ENaC Activity Research Articles

Salt Sensitivity of Blood Pressure in Women.

Several clinical and large population studies indicate that women are more salt-sensitive than men, yet the precise mechanisms by which the sexually dimorphic onset manifests remains incompletely understood. Here, we evaluate recent epidemiological data and highlight current knowledge from studies investigating sex-specific mechanisms of salt-sensitive blood pressure (SSBP). Emerging evidence indicates that women of all ethnicities are more salt-sensitive than men, at all ages both premenopausal and postmenopausal. However, menopause exacerbates severity and prevalence of SSBP, suggesting that female sex chromosomes predispose to and female sex hormones mitigate SSBP. Results from both human and rodent studies support the contribution of enhanced and inappropriate activation of the aldosterone-ECMR (endothelial cell mineralocorticoid receptor) axis promoting vascular dysfunction in females. Increases in adrenal response to angiotensin II, in association with higher ECMR expression and activation of endothelial ENaC (epithelial sodium channel) in females compared to males, are emerging as central players in the development of endothelial dysfunction and SSBP in females. Female sex increases the prevalence and susceptibility of SSBP and sex hormones and sex chromosome complement may exert antagonistic effects in the development of the female heightened SSBP.

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.

Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2

Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.

Role of microRNAs in aquaporin 2 regulation.

The current review aims to present the most recent achievements on the role of microRNAs (miRNAs) on the kidney function to stimulate research in the field and to expand new emerging concepts. The focus is on the role of miRNAs in intercellular communication along the segments of the nephron and on the epi-miRNAs, namely the possibility of some miRNAs to modulate the epigenetic machinery and so gene expression. Indeed, recent evidence showed that miRNAs included in exosomes and released by proximal tubule cells can modulate ENaC activity on cells of collecting duct. These data, although, from in-vitro models open to a novel role for miRNAs to participate in paracrine signaling pathways. In addition, the role of miRNAs as epigenetic modulators is expanding not only in the cancer field, but also in the other kidney diseases. Recent evidence identified three miRNAs able to modulate the AQP2 promoter metilation and showing an additional level of regulation for the AQP2. These evidence can inspire novel area of research both for renal physiology and drug discovery. The diseases involving the collecting duct are still missing disease modifying agents and the expanding miRNAs field could represent an opportunity.

Mineralocorticoid Receptor Antagonists Cause Natriuresis in the Absence of Aldosterone.

MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. We studied AS (aldosterone synthase)-deficient (AS-/-) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR-/-) mice. KS-MR-/- mice were used to compare MR expression and ENaC localization and cleavage with AS-/- mice. MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR-/- mice but were fully preserved along DCT2 and were partially reduced along CCD in AS-/- mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS-/- mice and were not increased along CCD after low salt. AS-/- mice exhibited transient Na+ wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS-/- mice led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR-/- mice. Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS-/- mice.

Kidney-Specific CAP1/Prss8-Deficient Mice Maintain ENaC-Mediated Sodium Balance through an Aldosterone Independent Pathway.

The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.

COVID-19 and Liquid Homeostasis in the Lung-A Perspective through the Epithelial Sodium Channel (ENaC) Lens.

Infections with a new corona virus in 2019 lead to the definition of a new disease known as Corona Virus Disease 2019 (COVID-19). The sever cases of COVID-19 and the main cause of death due to virus infection are attributed to respiratory distress. This is associated with the formation of pulmonary oedema that impairs blood oxygenation and hypoxemia as main symptoms of respiratory distress. An important player for the maintenance of a defined liquid environment in lungs needed for normal lung function is the epithelial sodium channel (ENaC). The present article reviews the implications of SARS-CoV-2 infections from the perspective of impaired function of ENaC. The rationale for this perspective is derived from the recognition that viral spike protein and ENaC share a common proteolytic cleavage site. This cleavage site is utilized by the protease furin, that is essential for ENaC activity. Furin cleavage of spike ‘activates’ the virus protein to enable binding to host cell membrane receptors and initiate cell infection. Based on the importance of proteolytic cleavage for ENaC function and activation of spike, it seems feasible to assume that virus infections are associated with impaired ENaC activity. This is further supported by symptoms of COVID-19 that are reminiscent of impaired ENaC function in the respiratory tract.

