从CDO1介导AQP2泛素化/磷酸化“串扰”促进其内吞转移探讨水臌贴“从肾论治肝硬化腹水”的机制

腹水是肝硬化失代偿期最突出的临床症状之一，其迁延难愈、反复发作易肝病及肾，首损肾阳致气不化水，使病情加重而威胁患者生存率。课题组临证以水臌贴消水除胀疗效良好，高通量筛选验证其可能靶向肾脏AQP2；预实验表明桂皮酮（水臌贴主要透皮成分之一）促进肾小管上皮细胞中AQP2内吞转移并复杂翻译后修饰。由此假设：水臌贴治疗肝硬化腹水与其靶向促进肾小管上皮细胞AQP2质膜转移有关；透皮后，其活性成分调控AQP2的泛素化/磷酸化串扰，发挥“温肾化气”作用。本项目拟利用基因敲减、表面等离子共振、GST-tag标签亲和纯化、定点突变等方法，从动物和细胞水平研究水臌贴通过CDO1/AQP2轴治疗肝硬化腹水效果，及透皮后调控AQP2内吞转移和双重修饰间串扰关系的机制，以阐明水臌贴“利水消胀”获效机制和“温肾化气”物质基础，对水臌贴“从肾论治肝硬化腹水”提供理论支持，并促进中医敷贴特色疗法防治肝硬化腹水的临床推广。

Ascites is one of the most prominent clinical symptoms in the decompensated stage of cirrhosis, the repeating and lingering pathological process tend to cause the liver disease affecting the kidney. In the meanwhile, ascites will deteriorate kidney yang causing unhealed ascites and further aggravation, threatening patient’s survival rate. We previously used Shuigu Patch in the treatment of cirrhosis ascites and gained a positive effect. We then analyzed the genomics differences in kidney of cirrhotic ascites rats and found that AQP2 one possible target of Shuigu Patch. Further we proved that Cinnamone, one of the main transdermal components of Shuigu Patch, may promote endocytosis transfer of AQP2 together with ubiquitination and phosphorylation modification in tubular epithelial cells. Therefore, a hypothesis is proposed: the effects of Shuigu Patch on cirrhosis ascites is related to its targeted promotion of AQP2 transfer from plasma to membrane; after the Shuigu Patch’s transdermal application, it interferes CDO1-mediated endocytosis of AQP2 with crosstalk between ubiquitination and phosphorylation, which may play an important role in nouring and warming the kidney thus facilitating diuresis reduction. This project intends to study the effect of Shuigu Patch on cirrhosis ascites and the role CDO1/AQP2 axis in vivo and in vitro. We will employ gene knockdown, surface plasmon resonance technology, GST-tag tag affinity purification, site-directed mutation of genes, etc. to study the complex modification mechanism of AQP2, especially its crosstalk between ubiquitination and phosphorylation. This research will elucidate the molecular mechanism of Shuigu Patch on "diuresis and dilation" and "warming kidney and facilitating qi", and also provide support for cirrhosis ascites treatment based on the kidney function, which is benefit for promoting clinical popularization of TCM application therapy in cirrhosis ascites treatment.