Epithelial Na(+) Channel Expression Research Articles

Defective natriuresis contributes to hyperkalemia in db/db mice during potassium supplementation.

Potassium supplementation reduces blood pressure and the occurrence of cardiovascular diseases, with K+-induced natriuresis playing a potential key role in this process. However, whether these beneficial effects occur in diabetes remains unknown. In this study, we examined the impact of high-K+ intake on renal Na+/K+ transport by determining the expression of major apical Na+ transporters, diuretics responses (as a proxy for specific Na+ transporter function), urinary Na+/K+ excretion, and plasma Na+/K+ concentrations in db/db mice, a model of type 2 diabetes mellitus. Although db/m mice exhibited increased fractional excretion of sodium (FENa) and fractional excretion of potassium (FEK) under high-K+ intake, these responses were largely blunted in db/db mice, suggesting impaired K+-induced natriuresis and kaliuresis in diabetes. Consequently, high-K+ intake increased plasma K+ levels in db/db mice, which could be attributed to the abnormal activity of sodium-hydrogen exchanger 3 (NHE3), sodium-chloride cotransporter (NCC), and epithelial Na+ channel (ENaC), as high-K+ intake could not effectively decrease NHE3 and NCC and increase ENaC expression and activity in the diabetic group. Inhibition of NCC by hydrochlorothiazide could correct the hyperkalemia in db/db mice fed a high-K+ diet, indicating a key role for NCC in K+-loaded diabetic mice. Treatment with metformin enhanced urinary Na+/K+ excretion and normalized plasma K+ levels in db/db mice with a high-K+ diet, at least partially, by suppressing NCC activity. Collectively, the impaired K+-induced natriuresis in diabetic mice under high-K+ intake may be primarily attributed to impaired NCC-mediated renal K+ excretion, despite the role of NHE3.

The Epithelial Na+ Channel Is a Zn2+ Sensitive Renal Na+ Reabsorption Pathway that Mediates Zn2+ Deficiency-induced Hypertension

Background: Zinc (Zn2+) deficiency (ZnD) is comorbid with many chronic diseases including kidney disease and diabetes. Individuals in these vulnerable populations have a higher prevalence of hypertension. Recently, we reported that ZnD induces hypertension by promoting renal Na+ reabsorption. However, the renal Na+ reabsorption mechanisms involved continue to be identified. The renal epithelial Na+ channel (ENaC) plays an essential role in regulating whole-body Na+ and water balance and is involved in hypertension. We hypothesize that ENaC is a Zn2+-sensitive channel that mediates ZnD-induced hypertension. Experimental Design: To establish the role of ENaC in ZnD-induced hypertension, WT C57Bl/6 mice were switched from a Zn2+ adequate to a Zn2+ deficient diet for 10 weeks. During weeks 9 and 10, mice were treated with benzamil, an ENaC inhibitor. Systolic blood pressure (BP) was monitored by tail-cuff plethysmography; Renal ENaC expression was examined by immunohistochemistry and Western blot. To authenticate the Zn2+ sensitivity of ENaC, mouse distal convoluted tubule cells (mDCT) were treated with a Zn2+ chelator (TPEN) or vehicle (ethanol) for 24 hours. A subset of TPEN-treated cellular monolayers were supplemented with Zn2+ for 24 hours to replete intracellular Zn2+. Cellular ENaC expression was examined by Western blot analysis. Results: 1) Blood pressure: Compared to Zn2+ adequate mice (101.4 mm Hg ± 4.225), Zn2+ deficient mice had increased BP (154.6 mm Hg ± 2.422). However, benzamil treatment lowered BP (115.0 mm Hg ± 4.626) of Zn2+ deficient mice. 2)Immunohistochemistry: Next, Zn2+ effects on ENaC expression were examined. In Zn2+ deficient mice, αENaC protein abundance significantly increased compared with Zn2+ adequate mice. 3) Western blot analysis: To directly investigate Zn2+effects on ENaC expression, in vitro studies were performed using mDCT cells. Western blot analysis showed that TPEN-induced ZnD stimulated both αENaC and βENaC protein expression. Conclusion: Collectively, our findings reveal that (1) ENaC is a Zn2+ sensitive renal Na+ reabsorption pathway and (2) ENaC mediates ZnD-induced hypertension. Significance: Understanding the renal mechanisms by which Zn2+ regulates blood pressure may have an important therapeutic impact on hypertension. Funding: R21 DK119879, R01 DK-133698 (National Institute of Diabetes and Digestive and Kidney Diseases); R25DK078381 (NIH NIDDK STEP-UP program);1742339 (National Science Foundation ASK Program). 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.

