Increased ENaC Activity Research Articles

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

Background: The renal epithelium is highly sensitive to changes in blood potassium (K+) levels. We have a detailed understanding of distal K+-sensitive signaling pathways, yet proximal mechanisms are less well-defined. Results: Mice lacking the basolateral proximal tubule inwardly rectifying K+ channel, Kir4.2, (Kir4.2−/−) demonstrated normal blood and urine electrolytes at baseline. Upon removal of K+ from their diet, Kir4.2−/− animals decompensated as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis compared to control animals (Kir4.2+/+). Potassium wasting in Kir4.2−/− mice was not proximal in origin, but was dependent upon increased ENaC activity along the connecting segment. This was demonstrated by the correction of K+ wasting in knockouts treated with the ENaC inhibitor, amiloride. Using lithium clearance studies, we further demonstrated increased distal sodium delivery in Kir4.2−/− animals due to reduced proximal tubule sodium reabsorption. Optical clearing and three-dimensional confocal imaging reveal Kir4.2−/− mice failed to undergo proximal tubule hypertrophy when fed a low K+ diet, while the distal convoluted tubule response was exaggerated in knockouts compared to control animals. A phosphoproteomics mass spectrometry analysis identified increased mTOR/AKT signaling as mediating the proximal dietary response to K+ depletion in control animals, and changes to mTOR/AKT signaling was blunted in Kir4.2 knockouts. Lastly, we demonstrated in isolated renal tubules that AKT phosphorylation in response to low K+ depended upon mTORC2 activation by secondary reductions in intracellular Cl−. Conclusions: In this work we identify the basolateral inwardly rectifying K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Kir4.2 knockout mice increase their distal nephron transport burden to maintain electrolyte homeostasis in the face of deficient proximal tubule cell function. Data identify a conserved proximal role for cell Cl− which, as it does along the distal convoluted tubule, responds to extracellular K+ changes to activate intracellular kinase signaling. Along the proximal tubule, mTOR/AKT signaling is essential for the cell response to low K+. Proximal and distal nephron cell Cl− responses work in concert to preserve K+ homeostasis. These studies were supported by NIH grants DP5-OD033412 (AST), DK51265, DK95785, DK62794, DK7569, P30DK114809 (RCH, MZZ), and the Vanderbilt Center for Kidney Disease and Vanderbilt Diabetes Research and Training Center (DK020593) pilot and feasibility grant (JSD). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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

Vaping-Induced Proteolysis Causes Airway Surface Dehydration.

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

Extracellular vesicles from BALF of pediatric cystic fibrosis and asthma patients increase epithelial sodium channel activity in small airway epithelial cells

Extracellular Vesicles (EVs) are nanosized vesicles derived from all cell types. EV cargo allows for intercellular communication, intracellular signaling, and regulation of proteins in recipient cells. We tested the hypothesis that EVs isolated from the bronchoalveolar-lavage fluid (BALF) of pediatric cystic fibrosis (CF) or pediatric asthma patients increase epithelial sodium channel (ENaC) activity in normal human small airway epithelial cells (SAECs) and the mechanism involves specific EV lipids. We characterized EVs from BALF of pediatric CF and pediatric asthma patients by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. The CF and asthma pediatric groups were similar in BALF electrolytes concentration and cell count, except for neutrophils, which were higher in the CF group. Lipidomic analyses for each group of EVs were performed using targeted mass spectrometry. Phosphatidylethanolamine, sphingomyelins, and triacylglycerol were enriched in both groups, but phosphatidylcholine and phosphatidylinositol concentrations were greater in the CF group compared to the asthma group, and the opposite trend was found for phosphatidylserine. Endogenous ENaC activity, measured by the single-channel patch-clamp technique, increased in normal human SAECs after challenging SAEC with EVs from either the CF or asthma groups compared to control EVs. In conclusion, EVs isolated from BALF of pediatric patients with CF or asthma have unique lipid profiles. Despite the differences, both types of EVs increase ENaC activity in normal human SAECs compared to control EVs isolated from the conditioned media of these cells.

PS-B09-8: N-METHYL-D-ASPARTATE (NMDA) RECEPTORS INDUCE RENAL VASODILATION AND REGULATES BLOOD PRESSURE.

