title: NADPH oxidase-dependent formation of reactive oxygen species contributes to angiotensin II-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells.
abstract: The objective of the present study was to investigate the role of NADPH oxidase-dependent formation of reactive oxygen species (ROS) in the angiotensin II (Ang II)-induced epithelial-mesenchymal transition (EMT) and in the accumulation of extracellular matrix (ECM) in rat peritoneal mesothelial cells (RPMCs). Primary cultured RPMCs were incubated with serum-free media for 24 h in order to arrest and synchronize cell growth. The cells were treated with Ang II (10⁻⁷ M) up to 48 h. Cells were pretreated with an Ang II type I receptor antagonist (losartan, 10⁻⁵ M), or an inhibitor of NADPH, oxidase diphenyleneiodonium (DPI) (10⁻⁵ M), for 1 h before addition of Ang II. The dichlorofluorescein (DCF)-sensitive cellular ROS were measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for the NADPH oxidase subunit p47phox, plasminogen activator inhibitor-1 (PAI-1), α-smooth muscle actin (α-SMA) and E-cadherin. PAI-1, α-SMA and p47phox protein expression were examined by Western blot analysis. Ang II significantly induced the production of intracellular ROS. DPI and losartan inhibited Ang II-induced ROS generation by 86.8% and 77.4% (p<0.05), respectively. Ang II significantly stimulated NADPH oxidase subunit p47phox mRNA and protein expression in RPMCs. Both losartan and DPI inhibited Ang II-induced up-regulation of p47phox mRNA by 37.3% and 67.8% (p<0.05), respectively. Ang II also stimulated α-SMA mRNA and protein expression and down-regulated E-cadherin mRNA expression in RPMCs. This effect was suppressed by both losartan and DPI. Ang II significantly up-regulated PAI-1 mRNA and protein expression and these were significantly suppressed by both losartan and DPI. In conclusion, NADPH oxidase-dependent formation of ROS mediates Ang II dependent EMT and accumulation of ECM in rat peritoneal mesothelial cells. NADPH oxidase may represent a potential therapeutic target in the management of peritoneal fibrosis.
authors.affiliation: Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chang
authors.forename: Zongpei
authors.initials: Z
authors.lastname: Jiang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Hengmei
authors.initials: H
authors.lastname: Zhu
authors.forename: Shu-Feng
authors.initials: SF
authors.lastname: Zhou
authors.forename: Xueqing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000378:metabolism; D015536:Down-Regulation; D004847:Epithelial Cells / Q000187:drug effects*; D058750:Epithelial-Mesenchymal Transition; D005109:Extracellular Matrix / Q000378:metabolism; D019808:Losartan / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D009861:Onium Compounds / Q000494:pharmacology; D056627:Peritoneal Fibrosis / Q000235:genetics / Q000628:therapy; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: D000199:Actins; D047228:Angiotensin II Type 1 Receptor Blockers; D015820:Cadherins; D016895:Culture Media, Serum-Free; D009861:Onium Compounds; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D000804:Angiotensin II; C007517:diphenyleneiodonium; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D019808:Losartan