving the pneumococcal virulence factor pneumolysin (PLY). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of the epithelial sodium channel (ENaC) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function -measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans Blue Dye incorporation in mouse lungs- following infection with pneumococci. PLY-induced hyperpermeability in HL-MVEC monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by co-localization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α KO (enENaC-α KO) mice develop increased capillary leak upon i.t. instillation with PLY or pneumococci, compared to wild type (wt) animals. TIP peptide diminishes capillary leak in Sp-infected wt mice, without significantly increasing lung bacterial load. Lung slices from Sp-infected enENaC-α KO mice have a significantly increased endothelial NOX2 expression, as compared to infected CRE mice. In conclusion, endothelial ENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in ARDS, without impairing host defense.

Luminal Flow in the Connecting Tubule induces Afferent Arteriole Vasodilation.

Renal autoregulatory mechanisms modulate renal blood flow. Connecting tubule glomerular feedback (CNTGF) is a vasodilator mechanism in the connecting tubule (CNT), triggered paracrinally when high sodium levels are detected via the epithelial sodium channel (ENaC). The primary activation factor of CNTGF-whether NaCl concentration, independent luminal flow, or the combined total sodium delivery-is still unclear. We hypothesized that increasing luminal flow in the CNT induces CNTGF via O2- generation and ENaC activation. Rabbit afferent arterioles (Af-Arts) with adjacent CNTs were microperfused ex-vivo with variable flow rates and sodium concentrations ranging from <1 mM to 80 mM and from 5 to 40 nL/min flow rates. Perfusion of the CNT with 5 mM NaCl and increasing flow rates from 5 to 10, 20, and 40 nL/min caused a flow rate-dependent dilation of the Af-Art (p<0.001). Adding the ENaC blocker benzamil inhibited flow-induced Af-Art dilation, indicating a CNTGF response. In contrast, perfusion of the CNT with <1 mM NaCl did not result in flow-induced CNTGF vasodilation (p>0.05). Multiple linear regression modeling (R2=0.51;p<0.001) demonstrated that tubular flow (β=0.163 ± 0.04;p<0.001) and sodium concentration (β=0.14 ± 0.03;p<0.001) are independent variables that induce afferent arteriole vasodilation. Tempol reduced flow-induced CNTGF, and L-NAME did not influence this effect. Increased luminal flow in the CNT induces CNTGF activation via ENaC, partially due to flow-stimulated O2- production and independent of nitric oxide synthase (NOS) activity.

Low potassium activation of proximal mTOR/AKT signaling is mediated by Kir4.2

The renal epithelium is sensitive to changes in blood potassium (K+). We identify the basolateral K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Mice lacking Kir4.2 have a compensated baseline phenotype whereby they increase their distal transport burden to maintain homeostasis. Upon dietary K+ depletion, knockout animals decompensate as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis. Potassium wasting is not proximal in origin but is caused by higher ENaC activity and depends upon increased distal sodium delivery. Three-dimensional imaging reveals Kir4.2 knockouts fail to undergo proximal tubule expansion, while the distal convoluted tubule response is exaggerated. AKT signaling mediates the dietary K+ response, which is blunted in Kir4.2 knockouts. Lastly, we demonstrate in isolated tubules that AKT phosphorylation in response to low K+ depends upon mTORC2 activation by secondary changes in Cl- transport. Data support a proximal role for cell Cl- which, as it does along the distal nephron, responds to K+ changes to activate kinase signaling.

Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity.

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin complex (mTORC)1, partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (normal salt) or 4.0% NaCl (high salt) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide [inhibitors of epithelial Na+ channel (ENaC), Na-K-2Cl cotransporter (NKCC2), or Na-Cl cotransporter (NCC), respectively] either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.NEW & NOTEWORTHY This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na+ channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.