Introduction: N-methyl-D-aspartate receptors (NMDAr) are glutamate/glycine receptors expressed in the kidney. NMDAr induce renal vasodilation through an unknown mechanism. Connecting tubule-glomerular feedback (CNTGF) is a vasodilator feedback mechanism, initiated by ENaC in the connecting tubule producing afferent arteriole (AffA) vasodilatation and promoting sodium excretion. We hypothesized that NMDAr activation stimulate CNTGF and that the inhibition of NMDAr decrease CNTGF vasodilation inducing hypertension. Methods: We explore the presence of NMDAr receptors in mouse by transcriptome analysis of microdisseted tubules segments, total kidney western blot and immunofluorescence. We used mpkCCD cells (mouse principal cells) to explore the interaction between NMDAr and ENaC in PC using trasnwell current analysis and split-open tubule using patch clamp method. To determine the effect of NMDAr on CNTGF, we measured CNTGF-mediated AffA dilation in the presence or absence of NMDAr agonists glutamate and glycine with and without NMDAr antagonist (MK801), using a double-microperfusion method in rabbits, and in-vivo by micropunture technique in rats. To evaluate the effect of NMDAr on blood pressure (BP), we measure BP in NMDA 2C global knock out mice. BP was measure using tail cuff methods. To confirm the effect of NMDAr on BP we infused subcutaneously NMDAr inhibitor MK801 for 7 days in a gain of function β ENaC mutated allele mice on129/Sv background on normal salt diet. Results: NMDAr was found to be expressed in connecting tubule and co-localized with AQP2 and ENaC. In mpkCCD cells and open split tubules, NMDAr activation increase ENaC activity, while NMDAr inhibition decrease ENaC activity (p < 00.1). In-vitro, tubular microperfusion of NMDAr agonist increased AffA dilation (p < 0.001). NMDAr agonist-induced dilation was blunted with MK-801 (NMDAr blocker) and totally blocked with benzamil (CNTGF inhibitor). In vivo, NMDAr agonist increase CNTGF mediated vasodilation (P < 0.01). NMDAr 2C knock out mice showed high BP in comparison with wildtype (SBP 114.6 ± 7.3 vs 100.4 ± 2.2 mmHg, respectively, p = 0.01). In β ENaC mutated mice, the infusion of NMDAr inhibitor (Mk-801) induces hypertension (SBP 146 ± 8 mmHg) while the vehicle infused mice remained normotensives (110.7 ± 7.7 mmHg; p < 0.01). Conclusions: NMDAr in the connecting tubule increase renal vasodilation by activating CNTGF. The genetic ablation of NMDAr 2C or the pharmacological inhibition of NMDAr increase blood pressure. Future studies need to confirm the role of NMDAr in the pathogenesis of hypertension.

Renal Tubular IL‐1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages

We recently showed that interleukin (IL)‐6 induces salt‐sensitivity in diabetic mice by dysregulating the epithelial sodium channel (ENaC). After 4 weeks on a high salt diet, male db/db mice displayed salt sensitivity, the accumulation of proinflammatory (CD80+) macrophages, and higher levels of IL‐6 and IL‐1β in the kidney compared to db/+ controls. However, the origin of this inflammatory response is unknown. We hypothesize that, during diabetes, renal tubular epithelial cells release IL‐1β which activates renal macrophages to produce IL‐6 and impair kidney function. Immunofluorescence analysis showed that the higher IL‐1β expression of db/db mice is exclusively localized in the renal tubules. Flow cytometry analysis of renal cells showed that db/db mice have higher IL‐1β expression in tubular (E‐cadherin+) cells (21 ± 2 vs. 8 ± 3 % of E‐cadherin+ cells, P<0.001) compared to db/+ controls. To evaluate whether high glucose triggers IL‐1β synthesis in tubular cells, a primary culture of renal tubular epithelial cells from wild‐type (WT) mice was exposed to either a low (LG, 5mM) or high (HG, 15mM) glucose media. HG‐treated cells released higher levels of IL‐1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and displayed higher expression of NLRP3 (4 ± 1‐fold increase, P<0.001) and Caspase‐1 (2.1 ± 0.2‐fold increase, P<0.001) compared to LG‐treated cells. These are major components of the NLRP3 inflammasome that synthesizes IL‐1β. No equivalent effect was observed using mannitol. To evaluate whether tubular cells can modify macrophage phenotype through IL‐1β, we co‐cultured tubular cells with bone marrow‐derived macrophages. Flow cytometry revealed that HG‐treated tubular cells induced a pro‐inflammatory polarization of macrophages not observed with LG‐treated cells (50 ± 4 vs. 15 ± 2 CD80+ macrophages, P<0.001). This effect was blunted when macrophages were obtained from IL‐1 receptor KO mice (IL1RKO) confirming a role of IL‐1β. To evaluate whether IL‐1β‐mediated macrophage activation plays a role in vivo, 2‐mo‐old diabetic db/db mice (n=6) were transplanted with a bone marrow of IL1RKO mice. These mice, named db/db‐IL1RKO, cannot respond to IL‐1β. db/db receiving wild‐type (WT) bone marrow (db/db‐WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to high salt diet for 4 weeks. Compared to db/db‐WT, db/db‐IL1RKO did not develop salt‐sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal CD80+ macrophages, (21 ± 4 vs. 54 ± 5 CD80+ macrophages, P<0.01), less renal IL‐6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05), and lower ENaC activity (65% decrease by amiloride test, P<0.05) despite an accumulation of tubular IL‐1β. In conclusion, our data show that tubular epithelial cells are a major source of IL‐1β under high glucose conditions. This cytokine promotes a pro‐inflammatory phenotype of macrophages resulting in higher renal IL‐6 levels which increases ENaC activity leading to salt sensitivity.

