Magnesium handling by the distal nephron

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Project summary Magnesium (Mg 2+ ) is an essential cofactor in many cellular processes, and disruption of Mg 2+ homeostasis can be life-threatening. Serum Mg 2+ is maintained within a narrow normal range by regulated reabsorption in the kidney. Mg The distal 2+ channel TRPM6/7, but reabsorption convoluted tubule (DCT) reabsorbs only a small fraction of the filtered Mg via the is tightly regulated in this segment. Most genetic causes of 2+ (10%) hypomagnesemia affecting DCT Mg2+ reabsorption. In (NCC) remain from diuretics. Mg NCC species that secretion, Familial changes the DCT, Na + reabsorption via the NaCl cotransporter plays a critical role in transcellular Mg + reabsorption, but the mechanisms linking the two processes unclear. Hypomagnesemia is observed in Gitelman and EAST syndromes, both of which ultimately arise loss of NCC activity, and following pharmacological NCC blockade with t he commonly used thiazide Chronically, DCT atrophy is observed with loss of NCC activity, and the resulting l oss of capacity for 2+ eabsorption s believed to be the major mechanism of renal Mg 2+ wasting. However, the early effects of inhibition on renal Mg 2+ handling are less clear. Our preliminary data in mice support findings in other that thiazides transiently lower, rather than increase, urinary Mg 2+ excretion. Based on this we propose reduced Mg 2+ excretion occurs following an acute K + load, which inhibits NCC to promote downstream K + serving to preserve serum Mg 2+ . In contrast, renal Mg 2+ handling appears norma in the disease Hyperkalemic Hypertension (FHHt), in which NCC is hyperactivated. Aim 1 will test the hypothesis that in DCT 2 r i l Na+ reabsorption modify Mg2+ handling prior to DCT remodeling, and this is physiologically relevant. We will use inducible mouse models of Gitelman syndrome, EAST syndrome, thiazide administration, and K+ loading to test this. We will perform time-course analyses and measure changes in electrolyte handling, and in DCT structure with optical tissue clearing and 3-D imaging. Hypomagnesemia and hypokalemia are commonly seen together clinically e.g. following cisplatin chemotherapy. Hypokalemia is often refractory to K+ supplementation unless hypomagnesemia is resolved, but the underlying mechanisms have not been determined. Aim 2 will test two proposed mechanisms that promote K+ secretion along the connecting segment and are supported by our preliminary data: (i) Mg2+-dependent disinhibition of the K+ channel ROMK and (ii) increased Na+ delivery from DCT. We will determine NCC, ENaC, and ROMK activities in hypomagnesemic mice and a new mouse model of hypomagnesemia/hypokalemia by performing diuretic response tests. We will also test whether Mg2+ supplementation can mitigate K+ losses in mouse models with inducible NCC inhibition. The apical K+ channel Kv1.1 has been proposed to generate the membrane potential that provides the drive for Mg2+ entry along the DCT, since human Kv1.1 mutations cause hypomagnesemia. However, experimental evidence is lacking. To test this, In Aim 3 we will phenotype a novel renal tubule-specific Kv1.1 knockout mouse, and determine whether Kv1.1 determines the apical membrane potential in the early DCT.