Hydrogen Peroxide-induced Alterations in Na+-phosphate Cotransport in Renal Epithelial Cells
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Abstract
This study was undertaken to examine the effect of oxidants on membrane transport function in renal epithelial cells. Hydrogen peroxide (H2O2) was used as a model oxidant and the membrane transport function was evaluated by measuring Na+-dependent phosphate (Na+-Pi) uptake in opossum kidney (OK) cells. H2O2 inhibited Na+-Pi uptake in a dose-dependent manner. The oxidant also caused loss of cell viability in a dose-dependent fashion. However, the extent of inhibition of the uptake was larger than that in cell viability. H2O2 inhibited Na+-dependent uptake without any effect on Na+-independent uptake. H2O2-induced inhibition of Na+-Pi uptake was prevented completely by catalase, dimethylthiourea, and deferoxamine, suggesting involvement of hydroxyl radical generated by an iron-dependent mechanism. In contrast, antioxidants Trolox, N,N’-diphenyl-p-phenylenediamine, and butylated hydroxyanisole did not affect the H2O2 inhibition. Kinetic analysis indicated that H2O2 decreased Vmax of Na+-Pi uptake with no change in the Km value. Phosphonoformic acid binding assay did not show any difference between control and H2O2-treated cells. H2O2 also did not cause degradation of Na+-Pi transporter protein. Reduction in Na+-Pi uptake by H2O2 was associated with ATP depletion and direct inhibition of Na+-K+-ATPase activity. These results indicate that the effect of H2O2 on membrane transport function in OK cells is associated with reduction in functional Na+-pump activity. In addition, the inhibitory effect of H2O2 was not associated with lipid peroxidation.
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