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

Background. 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. Methods. 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. Results. 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 nucle-obase/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. Conclusions. 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. a fixed CsA dose of 10 000 nM and a time exposure of 24 h, with cycle phase and cell type as factors accounting for the interaction between them. Serum urea nitrogen and creatinine differences between CsA-treated and control mice were studied using Student’s t -test. Abstract Background. We examined whether dedifferentiation is necessary for cell division of proximal tubule (PT) cells after acute PT injury. Methods. Rats were injected with a low (0.2 mg/kg) or high (4 mg/kg) dose of uranyl acetate (UA) to induce acute PT injury. Proliferating PT cells were labelled with bromod-eoxyuridine (BrdU) before sacrifice. Renal tissues were examined by double labelling of BrdU and megalin, aqua-porin 1 (AQP1), Na + –K + ATPase or vimentin, and by immunoelectron microscopy for BrdU + cells. Results. Under normal conditions, BrdU + PT cells were positive for the PT phenotype (megalin-, AQP1- and Na + –K + ATPase positive and vimentine negative, a mes-enchymal marker). Low-dose UA induced focal PT injury, and BrdU + initially proliferating PT cells were found in the proximal three quarters of the S3 segment of nephron as early as 12 h, which maintained the PT phenotype and were vimentin negative. Proliferating PT cells showed low expression of the PT cell protein phenotype from Day 2 to Day 5 with vimentin expression from Day 2. High-dose UA induced severe PT injury in the proximal three quarters of the S3 segment by Day 5. BrdU + initially proliferating PT cells, which were found in distal areas of the S3 segment as early as Day 2, showed low