FRI389 Granulosa Cell-specific Knockout Of Scnn1a Disturbs Estrus Cycle And Impairs Estrogen Production In Mice

Abstract Disclosure: X. Ma: None. R. Xu: None. Y. Que: None. J. Chen: None. Y. Ruan: None. Estrogens are produced primarily by ovarian granulosa cells in females, the homeostasis of which is not only a prerequisite for female reproduction but also essential to the overall health. Studies suggested Na+ environment/intake in relation to ovarian functions, although the underlying mechanism remains unclear. In the present study, we explored possible expression and function of the epithelial Na+ channel (ENaC) in granulosa cells. We analyzed an available single-cell RNA sequencing database of human ovarian cells, mouse ovarian tissues as well as a human granulosa cell line (KGN), which showed ENaC expression and its channel activities (measured by patch-clamp) in granulosa cells. Since ENaCα (Scnn1a) is the rate-limiting subunit for ENaC to function, we established a granulosa cell-specific Scnn1a knockout mouse model (Scnn1afl/fl, Cyp19a1-Cre). In such a conditional knockout (cKO) model, the estrus cycle of the female mice at reproductive ages of 8 to 10-week-old was found to be disturbed with the ratio of proestrus/estrus versus metestrus/diestrus significantly higher (t-test, p < 0.001) in cKO (1.8 ± 0.1, n = 8) compared to that of the Cre-negative control (Scnn1afl/fl, 1.0 ± 0.1, n = 8) mice. Histological analysis of the ovarian tissues showed a fewer (t-test, p < 0.05) number of corpus luteum in cKO mice (1.9 ± 0.8 per ovary, n = 8) than that of the control mice (6.2 ± 1.5 per ovary, n = 6). We next isolated the granulosa cells from the mouse model and tested their responses to gonadotropins in vitro. In granulosa cells from the control mice, the estradiol level in the culture medium (measured by ELISA) was significantly increased by the treatment of FSH (100 ng/ml, 48 hours) and subsequent LH (100 ng/ml, 18 hours) as compared to that of cells without FSH/LH treatment (33.0 ± 12.3 vs. 5.1 ± 0.9 ng/ml, t-test, p < 0.05, n = 6). Whereas, in cKO granulosa cells, no such elevation of estradiol level in response to FSH/LH was detected (7.6 ± 1.8 vs. 6.5 ± 1.3 ng/ml, t-test, p > 0.05, n = 6), suggesting impaired estrogen production with ENaCα knockout. Consistently, quantitative PCR results showed significant downregulation of Cyp19a1 (-96.1 ± 0.9%), Lhcgr (-99.5 ± 0.1%) and Fshr (- 67.9 ± 7.2%), three key steroidogenic genes, in FSH/LH-treated cKO cells, as compared to that of FSH/LH-treated control cells (n = 6, t-test, p < 0.05). Taken together, these results have suggested a previously undefined role of ENaC in granulosa cells for estrogen production in response to gonadotrophins maintaining female cycle homeostasis. This work was supported by National Natural Science Foundation of China (82071599). Presentation: Friday, June 16, 2023

Altered colonocyte differentiation may lead to decreased electrolyte and fluid absorption in the ileocolon of IBD patients by affecting the expression pattern of the responsible ion transporters

