Epithelial Na(+) Channel Research Articles

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.

Early Interleukin-6 Mediated Activation of SGK1 is Primarily via Variant 6

Hypertension is associated with inflammation, including elevated interleukin-6 (IL-6) levels. IL-6 is an inflammatory cytokine, which we have previously shown to activate the mineralocorticoid receptor (MR) and the downstream sodium (Na) transporters- Na-chloride cotransporter (NCC) and the epithelial Na channel (ENaC). However, how IL-6 activates the MR is still unknown. IL-6 is also linked to Serum and Glucocorticoid-regulated Kinase 1 (SGK1), which is a downstream effector of the MR. SGK1 regulates both NCC and ENaC, increasing activity and tubular reabsorption of Na. This increased Na reabsorption favors water retention, thus increasing blood pressure. Previously, IL-6 has also been linked with SGK1 activation. We hypothesize that IL-6 increases SGK1 mRNA and protein expression in renal epithelial cells. To test this, we used in vitro renal epithelial cell models of both the late distal convoluted tubule (DCT2) and inner medullary collecting duct (IMCD), mDCT15 and IMCD3, respectively. Cells were treated with an IL-6 time-course (100ng/ml for 1, 3 and 6 hours). There are 6 known transcriptional variants for SGK1. We designed variant-specific PCR primers and, a pilot study based on regular PCR concluded that expression levels of only variants 5 and 6 (V5, V6) are significant. Using Real Time quantitative RT-PCR, we were then able to quantify transcript levels of both SGK1 V5 and V6 variants. With IL-6 treatment, we observed a 160%-211% increase at early time points (30mins-1hr) in SKG1 V6 and a lesser response (26%-88% increase) with V5 as compared to vehicle treated mDCT15 cells. Since SGK1 has been observed in the membrane fraction and in the cytoplasm, protein extracts from cells were fractionated (mPER kit) to assess membrane and cytosolic SGK1 protein levels using immunoblots. Densitometric analysis of immunoblots demonstrated a 5.5- and 3.1-fold increase in SGK1 protein expression at 1hr in mDCT15 and IMCD3 cells, respectively. These data suggest that IL-6 induces increases in SGK1 transcription and translation. Interestingly, we also found that majority of SGK1 was in the membrane fraction and the level was consistent. Together, these data suggest that SGK1 V6 may be responsible for the increased sodium reabsorption in response to IL-6 stimulation in renal epithelial cells. NIH funding 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.

G Protein-coupled Estrogen Receptor 1 Signaling Downregulates Epithelial Na Channel Activity in the Collecting Duct

