Effects of peroxisome proliferators-activated receptor γ agonists on transforming growth factor-β1 and Smads signal pathway: experiment with rat renal fibroblasts

Objective To study the effects of peroxisome proliferators-activated receptor γ (PPARγ) agonists on transforming growth factor (TGF)-β1-induced fibrotic responses in renal fibroblasts, so as to investigate its effects in prevention of tubulointerstitial fibrosis. Methods Rat renal fibroblasts of the line NRK/49F were cultured and divided into groups. In group 1 TGFβ1 of the concentrations of 0, 1, 2, 5, and 10 ng/ml was added and co-cultured for 24 h. In group 2 TGFβ1 of the concentration of 5 ng/ml was added and co-cultured for 0, 6, 12, and 24 h respectively. Groups 3, 4, and 5 were pretreated with 10 μmol/L15d-PGJ2, PPARγ ligand, 10 μmol/L troglitazone, agonist of, and 10 μmol/L ciglitazone, both PPARγ agonists, respectively for 2 h, then treated with 5 ng/ml TGFβ1. A blank control group was set up. The cultured cells were collected. RT-PCR was used to detect the mRNA expression of TGF-β1-indued fibronectin (FN). Western blotting was used to detect the expression of TGF-β1-induced FN, Smad, and phosphorylated Smad (p-Smad) proteins. Results TGF-β1 enhanced the FN mRNA expression in a dose- and time-dependent manner. The FN mRNA expression of the 5 ng/ml TGF-β1 group was 3.7 times that of the control group (P0.05). The FN mRNA expression of the 15d-PGJ2, troglitazone-, and ciglitazone-pretreated groups were lower than that of the 5 ng/ml TGF-β1 group by 37.3%, 41.5%, and 22.7% respectively (all P0.05). The p-Smad2/3 protein expression levels of the TGF-β1 group began to increase 15 minutes after stimulation, increased in a time-dependent manner, peaked 1 hour after, and began to decrease 2 hours later. However, the levels of protein expression of total Smad2 and Smad3 did not change significantly in all groups. Both 2 ng/ml TGFβ1 and 5 ng/mlTGFβ1 significantly induced the increase of protein expression of p-Smad2/3 (all P0.05). The levels of protein expression of p-Smad2 and p-Smad3 of the 5 ng/mlTGFβ1 group were 3.42 and 0.97 times those of the 2 ng/mlTGFβ1 (both P0.05). The levels of protein expression of p-Smad2/3 of the 15d-PGJ2, troglitazone-, and ciglitazone -pretreated groups were all significantly lower than that of the 5 ng/mlTGFβ1 group by 61.2%, 53.0%, and 59.5%(all P0.05), However, there was no significant difference among different drug-treated groups (all P0.05). Conclusion Possibly through abrogating TGF-β1/Smads signaling pathway, PPARγ agonists inhibit TGF-β1-induced renal fibroblast extracellular matrix synthesis and may play a potential role in preventing tubulointerstitial fibrosis as a novel approach to prevent the progress of end stage renal dysfunction.