Sphingomyelin‐enriched extracellular vesicles from hypertensive diabetic mice regulate calcium mobilization and amiloride‐sensitive transepithelial current in mouse cortical collecting duct cells

Uncontrolled hypertension (HTN) and diabetes mellitus (DM) are two major public health burdens contributing to increased hospitalization, readmission costs, and adverse clinical outcomes. Moreover, DM is a leading cause of death and long‐term morbidity. Overexpression and activity of epithelial sodium channels (ENaC) in the kidney results in hypertension, but the mechanism for its regulation in the diabetic kidney is not entirely understood. Cholesterol is known to increase ENaC activity in cultured kidney cells in a calcium and PI(4,5)P2‐dependent mechanism. Presumably ENaC is regulated by other lipids in pathophysiology. Extracellular vesicles (EVs) are nano‐sized vesicles that play an important role in intercellular communication and intracellular signaling. Here, we investigated whether EVs isolated from the urine of hypertensive diabetic db/db mice or diabetic db/db mice augment ENaC activity by inhibiting calcium mobilization in mpkCCD cells. Adult diabetic db/db mice were salt loaded (4% NaCl) for 7 days to induce hypertension. Blood pressure was measured by tail‐cuff, and urine from db/db mice with systolic blood pressure of greater than 140mmHg or from normotensive db/db mice were used to isolate EVs. Nanoparticle tracking analysis was performed to determine EV concentration. EVs from hypertensive diabetic db/db mice (N=5) compared to diabetic db/db mice (N=5) suppressed ionomycin‐induced calcium mobilization in mpkCCD cells cultured on glass bottom 35 mm dishes (N=3 experiments, p<0.05) which was measured by confocal microscopy and augmented amiloride‐sensitive transepithelial current in mpKCCD cells cultured in 12mm diameter permeable supports (N=6 permeable supports per group) measured by an epithelial volt ohm meter (EVOM). Sphingomyelin were found to be enriched in urinary EVs from hypertensive diabetic db/db mice compared to diabetic only db/db mice. Exogenous application of sphingomyelin to mpkCCD cells mimicked the suppression of calcium mobilization and increase in amiloride‐sensitive transepithelial current in these cells. Taken together, these data suggest differences in the packaged cargo of urinary EVs between HTN and DM mice that presumably plays a role in the regulation of ENaC activity.

Visualization of Ectopic Serine Protease Activity by Förster Resonance Energy Transfer-Based Reporters.

