Regulation of Renal Tubular Mg2+ Handling by MUC1 and Lactation - PROJECT SUMMARY Magnesium is critical for physiology. Deficiency influences bone health, promoting osteoporosis. Despite its importance, and in contrast to other essential minerals, little is known about systemic regulation of Mg2+ balance. Our goal is to identify mechanisms used by the kidney to modify urinary Mg2+ excretion. Lactation depletes bodily Mg2+ stores and activates Mg2+ conservation in the kidney. As such, lactation provides a unique setting for exploring mechanisms modulating Mg2+ reabsorption in the nephron. This proposal explores mechanisms of lactation-associated Mg2+ conservation (LAMC) at systemic, tissue, cellular, and molecular levels. At the systemic level, we hypothesize that renal tubular parathyroid hormone receptor (PTHR) signaling stimulates renal tubular changes in lactation. Lactation-induced changes in gene expression in the nephron are highest for genes in segments expressing the PTHR. PTHR signaling in lactation is stimulated by PTH-related peptide secreted by the mammary gland. We will determine whether lactation-associated kidney changes depend upon PTHR signaling using a selectively kidney PTHR-deficient mouse. At the tissue level, we hypothesize that lactation remodels the nephron. In lactation, cells in a Mg2+-transporting nephron segment, the distal convoluted tubule, enhance expression of cyclin D1, suggesting cell proliferation. This could enhance surface area for Mg2+ reabsorption. Lactation-induced changes in tubule segment length and cell number will be explored using 2D stereometry, 3D microscopy, and single nuclear RNA-Seq. At the cellular level, we hypothesize that LAMC depends upon expression of the Mg2+-selective ion channel, TRPM6. Among known magnesiotropic genes, Trpm6 is unique in being upregulated in lactation. We will determine whether increased Trpm6 mRNA reflects a larger TRPM6-expressing cell population versus increasing the number of copies per cell. We will determine whether lactation enhances TRPM6 ion channel activity. A signaling pathway that influences LAMC is indicated by our observation that the glycoprotein MUC1, expressed in the distal nephron, is required for LAMC. A human MUC1 variant is associated with hypomagnesemia. We find that this variant impairs stimulation of EGFR activity by MUC1. Because EGFR stimulation is known to enhance TRPM6 activity, we will determine whether MUC1 modulates TRPM6 activity through the EGFR signaling pathway. At the molecular level, we have determined that inhibition of AKT, an effector within the EGFR signaling pathway, impairs activation of TRPM6. We have identified putative phosphorylation sites within TRPM6, and we will determine whether mutation of these sites influences stimulation of TRPM6 by MUC1 and EGFR signaling. These studies will provide insights regarding mechanisms that drive Mg2+ reabsorption in the kidney tubule and interventions to improve Mg2+ balance to improve bone health.