Balancing sodium and potassium: Implication of mTORC2-mediated ENaC activation in DCT2 and early CNT for potassium balance in high dietary potassium

Background: The activity of the epithelial sodium ion channel, ENaC, and ENaC-dependent potassium (K+) secretion through ROMK are influenced by both aldosterone-dependent and independent mechanisms in response to dietary potassium (K+). While ENaC activity in the late connecting tubule (CNT) and cortical collecting duct (CCD) depends on aldosterone, it is aldosterone-independent in the late distal convoluted tubule (DCT2) and early CNT (eCNT). Aldosterone-stimulated ENaC activity relies on SGK1, transcriptionally upregulated by aldosterone and subsequently phosphorylated and activated by mTOR complex-2 (mTORC2). Recent evidence suggests that high potassium can rapidly activate mTORC2/SGK1 signaling to stimulate ENaC. In this study, we explore mTORC2’s role in K+ secretion and aldosterone-independent ENaC regulation. Methods: Rictor, a core component of mTORC2, was selectively knocked out (KO'd) in DCT2, CNT and CD by utilizing Calbindin as Cre-driver (CRKO mice) or late CNT and CD, by employing AQP2 as the Cre-driver (ARKO mice). Both wild-type (WT) and KO mice were subjected to a high potassium (HK) diet for short-term (4 hours) or medium-term (48 hours) periods, enabling the monitoring of both the early response and prolonged adaptation to the HK diet. Parameters assessed include urinary and blood electrolyte levels, renal transporter expression, activity and localization, and mTORC2 target phosphorylation. Results: On a normal K+ diet, both KO mice maintained Na+ and K+ balance but CRKO mice had elevated aldosterone levels. After short-term (4-hour) HK intake, CRKO mice had disrupted Na+ and K+ balance, reduced K+ excretion and hyperkalemia. In contrast, ARKO mice maintained Na+ and K+ balance with elevated aldosterone. On a prolonged HK diet (48 hours), CRKO mice had severe hyperkalemia, reduced GFR and significantly elevated aldosterone level. Immunofluorescence studies indicated markedly reduced apical localization of active ENaC in DCT2/eCNT of CRKO mice. Conversely, ENaC apical localization in the late CNT and CCD was mostly unaffected. Phosphorylation of mTORC2 targets involved in ENaC regulation, such as SGK1, and SGK1’s target, Nedd4-2, was also reduced in CRKO mice. ARKO mice maintained ion balance with normal K+ levels but significantly increased aldosterone levels and maintained ENaC apical localization in the late CNT and CCD. Conclusions: The data suggest that high aldosterone can compensate mTORC2 deficiency and maintain ENaC activity in the late CNT and CCD. However, mTORC2 activity is crucial for maintaining ENaC activity in the late DCT and early CNT (aldosterone-independent) and preserving potassium balance during high dietary potassium intake. National Institutes of Health (R01- DK56695), James Hilton Manning and Emma Austin Manning Foundation. 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.

Humanized Diets Define Optimal Pressure Lowering Response to Dietary Potassium in Mice

High potassium (K) diets are known to lower blood pressure (BP) in rodents and humans but can also raise BP when consumed in extreme. Interestingly, this response also appears to be influenced by dietary sodium (Na) such that when Na intake is high, high K lowers BP, whereas when sodium intake is lower, high K increases BP (Little et al, Hypertension, 2023). We recently reported (Little et al, JCI Insight, 2023) that the K-mediated BP lowering response, driven by the inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC), is offset by an increase in ENaC (epithelial sodium channel) when an extremely high K diet is chronically consumed. Here, we define the optimal dietary K level needed to inactivate NCC without activating ENaC in the high Na setting and show this K level approximates the recommended level for humans by the DASH (Dietary Approaches to Stop Hypertension) trials. Mice were fed diets containing 6 different K contents, all with high Na (1.7%), so the effects of the control diet (1% K, 1.7% Na) could be compared to traditional extremes (0.1% and 5% K) and others (0.35%, 0.7%, 1.7%) that mimic typical variation in human diets (30-120 mEq/d) when mouse consumption is normalized for caloric intake relative to body mass. Metabolic cage and renal clearance studies were used to assess electrolyte and aldosterone (aldo) excretion as well as NCC- (hydrochlorothiazide (HCTZ) sensitive) and ENaC- (benzamil sensitive) dependent sodium handling. Blood potassium (PK) was measured using iSTAT. BP was measured hourly for 10 days using DSI telemetry probes in male mice (C57Bl6J). The BP response to the diuretics was also measured. PK levels changed significantly with each diet relative to the control diet from 3.2mM to 4.8mM. Systolic blood pressure (SBP; 12-hour active period average) also changed significantly with the different K diets. Extreme high or low K diets (0.1% and 5%) increased SBP by 7mmHg, 0.35% and 0.7% K diets increased SBP by 6 and 3mmHg, while the 1.7% K diet lowered SBP by 4mmHg relative to control (1% K, 132 mmHg). Thus, the relationship between SBP and PK is ‘U-shaped’. Diuretic studies revealed that HCTZ reduces SBP in the 0.1%, 0.35%, 0.7% K groups but had minimal effect in the 1.7% and 5% K groups. Conversely, inhibiting ENaC activity with benzamil normalized SBP in the 5% K group but had a minimal effect in the 1.7% group and no BP effect in the other groups. The BP-lowering effect of each diuretic correlated with increased natriuresis suggesting dietary K influences blood pressure by affecting distal nephron sodium transport. Lastly, urinary aldo levels increased with increasing dietary K, but compared to the control group, aldo levels were significantly lower in the 0.1%, 0.35%, and 0.7% groups, significantly higher in the 5% group, and not different in the 1.7% group. As previously reported, our results demonstrate that extremes in dietary intake can elevate blood pressure by stimulating distal nephron sodium transport proteins with low K inducing NCC activation and high K stimulating ENaC with elevated aldosterone. An optimal dietary K level (1.7%) that maximally reduces BP in mice was identified, suppressing NCC activity while limiting aldosterone production and ENaC activity. Remarkably, this diet mimics the human DASH diet in K content and further supports its effcacy as a blood pressure-lowering modality. DE (DK054231, DK51496); PW (DK054231, DK093501, DK110375): Leducq Foundation U54 DK137331-01. 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.

