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

尿调节蛋白在肾钙通道 TRPV5 调节中的作用

• 批准号: 8507230
• 负责人: Matthias Tilmann Florian Wolf
• 金额: $ 14.53万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8507230
• 关键词: Acetylglucosamine; Alleles; Animals; Basic Science; Binding; Blood; Ca(2+)-Transporting ATPase; Calcium; Calcium Channel; Calculi; Carrier Proteins; Cell surface; Cleaved cell; Crystal Formation; Cultured Cells; Data; Development; Development Plans; Disease; Distal convoluted renal tubule structure; Down-Regulation; Electrophysiology (science); Endocrine; Endocytosis; Epithelial; Excision; Excretory function; Extracellular Domain; Fellowship; Galactose; Galectin 1; Genes; Genetic; Goals; Homeostasis; Hormones; Human; Impairment; In Vitro; Ion Transport; K-Series Research Career Programs; Kidney; Kidney Calculi; Knowledge; Lead; Lectin; Limb structure; Link; Mediating; Membrane Proteins; Mentors; Molecular; Mus; Mutant Strains Mice; Mutation; Nephrolithiasis; Neuraminidase; Pathway interactions; Physicians; Physiological; Physiology; Polysaccharides; Probability; Protein Biochemistry; Proteins; Regulation; Research; Research Personnel; Risk Factors; Role; Scientist; Sialic Acids; Side; Sodium Chloride; Staining method; Stains; Surface; System; Testing; Thick; Training; Training Programs; Transgenic Mice; Tubular formation; UMOD gene; United States National Institutes of Health; Up-Regulation; Urine; Water; Work; absorption; anti aging; apical membrane; base; basolateral membrane; calbindin-D28K; calcium absorption; career development; extracellular; genome wide association study; glycosylation; hypercalciuria; improved; in vivo; mouse model; mutant; novel; overexpression; paracrine; patch clamp; protective effect; protein expression; research study; treatment strategy; urinary

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.

描述（由申请人提供）：这项NIH指导的职业发展奖提案描述了为候选人提供的为期五年的培训计划，该计划的医生科学家的长期目标是成为独立的学术研究员，研究重点是与肾脏生理学和疾病有关的上皮离子运输。为了实现这些目标，候选人及其导师制定了一项综合计划，其中包括新颖的科学思想，基础科学研究的高级培训以及详细的职业发展计划。该候选人提议在其博士后研究和研究金培训期间开发的基础研究的背景基础上，尤其是他先前在尿液统治（UMOD）（UMOD）方面的发现，这是一种蛋白质，该蛋白质会导致小鼠中引起肾结石和高钙尿症。大多数管状Ca2+通过细胞细胞机制在近端小管中重新吸收。但是，在远端曲折小管（DCT）和连接小管（CNT）中，Ca2+通过CA2+通道TRPV5通过跨侧运输吸收。突变体UMOD小鼠中的尿分析表明，在DCT和CNT中，TRPV5是最丰富的钙通道中的钙吸收受损。 TRPV5由抗衰老蛋白Klotho调节。 klotho充当唾液酸酶，在N-糖基化的TRPV5通道处暴露于半乳糖-N-乙酰葡萄糖胺，然后与无处不在的凝集素结合到Galectin-1。这会导致TRPV5内吞作用损害和TRPV5表面丰度增加。我的初步数据表明，UMOD上调TRPV5，并且在这方面，UMOD和Klotho在同一途径中起作用。该提案旨在提高UMOD如何干扰肾脏Ca2+吸收的理解。我的目的是分析UMOD的TRPV5上调的分子机制；检查UMOD在调节体内肾脏Ca2+排泄中的生理作用；并通过UMOD和Klotho检查TRPV5共同调节的机制，并在体内测试显着性。我将利用电生理学，蛋白质生物化学和小鼠遗传学测试我的假设。我将通过测试UMOD对TRPV5内吞作用，TRPV5单通道开放概率和电导的影响来分析UMOD如何增加TRPV5活性。我将测试突变UMOD的分泌减少是否可能导致TRPV5激活较少，从而导致高钙尿症。我将通过分析来自突变体UMOD和野生型动物的分离天然小管中的TRPV5表达和活性来测试我体外发现的重要性。我将检查UMOD和Klotho如何共同调节TRPV5的机制。为了分析UMOD和Klotho是否在体内调节肾脏Ca2+吸收，我将测试Klotho过表达是否可以挽救因突变体UMOD引起的高钙。交叉的突变体；过表达的Klotho小鼠将分析DCT/CNT中的肾脏Ca2+排泄，TRPV5表达和活性。这些实验获得的结果将改善高钙尿的知识，这是肾结石的重要危险因素，并可能有助于开发肾石石症中新型治疗策略。