Toxicity of phosphonoformic acid in vascular smooth muscle cells: relationship to vascular calcification

Abstract

Medial vascular calcification (VC) develops as a consequence of hyperphosphatemia secondary to chronic kidney disease. The role of inorganic phosphate (Pi) transporters in the pathogenesis of VC has been previously determined by interference of RNA and inhibition by phosphonoformic acid (PFA). We have found that inhibition of Pi transport by PFA in vascular smooth muscle cells (VSMC) from rat aorta showed a Ki of 2.6 mM, while the Km for Pi was 0.1 mM. VSMC express both type III Pi transporters, Pit1 and Pit2. When expressed in Xenopus laevis oocytes Pit1 and Pit2 were inhibited with PFA with Ki values of 2.7 and 4.6 mM, respectively, while they showed affinities of 0.1 and 0.2 mM. PFA at either 1 or 5 mM completely prevented calcification of VSMC induced with 2 mM phosphate for 5 days. Phase-contrast microscopy revealed that cells treated with either 1 or 5 mM PFA in either normal (1 mM) or high (2 mM) Pi concentration showed a different morphology and evidence of cell death. Cytotoxicity was analyzed as a function of cytosolic lactate dehydrogenase activity, revealing a mean lethal concentration (LC50) of 0.38 mM. Therefore the prevention calcification was not due to inhibition of Pi transport but to cytotoxicity of PFA that prevented trans-differentiation of VSMC into osteogenic-like cells. Further experiments have been developed to characterize the type of cell death, including apoptosis.