Phosphate depletion diminishes Mg2+ uptake in mouse distal convoluted tubule cells

Abstract

Hypophosphatemia caused by phosphate depletion is associated with renal magnesium wasting. The cellular mechanisms of phosphate depletion were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration, [Mg2+](i), was determined by microfluorescence. Mg2+ transport was assessed as a function of change in [Mg2+](i) with time following placement of Mg2+-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+ into Mg2+-depleted cells cultured in normal phosphate, 1.0 mM, was 175 ± 21 nM/second. Depletion of phosphate in the culture media was associated with a significant decrease in Mg2+ uptake, which was dependent on the degree of phosphate depletion and on the time cultured in hosphate-deficient media. Cells cultured for 16 hours in 0.3 mM and 0 mM phosphate possessed Mg2+ uptake rates of 105 ± 18 nM/second and 15 ± 12 nM/second, respectively. Diminished Mg2+ uptake was rapidly induced following placement in low phosphate and was fully reversed following readdition of phosphate to the culture media. The effects of phosphate depletion on Mg2+ uptake was post-translational in nature as fully up-regulated MDCT cells with maximal Mg2+ uptake was associated with a rapid decrease (within 30 min) in Mg2+ transport when placed in phosphate-deficient media. Although Mg2+ uptake is altered by the transmembrane voltage, diminished Mg2+ uptake associated with phosphate depletion was not dependent on changes in membrane voltage. Further, it was not associated with a sustained increase in intracellular Ca2+ concentration. Chlorothiazide, probably through hyperpolarization of the plasma membrane, stimulates Mg2+ uptake in normal, 283 ± 23 nM/second, and phosphate-depleted cells, 203 ± 29 nM/second, but failed to entirely correct the defective transport. These studies demonstrate that magnesium wasting associated with hypophosphatemia and phosphate depletion is due, in part, to diminished Mg2+ transport in the distal convoluted tubule. The evidence is that the actions of phosphate deficiency are through alterations of Mg2+ transport across the luminal membrane of the distal convoluted tubule cell.