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) characterized by disturbed homeostasis of the immune system, the intestinal mucosa and the microbiome, causing ‘leaky’ intestinal epithelial barrier with altered fluid and electrolyte absorption leading to diarrhea. Fluid absorption in the ileocolonic region is predominantly mediated by the Cl-/HCO3 - exchanger SLC26A3 (DRA) and the Na+/H+ exchanger SLC9A3 (NHE3), while in the distal colon, the epithelial Na+ channel (ENaC) also plays a role. The objective of the current study is to evaluate if the downregulation of specific absorptive ion transporters or general defect of the enterocyte differentiation correlate with the degree of diarrhea and inflammation. For this reason, we have analyzed the mRNA expression and/or protein abundance of DRA, NHE3 and ENaC, as well as a panel of genes that correlate with the differentiation state of the colonic epithelium, in different segments of the ileocolon of healthy individuals and IBD patients with an acute flare or in remission. Endoscopy and histopathologic scoring and TNFα mRNA levels were used to grade inflammation, clinical scores for disease activity and diarrhea. mRNA expression and immunohistochemical staining of DRA and NHE3 in healthy colonic tissue revealed differences in the expression pattern of DRA and NHE3 along the human colon, with DRA being most abundant in the transverse and descending colon and NHE3 in the ascending colon (n=10). The reduction of DRA and NHE3 mRNA expression/abundance in the membrane correlated with the degree of inflammation as well as the diarrhea score (n=42), while in inactive disease the expression and abundance of DRA and NHE3 did not differ from healthy controls (n=13). Furthermore, significant decrease of the ENaC subunit ENaCγ expression was detected in inflamed colon sigmoideum (n=30). Since the addressed ion transporters are expressed on differentiated intestinal epithelial cells, we further investigated if their altered expression is linked to a possible defect of the epithelial differentiation program. It was found that the mRNA expression of the absorptive enterocyte differentiation markers intestinal alkaline phosphatase (iALP), villin and HES1 was significantly decreased in inflamed colon, while the expression of the proliferative marker Ki67, HOPX, BM1 and Claudin2 was significantly increased. However, the expression of the stem cell marker LGR5 was not altered (n=30). The results showed a disrupted transcriptional regulation of the epithelial ion transporters DRA, NHE3 and ENaCγ in the inflamed epithelium of IBD patients, which will lead to their reduced function and cause electrolyte/water accumulation leading to diarrhea. The decreased expression of the epithelial ion transporters could be a consequence of the altered enterocyte differentiation of the inflamed epithelium, most likely due to signaling pathways triggered by the proinflamatory cytokines. Grant Support: DFG SE 460/21-1 and 22-1, and VW Z1953. 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.

Involvement of miR-495 overexpression in the pathogenesis of bronchopulmonary dysplasia in preterm infants via the targeting of NEDD4L-ENaC pathway.

Bronchopulmonary dysplasia (BPD) is a severe pulmonary complication causing morbidity and mortality in preterm infants. A key histopathological feature of BPD is late lung growth retardation, in which the process of alveolarization is hindered and the mechanism of which is unclear. Emerging evidence indicates that microRNAs (miRNAs) promote the development of BPD via the inhibition of their target genes. MiR-495 has been reported to be involved in various lung diseases. However, the physiological function of miR-495 in BPD has not yet been fully understood. Differentially expressed miRNAs in peripheral blood of patients with BPD were compared with those of normal controls. A dual-luciferase reporter assay was performed to identify the target genes of miR-495. A BPD neonatal rat model was established by injecting lipopolysaccharide (LPS) in the amniotic sac of pregnant rats. The morphology of the lungs was observed using hematoxylin and eosin (HE) staining. The expression of miR-495, neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L), and epithelial Na+ channel (ENaC) was tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, and immunofluorescent (IF) staining. The expression of miR-495 was significantly increased in the peripheral blood samples of premature infants with BPD and verified using qRT-PCR. NEDD4L was proven to be the target gene of miR-495. Additionally, miR-495 expression was also increased in the lungs of rat pups with BPD at postnatal day (P) 3 compared with the control group. qRT-PCR and Western blot results showed that NEDD4L expression was decreased while ENaC expression was increased at the transcriptional and translational levels. IF staining results showed that NEDD4L level was decreased while ENaC level was increased in the LPS-induced BPD rat model, which was consistent with abnormal changes in alveolar structure. The aberrant overexpression of miR-495 may contribute to the development of BPD by targeting NEDD4L-ENaC pathway, implying an imbalance in lung fluid clearance.

