Molecular mechanisms of WNK-SPAK/OSR1 regulation of transepithelial ion transport in the Drosophila renal tubule

WNK-SPAK/OSR1调节果蝇肾小管跨上皮离子转运的分子机制

• 批准号: 9480212
• 负责人: AYLIN RACHEL RODAN
• 金额: $ 33.98万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-08 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9480212
• 关键词: Molecular; mechanisms; WNK; SPAK; OSR1

PROJECT SUMMARY Disorders of transepithelial ion transport underlie clinical disorders of extracellular volume, blood pressure, and electrolytes, but molecular mechanisms of transepithelial ion transport are difficult to directly examine in the mammalian nephron. The applicants' long-term goal is to better understand epithelial ion transport mechanisms relevant to human kidney function, in sufficient molecular detail to define new therapeutic strategies. The overall objective of this application is to identify regulators of a kinase cascade, consisting of WNK (With No Lysine) and SPAK/OSR1 (Ste20-related proline alanine rich kinase/oxidative stress response) kinases, that plays an essential role in sodium and potassium homeostasis through the regulation of renal transepithelial ion transport. The application builds on three recent findings: Cl- directly binds to the WNK kinase domain to inhibit autophosphorylation and activation; the scaffold protein Mo25 (Mouse protein 25/Cab39) enhances the activity of SPAK/OSR1; and low potassium diet activates WNK-SPAK/OSR1 signaling. The central hypothesis is that transepithelial ion flux is directly regulated by transported ions (Cl- and K+) through modulation of WNK-SPAK/OSR1 signaling, while Mo25 provides additional regulatory control. The rationale is that better understanding of these molecular mechanisms will allow the design of novel therapeutics with fewer off-target effects. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the roles of Cl- and K+ in the regulation of WNK isoforms in transepithelial ion transport; 2) Determine the role of Mo25 in WNK signaling in a transporting epithelium; and 3) Probe tubule physiology using newly developed chemical WNK inhibitors. The approach is innovative by bridging fundamental molecular insights gained from biophysical studies, with the functional physiological roles of those molecular mechanisms, using newly developed platforms and tools to probe questions of transporting epithelium biology. Assays have been established, and demonstrated feasible in the investigators' hands, to examine regulation of Drosophila and mammalian WNKs by Cl- and K+ in vitro and in the fly renal tubule, and to measure intracellular Cl- in live tubules, with temporal resolution; and to measure transepithelial ion flux in genetically modified, or pharmacologically treated, tubules. The proposed research is significant, because it is expected to advance understanding of molecular mechanisms of WNK-SPAK/OSR1 regulation in a transporting renal epithelium. The studies will determine: 1) how quickly changes in intracellular Cl- change WNK activity; 2) whether WNKs act as K+ sensors; and 3) the role of Mo25 in transepithelial ion transport. In addition, these studies will further develop recently identified pharmacological WNK inhibitors, which will be a useful tool for further probing the biology of WNK-SPAK/OSR1 signaling in Drosophila and mammalian systems, and potentially serve as the basis for future development of therapeutic compounds for the treatment of volume overload, hypertension and hyperkalemia.

项目摘要 细胞外体积，血压和 电解质，但是很难直接检查旋转离子转运的分子机制 哺乳动物肾单位。申请人的长期目标是更好地了解上皮离子运输 与人肾功能相关的机制，有足够的分子细节来定义新的治疗 策略。该应用的总体目的是确定激酶级联反应的调节剂，包括 WNK（无赖氨酸）和SPAK/OSR1（Ste20相关的丙氨酸丙氨酸激酶/氧化应激反应） 激酶，通过调节肾脏在钠和钾稳态中起着至关重要的作用 transepithialial离子运输。该应用程序建立在最近的三个发现：Cl-直接与WNK结合 激酶结构域抑制自磷酸化和激活；支架蛋白MO25（小鼠蛋白 25/cab39）增强了SPAK/OSR1的活性；低钾饮食激活WNK-Spak/OSR1 信号。中心假设是旋转离子通量直接由转运离子（CL-和CL-和 K+）通过调制WNK-Spak/OSR1信号传导，而MO25提供了额外的调节控制。这 理由是更好地理解这些分子机制将允许设计新颖 具有更少脱靶效果的治疗剂。在强大的初步数据的指导下，中心假设将是 通过追求三个特定目的测试：1）确定Cl-和K+在WNK同工型调节中的作用 在transepithitial离子运输中； 2）确定MO25在WNK信号传导中的作用； 3）使用新开发的化学WNK抑制剂的探针小管生理。这种方法是创新的 通过弥合从生物物理研究中获得的基本分子见解，并具有功能生理学 这些分子机制的作用，使用新开发的平台和工具来探究问题 运输上皮生物学。已经建立了测定，并在调查人员中证明了可行 手，检查果蝇和哺乳动物WNK的调节，并在苍蝇肾脏中进行 小管，并测量具有时间分辨率的活小管中的细胞内Cl-；并测量跨度 离子通量在基因修饰或药理处理的小管中。拟议的研究很重要， 因为有望提高对WNK-Spak/OSR1调控分子机制的理解 运输肾上皮。研究将确定：1）细胞内CL-变化的变化速度如何 WNK活动； 2）WNK是否充当K+传感器； 3）MO25在旋转离子转运中的作用。在 此外，这些研究还将进一步开发最近确定的药理学WNK抑制剂，这将是 有用的工具，用于进一步探测果蝇和哺乳动物中WNK-Spak/OSR1信号传导的生物学 系统，并有可能作为治疗的未来治疗化合物的基础 体积过载，高血压和高钾血症的体积。