Channel-activating proteases (CAPs) play a fundamental role in the regulation of sodium transport across epithelial tissues mainly via cleavage-mediated fine-tuning of the activity of the epithelial sodium channel (ENaC). Hyperactivity of CAPs and subsequently increased ENaC activity have been associated with various diseases, including cystic fibrosis (CF). To date, there is only a limited number of tools available to investigate CAP activity. Here, we developed ratiometric, peptide-based Förster resonance energy transfer (FRET) reporters useful to visualize and quantify the activity of ectopic serine proteases including the CAPs prostasin and matriptase in human and murine samples in a temporally and spatially resolved manner. Lipidated varieties were inserted into the outer leaflet of the plasma membrane to detect enzyme activity on the surface of individual cells, that is, close to the protease substrates. The FRET reporters (termed CAPRee) selectively detected the activity of ectopic serine proteases such as CAPs in solution and on the surface of human and murine cells. We found increased CAP activity on the surface of cells with a genetic background of CF. The new reporters will contribute to a better understanding of ectopic serine protease activity and their regulation under physiological and pathophysiological conditions.

Abstract MP32: Renal Tubular Epithelial Cell-derived IL-1β Triggers The Inflammatory Response That Induces Salt Sensitivity In Diabetes

Renal inflammation, and specifically interleukin (IL)-6, induces salt-sensitivity in diabetic mice by upregulating the epithelial sodium channel (ENaC). However, the origin of this inflammatory response is still unknown. We hypothesize that, during diabetes, tubular epithelial cells initiate renal inflammation by releasing IL-1β that further activates renal macrophages to produce IL-6 and impair kidney function. A primary culture of renal tubular epithelial cells from wild-type (WT) mice was exposed to either low (LG, 5mM) or high (HG, 15mM) glucose media. HG-treated cells released higher levels of IL-1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and have higher expression of NLRP3 (4 ± 1 fold increase, P<0.001) and Caspase-1 (2.1 ± 0.2 fold increase, P<0.001) compared to LG-treated cells. No effect was observed using mannitol as an osmotic control. Tubular cells were also co-cultured with bone marrow-derived macrophages. Flow cytometry analysis revealed that HG-treated tubular cells induced an M1-type polarization of macrophages. This effect was blunted when macrophages were obtained from IL-1 receptor KO mice (IL1RKO) confirming a role of IL-1β. Here, the percentage of M1 macrophages (CD45 + F4/80 + CD80 + ) of total macrophages (CD45 + F4/80 + ) decreased from 31 ± 4% to 10 ± 3% (P<0.01). To evaluate whether IL-1β-mediated macrophage activation plays a role in vivo, 2-mo-old diabetic db/db mice (n=6) were transplanted with bone marrow from IL1RKO mice. In these mice, named db/db-IL1RKO, immune cells cannot respond to IL-1β. Db/db receiving wild-type (WT) bone marrow (db/db-WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to a high salt diet (NaCl 4% w/w) for 4 weeks. Db/db-IL1RKO did not develop salt-sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal IL-6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05) and lower ENaC activity (0.4-fold decrease by amiloride test, P<0.05) compared to db/db-WT. In conclusion, our data show that tubular epithelial cells are a major source of IL-1β under high glucose conditions. This cytokine promotes M1 macrophage polarization resulting in higher renal IL-6 levels which increases ENaC activity leading to salt sensitivity.

Probenecid inhibits cysts development in Pkd1 RC/RC mice

Background ADPKD cysts contain high levels of ATP that contribute to cyst enlargement. Among other effects, ATP excess leads to a reduced reabsorption in cyst-lining cells and cyst fluid accumulation. We demonstrated that Pkd1RC/RC mice, a model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. Probenecid, a uricosuric agent, is also used as a pannexin-1 blocker reducing ATP release. We studied therapeutic potential of probenecid in Pkd1RC/RC mice and its effect on sodium reabsorption. Methods : Pkd1RC/RC mice, a hypomorphic model of ADPKD, were aged till 10.5 months and osmotic minipumps were implanted to deliver probenecid for 42 days. After treatment, 1 year old conscious mice were subjected to a measurement of glomerular filtration rate (GFR) and kidneys were collected for histomorphological studies. Effect of probenecid on Na reabsorption was tested on mpkCCD cells seeded onto permeable supports with open-circuit current measurements. Results In vivo inulin clearance study demonstrates that Pkd1RC/RC mice have normal GFR 1.05±0.09 ml/min/100g at the 6 months old age (n=15) whereas GFR in C57BL/6 mice - 0.94±0.1 ml/min/100g (n=8). With disease progression, GFR in Pkd1RC/RC mice reduces to 0.36±0.08 ml/min/100g at 12 months age. 42 days long probenecid treatment (15.9 mg/kg/day) significantly improves GFR to 1.43±0.11 ml/min/100g (p<0.001). Probenecid treatment also reduced kidney hypertrophy: kidney/TBW ratio in vehicle group was 2.54±0.17% vs 1.76±0.05% probenecid (p<0.05). Histological study on sectioned kidneys revealed that probenecid significantly reduces cyst size. Cyst to total slice area ratio was 13.9±2.7% (vehicle) vs 3.4±0.8% (probenecid) (n=5 each group). Earlier we have shown that probenecid decreases luminal ATP release in immortalized CD cell culture. As ATP is capable of downregulating Na reabsorption via the epithelial sodium channel we tested if probenecid increases ENaC activity. We applied probenecid to mpkCCD cell monolayer and found that the drug causes a bell-shaped dose-dependent increase of amiloride-sensitive transepithelial flux with maximal effect at 50 μM. Conclusion : Probenecid demonstrates therapeutic potential against ADPKD cyst progression in a Pkd1RC/RC mouse model by reducing cyst size, renal hypertrophy and supporting GFR and reabsorption from the cyst space.

Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis

Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency.

Endothelial epithelial sodium channel involves in high-fat diet-induced atherosclerosis in low-density lipoprotein receptor-deficient mice

We previously showed that increased epithelial sodium channel (ENaC) activity in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) contributes to vasculature dysfunction. Here, we investigated whether ENaC participates in the pathological process of atherosclerosis using LDL receptor-deficient (LDLr−/−) mice. Male C57BL/6 and LDLr−/− mice were fed a normal diet (ND) or high fat diet (HFD) for 10 weeks. Our data show that treatment of LDLr−/− mice with a specific ENaC blocker, benzamil, significantly decreased atherosclerotic lesion formation and expression of matrix metalloproteinase 2 (MMP2) and metalloproteinase 9 (MMP9) in aortic arteries. Furthermore, benzamil ameliorated HFD-induced impairment of aortic endothelium-dependent dilation by reducing expression of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6 and production of adhesion molecules including VCAM-1 and ICAM-1 in both C57BL/6 and LDLr−/− mice fed with HFD. In addition, HFD significantly increased ENaC activity and the levels of serum lipids, including ox-LDL. Our in vitro data further demonstrated that exogenous ox-LDL significantly increased the production of TNF-α, IL-1β, IL-6, VCAM-1 and ICAM-1. This ox-LDL-induced increase in inflammatory cytokines and adhesion molecules was reversed by γ-ENaC silencing or by treatment with the cyclooxygenase-2 (COX-2) antagonist celecoxib. Benzamil inhibited HFD-induced increase in COX-2 expression in aortic tissue in both C57BL/6 and LDLr−/− mice, and γ-ENaC gene silencing attenuated ox-LDL-induced COX-2 expression in HUVECs. These data together suggest that HFD-induced activation of ENaC stimulates inflammatory signaling, thereby contributes to HFD-induced endothelial dysfunction and atherosclerotic lesion formation. Thus, targeting endothelial ENaC may be a promising strategy to halt atherogenesis.

SAT-LB127 Regulation of ENaC by Exosomal Lipids in the Diabetic Kidney

Effective treatment of hypertension (HTN) in patients with diabetes may help to significantly reduce the risk of those patients developing additional complications including vascular disease and diabetic nephropathy. Blockers of the renin-angiotensin system including angiotensin converting enzyme inhibitors and angiotensin receptor blockers are not always effective in treating HTN in diabetic patients. Therefore, the aim of this study was to use an animal model of type 2 diabetes to investigate a novel mechanism of diabetes associated HTN involving exosomal lipids in the upregulation of epithelial sodium channel (ENaC) activity in the kidney. We performed metabolic cages studies using male and female hypertensive (salt-loaded induced) diabetic db/db mice and healthy age-matched wild-type control mice in order to isolate and characterize urinary exosomes from each group by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. Our mass spectrometry based lipidomic studies identified key lipids that were differentially expressed in the kidney derived exosomes from the hypertensive diabetic mice compared to control mice. Sphingomyelin quantification assays showed total sphingomyelin content was elevated in the exosomes from the hypertensive diabetic mice compared to the control group. Single channel patch clamp studies showed urinary exosomes enriched in sphingomyelins from hypertensive diabetic mice compared to controls increase ENaC activity (at the level of channel density and open probability) in cultured distal tubule renal epithelial cells. Moreover, exogenous application of sphinomyelin-6 to cultured mouse cortical collecting duct (mpkCCD) cells resulted in an increase in amiloride-sensitive transepithelial current. Taken together, our data show various lipids are enriched in exosomes from hypertensive diabetic mice compared to controls and these exosomes positively regulate ENaC activity in distal tubule and collecting duct cells.

