Hydrogen Sulfide Prevents Hydrogen Peroxide-Induced Activation of Epithelial Sodium Channel through a PTEN/PI(3,4,5)P₃ Dependent Pathway

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.肾脏远端节段经上皮钠通道（epithelial sodium channel, ENaC）介导的钠重吸收，在盐敏感性高血压的发生发展中发挥关键作用。本团队此前报道，过氧化氢（hydrogen peroxide, H₂O₂）可通过提升细胞膜顶膜区域的磷脂酰肌醇3,4,5-三磷酸（phosphatidylinositol 3,4,5-trisphosphate, PI(3,4,5)P3）水平，激活A6远端肾单位细胞中的ENaC。本研究证实，硫化氢（hydrogen sulfide, H₂S）可拮抗H₂O₂诱导的A6细胞中ENaC激活。细胞贴附式膜片钳实验数据显示，外源性H₂O₂可显著提升ENaC的开放概率（open probability, PO），这与本团队此前的研究结果一致。0.1 mM硫氢化钠（NaHS）介导的H₂S处理，可完全阻断外源性H₂O₂诱导的ENaC异常激活。单独用H₂S预处理A6细胞，仅会轻微降低ENaC的开放概率；但经此处理后的细胞，H₂O₂无法再提升ENaC的开放概率。共聚焦显微镜成像结果显示，向A6细胞施加外源性H₂O₂，可显著升高细胞内活性氧（reactive oxygen species, ROS）水平，并诱导PI(3,4,5)P3在细胞膜顶膜区域聚集。H₂S处理可几乎完全抵消外源性H₂O₂对细胞内ROS水平及顶膜PI(3,4,5)P3水平的上述影响。此外，H₂S可显著抑制H₂O₂诱导的肿瘤抑制因子张力蛋白同源物（phosphatase and tensin homolog, PTEN）的氧化失活，而PTEN正是PI(3,4,5)P3的负调控因子。此外，作为PTEN特异性抑制剂的BPV(pic)，可通过类似H₂O₂的方式提升A6细胞中PI(3,4,5)P3水平与ENaC活性。本研究数据首次证实，H₂S可通过依赖PTEN-PI(3,4,5)P3的信号通路，阻断H₂O₂诱导的ENaC激活。