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

描述（由申请人提供）：这份由NIH指导的职业发展奖提案描述了一个为期五年的培训计划，该候选人是一名内科科学家，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运输。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博士后研究和奖学金培训期间的基础研究背景上，特别是他之前在尿调素（UMOD）的遗传学和表达方面的发现，UMOD是一种蛋白质，如果改变会导致肾结石和小鼠高钙尿症。大多数小管Ca2+通过细胞旁机制在近端小管中被重吸收。然而，在远曲小管（DCT）和连接小管（CNT）中，Ca2+通过Ca2+通道TRPV5通过上皮运输被吸收。突变型Umod小鼠的尿液分析表明，DCT和CNT的钙吸收受损，其中TRPV5是最丰富的钙通道。TRPV5受抗衰老蛋白Klotho调控。Klotho作为唾液酸酶发挥作用，在n-糖基化的TRPV5通道上暴露半乳糖- n-乙酰氨基葡萄糖，然后结合半乳糖凝集素-1，一种普遍存在的凝集素。这导致TRPV5内吞作用受损，TRPV5表面丰度增加。我的初步数据表明，UMOD上调TRPV5，并且UMOD和Klotho在这方面的相同途径上起作用。该建议旨在提高对UMOD如何干扰肾脏Ca2+吸收的理解。我的目的是分析UMOD上调TRPV5的分子机制；探讨UMOD在体内调节肾脏Ca2+排泄中的生理作用；探讨UMOD和Klotho共同调控TRPV5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。我将通过测试UMOD对TRPV5内吞作用、TRPV5单通道打开概率和电导的影响来分析UMOD是如何增加TRPV5活性的。我将测试突变型UMOD分泌减少是否会导致TRPV5激活减少，从而导致高钙尿症。我将通过分析突变型Umod和野生型动物分离的天然小管中TRPV5的表达和活性来验证我的体外研究结果的意义。我将研究UMOD和Klotho共同调控TRPV5的机制。为了分析UMOD和Klotho是否在体内共同调节肾脏Ca2+吸收，我将测试Klotho过表达是否可以挽救突变型UMOD引起的高钙尿症。杂交突变体Umod；我们将分析过表达Klotho小鼠的肾脏Ca2+排泄、TRPV5的表达和DCT/CNT的活性。这些实验的结果将提高人们对高钙尿症（肾结石的重要危险因素）的认识，并可能有助于开发新的肾结石治疗策略。

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.