Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells.
ABSTRACT Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2*) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 microM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2* (with the O2* scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 +/- 0.03 to 0.03 +/- 0.01. We also found that increasing endogenous O2* in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 +/- 0.05) after superoxide dismutase and 1.5 microM NO coincubation (0.21 +/- 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2* by approximately 30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2* alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.
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ABSTRACT: Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO ) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC PO ; however, in these cells H2O2 failed to elevate ENaC PO . Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.PLoS ONE 10/2013; 8(5):e64304. · 3.53 Impact Factor
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ABSTRACT: The significance of basal renal nitric oxide (NO) availability in the regulation of renal perfusion and sodium excretion in human congestive heart failure (CHF) has not been described previously. We studied the effects of acute systemic NO synthesis inhibition with N(G)-monomethyl-L-arginine (L-NMMA) in 12 patients with CHF and 10 healthy control subjects (CON) in a randomized placebo-controlled study. Effect parameters were renal plasma flow (RPF), renal vascular resistance (RVR), glomerular filtration rate (GFR), urine sodium excretion and plasma levels of vasoactive hormones. L-NMMA was associated with a significant decrease in RPF (CON-LNMMA: -13 ± 3% [P = .014]; CHF-LNMMA: -17 ± 7% [P = .017]) and a profound increase in RVR in both CHF and CON (CON-LNMMA: +26 ± 6% [P = .009]; CHF-LNMMA: +37 ± 70% [P = .005]). Significant decreases in sodium excretion were found in both CHF-LNMMA and CON-LNMMA. Relative changes from baseline were not statistically different between CHF-LNMMA and CON-LNMMA. After L-NMMA, RPF values correlated inversely with plasma aldosterone in CHF-LNMMA (P = .01). L-NMMA induced an increase in A-type natriuretic peptide (ANP) only in CHF-LNMMA (+18 ± 8%; P = .035), which correlated significantly with basal ANP levels (P = .034). There was no difference in the renal response to L-NMMA in CHF vs CON, suggesting that the impact of NO on renal perfusion and sodium excretion is maintained in stable CHF. We suggest that NO influences the release of ANP during high levels of atrial stretch in CHF.Journal of cardiac failure 11/2013; 19(11):776-785. · 3.07 Impact Factor
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ABSTRACT: Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel anti-oxidant drugs, such as Nox4-inhibitor compounds, are being developed. There is however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single channel patch clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04±0.18 (mean±SE) to 0.25±0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic-cell stretch response of A6 cells; indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertention.AJP Renal Physiology 07/2013; · 4.42 Impact Factor