Epithelial Na(+) Channel Activity Research Articles

Aldosterone applied mucosally or serosally is equally effective in stimulating ENaC activity in rat distal colon

Background: Aldosterone (ALDO), a mineralocorticoid hormone, is essential for regulating blood volume and electrolyte homeostasis. ALDO acts via intracellular mineralocorticoid receptors to stimulate epithelial Na+ channel (ENaC)-mediated Na+ absorption and large conductance K+ (BK) channel-mediated K+ secretion in both kidney and distal colon. We recently showed that ALDO stimulates iron absorption in rat distal colon and SK-CO15 cells (human colon cancer cell line), raising the prospect that ALDO might be used as adjuvant therapy in iron-deficient patients refractory to oral iron. Since, parenteral ALDO (or Fludrocortisone, a synthetic mineralocorticoid agonist) would adversely affect cardiovascular function, an alternative route of administration is required. Parenteral ALDO gains access to target epithelial cells via their serosal (basolateral) membranes, and in vitro studies have always employed serosally applied ALDO to determine its effects on colonic ion transport, but it is not known if mucosally (apically) applied ALDO has similar effects. Hypothesis: We hypothesized that like serosal ALDO, mucosal ALDO would diffuse across apical cell membranes and induce ENaC activity in normal rat distal colon. Aim: To determine whether mucosally applied ALDO stimulates amiloride-sensitive electrogenic Na+ transport in rat distal colon. Methods: Muscularis stripped normal rat (Sprague-Dawley; 126 – 150 g) distal colon was mounted under voltage clamp condition in Ussing chambers. ALDO (10 mM) was added to either the mucosal or serosal bath. ENaC activity was measured as the change in short-circuit current (Isc) following the mucosal addition of amiloride (10 mM). ALDO in mucosal and serosal bath solutions was measured using mass-spectroscopy. Results: With serosal addition of ALDO, transcriptionally induced ENaC activity began to increase after 4 hr and reached maximum/plateau by 5 hr. By contrast, with mucosal addition of ALDO, ENaC activity began to increase after 8 hr and slowly reached maximum/plateau by 9.5 hr. Maximal amiloride-sensitive ENaC activities were similar irrespective of whether ALDO was applied mucosally or serosally. Following addition of ALDO to the mucosal or serosal bath, a similar amount (40%) of ALDO had moved from mucosa to serosa or serosa to mucosa after 24 hr. Conclusions: We conclude that: (1) ALDO induces ENaC activity to the same extent, whether applied mucosally or serosally, and (2) the delayed effect of mucosally applied ALDO may reflect slower diffusion across apical membranes than across basolateral membranes. We speculate that developing a colon-specific delivery system may avoid the adverse cardiovascular effects of oral mineralocorticoid agonists (e.g. Fludrocortisone) if used to enhance colonic iron absorption in iron-deficient patients refractory to oral iron. Transition GrantSupport; Offce of Research and Graduate Education, WVU Health Sciences Center. 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.

Hypomagnesemia-induced Hypokalemia Is Associated with Higher Activities of ENaC and ROMK