Role of AVP in the Pathology of Liddle’s Syndrome

The epithelial Na+ channel, ENaC, is expressed in the apical plasma membrane of renal principal cells. ENaC activity is rate limiting for sodium reabsorption across the distal nephron and consequently, is the final arbiter of renal sodium excretion. Gain of function mutations in ENaC cause Liddle’s syndrome: an autosomal dominant form of inheritable hypertension. The hypertension of Liddle’s syndrome results from uncontrolled sodium reabsorption via hyperactive ENaC. We hypothesized that improper sodium reabsorption during Liddle’s syndrome may elevate plasma tonicity enough to evoke release of vasopressin (AVP). Indeed, AVP levels are elevated in Liddle’s mice, which have increased ENaC activity as assessed by patch clamp electrophysiology of isolated CNT/CCD. Tolvaptan, an AVP V2 receptor blocker, decreased ENaC activity in Liddle’s mice. Exogenous desmopressin (DDAVP), a V2 receptor specific agonist, had no further effect on ENaC activity in Liddle’s mice. However, DDAVP further increases blood pressure in Liddle’s mice, and this high blood pressure decreases with administration of benzamil, an ENaC blocker. We conclude from these results that ENaC activity is saturated in Liddle’s mice, in part due to elevated AVP levels, but that saturated ENaC activity is still necessary for additional effects of DDAVP on blood pressure in the Liddle’s model. Because DDAVP increases blood pressure but not ENaC activity in Liddle’s mice, we speculate that additional DDAVP‐stimulated water reabsorption further increases blood pressure in this model. Thus, in the Liddle’s mouse, while ENaC activity is saturated, water reabsorption can still respond to additional AVP signaling. Experiments investigating DDAVP effects on AQP2 and AQP3 expression are ongoing and will test whether the hyperosmotic draw of solute from tubular fluid across the collecting duct is saturated in the Liddle’s mouse.Support or Funding InformationThis research was supported by NIH/NIDDK grants R01DK113816 to JDS and R01DK103758 to AP and JDS.

Proteolytic activation of the epithelial sodium channel: role of pro‐protein convertases and prostasin

The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro‐protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC.We used the M‐1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro‐protein convertase inhibitor decanoyl‐RVKR‐chloromethyl ketone (dec‐RVKR‐cmk) and serine protease inhibitor camostat mesilate (CM). Knockout cell lines of prostasin and three PCSKs were generated with the CRISPR/Cas9 system in M‐1 cells. As an index of ENaC activity, amiloride‐sensitive short‐circuit current (ISC) and transepithelial voltage (VTE) was measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane levels and cleavage were tested by western blotting of surface biotinylated M‐1 cells with an antibody against the C‐terminal of γENaC. All values are means SE, compared with students t‐test or ANOVA as appropriate.Pro‐protein convertase inhibition decreased the amiloride‐sensitive ISC in M‐1 cells compared with controls (25.9 1.9 μA, n=5, vs. 45.7 7.0 μA, n=6, p&lt;0.05). Similarly, serine protease inhibition decreased VTE (−34.5 7.8 mV, n=3, vs. −74.2 0.9 mV, n=3, p&lt;0.05) but ISC was not significantly lower (68.0 8.5, n=3, vs. 74.0 2.3, n=3, n.s.). Preliminary evidence suggests that although both inhibitors decreased membrane expression of γENaC, only serine protease inhibitor treatment reduced γENaC cleavage (n=1). Knockout of the Psck3 (furin) gene by CRISPR completely abolished ENaC activity, while knockout of Pcsk4, Pcsk7, and prostasin all showed decreased ENaC activity compared to wild‐type M‐1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin reduced γENaC cleavage.We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.Support or Funding Information The Danish Medical Research Council

Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome.