Hypertension is more prevalent in men than women. Sodium (Na+) homeostasis is critical for blood pressure control. Recent studies in our lab showed that activation of G-protein coupled estrogen receptor 1 (GPER1) in the renal medulla evokes natriuresis in female, but not male, Sprague Dawley (SD) rats via endothelin 1 (ET-1) signaling. ET-1 has been shown to regulate epithelial Na+ channel (ENaC) which plays a crucial role in Na+ handling in the collecting duct (CD) and thus blood pressure. Whether ENaC contributes to GPER1-evoked natriuresis is unknown. We hypothesized that GPER1 activation suppresses ENaC activity, and this interaction may play a role in promoting Na+ excretion in females. To evaluate this, we investigated the tubular mRNA expression pattern of GPER1 and αENaC by RNAscope in male and female SD rat kidneys. We also infused G1 (GPER1 agonist, 5 pmol/kg/min) and/or amiloride (AML, ENaC inhibitor, 0.67 μg/kg/min) into the renal medulla of anesthetized female SD rats then measured urinary Na+ excretion. To further identify GPER1/ENaC interaction, we generated mice with deletion of GPER1 in principal cells (PC) of the CD by crossing GPER1 floxed and aquaporin 2 cre mice. Urinary ET-1 was assessed in WT and PC-GPER1 KO mice. Finally, patch clamp electrophysiology was performed on freshly-isolated CDs to determine ENaC activity in response to G1 activation and genetic deletion of GPER1. RNAscope revealed that GPER1 colocalizes with αENaC in male and female rat kidneys. The female kidney elicited a trend towards more outer medullary tubules co-expressing αENaC and GPER1 than males (43.3 ± 2.8 vs 34.7 ± 3.5 % of tubules positive for either, respectively P=0.0826). When infused into the female rat medulla, G1 and AML increased urinary Na+ excretion to a comparable extent (69.8 ± 22.2 vs 77.6 ± 31.0 % change from baseline, respectively P=0.9765). No additional natriuretic response was observed when G1 and AML were coadministered. Urinary ET-1 excretion was greater in female WT mice compared to male WT (1.95 ± 0.27 vs 1.04 ± 0.10 pg/mg creatinine, respectively P=0.0155). Genetic deletion of GPER1 in PC reduced ET-1 excretion in female, but not male, mice (female KO; 1.08 ± 0.18 pg/mg creatinine P=0.0166). Importantly, genetic deletion of GPER1 in PC upregulated ENaC open probability (Po) compared to WT (0.280 ± 0.024 vs 0.194 ± 0.024 Po, respectively P=0.0462). Similarly, treatment of WT CDs with G1 (1 μM) downregulated ENaC Po (G1 vs control; 0.064 ± 0.017 vs 0.194 ± 0.024 Po P=0.0024). In conclusion, our data demonstrate GPER1-αENaC colocalization in renal tissues, with a trend towards higher colocalization in the outer medulla of the female kidney. CD-GPER1 is required for the greater ET-1 excretion in female mice. Further, our data uncover a direct regulatory effect for GPER1 on ENaC activity in the CD. Improving our understanding of the GPER1/ET-1/ENaC natriuretic axis could reveal future sex-specific antihypertensive therapeutics. NIH/NIDDK R00 DK119413 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.

