Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium

Abstract

The uptake of labeled inorganic phosphate into primary rabbit kidney proximal tubule cells has been examined. Phosphate was accumulated into the primary proximal tubule cells against a concentration gradient. This accumulation was sensitive to inhibition by metabolic inhibitors. The dependence of phosphate uptake on the extracellular phosphate concentration was examined. Similarities were observed between primary proximal tubule cells and the LLC-PK1 cell line in these regards. These phosphate uptake data were then plotted on a Lineweaver-Burke plot. A nonlinear plot was obtained, which suggested that phosphate uptake occurs by means of a Na+ dependent, carrier mediated process, as well as by another Na+ independent mechanism. The pH dependence of phosphate uptake was also examined. Unlike previous observations with LLC-PK1 cells, optimal phosphate uptake occurred at pH 6.5. However, this difference between the two cell culture systems may possibly be explained by differences in uptake conditions. The dependence of phosphate uptake on the extracellular NaCl concentration was examined at three different pH values. The rate of phosphate uptake at pH 7.0 was observed to saturate at a lower NaCl concentration than at either pH 6.0 or pH 6.5. Furthermore, the optimal rate of phosphate uptake at pH 7.0 was observed to be higher than at the other two pH values studied when the NaCl concentration was below 120 mM. However, when the NaCl concentration was raised to 150 mM, optimal phosphate was observed to occur at pH 6.5 rather than at pH 7.0. These observations may be explained if the pH affects not only the rate of phosphate uptake but also the affinity of the phosphate uptake system for sodium. Phosphate uptake was also observed to be sensitive to several agents, Na2 X SO4 and NaSCN, which affect the membrane potential. As observed with phosphate uptake by LLC-PK1 (and renal brush border membrane vesicles), phosphate uptake was highly sensitive to inhibition by the phosphate analogue arsenate. Novel observations were that the phosphate analogue vanadate and its cellular metabolite vanadyl stimulated the initial rate of phosphate uptake.