Rodent models to study sodium retention in experimental nephrotic syndrome.

Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown. With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

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.

Effect of SGLT2 Inhibition on the Development of Salt‐sensitive Hypertension, RAAS, and Sodium Channels and Transporters in Dahl SS Rats

Hypertension is common among patients with type 2 diabetes mellitus (T2DM). The combination of diabetes and hypertension is associated with high morbidity and mortality because of an increased risk of cardiovascular and renal complications. Effective control of blood glucose levels and blood pressure is essential to prevent the development and progression of diabetic nephropathy in T2DM. Sodium‐Glucose Cotransporter‐2 (SGLT2) inhibitors are the new highly effective drugs for the treatment of T2DM and some cardiovascular diseases. Despite all the remarkable benefits on cardiovascular and renal systems, the molecular mechanisms of how SGLT2 inhibition affects water and electrolyte balance are incompletely understood. We hypothesized that SGLT2 inhibition alters blood pressure via affecting Renin‐Angiotensin‐Aldosterone System (RAAS), sodium channels and transporters. Dahl Salt‐Sensitive (SS) rat, a model for salt‐induced hypertension, was used to test this hypothesis in a hypertensive, non‐diabetic environment. We found that administration of dapagliflozin at a concentration of 2 mg/kg/day via drinking water during 3 weeks blunts the development of salt‐induced hypertension with no changes in the heart rate. Mean arterial pressure in the dapagliflozin treated group compared to the vehicle‐treated group during high salt (4% NaCl) challenge was significantly lower (136 ± 4 vs. 157 ± 6 mmHg; P<0.0001 on 21st day of the experiment). SGLT2 inhibition caused a significant diuretic effect (67 ± 6 vs. 40 ± 4 ml/day, P<0.001 on the 21st day of the experiment). Glucose excretion was dramatically increased in inhibitor‐treated rats compared to vehicle‐treated (747 ± 44 vs. 12 ± 2 mg/dL, P<0.0001 on the 21st day). We also observed increased Na+/creatinine ratios (138 ± 10 vs. 110 ± 6, P<0.004 on the 21st day) and Cl‐/creatinine ratios (133 ± 9 vs. 108 ± 6, P<0.03 on the 21st day). To define potential mechanisms, we first measured levels of hormones involved in the RAAS by using LC‐MS/MS. Despite effects on Na+/creatinine and Cl‐/creatinine ratios, dapagliflozin treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of SGLT2 inhibition on renal Na+ channels and transporters using patch‐clamp, Western blotting, and RT‐PCR. Neither mRNA nor protein expressions levels of all tested Na+ transporters (SGLT2, SGLT1, NHE3, NKCC2, NCC, and α‐, β‐, γ‐ ENaC subunits) were different between groups. Electrophysiological studies did not reveal any difference of SGLT2 inhibition on the conductance and activity of ENaC. Therefore, our data suggest that SGLT2 inhibition blunts the development of salt‐induced hypertension, causes glucosuria and natriuresis but does not affect RAAS and expression and activity of Na+ channels and transporters in a model of non‐diabetic, salt‐sensitive 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.

Interrogation of Novel Aldosterone‐induced Genes Associated With Acute Stimulation Of ENaC In Primary Collecting Duct Cells