Background: Hypokalemia occurs in 40 to 60% of patients with hypomagnesemia, and can result in cardiac arrhythmia and sudden death. Along the distal nephron, a decrease in intracellular Mg2+ has been proposed to release Mg2+-mediated inhibition of renal outer medullary K+ (ROMK) channels, increasing urinary K+ excretion. However, hypomagnesemia alone is not suffcient to cause hypokalemia. Higher activity of the epithelial Na+ channel (ENaC), which provides the driving force for K+ secretion via ROMK, has been proposed to be an additional requirement. We tested the hypothesis that hypomagnesemia-induced hypokalemia is associated with higher activities of ENaC and ROMK. Methods: C57/BL6 mice were fed normal (NL) [Mg2+ 0.15%/NaCl ~0.49% (wt/wt)], low Mg2+ (LM) [Mg2+ 0.0015– 0.003% (wt/wt); NaCl ~0.49%], low sodium (LS) [Na+ <0.001% (wt/wt)], or combined low sodium and low Mg2+ (LS/LM) [Mg2+ 0.0015-0.003%/Na+ <0.001% (wt/wt)] diets for 3 or 7 days. All diets contained diet [K+ ~0.8% (wt/wt)]. Kidneys and blood were harvested for Western blotting and measurement of plasma [K+]. DCT2/CNT from mice on NL or LS/LM diet were microdissected for measurement of ROMK activity via patch-clamping of split open tubules. Results: Plasma [K+] was only significantly lower than in mice on NL diet in mice on LS/LM diet. Abundances of cleaved α- and γ-ENaC, which correlate with ENaC activity, were lower on a LM diet compared with NL diet. Consistent with lower ENaC activity, the urinary Na+:K+ ratio following acute amiloride administration was lower in mice on LM diet than in mice on NL. While abundances of cleaved α- and γ-ENaC abundances were higher on LS diet than on LS/LM or NL diets, they did not differ between mice on LS/LM or NL diets. As expected, mice on LS diet displayed higher plasma aldosterone than mice on NL diet, but plasma aldosterone did not differ between mice on LS/LM or control diets. ROMK activity along DCT2/CNT was higher in mice on LS/LM diet than compared with mice on NL diet. Conclusions: While LM diet lowered and LS diet stimulated ENaC cleavage compared with controls, ENaC cleavage was similar in mice on LS/LM or NL diets, suggesting dietary Na+ and Mg2+ deficiency counteract each other with regard to ENaC activation. Other studies have shown that the inhibitory effect of hypokalemia on aldosterone secretion may be more potent than the effect of volume depletion on promoting aldosterone secretion, which may explain the lower plasma aldosterone in mice on LS/LM diet than in mice on LS diet. Overall, our data suggest that both ENaC and disinhibition of ROMK are required for the development of hypokalemia in the context of hypomagnesemia. Y.M., Uehara Foundation; J.M., NIDDK DK132066; W-H.W., NIDDK DK133220. 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.

Functional in vivo effects of an ENaC Na+ self inhibition-enhancing mutation

Background: Epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron is a critical regulator of extracellular fluid volume and K+ homeostasis. Physiologic concentrations of extracellular Na+ reduce channel activity (open probability; PO) via an allosteric mechanism termed “Na+ self-inhibition,” which involves Na+ ions binding to specific sites in the channel’s extracellular domains. Recent studies have identified human ENaC mutations that alter the Na+ self-inhibition response when assayed in vitro. Such mutations may confer sensitivity or resistance to salt-induced blood pressure changes. Using CRISPR-Cas9 technology, we generated mice carrying a mutation previously shown to enhance Na+ self-inhibition (αH282R) to assess the effects on ENaC-dependent Na+ transport in vivo. We predicted that ENaC activity would be reduced in these mice, leading to enhanced sensitivity to Na+ deprivation or K+ loading, along with accompanying compensatory changes to overcome the predicted loss of function. Methods: We measured urinary electrolyte handling in wild type (WT) and αH282R mice maintained on normal, low Na+, or high K+ diets using metabolic cages. We assessed ENaC activity ex vivo using patch clamp and Ussing chamber methods. We also measured blood chemistry and aldosterone levels in WT and αH282R mice under the dietary conditions described above. Results: When maintained on a normal chow diet (0.3% NaCl), αH2832R mice expressed ENaCs in the distal nephron with reduced PO compared to WT littermates (0.026 vs. 0.067; p<0.01), while ENaC subunit protein expression was unaltered. Amiloride-sensitive short-circuit currents were reduced in the distal colons of αH283R mice versus WT littermates (-12.6 vs. -3.9 μA/cm2; p<0.01). Plasma aldosterone levels were also higher in αH283R versus WT mice on a control diet (610 vs 288 pg/mL; p=0.02), while no other differences in baseline blood chemistry were observed. When challenged with either dietary Na+ restriction (<0.01% NaCl) or K+ loading (10% KCl), WT and αH283R mice responded similarly in terms of body weight maintenance, urinary Na+ and K+ excretion, and systemic electrolyte and metabolic parameters, with the exception of higher aldosterone levels in Na+-restricted αH282R mice compared to WT littermates (2,759 vs. 1271 pg/mL; p<0.01). Conclusions: The αH282R mutation, which enhances Na+ self-inhibition, confers reduced ENaC activity in mice. However, this loss of function may be compensated by ENaC-independent Na+/K+ handling mechanisms and/or increased aldosterone signaling during adaptation to dietary salt stressors. P30DK079307, T32DK061296. 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.

