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Fluorofenidone attenuates collagen I and transforming growth factor‐β1 expression through a nicotinamide adenine dinucleotide phosphate oxidase‐dependent way in NRK‐52E cells
Author(s) -
PENG ZHANGZHE,
HU GAOYUN,
SHEN HONG,
WANG LING,
NING WANGBIN,
XIE YANYUN,
WANG NASUI,
LI BINGXIN,
TANG YITING,
TAO LIJIAN
Publication year - 2009
Publication title -
nephrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 61
eISSN - 1440-1797
pISSN - 1320-5358
DOI - 10.1111/j.1440-1797.2009.01129.x
Subject(s) - nicotinamide adenine dinucleotide phosphate , nadph oxidase , p22phox , microbiology and biotechnology , losartan , oxidase test , reactive oxygen species , oxidative stress , western blot , nox4 , superoxide dismutase , chemistry , angiotensin ii , biochemistry , enzyme , biology , receptor , gene
SUMMARY: Aim: Fluorofenidone (1‐(3‐fluorophenyl)‐5‐methyl‐2‐(1H)‐pyridone) is a novel pyridone agent. The aim of the present study is to investigate the effects of fluorofenidone on angiotensin (Ang)II‐induced fibrosis and the involved molecular mechanism in rat proximal tubular epithelial cells. Methods: NRK‐52E cells, a rat proximal tubular epithelial cell line, were incubated with medium containing AngII, with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), losartan, fluorofenidone (2, 4 and 8 mmol/L) and pirfenidone (8 mmol/L) for 24 h. Cells in the serum‐free medium were controls. The expression of three subunits of NADPH oxidase, including p47phox, Nox‐4 and p22phox, were determined by real‐time reverse transcription polymerase chain reaction (RT–PCR) and western blot. NADPH oxidase activity was measured directly by superoxide dismutase (SOD) inhibitable cytochrome C reduction assay. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein fluorescence analysis. The mRNA and protein expression of collagen I and transforming growth factor (TGF)‐β1 were determined by real‐time RT–PCR and enzyme‐linked immunosorbent assay. Results: Fluorofenidone significantly inhibited TGF‐β1 and collagen I expression upregulation induced by AngII or TGF‐β1 respectively. Moreover, fluorofenidone greatly reduced the elevation of expression and activity of NADPH oxidase and inhibited ROS generation induced by AngII in rat proximal tubular epithelial cells. These responses were also attenuated by DPI, losartan, and pirfenidone. Conclusion: Fluorofenidone acted as an anti‐oxidative and anti‐fibrotic agent through the mechanisms of blocking NADPH oxidase‐dependent oxidative stress and inhibiting TGF‐β1 expression in rat proximal tubular epithelial cells.