The Role of Ubiquitin in Sodium Chloride Co-Transporter Regulation

描述（由申请人提供）： 高血压是一个主要的公共卫生问题。迄今为止，每种遗传性高血压都与肾脏中盐处理缺陷有关。本申请试图探索参与调节肾脏中的关键盐处理转运蛋白之一，位于远曲小管中的氯化钠协同转运蛋白（NCC）的机制。先前的研究已经报道了NCC活性降低，以响应WNK 4（一种与称为戈登综合征的遗传性高血压形式相关的激酶）和佛波酯（二酰基甘油的化学类似物，激素刺激的常见途径）的存在，但NCC活性降低的机制尚不清楚。本申请将试图证明WNK 4和佛波醇酯通过减少在细胞表面表达的NCC的量来降低NCC活性，该过程通过增强NCC的泛素化而发生，从而促进共转运蛋白的降解。实验模型将是利用具有天然NCC活性和完整调控途径的永生化小鼠远曲小管细胞系的细胞模型。通过放射性示踪剂摄取测量NCC活性，通过生物素化和免疫荧光测量NCC细胞表面表达，以及通过免疫沉淀和免疫印迹在WNK 4基因沉默或用佛波醇酯处理的细胞中测量泛素化，将建立活性、表面表达和泛素化之间的关联。NCC泛素化的机制也将被确定，包括泛素化的位点和所需的酶。这个项目将有助于培养本杰明高博士作为一个医生，科学家，并促进他过渡到一个独立的研究者和科学界的贡献成员。高血压是一个巨大的公共卫生问题，发病率和死亡率惊人。由于遗传性高血压与钠重吸收密切相关，并且我们的许多高血压治疗方法都抑制NCC活性，因此了解NCC活性的调节将使我们深入了解高血压的发病机制，并有一天可能提供进一步的治疗途径来控制高血压。

DESCRIPTION (provided by applicant): Hypertension represents a major public health concern. To date, each form of inherited hypertension has been linked to defective salt handling in the kidney. This application seeks to explore the mechanisms involved in the regulation of one of the key salt-handling transporters in the kidney, the sodium chloride co- transporter (NCC) located in distal convoluted tubule. Previous studies have reported decreased NCC activity in response to the presence of WNK4 (a kinase associated with a form of genetic hypertension called Gordon's Syndrome) and phorbol esters (chemical analogs of diacylglycerol, a common pathway for hormonal stimulation) but the mechanism by which NCC activity is reduced is unclear. This application will seek to demonstrate that WNK4 and phorbol esters decrease NCC activity by decreasing the amount of NCC expressed at the cell surface, a process that occurs by enhancing the ubiquitination of NCC, thereby promoting degradation of the co-transporter. The experimental model will be a cell model utilizing an immortalized mouse distal convoluted tubule cell line with native NCC activity and intact regulatory pathways. The association between activity, surface expression, and ubiquitination will be established by measuring NCC activity via radiotracer uptake, NCC cell surface expression via biotinylation and immunofluorescence, and ubiquitination via immunoprecipitation and immunoblotting in cells gene-silenced for WNK4 or treated with phorbol esters. The mechanisms of NCC ubiquitination will also be determined, including sites of ubiquitination and required enzymes. This project will help train Dr. Benjamin Ko as a physician-scientist and facilitate his transition to an independent investigator and contributing member of the scientific community. Public Health Relevance Hypertension represents a tremendous public health concern with staggering morbidity and mortality. As inherited hypertension is intimately tied to sodium reabsorption and many of our treatments for hypertension inhibit NCC activity, understanding the regulation of NCC activity will give us insight into the pathogenesis of hypertension and may one day provide further treatment avenues to control hypertension.