[NADPH oxidase-derived reactive oxygen species involved in angiotensin II-induced monocyte chemoattractant protein-1 expression in mesangial cells].
OBJECTIVE
To investigate the origin of oxidative stress induced by angiotensin II (AngII) in human mesangial cells and the role of reactive oxygen species (ROS) in AngII-induced monocyte chemoattractant protein-1 (MCP-1) expression.


METHODS
MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence. p47phox and p67phox translocation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups: the control, the AngIIinfusion [AngII 400 ng/(kg.min)], and the apocynin treatment. AngII was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA.


RESULTS
In cultured human mesangial cells, AngII induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. AngII increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent. Incubation with different dosages of AngII (1 nmol/L, 10 nmol/L, and 100 nmol/L AngII) for 60 min, ROS production increased at 1.82, 2.92, and 4.08 folds respectively. AngII-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI, 10 micromol/L) and apocynin (500 micromol/L), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex Iinhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect. AngII-induced ROS generation was inhibited by the AT1 antagonist losartan (10 micromol/L) but not the AT2 antagonist PD123319 (10 micromol/L). AngII treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished AngII-induced MCP-1 expression. AngII infusion increased urinary and p67 translocation by 2.69-, 2.97-, and 2.67-fold, respectively.


CONCLUSIONS
NADPH oxidase-derived ROS is involved in AngII-induced MCP-1 expression. Inhibition of NADPH oxidase alleviates AngII-induced renal injury.
Chen
Ying
Y
Department of Nephrology, Nanjing Children's Hospital, Affiliated to Nanjing Medical University, Nanjing 210008, China.
Zhang
Ai-hua
AH
Huang
Song-ming
SM
Ding
Gui-xia
GX
Zhang
Wei-zhen
WZ
Bao
Hua-ying
HY
Wu
Hong-mei
HM
Chen
Rong-hua
RH
D000098:Acetophenones / Q000494:pharmacology; D000804:Angiotensin II / Q000008:administration & dosage / Q000494:pharmacology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019808:Losartan / Q000494:pharmacology; D008297:Male; D050527:Mesangial Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D018384:Oxidative Stress; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport; D011897:Random Allocation; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000098:Acetophenones; D047228:Angiotensin II Type 1 Receptor Blockers; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; D000804:Angiotensin II; C007517:diphenyleneiodonium; C056165:acetovanillone; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019808:Losartan