Epithelial Sodium Channels in Monocyte‐Macrophage Migration and Regulation by Pro‐inflammatory Cytokines TNFα and IFNγ

Migration of monocytes‐macrophages play an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. While Epithelial Na+ Channels (ENaC) are required for normal migratory responses in glial, trophoblast, and vascular smooth muscle cells, their role in monocyte‐macrophage migration signaling is unknown. The Human Protein Atlas data base (www.proteinatlas.org) shows ENaC message in peripheral blood mononuclear cells and tissue resident macrophages in human populations. We used an in vitro model to determine the importance of ENaC in chemotactic migration of monocytes‐macrophages using a standard Boyden chamber assay. Cells were suspended in 0.4% FBS ± amiloride (0.1‐10 µM) or benzamil (0.01‐1 µM) to inhibit ENaC channels, then migrated for 4 hr towards a 10% FBS gradient. In mouse RAW cells, migration responses were inhibited by amiloride (65±6 – 46±4% of control at 0.1 and 10 µM, p=0.0001) and benzamil (55±6 – 55±5% of control at 0.01 and 1.0 µM, p<0.0001). In human THP‐1 cells, migration responses were similarly inhibited by amiloride (73±5 ‐ 40±6% of control at 0.1 and 10 µM, p<0.0001) and benzamil (68±7 – 53±4% of control at 0.01 and 1.0 µM, p<0.0001). Since benzamil and amiloride are selective for ENaC at sub and low micromolar concentrations, respectively, our findings indicate ENaC signaling is required for migration of unstimulated monocyte‐macrophage cells. To determine if pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interferon gamma (INFγ), regulate ENaC expression and function, we used TaqMan quantitative PCR, western blotting and chemotactic migration. Cells were exposed to a standard protocol to activate macrophages: INFγ for 48 hr plus TNFα (10 ng/mL each) during the last 24 hr. Expression of α and βENaC and GAPDH was calculated as fold change using the delta delta CT method (2‐∆∆CT). INFγ48hr + TNFα24hr increased α and βENaC message (9±2 and 5±1, n=4‐6, p=0.01). γENaC was undetectable. Despite increased message, whole cell αENaC protein expression was inhibited 50% by western blotting. The mechanism underlying message stimulation and protein inhibition remains to be determined. A similar effect on message upregulation and migration inhibition also occurred with 18 hr INFγ or TNFα alone (0‐10 ng/mL). Activation with INFγ/TNFα inhibited migration up to 70% and abolished the amiloride (1 µM) sensitive component. In unstimulated cells, amiloride (1 µM) coculture induced a rebound increase in migration following withdrawal, which was abolished by INFγ/TNFα treatment. This finding suggests cytokine induced ENaC loss mediates the migration inhibition. In summary, ENaC mediates monocyte migration, however, monocyte activation by INFγ/TNFα inhibits migration by inhibiting ENaC. Since pro‐inflammatory cytokines initiate monocyte differentiation and polarization to the phagocytic M1 phenotype, future studies will determine if ENaC is required for the phenotypic switch.

Effects of amiloride on acetylcholine-dependent arterial vasodilation evolve over time in mice on a high salt diet.

The maintenance of endothelial health is required for normal vascular function and blood pressure regulation. The epithelial Na+ channel (ENaC) in endothelial cells has emerged as a new molecular player in the regulation of endothelial nitric oxide production and vascular stiffness. While ENaC expression in the kidney is negatively regulated by high [Na+], ENaC expression in isolated endothelial cells has been shown to increase in response to a high extracellular [Na+]. In culture, this increased expression leads to cellular stiffening and decreased nitric oxide release. In vivo, the effects of high salt diet on endothelial ENaC expression and activity have varied depending on the animal model utilized. Our aim in the present study was to examine the role of endothelial ENaC in mediating vasorelaxation in the C57Bl/6 mouse strain. We utilized pressure myography to test the responsiveness of thoracodorsal arteries to acetylcholine in mice with increased sodium consumption both in the presence and absence of increased aldosterone. ENaC’s contribution was assessed with the use of the specific inhibitor amiloride. We found that while aldosterone had very little effect on ENaC's contribution to acetylcholine sensitivity, a high salt diet led to an amiloride‐dependent shift in the acetylcholine response of vessels. However, the direction of this shift was dependent on the length of high salt diet administration. Overall, our studies reveal that ENaC's role in the endothelium may be more complicated than previously thought. The channel does not simply inhibit nitric oxide generation, but instead helps preserve a homeostatic response.

Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension.

Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. Results: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1-/-versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1-/- females. In response to an acute salt load, Na+ excretion was similar in wild-type and Noxa1-/- mice before and 14 days after Ang II infusion. However, Na+ excretion was delayed after 1-2 days of Ang II in male wild-type versus Noxa1-/- mice. Ang II increased epithelial Na+ channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1-/- mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. Innovation and Conclusion: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na+ reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550-566.

Deletion of the Gamma Subunit of ENaC in Endothelial Cells Does Not Protect against Renal Ischemia Reperfusion Injury.

Acute kidney injury due to renal ischemia-reperfusion injury (IRI) may lead to chronic or end stage kidney disease. A greater understanding of the cellular mechanisms underlying IRI are required to develop therapeutic options aimed at limiting or reversing damage from IRI. Prior work has shown that deletion of the α subunit of the epithelial Na+ channel (ENaC) in endothelial cells protects from IRI by increasing the availability of nitric oxide. While canonical ENaCs consist of an α, β, and γ subunit, there is evidence of non-canonical ENaC expression in endothelial cells involving the α subunit. We therefore tested whether the deletion of the γ subunit of ENaC also protects mice from IRI to differentiate between these channel configurations. Mice with endothelial-specific deletion of the γ subunit and control littermates were subjected to unilateral renal artery occlusion followed by 48 h of reperfusion. No significant difference was noted in injury between the two groups as assessed by serum creatinine and blood urea nitrogen, levels of specific kidney injury markers, and histological examination. While deletion of the γ subunit did not alter infiltration of immune cells or cytokine message, it was associated with an increase in levels of total and phosphorylated endothelial nitric oxide synthase (eNOS) in the injured kidneys. Our studies demonstrate that even though deletion of the γ subunit of ENaC may allow for greater activation of eNOS, this is not sufficient to prevent IRI, suggesting the protective effects of α subunit deletion may be due, in part, to other mechanisms.

Abstract 06: Mechanisms And Consequences Of Casein Kinase II And Ankyrin 3 Regulation Of The Epithelial Sodium Channel

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

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.

Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum-resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate-induced activation of αβS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

Aldosterone Mediated Regulation of Epithelial Sodium Channel (ENaC) Subunits in the Rat Hypothalamus

Current evidence suggests that the epithelial Na+ channel (ENaC) in the brain plays a significant role in the development of hypertension. ENaC is present in vasopressin (VP) neurons in the hypothalamus, suggesting that ENaC in VP neurons is involved in the regulation of blood pressure. Our recent study demonstrated that high dietary salt intake caused an increase in the expression and activity of ENaC that were responsible for the more depolarized basal membrane potential in VP neurons. A known regulator of ENaC expression, the mineralocorticoid receptor (MR), is present in VP neurons, suggesting that ENaC expression in VP neurons is regulated by aldosterone. In this study, the effects of aldosterone and corticosterone on ENaC were examined in acute hypothalamic slices. Real-time PCR and Western blot analysis showed that aldosterone and corticosterone treatment resulted in a significant increase in the expression of γENaC, but not α- or βENaC, and that this expression was attenuated by MR and glucocorticoid receptor (GR) antagonists. Moreover, chromatin immunoprecipitation demonstrated that the aldosterone-MR complex directly interacts with the promoter region of the γENaC gene. However, the treatment with aldosterone did not cause subcellular translocation of ENaC toward the plasma membrane nor an increase in ENaC Na+-leak current. These results indicate that expression of γENaC in VP neurons is induced by aldosterone and corticosterone through their MR and GR, respectively; however, aldosterone or corticosterone alone is not sufficient enough to increase ENaC current when they are applied to hypothalamic slices in vitro.

Loss of barrier integrity in alveolar epithelial cells downregulates ENaC expression and activity via Ca2+ and TRPV4 activation.

