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.
Low serum magnesium levels are associated with a higher risk to develop common disorders such as diabetes mellitus type 2 and hypertension, which is relevant for public health, as low magnesium levels occur in up to 15% of the population. Because urinary Mg2+ wasting contributes to low serum magnesium levels, we propose to study the regulation of the apical Mg2+ channel TRPM6 by the most abundant urinary protein Uromodulin (UMOD), which upregulates TRPM6 channels in the kidney. This application is relevant to the NIH?s mission as specific UMOD variants decrease urinary UMOD secretion, which may contribute to urinary magnesium wasting and low serum magnesium levels, thus predisposing carriers of these UMOD variants to a higher risk for developing diabetes mellitus type 2 and hypertension.
|Nie, Mingzhu; Bal, Manjot S; Liu, Jie et al. (2018) Uromodulin regulates renal magnesium homeostasis through the ion channel transient receptor potential melastatin 6 (TRPM6). J Biol Chem 293:16488-16502|
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|Wolf, Matthias Tilmann Florian (2017) Inherited and acquired disorders of magnesium homeostasis. Curr Opin Pediatr 29:187-198|