Acta Pharmacologica Sinica (2009) 30: 1601–1606; doi: 10.1038/aps.2009.155

Aim: To investigate the role of adenosine 5'-triphosphate (ATP)-induced generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the production of transforming growth factor-β1 (TGF-β1) in cultured rat glomerular mesangial cells under high-glucose conditions.

Methods: Subconfluent glomerular mesangial cells were serum-starved for 24 h and pretreated with suramin, diphenylenechloride iodonium (DPI) or PD98059 followed by stimulation with a high concentration of glucose (30 mmol/L D-glucose) or ATP (300 μmol/L).  Extracellular and total ATP and ROS production were detected using commercially available kits.  Phosphorylation of ERK1/2 was evaluated by Western blot.  TGF-β1 mRNA expression was examined by real-time PCR.

Results: Suramin had a dose-dependent inhibitory effect on the generation of ROS induced by high glucose.  Extracellular ATP production by mesangial cells increased markedly after a 2-h incubation with high glucose.  ROS production was upregulated in mesangial cells after 5 min incubation with 300 μmol/L ATP and was sustained for 120 min.  ERK1/2 was significantly activated after 5 min incubation of mesangial cells with ATP, this activation was partially inhibited by DPI.  The effects of high glucose on TGF-β1 mRNA were markedly inhibited by suramin, DPI or PD98059.

Conclusion: Our results suggest that a high concentration of glucose increases the extracellular levels of ATP in mesangial cells within a short time-frame.  ATP, in turn, activates ERK1/2, an effect which is at least partially dependent on ROS, which results in the upregulation of TGF-β1.

Keywords: diabetic nephropathy; reactive oxygen species; adenosine 5'-triphosphate; extracellular signal-regulated kinase 1/2; transforming growth factor-β1; mesangial cells