The Proximal Tubule Response to Low Potassium Is Mediated by mTOR/AKT Activation via Kir4.2

Background: The renal epithelium is highly sensitive to changes in blood potassium (K+) levels. We have a detailed understanding of distal K+-sensitive signaling pathways, yet proximal mechanisms are less well-defined. Results: Mice lacking the basolateral proximal tubule inwardly rectifying K+ channel, Kir4.2, (Kir4.2−/−) demonstrated normal blood and urine electrolytes at baseline. Upon removal of K+ from their diet, Kir4.2−/− animals decompensated as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis compared to control animals (Kir4.2+/+). Potassium wasting in Kir4.2−/− mice was not proximal in origin, but was dependent upon increased ENaC activity along the connecting segment. This was demonstrated by the correction of K+ wasting in knockouts treated with the ENaC inhibitor, amiloride. Using lithium clearance studies, we further demonstrated increased distal sodium delivery in Kir4.2−/− animals due to reduced proximal tubule sodium reabsorption. Optical clearing and three-dimensional confocal imaging reveal Kir4.2−/− mice failed to undergo proximal tubule hypertrophy when fed a low K+ diet, while the distal convoluted tubule response was exaggerated in knockouts compared to control animals. A phosphoproteomics mass spectrometry analysis identified increased mTOR/AKT signaling as mediating the proximal dietary response to K+ depletion in control animals, and changes to mTOR/AKT signaling was blunted in Kir4.2 knockouts. Lastly, we demonstrated in isolated renal tubules that AKT phosphorylation in response to low K+ depended upon mTORC2 activation by secondary reductions in intracellular Cl−. Conclusions: In this work we identify the basolateral inwardly rectifying K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Kir4.2 knockout mice increase their distal nephron transport burden to maintain electrolyte homeostasis in the face of deficient proximal tubule cell function. Data identify a conserved proximal role for cell Cl− which, as it does along the distal convoluted tubule, responds to extracellular K+ changes to activate intracellular kinase signaling. Along the proximal tubule, mTOR/AKT signaling is essential for the cell response to low K+. Proximal and distal nephron cell Cl− responses work in concert to preserve K+ homeostasis. These studies were supported by NIH grants DP5-OD033412 (AST), DK51265, DK95785, DK62794, DK7569, P30DK114809 (RCH, MZZ), and the Vanderbilt Center for Kidney Disease and Vanderbilt Diabetes Research and Training Center (DK020593) pilot and feasibility grant (JSD). 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.

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

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969 binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of β-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics (MD) simulations predicted critical residues in the thumb domain of β-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the β-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. MD simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (βR437C – γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

Select amino acids recover cytokine-altered ENaC function in human bronchial epithelial cells.

