Differential proteomic analysis of cyclosporine A-induced toxicity in renal proximal tubule cells.

Abstract

The use of cyclosporine A (CsA) as a potent immunosuppressant has been limited by its severe nephrotoxic effects. The mechanisms involved are haemodynamic but also related to direct toxic effects of CsA on proximal tubule epithelial cells. We focused on defining a proteomic profile in CsA-treated proximal tubule cells to distinguish the direct impact of CsA on these cells from overlapping haemodynamically mediated phenomena that occur in an in vivo system.
By means of high-throughput differential proteomic analyses and mass spectrometry techniques in CsA and vehicle-treated proximal tubule-derived cell lines of human and mouse origin, we determined proteins that change their expression in the presence of CsA.
CsA-induced toxicity analyses revealed that 10 mM CsA for 24 h was the threshold condition to induce significant changes in cell viability and proteomic profile. We identified 38 differentially expressed proteins on CsA-treated mouse PCT3 and human HK-2 cells, related to protein metabolism, response to damage, cell organization and cytoskeleton, energy metabolism, cell cycle and nucleobase/nucleoside/nucleotidic metabolism. 1D and 2D western blot assays in crude extracts from CsA-treated cells or kidneys with impaired function upon CsA treatment revealed a correlation with proteomic changes or differential isoform expression, in randomly selected proteins.
Proteins identified in this work might be useful markers to eventually distinguish CsA toxicity from chronic allograft nephropathy in protocol biopsies of transplanted patients, facilitating the adjustment of CsA doses to non-toxic ranges, as well as to study the impact of potential therapeutic interventions in an animal model.