Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells.
BACKGROUND
The nephrotoxicity induced by immunosuppressant FK506 remains a serious clinical problem, and the underlying mechanism has not been completely understood. The present study was undertaken to determine the role of hydrogen peroxide in FK506-mediated cytotoxicity in a porcine renal proximal tubular cell line, LLC-PK1 cells, and human embryonic kidney (HEK293) cells.


METHODS
Cytotoxicity was estimated by crystal violet and lactate dehydrogenase release assays. The activity of reactive oxygen species (ROS) was detected by flow cytometry. FK506-induced cell death was examined in the presence of the hydrogen peroxide scavenger, catalase, or a scavenger of hydroxyl radicals, sodium benzoate. As a control, FK506-induced cell death was also measured in the presence of superoxide anion inhibitor, 4,5-dihydroxy-1,2-benzene disulfonic acid (Tiron), TEMPO, or overexpressed human manganese superoxide dismutase (MnSOD). Catalase was also used in tumor necrosis factor-alpha (TNF-alpha)-induced cell injury to determine whether the enzyme specifically protected cells against FK506-mediated cytotoxicity.


RESULTS
FK506 induced cell death in a dose-dependent manner and coincided with a dose-dependent increase in ROS activity. Abrogation of FK506-mediated ROS by catalase and N-acetylcysteine blunted FK506-induced cell death. Furthermore, overexpression of catalase, sodium benzoate, and deferoxamine inhibited the cytotoxic effect of FK506. In contrast, Tiron, TEMPO, or overexpression of human MnSOD failed to show cytoprotection. In fact, TEMPO or expression of MnSOD enhanced the effect of FK506. Catalase did not significantly affect TNF-alpha-induced cell injury.


CONCLUSIONS
Catalase is uniquely required in cellular protection against FK506 cytotoxicity, which suggests an important role for hydrogen peroxide in the cellular actions of FK506.
Zhou
Xiaoming
X
Division of Nephrology, Department of Medicine, Uniformed Services University, Bethesda, Maryland 20814, USA. xiazhou@usuhs.mil
Yang
Guang
G
Davis
Christopher A
CA
Doi
Sonia Q
SQ
Hirszel
Przemyslaw
P
Wingo
Charles S
CS
Agarwal
Anupam
A
D000818:Animals; D002374:Catalase / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000633:toxicity*; D007668:Kidney / Q000166:cytology*; D018374:LLC-PK1 Cells; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D016559:Tacrolimus / Q000633:toxicity*; D014162:Transfection
D007166:Immunosuppressive Agents; D017382:Reactive Oxygen Species; D013481:Superoxides; D006861:Hydrogen Peroxide; D002374:Catalase; D016559:Tacrolimus