The airway epithelium plays a pivotal role in regulating mucosal immunity and inflammation. Epithelial barrier function, homeostasis of luminal fluid, and mucociliary clearance are major components of mucosal defense mechanisms. The epithelial sodium channel (ENaC) is one of the key players in controlling airway fluid volume and composition, and characteristic cytokines cause ENaC and barrier dysfunctions following pulmonary infections or allergic reactions. Given the limited understanding of the requisite duration and magnitude of cytokines to affect ENaC and barrier function, available treatment options for restoring normal ENaC activity are limited. Previous studies have demonstrated that distinct amino acids can modulate epithelial ion channel activities and barrier function in intestines and airways. Here, we have investigated the time- and concentration-dependent effect of representative cytokines for Th1- (IFN-γ and TNF-α), Th2- (IL-4 and IL-13), and Treg-mediated (TGF-β1) immune responses on ENaC activity and barrier function in human bronchial epithelial cells. When cells were exposed to Th1 and Treg cytokines, ENaC activity decreased gradually while barrier function remained largely unaffected. In contrast, Th2 cytokines had an immediate and profound inhibitory effect on ENaC activity that was subsequently followed by epithelial barrier disruption. These functional changes were associated with decreased membrane protein expression of α-, β-, and γ-ENaC, and decreased mRNA levels of β- and γ-ENaC. A proprietary blend of amino acids was developed based on their ability to prevent Th2 cytokine-induced ENaC dysfunction. Exposure to the select amino acids reversed the inhibitory effect of IL-13 on ENaC activity by increasing mRNA levels of β- and γ-ENaC, and protein expression of γ-ENaC. This study indicates the beneficial effect of select amino acids on ENaC activity in an in vitro setting of Th2-mediated inflammation suggesting these amino acids as a novel therapeutic approach for correcting this condition.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Proteolytic Activation of the Epithelial Sodium Channel (ENaC): Its Mechanisms and Implications.

Epithelial sodium channel (ENaC) are integral to maintaining salt and water homeostasis in various biological tissues, including the kidney, lung, and colon. They enable the selective reabsorption of sodium ions, which is a process critical for controlling blood pressure, electrolyte balance, and overall fluid volume. ENaC activity is finely controlled through proteolytic activation, a process wherein specific enzymes, or proteases, cleave ENaC subunits, resulting in channel activation and increased sodium reabsorption. This regulatory mechanism plays a pivotal role in adapting sodium transport to different physiological conditions. In this review article, we provide an in-depth exploration of the role of proteolytic activation in regulating ENaC activity. We elucidate the involvement of various proteases, including furin-like convertases, cysteine, and serine proteases, and detail the precise cleavage sites and regulatory mechanisms underlying ENaC activation by these proteases. We also discuss the physiological implications of proteolytic ENaC activation, focusing on its involvement in blood pressure regulation, pulmonary function, and intestinal sodium absorption. Understanding the mechanisms and consequences of ENaC proteolytic activation provides valuable insights into the pathophysiology of various diseases, including hypertension, pulmonary disorders, and various gastrointestinal conditions. Moreover, we discuss the potential therapeutic avenues that emerge from understanding these mechanisms, offering new possibilities for managing diseases associated with ENaC dysfunction. In summary, this review provides a comprehensive discussion of the intricate interplay between proteases and ENaC, emphasizing the significance of proteolytic activation in maintaining sodium and fluid balance in both health and disease.

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

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

A Serine Protease Inhibitor, Camostat Mesilate, Suppresses Urinary Plasmin Activity and Alleviates Hypertension and Podocyte Injury in Dahl Salt-Sensitive Rats.

In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions.

Vaping-Induced Proteolysis Causes Airway Surface Dehydration.

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers' airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers' BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers' airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease.

Kidney-Specific Membrane-Bound Serine Proteases CAP1/Prss8 and CAP3/St14 Affect ENaC Subunit Abundances but Not Its Activity.

The serine proteases CAP1/Prss8 and CAP3/St14 are identified as ENaC channel-activating proteases in vitro, highly suggesting that they are required for proteolytic activation of ENaC in vivo. The present study tested whether CAP3/St14 is relevant for renal proteolytic ENaC activation and affects ENaC-mediated Na+ absorption following Na+ deprivation conditions. CAP3/St14 knockout mice exhibit a significant decrease in CAP1/Prss8 protein expression with altered ENaC subunit and decreased pNCC protein abundances but overall maintain sodium balance. RNAscope-based analyses reveal co-expression of CAP3/St14 and CAP1/Prss8 with alpha ENaC in distal tubules of the cortex from wild-type mice. Double CAP1/Prss8; CAP3/St14-deficiency maintained Na+ and K+ balance on a Na+-deprived diet, restored ENaC subunit protein abundances but showed reduced NCC activity under Na+ deprivation. Overall, our data clearly show that CAP3/St14 is not required for direct proteolytic activation of ENaC but for its protein abundance. Our study reveals a complex regulation of ENaC by these serine proteases on the expression level rather than on its proteolytic activation.