Acid-sensing (proton-gated) ion channels (ASICs) in GtoPdb v.2023.1

Acid-sensing ion channels (ASICs, nomenclature as agreed by NC-IUPHAR [48, 2, 3]) are members of a Na+ channel superfamily that includes the epithelial Na+ channel (ENaC), the FMRF-amide activated channel (FaNaC) of invertebrates, the degenerins (DEG) of Caenorhabitis elegans, channels in Drosophila melanogaster and 'orphan' channels that include BLINaC [70] and INaC [72] that have also been named BASICs, for bile acid-activated ion channels [90]. ASIC subunits contain 2 TM domains and assemble as homo- or hetero-trimers [45, 41, 7, 94, 93, 77] to form proton-gated, voltage-insensitive, Na+ permeable, channels that are activated by levels of acidosis occurring in both physiological and pathophysiological conditions with ASIC3 also playing a role in mechanosensation (reviewed in [44, 89, 48, 69, 23]). Splice variants of ASIC1 [termed ASIC1a (ASIC, ASICα, BNaC2α) [84], ASIC1b (ASICβ, BNaC2β) [19] and ASIC1b2 (ASICβ2) [79]; note that ASIC1a is also permeable to Ca2+], ASIC2 [termed ASIC2a (MDEG1, BNaC1α, BNC1α) [66, 85, 40] and ASIC2b (MDEG2, BNaC1β) [56]] differ in the first third of the protein. Unlike ASIC2a (listed in table), heterologous expression of ASIC2b alone does not support H+-gated currents. A third member, ASIC3 (DRASIC, TNaC1) [83] is one of the most pH-sensitive isoforms (along with ASIC1a) and has the fastest activation and desensitisation kinetics, however can also carry small sustained currents. ASIC4 (SPASIC) evolved as a proton-sensitive channel but seems to have lost this function in mammals [58]. Mammalian ASIC4 does not support a proton-gated channel in heterologous expression systems but is reported to downregulate the expression of ASIC1a and ASIC3 [1, 43, 34, 54]. ASICs channels are primarily expressed in central (ASIC1a, -2a, 2b and -4) and peripheral neurons including nociceptors (ASIC1-3) where they participate in neuronal sensitivity to acidosis. Humans express, in contrast to rodents, ASIC3 also in the brain [27]. ASICs have also been detected in taste receptor cells (ASIC1-3)), photoreceptors and retinal cells (ASIC1-3), cochlear hair cells (ASIC1b), testis (hASIC3), pituitary gland (ASIC4), lung epithelial cells (ASIC1a and -3), urothelial cells, adipose cells (ASIC3), vascular smooth muscle cells (ASIC1-3), immune cells (ASIC1,-3 and -4) and bone (ASIC1-3) (ASIC distribution is reviewed in [55, 28, 42]). A neurotransmitter-like function of protons has been suggested, involving postsynaptically located ASICs of the CNS in functions such as learning and fear perception [35, 50, 97], responses to focal ischemia [91] and to axonal degeneration in autoimmune inflammation in a mouse model of multiple sclerosis [39], as well as seizures [98] and pain [89, 29, 30, 13, 32]. Heterologously expressed heteromultimers form ion channels with differences in kinetics, ion selectivity, pH- sensitivity and sensitivity to blockers that resemble some of the native proton activated currents recorded from neurones [56, 5, 38, 11]. In general, the known small molecule inhibitors of ASICs are non-selective or partially selective, whereas the venom peptide inhibitors have substantially higher selectivity and potency. Several clinically used drugs are known to inhibit ASICs, however they are generally more potent at other targets (e.g. amiloride at ENaCs, ibuprofen at COX enzymes) [68, 63]. The information in the tables below are for the effects of inhibitors on homomeric channels, for information of known effects on heteromeric channels see the comments below.

Critical contribution of the intracellular C-terminal region to TRESK channel activity is revealed by the epithelial Na+ current ratio method

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology, and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in a low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function.

Aldosterone: Renal Action and Physiological Effects.

Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na+ ) or potassium (K+ ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na+ intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na+ absorption in part via the epithelial Na+ channel (ENaC), the principal channel responsible for the fine-tuning of Na+ balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists. © 2023 American Physiological Society. Compr Physiol 13:4409-4491, 2023.

Nicotine aerosols diminish airway CFTR function and mucociliary clearance.

Electronic cigarettes (e-cigs) are often promoted as safe alternatives to smoking based on the faulty perception that inhaling nicotine is safe until other harmful chemicals in cigarette smoke are absent. Previously, others and we have reported that, similar to cigarette smoke, e-cig aerosols decrease CFTR-mediated ion transport across airway epithelium. However, it is unclear whether such defective epithelial ion transport by e-cig aerosols occurs in vivo and what the singular contribution of inhaled nicotine is to impairments in mucociliary clearance (MCC), the primary physiologic defense of the airways. Here, we tested the effects of nicotine aerosols from e-cigs in primary human bronchial epithelial (HBE) cells and two animal models, rats and ferrets, known for their increasing physiologic complexity and potential for clinical translation, followed by in vitro and in vivo electrophysiologic assays for CFTR activity and micro-optical coherence tomography (μOCT) image analyses for alterations in airway mucus physiology. Data presented in this report indicate nicotine in e-cig aerosols causes 1) reduced CFTR and epithelial Na+ channel (ENaC)-mediated ion transport, 2) delayed MCC, and 3) diminished airway surface hydration, as determined by periciliary liquid depth analysis. Interestingly, the common e-cig vehicles vegetable glycerin and propylene glycol did not affect CFTR function or MCC in vivo despite their significant adverse effects in vitro. Overall, our studies contribute to an improved understanding of inhaled nicotine effects on lung health among e-cig users and inform pathologic mechanisms involved in altered host defense and increased risk for tobacco-associated lung diseases.