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.

Postprandial Effects on ENaC‐Mediated Sodium Absorption

Recent studies have suggested that postprandial increases in insulin directly contribute to reduced urinary sodium (Na+) excretion by altering renal Na+ transport. While there are a number of renal transporters that respond to insulin stimulation, which of these transporters are involved in this Na+ sparing mechanism under normal physiological conditions is not fully established. The epithelial sodium channel (ENaC) is the rate limiting step for sodium reabsorption in the distal nephron and its regulation by insulin is well established; however, the regulatory effect of acute hyperinsulinemia (as would result from a meal) on ENaC under normal conditions is still being debated. The main objective of this study was to test the contribution of ENaC as well as other Na+ transporters to the increase in renal Na+ reabsorption following a meal. To test this, we used fasted Sprague Dawley rats that had been given a high carbohydrate supplement to simulate a meal and analyzed changes to ENaC activity as well as expression of other renal transporters involved in sodium homeostasis. Postprandial changes in circulating renin‐angiotensin‐aldosterone system (RAAS) hormones known to enhance ENaC activity were also tested. We found that 4 hours post carbohydrate stimulus, ENaC open probability increased relative to controls in split‐open isolated collecting duct tubules (0.34 ±0.06 vs. 0.70 ± 0.07, n≥5, p&lt;0.01), while the number of channels at the surface and total ENaC α‐ and β‐subunit levels remained unchanged. We also examined expression levels of the Na+‐Cl− cotransporter (NCC), Na+‐K+‐2Cl− cotransporter (NKCC2), and Na+‐K+‐ATPase (NKA). While there were no significant differences in expression of NCC or NKCC2 in either group, NKA expression in the postprandial group was significantly increased (1.0 ± 0.08 vs. 1.62 ± 0.10, n=5, p&lt;0.001). Additionally, using mass spectrometry‐based analysis, we interrogated different metabolites of RAAS signaling between the stimulated and fasted rats and found no significant changes in aldosterone or angiotensin metabolite levels. These results suggest that acute hyperinsulinemia following a meal increases ENaC activity independent of the RAAS signaling cascade. Our study demonstrates that ENaC and NKA regulation via insulin is a likely contributor to an evolutionary mechanism for preserving sodium and volume loss after a meal, and that this regulation is distinct from classical ENaC regulation by RAAS.Support or Funding InformationT32 HL134643; R35 HL135749, HL56259This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Postprandial Effects on ENaC-Mediated Sodium Absorption

Recent studies have suggested that postprandial increases in insulin directly contribute to reduced urinary sodium excretion. An abundance of research supports the ability of insulin to augment epithelial sodium channel (ENaC) transport. This study hypothesized that ENaC contributes to the increase in renal sodium reabsorption following a meal. To test this, we used fasted or 4 hour postprandial Sprague Dawley rats to analyze ENaC expression and activity. We also assessed total expression of additional sodium transporters (Na+-Cl− cotransporter (NCC), Na+-K+-2Cl− cotransporter (NKCC2), and Na+-K+-ATPase (NKA)) and circulating hormones involved in the renin-angiotensin-aldosterone system (RAAS). We found that after carbohydrate stimulus, ENaC open probability increased in split-open isolated collecting duct tubules, while ENaC protein levels remained unchanged. This was supported by a lack of change in phosphorylated Nedd4-2, an E3 ubiquitin ligase protein which regulates the number of ENaCs at the plasma membrane. Additionally, we found no differences in total expression of NCC, NKCC2, or NKA in the postprandial rats. Lastly, there were no significant changes in RAAS signaling between the stimulated and fasted rats, suggesting that acute hyperinsulinemia increases ENaC activity independent of the RAAS signaling cascade. These results demonstrate that insulin regulation of ENaC is a potential mechanism to preserve sodium and volume loss following a meal, and that this regulation is distinct from classical ENaC regulation by RAAS.

