Cisplatin-induced Alterations of Na+-dependent Phosphate Uptake in Renal Epithelial Cells

Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on Na+-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using [32P]-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, [14C]phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the Na+-dependent uptake without altering Na+-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of Na+-dependent phosphate uptake without any change in the Km value. Na+-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of Na+-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.

Changes in phosphate transporter activity evaluated by phosphonoformic acid binding in cadmium-exposed renal brush-border membranes

Direct exposure of renal tubular brush-border membranes (BBM) to free cadmium (Cd) causes a reduction in phosphate (Pi) transport capacity. Biochemical mechanism of this reduction was investigated in the present study. Renal proximal tubular brush-border membrane vesicles (BBMV) were isolated from rabbit kidney outer cortex by Mg precipitation method. Vesicles were exposed to 50~200 muM CdCl2 for 30 min, then the phosphate transporter activity was determined. The range of Cd concentration employed in this study was comparable to that of the unbound Cd documented in renal cortical tissues of Cd-exposed animals at the time of onset of renal dysfunction. The rate of sodium-dependent phosphate transport (Na+-Pi cotransport) by BBMV was determined by 32P-labeled inorganic phosphate uptake, and the number of Na+/-Pi cotransporters in the BBM was assessed by Pi-protectable 14C-labeled phosphonoformic acid ((14C)PFA) binding. The exposure of BBMV to Cd decreased the Na+-Pi cotransport activity in proportion to the Cd concentration in the preincubation medium, but it showed no apparent effect on the Pi-protectable PFA binding. These results indicate that an interaction of renal BBM with free Cd induces a reduction in Na+-Pi cotransport activity without altering the carrier density in the membrane. This, in turn, suggest that the suppression of phosphate transport capacity (Vmax) observed in Cd-treated renal BBM is due to a reduction in Na+-Pi translocation by existing carriers, possibly by Cd-induced fall in membrane fluidity.

Effect of phosphonoformic acid on vascular smooth muscle cell calcification induced by elevated phosphate / 中国药理学通报

AIM To observe the effect of phosphonoformic acid (PFA) in different concentration on vascular calcification induced by elevated phosphate. METHODS The cell culture of bovine aortic smooth muscle were performed. Calcium deposition in different phosphate and PFA concentrations were determined by O cresolphthalein complexone method, and osteocalcin expression by radioimmunity and RT PCR. RESULTS Compared to Pi 1 5 mmol?L -1 group,bovine smooth muscle cells (BSMC) cultured in medium containing Pi 2 0 mmol?L -1 phosphate level increased calcium deposition 〔On day 6, (77 187?11 692) mg?g -1 Pro versus (25 768?1 750) mg?g -1 Pro, P