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.

Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and protein kinase A (PKA)-regulated channel, expressed on the luminal surface of secretory and absorptive epithelial cells. CFTR has a complex, cell-specific regulatory network playing a major role in cAMP- and Ca2+-activated secretion of electrolytes. It secretes intracellular Cl– and bicarbonate and regulates absorption of electrolytes by differentially controlling the activity of the epithelial Na+ channel (ENaC) in colon, airways, and sweat ducts. The CFTR gene expression is regulated by cell-specific, time-dependent mechanisms reviewed elsewhere [1]. This review will focus on the transcriptional, post-transcriptional, and translational regulation of CFTR by cAMP-PKA, non-coding (nc)RNAs, and TGF-β signaling pathways in cystic fibrosis (CF) cells.

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.

PS-BPR03-5: A CASE OF LIDDLE SYNDROME-LIKE REFRACTORY HYPERTENSION WITH TREATMENT-RESISTANT HYPOKALEMIA COMPLICATED BY NEPHROTIC SYNDROME DUE TO DIABETIC NEPHROPATHY

A 62-year-old woman had been treated for diabetic nephrotic syndrome, hypokalemia, and edema with perindopril, furosemide, potassium L-aspartate, valsartan, and spironolactone since January, X. On May 15, X, fatigue and nausea appeared, and on the morning of May 20, these symptoms increased. On the morning of May 20, she visited the hospital. She was urgently admitted to the hospital because of prominent edema in both lower limbs, elevated blood pressure (164/83 mmHg), marked hypokinemia (K 2.4 mEq/L), and metabolic alkalosis (pH 7.612, HCO3- 31.3 mmol/L) in the arterial blood gas. Post-hospitalization examination revealed hyporeninemia and hypoaldosteronemia. Since administration of spironolactone in addition to previous medications failed to improve the hypokaremia and hypertension, triamterene 100 mg was added. The treatment was resulted in rapid hypotension, accompanied by improvement of hypokalemia and metabolic alkalosis. The dose of triamterene was later reduced to 50 mg, and excess blood pressure lowering was improve. Genetic testing did not identify any mutations in the beta or gamma subunits of the ENaC gene. Discussion: In this patient, no hypokalemia was observed in previous examinations. The hypokalemia in this patient was spironolactone-resistant, and the fact that triamterene, a distal tubular epithelial Na channel (ENaC) inhibitor, was effective in this patient; furthermore, no genetic abnormality in ENaC, suggests the possibility of an acquired ENaC abnormality similar to the Liddle syndrome.

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.

Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non-canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope-tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti-ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well-characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non-epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti-α subunit antibody recognizes an intense non-specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone-induced full length α-subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β- and γ-subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti-ENaC antibodies.

PS-B05-6: FABRY DISEASE MOUSE IS RESISTANT TO HIGH-SALT DIET-INDUCED HYPERTENSION PROBABLY VIA DYSFUNCTIONAL SODIUM TRANSPORTS AND AQUAPORIN 2

Background: Fabry disease is a rare X-linked lysosomal storage disorder resulting from an error in glycosphingolipid metabolism caused by the GLA gene mutation. Patients with Fabry disease often have near normal or even low systemic blood pressure although the mechanism underlying such observed finding remains unexplained. This study examined whether a high salt intake could affect transport of sodium and water and induce hypertension in mice with Fabry disease. Methods: Fabry disease model mice (B6;129-Glatm1Kul/J) and wild-type (WT) mice received 8% or normal NaCl salt diet for 2 weeks. Results: A high-salt diet for 2 weeks generated higher blood pressure in WT mice but not in Fabry disease mice. Free water clearance (FWC) and electrolyte free water clearance (EFWC) in groups fed with a high salt diet were lower than those in groups fed with a normal salt diet. Both high salt-fed WT and Fabry disease mice had decreased gamma-subunit of epithelial Na channel (ENaC) compared with normal salt-fed groups. In Fabry disease mice fed a high-salt diet, there was an decrease in renal expressions of Na+/H+ exchanger isoform 3 (NHE3) and Na+-Cl- cotransporter (NCC) compared with WT mice fed a high-salt diet. Although renal expression of vasopressin V2 receptor (V2R) was upregulated in Fabry mice fed with a high salt diet, renal aquaporin 2 (AQP2) level failed to increase up to the level in WT mice fed with a high salt diet. Conclusion: These findings suggest that the decreased expression of NHE3 and NCC and impaired response of AQP2 in kidneys of Fabry disease to a salt load could be one of mechanisms by which Fabry disease could have resistance to the development of hypertension.