Aldosterone (ALDO) is a key volume‐regulating hormone which promotes Na+ reabsorption in the distal nephron. ALDO sensitivity is conferred by the enzyme 11βHSD2 which breaks down the related more abundant steroid, cortisol. Mutations in Hsd11b2 give rise to the syndrome of apparent mineralocorticoid excess, where hypertension is associated with excessive renal Na+ reabsorption. In the distal nephron corticosteroid‐induced Na+ transport, via the epithelial Na+ channel ENaC, is due to transcriptional processes and previous studies have identified key genes including Sgk1. Our lab carried out transcriptomic analysis of mCCDcl1 cells following acute corticosteroid induced ENaC‐mediated Na+ transport, identifying several novel ALDO induced genes. The present study aimed to elucidate the effects of corticosteroids on these target genes in the collecting duct (CD), utilising an in vitro and in vivo approach.A CD‐specific reporter mouse was generated using Aqp2Cre to express eGFP. Primary cells were isolated by FACS and either grown in culture or RNA was extracted immediately. Cells were grown on filter membranes to allow development of polarised monolayers and electrometric properties were assessed using an epithelial volt‐ohm‐meter. Target gene expression was determined by qRT‐PCR.Assessment of nephron segment‐specific gene expression revealed an enrichment of genes encoding α‐ENaC, ROMK and 11βHSD2 but not NHE3 or NKCC2. NCC was detected but parvalbumin was absent, demonstrating isolation of cells from the late distal tubule onwards. In addition to genes associated with principal cells we also detected genes encoding pendrin and AE1, indicative of intercalated cells therefore this population reflects CD cells. When grown in culture for 9‐11 days, these cells exhibited Vt and Rt of ‑20.3±3.1 mV and 5.1 ±0.1 kΩ∙cm2, respectively. This gave rise to an Ieq of ‑3.9±0.6 μA·cm‑2 which was near abolished with 10 µM amiloride – indicating Na+ transport via ENaC.Acute application of steroid hormones to cells for 3h: ALDO (3 nM), DEX (100 nM) and CORT (10 nM) – the latter only when 11βHSD2 activity was inhibited using carbenoxolone (CBX, 10 µM) – stimulated amiloride‐sensitive currents by 2.0‐, 3.6‐ and 2.4‐fold, respectively. Analysis of ALDO‐induced genes revealed both ALDO and CORT upregulated Sgk1, Zbtb16 and Rasd1. ALDO also upregulated Sult1d1 whereas CORT also upregulated an unannotated gene Gm43305. DEX upregulated all of these as well as 2 additional genes previously detected: Defb1 and Gm16178.To determine the relevance of these findings in vivo reporter mice were administered ALDO, vehicle or CORT, by ip injection, the latter under control conditions or following CBX treatment for 8 days. Cells were isolated after 3h. The previously identified Zbtb16, Rasd1, Gm43305 and Sult1d1 were upregulated in both ALDO and CBX+CORT groups, though no change was detected in either Sgk1 or Defb1. Scnn1a was also upregulated in both groups and additionally Per1 and Sult1a1 were upregulated in mice treated with CBX‐CORT.Together these data confirm 4 novel corticosteroid‐induced genes in primary CD cells associated with acute stimulation of ENaC activity.

Increase in Salt Concentration in the Culture Media Enhances Protein Expression of Tumor Necrosis Factor‐Alpha Receptor Type 1 in Cultured Renal Cortical Collecting Duct Cells But Not in Proximal Tubular Cells