Luminal flow-stimulated superoxide participates in connecting tubule-glomerular feedback

Increasing NaCl concentration in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art) through the connecting tubule-glomerular feedback (CNTGF). CNTGF is mediated by Na+ transport via epithelial Na+ channels (ENaC) in the CNT. It is unknown if CNTGF is mainly activated by NaCl concentration or sodium delivery or if there is an independent luminal flow effect. Previous studies showed that luminal flow stimulates superoxide (O2−), and O2− can activate ENaC. Thus we hypothesized that increasing luminal flow in the CNT induces CNTGF via O2− generation and ENaC activation. Rabbit Af-Arts with the adjacent CNTs were microdissected and perfused in vitro. Protocols consist of two consecutive dose-response curves to flow rate in the same preparation. When the CNT was perfused with 5mM NaCl, increasing flow rates from 5 to 10, 20, and 40 nL/min caused flow rate-dependent Af-Art dilatation (p<0.01). Adding an ENaC blocker benzamil to the perfusate blocked flow-induced Af-Art dilatation, indicating a CNTGF response. Using mathematical modeling, an increased tubular flow controlled by NaCl delivery into the CNT independently increases CNTGF vasodilation. However, when CNT was perfused with 0 mM NaCl, increasing the flow rate did not induce CNTGF vasodilation. The O2− dismutase mimetic tempol reduced flow-induced CNTGF. In the presence of L-NAME (NOS inhibitor), the reduced CNTGF mediated by tempol was unaffected. We concluded that increasing luminal flow in CNT induces CNTGF activation via ENaC, partly due to flow-stimulated O2− production and independently of NOS. 1K01HL155235. 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.

Activation of renal epithelial Na+ channels (ENaC) in infants with congenital heart disease.

This study was designed to measure the concentration and activity of urinary proteases that activate renal epithelial sodium channel (ENaC) mediated Na+ transport in infants with congenital heart disease, a potential mechanism for fluid retention. Urine samples from infants undergoing cardiac surgery were collected at three time points: T1) pre-operatively, T2) 6-8 h after surgery, and T3) 24 h after diuretics. Urine was collected from five heathy infant controls. The urine was tested for four proteases and whole-cell patch-clamp testing was conducted in renal collecting duct M-1 cells to test whether patient urine increased Na+ currents consistent with ENaC activation. Heavy chain of plasminogen, furin, and prostasin were significantly higher in cardiac patients prior to surgery compared to controls. There was no difference in most proteases before and after surgery. Urine from cardiac patients produced a significantly greater increase in Na+ inward currents compared to healthy controls. Urine from infants with congenital heart disease is richer in proteases and has the potential to increase activation of ENaC in the nephron to enhance Na+ reabsorption, which may lead to fluid retention in this population.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Reverse Phenotypes of Patients with Genetically Confirmed Liddle Syndrome.