Neuropeptides and degenerin/epithelial Na+ channels: a relationship from mammals to cnidarians.

Ion channels of the degenerin (DEG)/epithelial Na+ channel (ENaC) family serve diverse functions ranging from mechanosensation over Na+ reabsorption to H+ sensing and neurotransmission. However, several diverse DEG/ENaCs interact with neuropeptides; some are directly activated, whereas others are modulated by neuropeptides. Two questions arise: does this interaction have a common structural basis and does it have an ancient origin? Current evidence suggests that RFamide neuropeptides activate the FMRFamide-activated Na+ channels (FaNaCs) of invertebrates via binding to a pocket at the external face of their large extracellular domain. It is likely that RFamides might activate DEG/ENaCs from the freshwater polyp Hydra (the HyNaCs) via binding to a similar pocket, although there is not yet any experimental evidence. In contrast, RFamide neuropeptides modulate acid-sensing ion channels (ASICs) from vertebrates via binding to a central cavity enclosed by β-sheets of the extracellular domain. Dynorphin opioid peptides, for their part, bind to the acidic pocket of ASICs, which might be evolutionarily related to the peptide binding pocket of FaNaCs, but instead of opening the channels they work as antagonists to stabilize its closed state. Moreover, peptides interacting with DEG/ENaCs from animals of different phyla, although having similar sequences, are evolutionarily unrelated to each other. Collectively, it appears that despite a seemingly similar interaction with similar peptides, the interaction of DEG/ENaCs with neuropeptides has diverse structural bases and many origins.

The effect of high-dietary K+ (HK) on Kir4.1/Kir5.1 and ROMK in the distal convoluted tubule (DCT) is not affected by gender and Cl- content of the diet.

Basolateral potassium channels in the distal convoluted tubule (DCT) are composed of inwardly-rectifying potassium channel 4.1 (Kir4.1) and Kir5.1. Kir4.1 interacts with Kir5.1 to form a 40pS K+ channel which is the only type K+ channel expressed in the basolateral membrane of the DCT. Moreover, Kir4.1/Kir5.1 heterotetramer plays a key role in determining the expression and activity of thiazide-sensitive Na-Cl cotransport (NCC). In addition to Kir4.1/Kir5.1, Kir1.1 (ROMK) is expressed in the apical membrane of the late DCT (DCT2) and plays a key role in mediating epithelial Na+ channel (ENaC)-dependent K+ excretion. High dietary-K+-intake (HK) stimulates ROMK and inhibits Kir4.1/Kir5.1 in the DCT. Inhibition of Kir4.1/Kir5.1 is essential for HK-induced suppression of NCC whereas the stimulation of ROMK is important for increasing ENaC-dependent K+ excretion during HK. We have now used the patch-clamp-technique to examine whether gender and Cl- content of K+-diet affect HK-induced inhibition of basolateral Kir4.1/Kir5.1 and HK-induced stimulation of ROMK. Single-channel-recording shows that basolateral 40pS K+ channel (Kir4.1/Kir5.1) activity of the DCT defined by NPo was 1.34 (1% KCl, normal K, NK), 0.95 (5% KCl) and 1.03 (5% K+-citrate) in male mice while it was 1.47, 1.02 and 1.05 in female mice. The whole-cell recording shows that Kir4.1/Kir5.1-mediated-K+ current of the early-DCT (DCT1) was 1,170pA (NK), 725pA (5% KCl) and 700pA (5% K+-citrate) in male mice whereas it was 1,125pA, 674pA and 700pA in female mice. Moreover, K+-currents (IK) reversal potential of DCT (an index of membrane potential) was -63mV (NK), -49mV (5% KCl) and -49mV (5% K-citrate) in the male mice whereas it was -63 mV, -50mV and -50mV in female mice. Finally, TPNQ-sensitive whole-cell ROMK-currents in the DCT2 /initial-connecting tubule (CNT) were 910pA (NK), 1,520pA (5% KCl) and 1,540pA (5% K+-citrate) in male mice whereas the ROMK-mediated K+ currents were 1,005pA, 1,590pA and 1,570pA in female mice. We conclude that the effect of HK intake on Kir4.1/Kir5.1 of the DCT and ROMK of DCT2/CNT is similar between male and female mice. Also, Cl- content in HK diets has no effect on HK-induced inhibition of Kir4.1/Kir5.1 of the DCT and HK-induced stimulation of ROMK in DCT2/CNT.

