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。到目前为止，每种形式的遗传性高血压都与肾脏盐处理缺陷有关。这项应用旨在探索肾脏中关键的盐分转运体之一--位于远端曲管的氯化钠共转运体(NCC)的调节机制。以前的研究已经报道了WNK4(一种与一种被称为戈登综合征的遗传性高血压有关的激酶)和佛波酯(一种二酰甘油的化学类似物，一种常见的激素刺激途径)的存在导致NCC活性降低，但NCC活性降低的机制尚不清楚。这项应用将试图证明WNK4和佛波酯通过减少细胞表面表达的NCC的量来降低NCC的活性，这一过程是通过增强NCC的泛素化从而促进辅助转运体的降解来实现的。实验模型将是利用永生化的小鼠远端曲管细胞系建立的细胞模型，该细胞系具有天然的NCC活性和完整的调控途径。在WNK4基因沉默或佛波酯处理的细胞中，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，通过免疫沉淀和免疫印迹测量泛素化，将建立活性、表面表达和泛素化之间的联系。还将确定NCC泛素化的机制，包括泛素化的位置和所需的酶。该项目将帮助培训本杰明·科博士成为一名内科科学家，并促进他转变为一名独立的研究员和科学界的贡献成员。高血压是一个巨大的公共卫生问题，其发病率和死亡率令人震惊。由于遗传性高血压与钠重吸收密切相关，而我们对高血压的许多治疗方法都抑制NCC活性，了解NCC活性的调节将使我们深入了解高血压的发病机制，并有可能有一天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.