胆固醇在肾集合管上皮细胞膜微结构水平调节ENaC通道的机制研究

Kidney transplantation and application of immunosuppressant-cyclosporine A (CsA) can induce refractory hypertension. However, the machanism remains unclear. Most studies considered that it relates to abnormal cholesterol metabolism. We have validated that CsA can stimulates ENaC by elevating membrane cholesterol in renal collecting duct epithelial cells. Moreover, ENaC is an important channel in regulation of blood pressure. Our preliminary studies suggest that lipid rafts which are rich in cholesterol are important regions for maintaining ENaC activity and mainly located in microvilli, but ENaC is not directly combined with cholesterol. In contrast, distribution of PIP2 which is a potent ENaC channel activator via a direction interaction and PI(4)P5K which is the kinase responsible for PIP2 synthesis are both modulated by cholesterol. So, we suppose that cholesterol may stimulates ENaC channel by indirectly promoting the assembling of ENaC and increasing the accumulation or synthesis of PIP2. We will use mouse collecting duct epithelial cells and isolated, split-open cortical collecting ducts combined with a variety of experimental methods, especially with scanning ion conductance microscopy (SICM) microstructure navigation patch- clamp technique to illuminate the pathway of ENaC regulation by cholesterol-dependent distribution of PIP2 and PI(4)P5K. This research will reveal the machanism of hypertension induced by lipid metabolism disorders, for instance, kidney transplantation and application of CsA and verify the attenuating effect of statins according to the hypothesis mechanism of this research , provide potential targets for clinical prevention and treatment.

接受肾移植及应用免疫抑制剂环孢霉素A(CsA)可诱发难治性高血压，但机制不明。研究表明可能与胆固醇代谢异常有关。我们已证实CsA可通过升高肾集合管上皮细胞胆固醇水平激活ENaC通道，而ENaC是血压调节的重要通道；预实验发现ENaC活性依赖于富含胆固醇的脂质筏环境并主要分布在细胞膜微绒毛中，但不与胆固醇直接结合，而ENaC直接激活因子PIP2及其合成前体PI(4)P5K受胆固醇调节分布，因此推测胆固醇是通过促进微绒毛上ENaC的组装及PIP2的聚集或合成增加来实现激活的。我们将应用鼠集合管上皮细胞及活体分离的小鼠集合管为研究对象结合多种实验手段并通过SICM定位膜片钳技术在膜微结构水平阐明PIP2、 PI(4)P5K的胆固醇依赖性分布对ENaC的调节途径。进一步揭示脂代谢异常，如肾移植及应用CsA导致高血压的发生机制，并验证他汀药在本研究假说机制上是否成立并起到逆转效果，提供临床防治靶点。

肾脏集合管主细胞的上皮钠离子通道（ENaC）对调节系统血压起到关键作用。我们之前的研究证实集合管上皮细胞顶膜上的胆固醇可以调节ENaC通道，但潜在机制仍不明确。我们应用膜片钳及共聚焦实验评估了ENaC通道的活性和表达密度，实验显示应用MbCD拔除膜上胆固醇可以显著降低阿米洛利-敏感的跨膜电流以及ENaC通道活性；同样的现象也出现在应用洛伐他汀减少膜上胆固醇合成后。我们已证实磷脂酰肌醇-4,5-二磷酸（PIP2）作为ENaC通道的直接激活剂主要分布在细胞顶膜特殊结构微绒毛上，我们应用共聚焦实验证实ENaC与PIP2共表达在微绒毛上，同样胆固醇也与PIP2共表达在微绒毛上，无论应用MbCD拔除膜上胆固醇还是应用洛伐他汀减少膜上胆固醇合成均可以显著降低微绒毛上胆固醇、PIP2和ENaC的表达。脂质结合实验显示PIP2并不与胆固醇直接结合，但PIP2合成前体PI(4)P5K受胆固醇直接调解。综上，本课题证实胆固醇是通过促进微绒毛上ENaC的组装及PIP2的合成增加来实现激活的。我们首次在膜微结构水平阐明PIP2、 PI(4)P5K的胆固醇依赖性分布对ENaC的调节途径。进一步揭示胆固醇代谢异常，如肾移植及应用CsA导致高血压的发生机制，并验证了他汀药在本研究机制上可起到逆转效果，提供临床防治靶点。