ENaC regulation by biliary factors

晚期肝病患者经常出现液体潴留和电解质紊乱 部分由于肾素-血管紧张素-醛固酮系统的激活。醛固酮激活 远端肾单位上皮Na+通道（ENaC），增强尿Na+潴留和K+ 排泄但许多肝病患者在没有明显激活的情况下经历液体潴留 血管紧张素-醛固酮系统。肝病患者也经常表现出 血浆胆汁酸升高和高胆红素血症。初步数据显示特定的胆汁酸 结合胆红素在体外激活ENaC，牛磺胆酸在体外激活ENaC。 孤立的集合管。我们建议的中心假设是尿胆汁酸和 可能结合胆红素直接激活ENaC，促进Na+和液体潴留以及K+ 排泄本提案的目的是确定这种调节是否在体内发生， 确定调控的分子机制。初步数据显示， 酒精性肝炎患者含有浓度的活化胆汁酸和结合胆汁酸。 胆红素足以激活通道，并且确实激活通道。我们将确定 酒精性肝炎患者的尿液是否激活实验细胞中的ENaC。我们进一步 假设胆汁因子升高激活远端肾单位ENaC，促进Na+ 滞留和K+排泄。我们将在孤立的集合管中检验这一假设。我们还将 通过长期给予胆汁因子并量化 体重、血K+、尿Na+和K+、血浆醛固酮和全身水。初步数据 提示牛磺胆酸促进Na+保留、K+损失和体积膨胀。我们将 通过使用药物阻断ENaC，以及通过以下方法确定ENaC对观察到的变化的贡献： 利用遗传变化增加通道对胆汁因子的敏感性或降低ENaC 对肾脏Na+和K+处理的贡献。实验还将研究分子 胆汁酸对ENaC的调节机制。我们假设激活胆汁酸 直接与通道连接以增强通道活动。我们将使用衍生胆汁酸来检测 直接结合，以及通道和胆汁酸的关键化学和生物物理特性 来剖析这种互动。关键实验将在多个细胞模型中进行，包括 爪蟾卵母细胞、培养的上皮细胞和小鼠集合管。直接激活ENaC， 以前没有研究过肝脏疾病中胆汁因素升高， 与肝脏疾病相关的容量超负荷、水肿和电解质失衡。

Patients with advanced liver disease often experience fluid retention and electrolyte disturbances due in part to activation of the renin-angiotensin-aldosterone system. Aldosterone activates the epithelial Na+ channel (ENaC) in the distal nephron, enhancing urinary Na+ retention and K+ excretion. But many liver disease patients experience fluid retention without apparent activation of the renin-angiotensin-aldosterone system. Liver disease patients also frequently exhibit elevated plasma bile acids and hyperbilirubinemia. Preliminary data show that specific bile acids and conjugated bilirubin activate ENaC in vitro and that taurocholic acid activates ENaC in isolated collecting ducts. The central hypothesis of our proposal is that urinary bile acids and possibly conjugated bilirubin directly activate ENaC, promoting Na+ and fluid retention and K+ excretion. The goal of this proposal is to determine whether this regulation occurs in vivo and to determine the molecular mechanism of regulation. Preliminary data suggest that urine from patients with alcoholic hepatitis contains concentrations of activating bile acids and conjugated bilirubin sufficient to activate the channel, and indeed activate the channel. We will determine whether urine from alcoholic hepatitis patients activates ENaC in experimental cells. We further hypothesize that elevated biliary factors activate ENaC in the distal nephron, promoting Na+ retention and K+ excretion. We will test this hypothesis in isolated collecting ducts. We will also test this hypothesis in vivo by chronically administering biliary factors and quantifying changes in weight, blood K+, urinary Na+ and K+, plasma aldosterone, and total body water. Preliminary data suggest that taurocholic acid promotes Na+ retention, K+ loss, and volume expansion. We will determine the contribution of ENaC to observed changes by using drugs to block ENaC, and by using genetic changes to increase the channel's sensitivity to biliary factors or to decrease ENaC's contribution to renal Na+ and K+ handling. Experiments will also investigate the molecular mechanism of ENaC regulation by bile acids. We hypothesize that activating bile acids interact directly with the channel to enhance channel activity. We will use derivatized bile acids to detect direct binding, and key chemical and biophysical properties of the both the channel and bile acids to dissect the interaction. Key experiments will be performed in multiple cell models, including Xenopus oocytes, cultured epithelial cells, and mouse collecting ducts. Direct ENaC activation by biliary factors elevated in liver disease has not been previously investigated, and may contribute to the volume overload, edema, and electrolyte imbalances associated with liver disease.