Renal Regulation of the Magnesium Channel TRPM6 by Uromodulin

项目摘要/摘要 高达15%的人口患有低镁血症，这与更高的发病风险有关。 常见疾病，如2型糖尿病和高血压。尿中镁离子流失导致 低镁血症，但对肾脏镁离子稳态的了解非常有限。这项建议旨在 提高对肾脏镁离子动态平衡的认识。调节镁离子重吸收的主要器官是 肾脏。尿镁最终浓度由远端上皮细胞镁离子通道TRPM6决定 曲管(DCT)。尿调蛋白(Umod)作为Umod基因可能是肾脏镁离子处理的新调节剂 在低镁血症小鼠中表达增加。我们的初步数据证实了UMOD患者的尿镁流失 敲除(Umod-/-)小鼠。此外，我们还发现TRPM6全细胞电流密度增加，TRPM6细胞 表面丰度与UMOD表达。我们这项申请的总体目标是研究 尿蛋白UMOD与镁离子通道TRPM6的调节我们的初步结果提高了 从TAL分泌的UMOD从管腔侧上调DCT中的TRPM6活性的新假说 通过损害TRPM6的内吞作用，从而增加通道细胞表面的丰度，从而增强 管状镁离子重吸收。我们假设，在低镁离子条件下，肾小管UMOD分泌增加 然后通过TRPM6加强肾脏对镁的重吸收，以减轻镁离子的损失。在目标1中，我们将测试这一点 通过分析细胞外UMOD调节TRPM6活性的机制，提出了体外假说。vbl.使用 全细胞膜片钳记录和培养细胞表达系统中的蛋白质生物化学，我们将测试 UMOD通过损害TRPM6的内吞作用增加TRPM6细胞表面的丰度，并检查哪个UMOD TRPM6的监管需要域名。在目标2中，我们将测试UMOD对肾脏镁离子的作用 在体内处理。我们将通过免疫荧光成像检查Umod-/-小鼠是否存在尿镁流失 降低心尖部TRPM6的表达。研究UMOD是否是肾脏反馈机制的一部分 对于系统性的镁离子变化，我们将分析野生型和Umod-/-小鼠对正常、 低镁和高镁日粮。鉴于我们在离子通道生理学、小鼠生理学、色氨酸通道方面的经验 UMOD，我们有很好的装备来进行这项研究。这项工作的贡献将是巨大的，因为 尿蛋白对肾脏镁离子的调节代表了一个新的概念。我们预计会产生积极的翻译影响 由于所提出的针对系统性镁离子稳态的反馈机制可能提供一种 对尿镁消耗倾向的新解释。UMOD基因多态性影响尿中UMOD 分泌物，我们将在未来的R01应用中在人类队列中测试UMOD多态是否有助于 UMOD分泌减少导致的尿镁排出减少以及这些UMOD基因是否与 常见病。我们假设特定的UMOD多态会降低个体对 调整到低镁条件，这可能是低镁血症和常见疾病之间的联系。

PROJECT SUMMARY/ABSTRACT Up to 15% of the population has hypomagnesemia which is associated with a higher risk of developing common disorders like diabetes mellitus type 2 and hypertension. Urinary Mg2+ losses contribute to hypomagnesemia, but knowledge about renal Mg2+ homeostasis is very limited. This proposal seeks to improve our understanding of renal Mg2+ homeostasis. The major organ for regulated Mg2+ reabsorption is the kidney. The final urinary Mg2+ concentration is determined by the epithelial Mg2+ channel TRPM6 in the distal convoluted tubule (DCT). Uromodulin (Umod) is a possible new modifier of renal Mg2+ handling as Umod gene expression is increased in hypomagnesemic mice. Our preliminary data confirm urinary Mg2+ wasting in Umod knock-out (Umod-/-) mice. In addition, we found increased TRPM6 whole-cell current density and TRPM6 cell surface abundance with UMOD expression. Our overall objective of this application is to study the effect of the urinary protein UMOD regarding the regulation of the Mg2+ channel TRPM6. Our preliminary results raise a novel hypothesis that UMOD, secreted from TAL, upregulates TRPM6 activity in the DCT from the luminal side by impairing TRPM6 endocytosis, thereby increasing channel cell surface abundance and so enhancing tubular Mg2+ reabsorption. We hypothesize that in low Mg2+ conditions, tubular UMOD secretion increases which then enhances renal Mg2+ reabsorption via TRPM6 to mitigate Mg2+ losses. In aim 1, we will test this hypothesis in vitro by analyzing the mechanism by which extracellular UMOD regulates TRPM6 activity. Using whole-cell patch-clamp recording and protein biochemistry in cultured cell expression systems, we will test if UMOD increases TRPM6 cell surface abundance by impairing TRPM6 endocytosis and examine which UMOD domains are required for TRPM6 regulation. In aim 2, we will test the role of UMOD regarding renal Mg2+ handling in vivo. We will examine by immunofluorescent imaging if urinary Mg2+ wasting in Umod-/- mice is due to decreased apical TRPM6 expression. To study if UMOD is part of a renal feedback mechanism responding to systemic Mg2+ changes, we will analyze the response of wild-type and Umod-/- mice challenged with normal, low and high Mg2+ diets. Given our experience in ion channel physiology, mouse physiology, TRP channels and UMOD, we are very well equipped to perform this study. The contribution of this work will be significant as renal Mg2+ regulation by a urinary protein represents a novel concept. We expect a positive translational impact of this project, as the proposed feedback mechanism targeting systemic Mg2+ homeostasis may provide a novel explanation for a predisposition for urinary Mg2+ wasting. As UMOD polymorphisms affect urinary UMOD secretion, we will test in a future R01 application in a human cohort if UMOD polymorphisms contribute to urinary Mg2+ wasting by decreased UMOD secretion and if these UMOD polymorphisms are associated with common disorders. We hypothesize that specific UMOD polymorphisms reduce an individual’s adaptability to adjust to low Mg2+ conditions which could provide a link between hypomagnesemia and common disorders.