Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells.
BACKGROUND
The clinical use of cyclosporine (CsA) is limited by its nephrotoxicity. 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 endothelial cells, or murine endothelial cells were cultured with CsA at final concentrations of 0 to 1000 ng/mL for 4 to 24 hours. As inhibitors of apoptosis, 0.01 mol/L L-nitromethylarginine (L-NAME) or 1 microg/mL cycloheximide (CHX) was added, whereas 0.01 mol/L sodium nitroprusside (as a nitric oxide 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 immunohistochemically 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. Fragmentation of DNA confirmed apoptosis. L-NAME and CHX inhibited the phenomenon, whereas sodium nitroprusside enhanced it. Each cell line significantly increased NOS activity in response to CsA, an effect blunted by L-NAME and CHX. Neither inhibitor modified the increased iNOS mRNA expression elicited by CsA. Positive staining for both iNOS and p53 proteins was observed in all cell lines incubated with CsA that were inhibited by CHX; L-NAME inhibited only p53 staining.


CONCLUSIONS
CsA induces apoptosis in various renal cell lines, and this effect is mediated by the induction of iNOS via p53. These effects may contribute to the acellular fibrosis characteristic of late CsA nephrotoxicity.
Amore
A
A
Nephrology and Dialysis Department, Central Laboratory, Regina Margherita Children's Hospital, Torino, Italy. amorenefro@email.oirmsantanna.piemonte.it
Emancipator
S N
SN
Cirina
P
P
Conti
G
G
Ricotti
E
E
Bagheri
N
N
Coppo
R
R
D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D009569:Nitric Oxide; D016572:Cyclosporine; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester