Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells

Nephrology (Carlton). 2009 Sep;14(6):565-72. doi: 10.1111/j.1440-1797.2009.01129.x.

Abstract

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)-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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Collagen Type I / antagonists & inhibitors*
  • Collagen Type I / genetics
  • Fibrosis / drug therapy*
  • Kidney / pathology
  • Kidney Tubules, Proximal / drug effects
  • Kidney Tubules, Proximal / metabolism
  • NADPH Oxidases / genetics
  • NADPH Oxidases / physiology*
  • Pyridones / pharmacology*
  • Rats
  • Superoxides / metabolism
  • Transforming Growth Factor beta1 / antagonists & inhibitors*
  • Transforming Growth Factor beta1 / genetics

Substances

  • 5-methyl-1-(3-fluorophenyl)-2-(1H)-pyridone
  • Collagen Type I
  • Pyridones
  • Transforming Growth Factor beta1
  • Superoxides
  • pirfenidone
  • NADPH Oxidases