The epithelial Na channel (ENaC) plays an essential role in lung physiology by modulating the amount of liquid lining the respiratory epithelium. Here, we tested the effect of breaking alveolar epithelial cell barrier integrity on ENaC expression and function. We found that either mechanical wounding by scratching the monolayer or disruption of tight junction with EDTA induced a ~ 50% decrease of α,β and γENaC mRNA expression and an 80% reduction of ENaC short-circuit current (Isc) at 6h. Scratching the cell monolayer generated a Ca2+ wave that spread from the margin of the scratch to distant cells. Pretreatment with BAPTA-AM, an intracellular Ca2+ chelator, abolished the effect of mechanical wounding and EDTA on αENaC mRNA expression, suggesting that [Ca2+]i is important for this modulation. We tested the hypothesis that a mechanosensitive channel such as TRPV4, a cationic channel known to increase [Ca2+]i, could mediate this effect. Activation of the channel with the TRPV4 specific agonist GSK-1016790A (GSK) decreased αENAC mRNA expression and almost completely abolished ENaC Isc. Pretreatment of alveolar epithelial cells with HC-067047 (HC0), a specific TRPV4 antagonist, reduced the extent of αENAC mRNA downregulation by mechanical wounding and EDTA. Altogether, our results suggest that mechanical stress induced by wounding or TRPV4-mediated loss of tight junction increases [Ca2+]i and elicits a Ca2+ wave that affects ENaC expression and function away from the site of injury. These data are important to better understand how Ca2+ signaling affects lung liquid clearance in injured lungs.

Effect of dietary salt intake on epithelial Na+ channels (ENaCs) in the hypothalamus of Dahl salt-sensitive rats.

All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) and the mineralocorticoid receptor (MR), a known regulator of ENaC, are located in vasopressin (VP) synthesizing magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. Our previous study showed that ENaC mediates a Na+ leak current that affects the steady‐state membrane potential of VP neurons. This study was conducted in Dahl salt‐sensitive (Dahl‐SS) rats to determine if any abnormal responses in the expression of ENaC subunits and MR occur in the hypothalamus and kidney in response to a high dietary salt intake. After 21 days of high salt consumption, Dahl‐SS rat resulted in a significant increase in γ ENaC expression and exhibited proteolytic cleavage of this subunit compared to Sprague–Dawley (SD) rats. Additionally, Dahl‐SS rats had dense somato‐dendritic γ ENaC immunoreactivity in VP neurons, which was absent in SD rats. In contrast, SD rats fed a high salt diet had significantly decreased α ENaC subunit expression in the kidney and MR expression in the hypothalamus. Plasma osmolality measured daily for 22 days demonstrated that Dahl‐SS rats fed a high salt diet had a steady increase in plasma osmolality, whereas SD rats had an initial increase that decreased to baseline levels. Findings from this study demonstrate that Dahl‐SS rats lack a compensatory mechanism to down regulate ENaC during high dietary salt consumption, which may contribute to the development of hypertension.

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

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

Regulation of the epithelial Na+ channel by paraoxonase-2

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.

Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats.

During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19–23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19–23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19–23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that modulates the postnatal increase in TRC ENaC and the neural and behavioral responses to NaCl.

Derlin-1 promotes ubiquitylation and degradation of the epithelial Na+ channel, ENaC.

Ubiquitylation of the epithelial Na+ channel (ENaC) plays a critical role in cellular functions, including transmembrane transport of Na+, Na+ and water balance, and blood pressure stabilization. Published studies have suggested that ENaC subunits are targets of ER-related degradation (ERAD) in yeast systems. However, the molecular mechanism underlying proteasome-mediated degradation of ENaC subunits remains to be established. Derlin-1, an E3 ligase mediator, links recognized target proteins to ubiquitin-mediated proteasomal degradation in the cytosol. In the present study, we found that derlin-1 suppressed the expression of ENaC at the protein level and that the subunit α-ENaC (also known as SCNN1A) physically interacted with derlin-1 at the membrane-anchored domains or the loop regions, and that derlin-1 initiated α-ENaC retrotranslocation. In addition, HUWE1, an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase, was recruited and promoted K11-linked polyubiquitylation of α-ENaC and, hence, formation of an α-ENaC ubiquitin-mediated degradation complex. These findings suggest that derlin-1 promotes ENaC ubiquitylation and enhances ENaC ubiquitin- mediated proteasome degradation. The derlin-1 pathway therefore may represent a significant early checkpoint in the recognition and degradation of ENaC in mammalian cells.