Renal Regulation of the Magnesium Channel TRPM6 by Uromodulin

尿调节蛋白对镁通道 TRPM6 的肾脏调节

• 批准号: 9225927
• 负责人: Matthias Tilmann Florian Wolf
• 金额: $ 8.1万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-18 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9225927
• 关键词: Address; Affect; Animals; Apical; Cations; Cell surface; Cells; Childhood; Chronic; Chronic Kidney Failure; Cultured Cells; Data; Development; Diet; Disease; Distal convoluted renal tubule structure; Endocytosis; Epithelial; Feedback; Future; Gene Expression; General Population; Genetic Polymorphism; Goals; Health; Homeostasis; Human; Hypertension; Hypocalcemia result; Hypomagnesemia; Image; Impairment; In Vitro; Individual; Ion Channel; Kidney; Knock-out; Knowledge; Limb structure; Link; Magnesium; Metabolic syndrome; Mission; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Organ; Outcome; Physiological; Physiology; Population; Predisposition; Protein Biochemistry; Proteins; Public Health; Regulation; Role; Serum; Serum magnesium level observed; Side; System; TRP channel; Testing; Thick; Time; Tubular formation; UMOD gene; United States National Institutes of Health; Up-Regulation; Urine; Variant; Wild Type Mouse; Work; absorption; acute symptom; cohort; density; experience; extracellular; high risk; improved; in vitro activity; in vivo; insight; novel; patch clamp; receptor; response; translational impact; urinary; wasting; Address; Affect; Animals; Apical; Cations; Cell surface; Cells; Childhood; Chronic; Chronic Kidney Failure; Cultured Cells; Data; Development; Diet; Disease; Distal convoluted renal tubule structure; Endocytosis; Epithelial; Feedback; Future; Gene Expression; General Population; Genetic Polymorphism; Goals; Health; Homeostasis; Human; Hypertension; Hypocalcemia result; Hypomagnesemia; Image; Impairment; In Vitro; Individual; Ion Channel; Kidney; Knock-out; Knowledge; Limb structure; Link; Magnesium; Metabolic syndrome; Mission; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Organ; Outcome; Physiological; Physiology; Population; Predisposition; Protein Biochemistry; Proteins; Public Health; Regulation; Role; Serum; Serum magnesium level observed; Side; System; TRP channel; Testing; Thick; Time; Tubular formation; UMOD gene; United States National Institutes of Health; Up-Regulation; Urine; Variant; Wild Type Mouse; Work; absorption; acute symptom; cohort; density; experience; extracellular; high risk; improved; in vitro activity; in vivo; insight; novel; patch clamp; receptor; response; translational impact; urinary; wasting

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.

项目摘要/摘要 多达15％的人口患有低磁性血症，这与较高的发展风险有关 常见疾病，例如2型糖尿病和高血压。尿Mg2+损失有助于 低磁性血症，但了解肾脏MG2+稳态非常有限。该提议试图 提高我们对肾脏MG2+稳态的理解。受调节的MG2+重吸收的主要器官是 肾。最终的尿Mg2+浓度由不同的上皮MG2+通道TRPM6确定 曲折管（DCT）。静脉复合蛋白（UMOD）是肾脏MG2+处理的新修饰符，为UMOD基因 低磁性小鼠的表达增加。我们的初步数据证实了UMOD中的尿Mg2+浪费 敲除（UMOD - / - ）小鼠。此外，我们发现TRPM6全细胞电流密度和TRPM6细胞增加 表面丰富，并表达UMOD。我们该应用的总体目标是研究 关于MG2+通道TRPM6的调节的尿蛋白UMOD。我们的初步结果提出了 TAL分泌的UMOD的新型假设在DCT中上调了TRPM6活性。 通过损害TRPM6内吞作用，从而增加了通道细胞表面丰度，从而增强 块茎MG2+重吸收。我们假设在低Mg2+条件下，块茎UMOD分泌增加 然后，通过TRPM6增强了肾脏MG2+重吸收，以减轻MG2+损失。在AIM 1中，我们将测试 通过分析细胞外UMOD调节TRPM6活性的机制，可以在体外假设。使用 全细胞斑块钳记录和蛋白质生物化学在培养的细胞表达系统中，我们将测试是否是否 UMOD通过损害TRPM6内吞作用来增加TRPM6细胞表面丰度并检查哪个UMOD TRPM6调节需要域。在AIM 2中，我们将测试UMOD在肾脏MG2+方面的作用 在体内处理。如果在UMOD - / - 小鼠中，我们将通过免疫荧光成像检查 改善顶端TRPM6表达。研究UMOD是否是肾脏反馈机制的一部分 对于系统性的MG2+变化，我们将分析野生型和UMOD - / - 小鼠的反应，该反应受到正常质疑的 低MG2+饮食。鉴于我们在离子通道生理学，鼠标生理学，TRP通道方面的经验 和UMOD，我们有足够的能力进行这项研究。这项工作的贡献将非常重要 肾脏蛋白的肾脏MG2+调节代表了一个新颖的概念。我们预计会产生积极的翻译影响 在这个项目中，正如针对全身MG2+稳态的拟议反馈机制可能提供的 尿液Mg2+浪费的易感性的新颖解释。由于UMOD多态性会影响尿液UMOD 分泌，如果UMOD多态性有助于 通过下降UMOD分泌，尿液MG2+浪费，如果这些UMOD多态性与 常见疾病。我们假设特定的UMOD多态性降低了个人对 适应低MG2+条件，可以提供低磁性血症和常见疾病之间的联系。