The Role of Uromodulin in the Regulation of the Renal Calcium Channel TRPV5

描述（由申请人提供）：这个NIH指导的职业发展奖提案描述了候选人的五年培训计划，候选人是一名医生科学家，其长期目标是成为一名独立的学术研究者，研究重点是与肾脏生理学和疾病相关的上皮离子转运。为了实现这些目标，候选人和他的导师制定了一个综合计划，包括新颖的科学思想，基础科学研究的高级培训和详细的职业发展计划。候选人建议建立在他的博士后研究和奖学金培训期间开发的基础研究的背景上，特别是他以前在尿调蛋白（UMOD）的遗传学和表达方面的发现，这种蛋白质如果改变会导致小鼠肾结石和高钙尿症。大部分肾小管钙离子通过细胞旁机制在近端小管重吸收。然而，在远曲小管（DCT）和连接小管（CNT）中，Ca 2+通过Ca 2+通道TRPV 5经由跨上皮转运被吸收。突变Umod小鼠的尿液分析表明DCT和CNT中的钙吸收受损，其中TRPV 5是最丰富的钙通道。TRPV 5受Klotho（一种抗衰老蛋白）调节。Klotho作为唾液酸酶发挥作用，将半乳糖-N-乙酰葡糖胺暴露于N-糖基化TRPV 5通道，然后与半乳糖凝集素-1（一种普遍存在的凝集素）结合。这导致TRPV 5内吞作用受损和TRPV 5表面丰度增加。我的初步数据表明，UMOD上调TRPV 5，并且UMOD和Klotho在这方面以相同的途径工作。该提案旨在提高对UMOD如何干扰肾Ca 2+吸收的理解。本研究的目的是分析UMOD上调TRPV 5的分子机制;研究UMOD在体内调节肾脏Ca 2+排泄中的生理作用;研究UMOD和Klotho共同调节TRPV 5的机制并检验其在体内的意义。我将利用电生理学，蛋白质生物化学和小鼠遗传学来验证我的假设。通过检测UMOD对TRPV 5内吞、TRPV 5单通道开放概率和电导的影响，分析UMOD如何增加TRPV 5活性。我将测试突变体UMOD的分泌减少是否可能导致TRPV 5活化减少，从而引起高钙尿症。我将通过分析突变型Umod和野生型动物分离的天然小管中TRPV 5的表达和活性来测试我的体外研究结果在体内的意义。我将研究UMOD和Klotho如何共同调节TRPV 5的机制。为了分析UMOD和Klotho是否在体内一起调节肾Ca 2+吸收，我将测试Klotho过表达是否可以挽救由于突变UMOD引起的高钙尿症。将分析交叉突变型Umod;过表达Klotho小鼠的肾脏Ca 2+排泄、TRPV 5表达和DCT/CNT中的活性。通过这些实验获得的结果将提高对高钙尿症的认识，高钙尿症是肾结石的一个重要危险因素，并可能有助于肾结石新治疗策略的发展。

DESCRIPTION (provided by applicant): This NIH mentored Career Development Award proposal describes a five year training program for the candidate, a physician scientist with the long-term goal of becoming an independent academic investigator with a research focus on epithelial ion transport related to kidney physiology and diseases. To accomplish these goals the candidate and his mentors developed an integrated plan encompassing novel scientific ideas, advanced training in basic science research and a detailed career development plan. The candidate proposes to build on a background in basic research developed during his postdoctoral research and fellowship training, specifically on his previous findings in genetics and expression of Uromodulin (UMOD), a protein which if altered causes kidney stones and hypercalciuria in mice. Most of the tubular Ca2+ is reabsorbed in the proximal tubule by paracellular mechanisms. However, in the distal convoluted tubule (DCT) and connecting tubule (CNT) Ca2+ is absorbed via transepithelial transport by the Ca2+ channel TRPV5. Urinary analysis in mutant Umod mice suggests impaired calcium absorption in the DCT and CNT where TRPV5 is the most abundant calcium channel. TRPV5 is regulated by Klotho, an anti-aging protein. Klotho functions as a sialidase, exposing galactose-N-acetylglucosamine at N-glycosylated TRPV5 channels, which then bind to galectin-1, a ubiquitous lectin. This results in impairment of TRPV5 endocytosis and increased TRPV5 surface abundance. My preliminary data indicate that UMOD upregulates TRPV5 and that UMOD and Klotho work in the same pathway in this respect. This proposal seeks to improve the understanding how UMOD interferes with renal Ca2+ absorption. My aims are to analyze what the molecular mechanism of TRPV5 upregulation by UMOD is; to examine the physiological role of UMOD in regulating renal Ca2+ excretion in vivo; and to examine the mechanism of TRPV5 co-regulation by UMOD and Klotho and test the significance in vivo. I will test my hypotheses utilizing electrophysiology, protein biochemistry and mouse genetics. I will analyze how UMOD increases TRPV5 activity by testing UMOD's impact on TRPV5 endocytosis, TRPV5 single channel open probability and conductance. I will test if decreased secretion of mutant UMOD may contribute to less TRPV5 activation thus causing hypercalciuria. I will test the significance of my in vitro findings in viv by analyzing TRPV5 expression and activity in isolated native tubules from mutant Umod and wild type animals. I will examine the mechanism how UMOD and Klotho co-regulate TRPV5. To analyze if UMOD and Klotho regulate renal Ca2+ absorption together in vivo I will test if Klotho overexpression can rescue hypercalciuria due to mutant UMOD. Crossed mutant Umod; overexpressing Klotho mice will be analyzed for renal Ca2+ excretion, TRPV5 expression and activity in the DCT/CNT. Results obtained by these experiments will improve the knowledge of hypercalciuria, a significant risk factor for kidney stones, and may contribute to the development of novel treatment strategies in nephrolithiasis.