Tumor necrosis factor‐alpha (TNFα) exerts its biological responses via interaction with its’ two cell surface receptors, type 1 (TNFR1) and type 2 (TNFR2) which are differentially expressed and regulated in the kidney. Previously, we have demonstrated that circulating TNFα exerts natriuresis via its action on TNFR1 but not on TNFR2 and such TNFR1 activation exerts inhibitory action on distal tubular sodium transport, particularly tubular ENaC activity. As TNFα level is elevated during dietary intake of high salt (HS), we hypothesized that increases in salt concentration in renal tissue enhances TNFα ‐TNFR1 activity in the renal tubules to induce natriuretic response to HS intake. To test this hypothesis, we examined the protein expression of TNFR1 and TNFR2 in cultured proximal (human kidney [HK‐2] cell line) and distal (murine cortical collecting duct [M‐1] cell line) tubular cells at different concentrations of NaCl in the culture media. Both these HK‐2 and M‐1 cells were incubated for 24 hours in control media with normal NaCl concentration (120 mM) and at media containing higher NaCl (140 mM and 160 mM) concentrations. TNF receptors protein expressions were analyzed by the Western Blot technique using appropriate antibodies for TNFR1 (rabbit anti‐TNFR1; Abcam cat # 19139) and TNFR2 (rabbit anti‐TNFR2; Abcam cat # 109322), which were normalized by expression of β‐actin or GAPDH proteins using appropriate antibodies. The blot image was quantified and measured as band intensity density with Image J software. The quantitative immunoblot analysis in HK‐2 cells showed that there were no significant changes in the band intensity unit (IU) in TNFR1 (120 mM, 70.1±12.8 IU; 140 mM, 73.7±9.9 IU; 160 mM, 57.2±9.8 IU; n=6 in each condition) or TNFR2 protein (120 mM, 83.9 ±6.3 IU; 140 mM, 66.8±14.1 IU; 160 mM, 77.8±2.3 IU; n=6 in each condition) levels. On the other hand, TNFR1 protein expression was significantly higher in M‐1 cells incubated in 160 mM NaCl conc. (135.6 ±7.8 IU, n=7; P< 0.005) than that in 120 mM (109.3±2.8 IU, n=7) and in 140 mM (91.1±10.8 IU, n=7) NaCl conc. However, TNFR2 protein expression in M‐1 cells did not significantly alter with the changes in NaCl conc. in the media (120 mM, 137±20.1 IU; 140 mM, 128±7.2 IU; 160 mM, 143±7.2 IU; n=7 in each condition). These data indicate that TNFR1 activity is upregulated in renal collecting duct cells (CCD) in response increases in tissue NaCl concentration in the kidney. Such upregulation of TNFR1 activity in the CCD induced by high salt concentration, exerts inhibitory action on distal sodium transport and plays a role in enhancing urinary sodium excretion during dietary HS intake.

PIP2 Interacts with MLP and ENaC Electrostatically In Renal Epithelial Cells

We examined the interaction of a membrane‐associated protein, MARCKS‐like Protein‐1 (MLP‐1), and an ion channel, Epithelial Sodium Channel (ENaC), with phosphatidylinositol 4, 5‐bisphosphate (PIP2). PIP2 activates ENaC in inside‐out patches (half activating concentration of 21 ± 1.17). There are several PIP2 binding sites on ENaC, but 2 binding sites with a large number of basic residues at the N‐terminus of ENaC b and g are critical sites for PIP2 regulation of ENaC. PIP2 is necessary to open ENaC, but there is a problem with a simple model of ENaC and PIP2 associating by membrane lateral diffusion. ENaC is a relatively rare protein (a few functional apical channels/m2). PIP2 is also a rare molecule: less than 1 in 1000 membrane lipids. Given the known abundance of PIP2 and ENaC and the diffusion constant of PIP2 in the apical membrane, the mean time it would take a PIP2 molecule to find an ENaC channel by random lateral diffusion is 6.3x102s or approximately 10 minutes. But, in normal epithelial cells expressing ENaC, the channel opens about every 2 seconds. We hypothesize that normal channel activity requires MLP‐1 associated with the inner leaflet of the cell membrane. MLP‐1’s strongly positively charged effector domain sequesters PIP2 electrostatically without direct covalent binding. This implies differences between PIP2 association with MLP‐1 and the binding of a ligand to a specific receptor. First, the effect of MLP‐1 charge acts at a distance. The coulombic force exerted by MLP‐1 on PIP2 does decrease as the square of the distance between MLP‐1 effector domain and PIP2, but the distances within the membrane are relatively small and the membrane dielectric constant is also very small so the forces can be significant over a large membrane area. In the absence of MLP‐1, PIP2 moves around the membrane lipid in a two‐dimensional random walk, but the presence of the charged effector domain biases the walk so that PIP2 always moves towards MLP‐1, at first slowly and then rapidly until it is in MLP‐1’s potential well. This implies that MLP‐1 increases the local concentration of PIP2 exponentially as electrostatic theory predicts. MLP‐1 membrane concentration is near 10mM which is comparable to the concentration of PIP2. Given PIP2‘s lateral mobility in the membrane of about 10‐8cm2s‐1, MLP‐1 would rapidly sequester most membrane PIP2. This would increase the local PIP2 concentration over a hundred‐fold. Our data shows that ENaC covalently binds MLP‐1, so this increased PIP2 concentration would be in close proximity to PIP2 binding sites on the cytosolic N terminus of ENaC. This binding is also electrostatic since the ENaC binding sites contain multiple basic residues. The advantage of electrostatic interaction is that PIP2 can remain in close proximity to both ENaC and MLP‐1. We have modified the charge structure of the PIP2 –binding domains of MLP‐1 and ENaC and showed that the changes affect membrane localization and ENaC activity consistent with electrostatic theory. Using STED FRAP, we also measured the diffusion rates of MLP‐1, ENaC, and PIP2 and ENaC and PIP2 in the absence of MLP‐1 that also support an electrostatic model.

Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel’s γ-subunit

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, β-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αβγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C–Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.

Downregulation of epithelial sodium channel (ENaC) activity in cystic fibrosis cells by epigenetic targeting

The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients’ treatment by epigenetic targeting.

Metformin suppresses epithelial sodium channel hyperactivation and its associated phenotypes in a mouse model of obstructive lung diseases

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the world, and has no radical treatment. Inhibition of amiloride-sensitive epithelial sodium ion channel (ENaC) has now been considered as a potential therapeutic target against COPD. One possible modulator of ENaC is AMP-activated protein kinase (AMPK), a key molecule that controls a wide variety of cellular signals; however, little is known about whether metformin, a clinically available AMPK activator, has a protective role against ENaC-associated chronic pulmonary phenotypes, such as emphysema and pulmonary dysfunction. We first used ENaC-overexpressing human bronchial epithelial cells (β/γENaC-16HBE14o-) and identified that Metformin significantly reduced ENaC activity. Consistently, in vivo treatment of ENaC-overexpressing COPD mouse model (C57BL/6-βENaC-Tg mice) showed improvement of emphysema and pulmonary dysfunction, without any detrimental effect on non-pulmonary parameters (blood glucose level etc.). Bronchoalveolar lavage fluid (BALF) and lung tissue analyses revealed significant suppression in the infiltration of neutrophils as well as the expression of inflammatory markers (KC), neutrophil gelatinase (MMP9) and macrophage elastase (MMP12) in metformin-treated C57BL/6-βENaC-Tg mice. Overall, the present study demonstrates that metformin directly inhibits ENaC activity in vitro and provides the first evidence of therapeutical benefit of Metformin for COPD with higher ENaC activity.

ENaC activation by proteases

Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.

Sodium retention in nephrotic syndrome is independent of the activation of the membrane-anchored serine protease prostasin (CAP1/PRSS8) and its enzymatic activity

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.

Atrial Natriuretic Peptide and the Epithelial Sodium Channel Contribute to Spinal Cord Injury-Induced Polyuria in Mice.

Polyuria is found in patients with spinal cord injury (SCI). However, the underlying cellular and molecular mechanism is unknown. Here, we show that mice had elevated urine for 7 days after T10 contusion. Using multi-photon confocal microscopy, we performed intra-vital imaging experiments to evaluate water reabsorption in kidney tubules by examining fluorescent intensity in the lumen of the distal tubule from live mice. The data show that SCI significantly reduced the concentrating function of kidney tubules. The reduced water reabsorption appears to be mediated by atrial natriuretic peptide (ANP) because SCI increased the expression levels of both ANP and natriuretic peptide receptor A (NPR-A) in the kidney cortex. Our patch-clamp single-channel recordings from split-open distal tubules show that SCI decreased the activity of the epithelial sodium channel (ENaC). Western blot combined with confocal microscopy data show that the levels of 70 kD γ-ENaC, which is an active isoform because of proteolytic cleavage, were significantly reduced in distal tubule principal cells. An NPR-A inhibitor (A71915) given intravenously eliminated the effects of SCI on ENaC and polyuria. These data together with previous studies suggest that SCI causes polyuria, probably by reducing ENaC activity through elevating ANP and NPR-A. Further investigation of the signal transduction pathways may provide useful information for discovering an efficient drug to treat SCI-induced polyuria.