Characterization of P2rX7 knockout in PCK (Polycystic Kidney) rats

Accumulating evidence suggests that the autocrine and paracrine effects of Adenosine‐3‐phosphate (ATP) could be detrimental for the progression of polycystic kidney diseases (PKD). P2X7 targeting in cell cultures and zebrafish was shown to decrease cyst progression. In this project, CRISPR/Cas9 approach was employed to knockout P2X7 receptor in the PCK rat strain, a model of autosomal recessive PKD (ARPKD), to study involvement of P2X7 in PKD progression in mammals. Mendelian distribution was observed in the litters showing no embryonic lethality. Adult knockout animals exhibited moderate hypertension (mean arterial blood pressure 123±6 and 132±5 mmHg at 9AM/9PM as registered with continuous DSI telemetry). General morphological evaluation of the kidneys revealed that total P2X7 knockout caused exacerbated cystogenesis compared to wild‐type littermates.Impaired renal function in the knockout group was also confirmed with metabolic studies and inulin clearance studies in conscious animals. Thirteen weeks old P2rx7−/+ and P2rx7−/− animals had higher body mass than P2rx7+/+ littermates (421±9g and 430±10.5g vs 392±12g; n=5–14). Plasma sodium and chloride (not potassium) were slightly but significantly lower in the knockout group. We observe significant proteinuria of 35–40 mg/day/100g body weight in both groups (at 13 weeks) but with aging (at 24 weeks), it reached 89±13 and 157±19 mg/day/100g in wild‐type and knockout animals, respectively. Glomerular filtration rate was also lower in the P2rx7−/− group (0.88±0.06 vs 1.22±0.11 ml/min/100g body weight) in young rats, however, with aging GFR significantly decreased in both groups and did not differ (0.42 and 0.49, respectively).Purinergic signaling is known as a powerful down‐regulator of ENaC‐mediated sodium reabsorption. Thus, patch clamp analysis revealed that acute basolateral application of 100μM α,β‐methylene‐ATP to M1 collecting ducts cell culture grown on permeable membranes decreases activity of apical ENaC (NPo from 0.5 ±0.15 to 0.19±0.12, n=10). At the next step we investigated if the P2X7 knockout affect ENaC activity. Patch clamp technique was also applied on three types of tubular epithelia isolated from knockout PCK rats and their wild‐type littermates: split‐opened non‐dilated collecting ducts, small developing cysts and large mature cysts. In these preparations we found that developing cysts exhibit increased ENaC activity whereas mature cysts had impaired channel activity (NPo= 0.79±0.12; 1.1±0.2 and 0.39±0.16). In all three tissue types P2X7 knockout increased ENaC activity (NPo=1.36±0.17; 2.3±0.36; 0.86±0.05), as expected.In conclusion, knockout of the P2rx7 gene in PCK rats increases cystogenesis and aggravates renal insufficiency. P2X7 deficiency also increases activity of ENaC in PCK rat collecting ducts and cysts.Support or Funding InformationSupported by K99/R00 HL116603, P30 DK090868 via Baltimore PKD Center P&amp;F Grant and R24 HL114474 via MCW Gene Editing Rat Resource CenterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclosporin A Induces Hypertension via a Cholesterol‐ and ENaC‐Dependent Mechanism

Hypertension related to the use of immunosuppressive drugs, particularly cyclosporin A (CsA), presents one of the major problems in the management of transplant recipients. Our recent studies suggest a hypothesis that CsA may cause hypertension by stimulating the renal epithelial sodium channel (ENaC) via a pathway associated with elevated intracellular cholesterol (Cho). To further determine the mechanism, we performed confocal microscopy and patch‐clamp experiments using cultured cortical collecting duct (CCD) principal cells and measured the blood pressure in mice. Confocal data show that CsA and Cho increased both the length of microvilli of CCD cells where ENaC is located and the levels of phosphatidylinositol‐4,5‐bisphosphate (PIP2) in microvilli. Conversely, inhibition of Cho synthesis with lovastatin decreased the levels of PIP2, a well‐known ENaC activator. Patch‐clamp data shows that Cho increased ENaC activity in a PIP2‐dependent manner. Interestingly, CsA elevated the blood pressure in mice and the elevation was abolished by either inhibition of Cho synthesis with lovastatin or blockade of ENaC with amiloride. These results suggest that CsA increases the blood pressure of mice by stimulating ENaC via a pathway associated with the elevation of intracellular Cho and PIP2 in the principal cells of CCD. However, it remains to be determined whether statins or amiloride can be used for the treatment of CsA‐induced hypertension.Support or Funding Information(Supported by NIH R01DK100582 to H.M.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.