Liddle syndrome was initially characterized by hypertension, hypokalemia, metabolic alkalosis, and suppressed plasma renin and aldosterone, resulting from gain-of-function variants in the epithelial Na + channel (ENaC). Efficient treatment with ENaC inhibitors is available, but the phenotypic spectrum of genetically confirmed Liddle syndrome is unknown, and some patients may remain undiagnosed and at risk of inefficient treatment. In this study, we used a reverse phenotyping approach to investigate the Liddle syndrome phenotypic spectrum and genotype-phenotype correlations. Pubmed, Embase, Scopus, and the Human Gene Mutation Database were searched for articles reporting Liddle syndrome variants. The genetic variants were systematically classified to identify patients with genetically confirmed Liddle syndrome. We identified 62 articles describing 45 unique variants within 86 Liddle syndrome families, and phenotypic data were pooled for 268 patients with confirmed Liddle syndrome. The Liddle syndrome variants localized to exon 13 of SCNN1B and SCNN1G , disrupting the PPPxY motif critical for downregulating ENaC activity. Hypertension sensitive to ENaC inhibition was present in 97% of adults carrying Liddle syndrome variants while hypokalemia, metabolic alkalosis, and plasma renin and aldosterone suppression showed incomplete penetrance. In addition, 95% and 55% of patients had a family history of hypertension or cerebrovascular events, respectively. The genotype had minor phenotypic effects; however, probands compared with relatives showed significant phenotypic discrepancies consistent with selection bias for initial genetic screening. Patients with genetically confirmed Liddle syndrome displayed a phenotypic spectrum, with ENaC-sensitive hypertension and family history of hypertension being the most common features. The phenotype seemed independent of the specific gene or variant type involved.

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced ENaC activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γ ENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR-Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared to wild type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo, but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 μM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 μM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, β-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11β2 (11β-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2, targeting ENaC for degradation. MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

P2Y2 receptor decreases blood pressure by inhibiting ENaC.

Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion, and blood pressure. Activating P2ry2 in littermate controls but not principal cell-specific P2ry2-knockout mice decreased the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolished increases in sodium excretion in response to stimulation of P2ry2 and compromised the normal ability to excrete a sodium load. Consequently, principal cell-specific knockout of P2ry2 prevented decreases in blood pressure in response to P2ry2 stimulation in the deoxycorticosterone acetate-salt (DOCA-salt) model of hypertension. In wild-type littermate controls, such stimulation decreased blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq-designer receptors exclusively activated by designer drugs and clozapine N-oxide decreased the activity of ENaC in renal tubules, promoting a natriuresis that lowered elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation and that inhibition of ENaC activity in response to P2ry2-mediated Gq signaling lowered blood pressure by increasing renal sodium excretion.

Effect of pregnancy and hypertension on kidney function in female rats: Modeling and functional implications.

Throughout pregnancy, the kidneys undergo significant adaptations in morphology, hemodynamics, and transport to achieve the volume and electrolyte retention required to support a healthy pregnancy. Additionally, during pregnancies complicated by chronic hypertension, altered renal function from normal pregnancy occurs. The goal of this study is to analyze how inhibition of critical transporters affects gestational kidney function as well as how renal function is affected during chronic hypertension in pregnancy. To do this, we developed epithelial cell-based multi-nephron computational models of solute and water transport in the kidneys of a female rat in mid- and late pregnancy. We simulated the effects of key individual pregnancy-induced changes on renal Na+ and K+ transport: proximal tubule length, Na+/H+ exchanger isoform 3 (NHE3) activity, epithelial Na+ channel activity (ENaC), K+ secretory channel expression, and H+-K+-ATPase activity. Additionally, we conducted simulations to predict the effects of inhibition and knockout of the ENaC and H+-K+-ATPase transporters on virgin and pregnant rat kidneys. Our simulation results predicted that the ENaC and H+-K+-ATPase transporters are essential for sufficient Na+ and K+ reabsorption during pregnancy. Last, we developed models to capture changes made during hypertension in female rats and considered what may occur when a rat with chronic hypertension becomes pregnant. Model simulations predicted that in hypertension for a pregnant rat there is a similar shift in Na+ transport from the proximal tubules to the distal tubules as in a virgin rat.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of ENaC in Salt-Sensitive Hypertension

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na + Channel (ENaC) is the final arbiter of Na + excretion in the kidneys. Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure. We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII-dependent ENaC-Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split-open collecting ducts from principal cell-specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank-3 binding to β-ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation. This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816. This is the full abstract presented at the American Physiology Summit 2023 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.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

