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 跨上皮离子转运障碍是细胞外容量、血压和血糖等临床疾病的基础。 电解质，但跨上皮离子转运的分子机制很难直接检查， 哺乳动物肾单位申请人的长期目标是更好地了解上皮细胞离子转运 与人类肾功能相关的机制，以足够的分子细节来定义新的治疗方法 战略布局本申请的总体目标是鉴定激酶级联的调节剂，其由以下组成： WNK（不含赖氨酸）和SPAK/OSR 1（Ste 20相关脯氨酸丙氨酸富集激酶/氧化应激反应） 激酶，通过调节肾脏的钠和钾稳态发挥重要作用 跨上皮离子转运该应用建立在三个最近的发现：Cl-直接结合到WNK 激酶结构域，以抑制自磷酸化和活化;支架蛋白Mo 25（小鼠蛋白 25/Cab 39）增强SPAK/OSR 1活性，低钾饮食激活WNK-SPAK/OSR 1 信号中心假设是跨上皮离子通量直接受转运离子（Cl-和Cl-）调节。 K+）通过调节WNK-SPAK/OSR 1信号传导，而Mo 25提供额外的调节控制。的 基本原理是，更好地理解这些分子机制将允许设计新的 具有更少脱靶效应的治疗剂。在强有力的初步数据的指导下，中心假设将是 通过追求三个具体目标进行测试：1）确定Cl-和K+在WNK亚型调节中的作用 2）确定Mo 25在转运上皮中WNK信号传导中的作用; 和3）使用新开发的化学WNK抑制剂探测小管生理学。方法是创新的 通过将从生物物理学研究中获得的基本分子见解与功能生理学研究联系起来， 这些分子机制的作用，使用新开发的平台和工具来探讨这些问题， 运输上皮生物学已经建立了检测方法，并在研究者的研究中证明了其可行性。 手，检查调节果蝇和哺乳动物WNK的Cl-和K+在体外和苍蝇肾 肾小管，并测量细胞内Cl-在肾小管，与时间分辨率;并测量跨上皮细胞 在遗传修饰的，或微生物处理的，小管中的离子通量。拟议的研究意义重大， 因为它有望促进对WNK-SPAK/OSR 1调节的分子机制的理解， 一种运输肾上皮。这些研究将确定：1）细胞内Cl-变化的速度 WNK活性; 2）WNK是否充当K+传感器;和3）Mo 25在跨上皮离子转运中的作用。在 此外，这些研究将进一步开发最近确定的药理学WNK抑制剂，这将是一个新的研究领域。 用于进一步探索果蝇和哺乳动物中WNK-SPAK/OSR 1信号传导的生物学的有用工具 系统，并可能作为基础，为未来的发展，治疗化合物的治疗 容量超负荷高血压和高钾血症