Rare Variants in Genes Encoding Subunits of the Epithelial Na+ Channel Are Associated With Blood Pressure and Kidney Function.

The epithelial Na+ channel (ENaC) is intrinsically linked to fluid volume homeostasis and blood pressure. Specific rare mutations in SCNN1A, SCNN1B, and SCNN1G, genes encoding the α, β, and γ subunits of ENaC, respectively, are associated with extreme blood pressure phenotypes. No associations between blood pressure and SCNN1D, which encodes the δ subunit of ENaC, have been reported. A small number of sequence variants in ENaC subunits have been reported to affect functional transport in vitro or blood pressure. The effects of the vast majority of rare and low-frequency ENaC variants on blood pressure are not known. We explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests. We found that variants in SCNN1A and SCNN1B were associated with diastolic blood pressure and mean arterial pressure (P<0.00625). Although SCNN1D is poorly expressed in human kidney tissue, SCNN1D variants were associated with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure (P<0.00625). ENaC variants in 2 of the 4 subunits (SCNN1B and SCNN1D) were also associated with estimated glomerular filtration rate (P<0.00625), but not with stroke. Our results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function.

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

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.

Teleostean fishes may have developed an efficient Na+ uptake for adaptation to the freshwater system.

Understanding Na+ uptake mechanisms in vertebrates has been a research priority since vertebrate ancestors were thought to originate from hyperosmotic marine habitats to the hypoosmotic freshwater system. Given the evolutionary success of osmoregulator teleosts, these freshwater conquerors from the marine habitats are reasonably considered to develop the traits of absorbing Na+ from the Na+-poor circumstances for ionic homeostasis. However, in teleosts, the loss of epithelial Na+ channel (ENaC) has long been a mystery and an issue under debate in the evolution of vertebrates. In this study, we evaluate the idea that energetic efficiency in teleosts may have been improved by selection for ENaC loss and an evolved energy-saving alternative, the Na+/H+ exchangers (NHE3)-mediated Na+ uptake/NH4 + excretion machinery. The present study approaches this question from the lamprey, a pioneer invader of freshwater habitats, initially developed ENaC-mediated Na+ uptake driven by energy-consuming apical H+-ATPase (VHA) in the gills, similar to amphibian skin and external gills. Later, teleosts may have intensified ammonotelism to generate larger NH4 + outward gradients that facilitate NHE3-mediated Na+ uptake against an unfavorable Na+ gradient in freshwater without consuming additional ATP. Therefore, this study provides a fresh starting point for expanding our understanding of vertebrate ion regulation and environmental adaptation within the framework of the energy constraint concept.