PL-4: INSIGHTS INTO MOLECULAR MECHANISMS OF HYPERTENSION FROM HUMAN GENETICS

Identification and investigation of rare patients with extreme forms of high and low blood pressure has led to the identification of specific genes whose mutation results in these outlier phenotypes. These rare mutations converge on genes that mediate or regulate the renal reabsorption of Na-Cl. Mutations that increase or decrease renal salt reabsorption respectively increase or decrease blood pressure. Mutations that cause hypertension include gain of function mutations in subunits of the epithelial Na+ channel (ENaC) and in any of eight genes that cause constitutive activity of ENaC by its stimulation via its major regulator, the mineralocorticoid receptor, the target of aldosterone signaling; these increase salt reabsorption in the distal nephron. Conversely, mutations that cause hypotension include loss of function mutations in ENaC subunits, but also mutations the thiazide-sensitive Na-Cl cotransporter (NCC) or a K+ channel required for cotransporter function that act in the distal convoluted tubule (DCT), or mutations in the Na-K-2Cl cotransporter as well as K+ and Cl- channels expressed in the thick ascending limb of Henle that are required for normal Na-Cl reabsorption in this nephron segment. ENaC, the Na-Cl cotransporter and the Na-K-2Cl cotransporter, as well as the mineralocorticoid receptor are all commonly used targets for treatment of hypertension and/or diuresis. In addition to these genes, genetic studies also identified a previously unrecognized pathway that regulates the balance between renal salt reabsorption and K+ secretion. Mutations in the kinases WNK1 or WNK4, or genes that mediate their degradation via ubiquitylation, CUL3 and KLHL3 cause hypertension with hyperkalemia. Activity of WNK1 and WNK4 stimulates activation of NCC promoting Na-Cl reabsorption in the DCT, reducing Na-Cl- delivery distally, thereby limiting ENaC activity and limiting K+ secretion in the distal nephron. Conversely, hyperkalemia inhibits WNK activity, promoting distal Na-Cl delivery and maximizing K+ secretion. Angiotensin II signaling promotes WNK activity, increasing Na-Cl reabsorption in the DCT and inhibiting K+ secretion, while hyperkalemia appears to inhibit WNK activity, providing distal Na+ delivery that is used to drive K+ secretion. The importance of this pathway is that it can explain the mechanism by which increased dietary K+ lowers blood pressure. Both reduced dietary salt intake and increased dietary K+ can reduce blood pressure in controlled settings, and recent large population studies have shown that increased dietary K+ and reduced Na-Cl intake not only lower blood pressure but reduce the incidence of cardiovascular disease and death.

NOXA1-dependent NADPH oxidase 1 signaling mediates angiotensin II activation of the epithelial sodium channel.

The activity of the epithelial Na+ channel (ENaC) in principal cells of the distal nephron fine-tunes renal Na+ excretion. The renin-angiotensin-aldosterone system modulates ENaC activity to control blood pressure, in part, by influencing Na+ excretion. NADPH oxidase activator 1-dependent NADPH oxidase 1 (NOXA1/NOX1) signaling may play a key role in angiotensin II (ANG II)-dependent activation of ENaC. The present study aimed to explore the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC in renal principal cells. Patch-clamp electrophysiology and principal cell-specific Noxa1 knockout (PC-Noxa1 KO) mice were used to determine the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC. The activity of ENaC in the luminal plasma membrane of principal cells was quantified in freshly isolated split-opened tubules using voltage-clamp electrophysiology. ANG II significantly increased ENaC activity. This effect was robust and observed in response to both acute (40 min) and more chronic (48-72 h) ANG II treatment of isolated tubules and mice, respectively. Inhibition of ANG II type 1 receptors with losartan abolished ANG II-dependent stimulation of ENaC. Similarly, treatment with ML171, a specific inhibitor of NOX1, abolished stimulation of ENaC by ANG II. Treatment with ANG II failed to increase ENaC activity in principal cells in tubules isolated from the PC-Noxa1 KO mouse. Tubules from wild-type littermate controls, though, retained their ability to respond to ANG II with an increase in ENaC activity. These results indicate that NOXA1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells. As such, NOXA1/NOX1 signaling in the distal nephron plays a central role in Na+ homeostasis and control of blood pressure, particularly as it relates to regulation by the renin-ANG II axis.NEW & NOTEWORTHY Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

Chronic activation of vasopressin-2 receptors induces hypertension in Liddle mice by promoting Na+ and water retention.

The renin-angiotensin-aldosterone and arginine vasopressin-V2 receptor-aquaporin-2 (AQP2) systems converge on the epithelial Na+ channel (ENaC) to regulate blood pressure and plasma tonicity. Although it is established that V2 receptors initiate renal water reabsorption through AQP2, whether V2 receptors can also induce renal Na+ retention through ENaC and raise blood pressure remains an open question. We hypothesized that a specific increase in V2 receptor-mediated ENaC activity can lead to high blood pressure. Our approach was to test effects of chronic activation of V2 receptors in Liddle mice, a genetic mouse model of high ENaC activity, and compare differences in ENaC activity, urine Na+ excretion, and blood pressure with control mice. We found that ENaC activity was elevated in Liddle mice and could not be stimulated further by administration of desmopressin (dDAVP), a V2 receptor-specific agonist. In contrast, Liddle mice showed higher levels of expression of AQP2 and aquaporin-3, but they could still respond to dDAVP infusion by increasing phospho-AQP2 expression. With dDAVP infusion, Liddle mice excreted smaller urine volume and less urine Na+ and developed higher blood pressure compared with control mice; this hypertension was attenuated with administration of the ENaC inhibitor benzamil. We conclude that V2 receptors contribute to hypertension in the Liddle mouse model by promoting primary Na+ and concomitant water retention.NEW & NOTEWORTHY Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

Deficient regulation of the epithelial Na+ channel (ENaC) activity and impaired urinary conservation of Na+ in Epac1 ‐/‐ and Epac2 ‐/‐ mice

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1‐/‐ and Epac2‐/‐ mice exhibit a delayed anti‐natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split‐opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the knockout mice fed with Na+ deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker, ESI‐05 and Epac1&2 blocker, ESI‐09 decreased ENaC activity in EpacWT mice kept on Na+ deficient diet but not on the regular diet. ESI‐09 injections led to natriuresis in EpacWT mice on Na+ deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate non‐redundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC over‐activation.

Activation by Cleavage of the Epithelial Na+ Channel α and γ Subunits Independently Coevolved with the Vertebrate Terrestrial Migration

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel’s α and γ subunits. Here we investigated the evolution of ENaC regulatory mechanisms to determine which features coevolved with the marine‐terrestrial transition. We consistently found both activating cleavage sites in the ENaC α and γ subunits of terrestrial vertebrates, while they appeared only sporadically in fishes. We confirmed that cleavage occurred at sites found in the γ subunit from Australian lungfish, leading to channel activation. Phylogenetic analysis and likelihood ratio tests showed that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation. They also showed a coevolutionary dependence of tandem polybasic tracts with terrestrial status and with lungs, coincident with the ENaC activator aldosterone. Amplification of transcripts by RT‐PCR in ray‐finned Polypteriformes and previously in lobe‐finned lungfish suggest that transcripts for ENaC subunits with cleavage sites are readily detected at important sites of ion exchange (gills and kidney), but are difficult to detect in lungs. Analysis of ancestral reconstructions strongly suggests that the polybasic tracts appeared independently in the α and γ subunits of ENaC. Similar analyses of the PY motif, required for Nedd4‐2 dependent regulation, showed no coevolutionary pattern and that the PY motif first arose in an ancient ancestral ENaC subunit. Our data suggest that changes associated with adaptation to terrestrial life provided selective pressure for the development of ENaC activation by proteolytic cleavage.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of the Epithelial Sodium Channel

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys (see attached Figure). Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin‐3 (Ank‐3). The binding of Ank‐3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank‐3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure (see attached Figure). We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII‐dependent ENaC‐Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split‐open collecting ducts from principal cell‐specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank‐3 binding to β‐ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank‐3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation.