Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells

Background The clinical use of cyclosporine (CsA) is limited by its nephrot oxicity. Apoptosis, perhaps instigated by increased nitric oxide synthase ( NOS) activity,may play a role in such toxicity. Methods. Human mesangial cells, human tubular cells, human umbilical vein e ndothelial cells, or murine endothelial cells were cultured with CsA at fin al concentrations of 0 to 1000 ng/mL for 4 to 24 hours. As inhibitors of ap optosis, 0.01 mol/L L-nitromethylarginine (L-NAME) or 1 mu g/mL cycloheximi de (CHX) was added, whereas 0.01 mol/L sodium nitroprusside (as a nitric ox ide donor) was used as a positive control. Apoptosis was assessed by using TUNEL method and by DNA fragmentation by electrophoresis. In addition, NOS enzymatic activity, Northern blots for inducible NOS (iNOS) mRNA, and immun ohistochemically demonstrable iNOS protein were evaluated. Results. Within 12 to 24 hours, CsA significantly increased the fraction (8 to 35%) of apoptotic cells in each cell line, according to the dose. Fragm entation of DNA confirmed apoptosis. L-NAME and CHX inhibited the phenomeno n, whereas sodium nitroprusside enhanced it. Each cell line significantly i ncreased NOS activity in response to CsA, an effect blunted by L-NAME and C HX. Neither inhibitor modified the increased iNOS mRNA expression elicited by CsA. Positive staining for both iNOS and p53 proteins was observed in al l cell lines incubated with CsA that were inhibited by CHX; L-NAME inhibite d only p53 staining. Conclusions. CsA induces apoptosis in various renal cell lines, and this ef fect is mediated by the induction of iNOS via p53. These effects may contri bute to the acellular fibrosis characteristic of late CsA nephrotoxicity.