Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells.
OBJECTIVE
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)-beta1 were determined by real-time RT-PCR and enzyme-linked immunosorbent assay.


RESULTS
Fluorofenidone significantly inhibited TGF-beta1 and collagen I expression upregulation induced by AngII or TGF-beta1 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.


CONCLUSIONS
Fluorofenidone acted as an anti-oxidative and anti-fibrotic agent through the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-beta1 expression in rat proximal tubular epithelial cells.
Peng
Zhang-Zhe
ZZ
Division of Nephrology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Hu
Gao-Yun
GY
Shen
Hong
H
Wang
Ling
L
Ning
Wang-Bin
WB
Xie
Yan-Yun
YY
Wang
Na-Sui
NS
Li
Bing-Xin
BX
Tang
Yi-Ting
YT
Tao
Li-Jian
LJ
D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000037:antagonists & inhibitors* / Q000235:genetics; D005355:Fibrosis / Q000188:drug therapy*; D007668:Kidney / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000502:physiology*; D011728:Pyridones / Q000494:pharmacology*; D051381:Rats; D013481:Superoxides / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors* / Q000235:genetics
C511655:5-methyl-1-(3-fluorophenyl)-2-(1H)-pyridone; D024042:Collagen Type I; D011728:Pyridones; D053773:Transforming Growth Factor beta1; D013481:Superoxides; C093844:pirfenidone; D019255:NADPH Oxidases