The NADPH oxidase inhibitor DPI can abolish hypoxia-induced apoptosis of human kidney proximal tubular epithelial cells through Bcl2 up-regulation via ERK activation without ROS reduction.
OBJECTIVE
Ischemia/reperfusion injury (IRI), resulting from hypoxic damage within a graft, is the leading cause of cell death and graft rejection. In this study, we investigated whether a HIF-1α inhibitor or various antioxidants were able to prevent ischemic injury in a cellular model in which experimental hypoxia was induced using CoCl2.


METHODS
The ischemic injury induced in HK-2 cells by CoCl2 was validated by increased reactive oxygen species (ROS) production, reduced cell viability, and increased apoptosis at different times and doses. The preventative effects of various anti-oxidants on ischemic injury were evaluated using ROS levels, cell viability, and apoptosis. The MAPK phosphorylation status and Bcl2/Bax expression levels were evaluated after treatment with various antioxidants.


RESULTS
The increase in ROS induced by hypoxia was significantly inhibited by NAC and CAPE, but not by any other treatment. The reduction in cell viability induced by CoCl2 was significantly inhibited by NAC and DPI, but not by any other treatment. The apoptosis induced by CoCl2 was also significantly inhibited by NAC and DPI, but not by any other treatment. Moreover, NAC and DPI prevented CoCl2-induced apoptosis in HK-2 cells in a dose- and time-dependent manner. Treatment of CoCl2 and HK-2 cells treated with DPI, but not NAC, significantly induced ERK activation and Bcl2 expression. NAC and DPI treatment prevented the apoptosis of cells cultured under hypoxic conditions.


CONCLUSIONS
Our results suggest that DPI should be investigated further as a novel protective agent that prevents kidney ischemia.
Departmentof Microbiology and Immunology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Hyunkeun
H
Song
Department of Thoracic and Cardiovascular Surgery, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Il-Yong
IY
Han
Departmentof Microbiology and Immunology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Yeonye
Y
Kim
Department of Plastic & Reconstructive Surgery, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Young Hwan
YH
Kim
Departmentof Microbiology and Immunology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Il-Whan
IW
Choi
Departmentof Microbiology and Immunology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea.
Su-Kil
SK
Seo
Department of Dermatology, College of Medicine, INJE University, Haeundaero 875, HaeundaeGu, Busan 612-896, Republic of Korea.
So Young
SY
Jung
Departmentof Microbiology and Immunology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea. Electronic address: micpsg@inje.ac.kr.
SaeGwang
S
Park
Department of Pathology, College of Medicine, INJE University, Bockjiro 75, BusanjinGu, Busan 614-735, Republic of Korea. Electronic address: pathkang@inje.ac.kr.
Mi Seon
MS
Kang
D016587:Antimutagenic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003035:Cobalt / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects*; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
D016587:Antimutagenic Agents; C494826:BAX protein, human; C000595707:BCL2 protein, human; D009861:Onium Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D003035:Cobalt; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D048049:Extracellular Signal-Regulated MAP Kinases; C018021:cobaltous chloride
Antioxidant; Apoptosis; Cell viability; Human renal proximal tubular epithelial cells; NADPH oxidase inhibitor; Reactive oxygen species; Renal ischemia–reperfusion injury