Abstract P418: Antigen-presenting Cell-specific MAP-kinase Signaling Contributes To Inflammation And Salt-sensitivity Of Blood Pressure In Humans

Salt-sensitivity of blood pressure (SSBP) is defined by the changes in blood pressure in concert with salt intake/depletion. SSBP affects 50 percent of hypertensive and 25 percent of normotensive individuals and is an independent risk for stroke and cardiovascular death in both groups. We recently found that phosphorylation of STAT3 and SMAD3 in antigen-presenting cells plays an important role in developing SSBP. We hypothesized that mitogen-activated protein kinases (MAPKs) signaling plays an important role in activating/phosphorylating JAK-STAT-SMAD and contributes to salt-sensitivity of blood pressure. We isolated monocytes from eleven healthy individuals and treated them with either normal salt (NS) or high salt (HS) and performed bulk-RNA-sequencing. The bulk-RNA transcriptomic data analysis suggested that out of 21 members of the MAPK family, eighteen were upregulated (1.7-fold) in monocytes exposed to high salt. Among these, MAPK7 (NS:818.18±54.96; HS:1473.18±151.50) and MAPKAPK2 (NS:3977.81±286.67; HS:6222.63±440.32) were significantly upregulated. In additional experiments, we enrolled eleven hypertensive patients and phenotyped them for salt-sensitivity using a rapid salt-load and depletion inpatient protocol. Blood was collected at baseline, after salt loading, and depletion, and CITE-Seq was performed on isolated PBMCs. The data analysis showed that MAPK7 and MAPKAPK2 expressions mirror the blood pressure changes following salt loading and depletion in salt-sensitive but not in salt-resistant patients. Moreover, we found a positive correlation between MAPKAPK2 expression and the pulse pressure ((PP) r=0.52, p=0.033) after salt-load. Epoxyeicosatrienoic acid (EET) has been shown to activate MAPKs resulting in inhibiting ENAC activity. The data showed a strong positive correlation between MAPK7 expression and levels of EETs in plasma (r=0.70, p=0.036), and urine (r=0.68, p=0.045) after salt-load. These results from bulk-RNA and single-cell CITE-Seq data indicate that MAPKs play an important role in human SSBP.

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.

Direct endothelial ENaC activation mitigates vasculopathy induced by SARS-CoV2 spike protein.

Although both COVID-19 and non-COVID-19 ARDS can be accompanied by significantly increased levels of circulating cytokines, the former significantly differs from the latter by its higher vasculopathy, characterized by increased oxidative stress and coagulopathy in lung capillaries. This points towards the existence of SARS-CoV2-specific factors and mechanisms that can sensitize the endothelium towards becoming dysfunctional. Although the virus is rarely detected within endothelial cells or in the circulation, the S1 subunit of its spike protein, which contains the receptor binding domain (RBD) for human ACE2 (hACE2), can be detected in plasma from COVID-19 patients and its levels correlate with disease severity. It remains obscure how the SARS-CoV2 RBD exerts its deleterious actions in lung endothelium and whether there are mechanisms to mitigate this. In this study, we use a combination of in vitro studies in RBD-treated human lung microvascular endothelial cells (HL-MVEC), including electrophysiology, barrier function, oxidative stress and human ACE2 (hACE2) surface protein expression measurements with in vivo studies in transgenic mice globally expressing human ACE2 and injected with RBD. We show that SARS-CoV2 RBD impairs endothelial ENaC activity, reduces surface hACE2 expression and increases reactive oxygen species (ROS) and tissue factor (TF) generation in monolayers of HL-MVEC, as such promoting barrier dysfunction and coagulopathy. The TNF-derived TIP peptide (a.k.a. solnatide, AP301) -which directly activates ENaC upon binding to its a subunit- can override RBD-induced impairment of ENaC function and hACE2 expression, mitigates ROS and TF generation and restores barrier function in HL-MVEC monolayers. In correlation with the increased mortality observed in COVID-19 patients co-infected with S. pneumoniae, compared to subjects solely infected with SARS-CoV2, we observe that prior intraperitoneal RBD treatment in transgenic mice globally expressing hACE2 significantly increases fibrin deposition and capillary leak upon intratracheal instillation of S. pneumoniae and that this is mitigated by TIP peptide treatment.

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.)