Effect of the Rho kinase inhibitor Y-27632 and fasudil on inflammation and fibrosis in human mesangial cells (HMCs) under high glucose via the Rho/ROCK signaling pathway

This study investigated the effect of the Rho kinase inhibitor Y-27632 and fasudil on the development of human mesangial cell (HMC) inflammation and fibrosis induced by high glucose and to clarify the contribution of the Rho/ROCK signaling pathway in the pathogenesis of diabetic kidney disease (DKD). High glucose (30 mmol/l) induced the Rho/ROCK signaling pathway. Western blotting was used to detect active and total RhoA, ROCK-I, tumor necrosis factor-alpha (TNF-alpha), connective tissue growth factor (CTGF) activation, and fibronectin (FN) up-regulation as assessed by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Lipofectamine (TM) 2000 was applied to transfect RhoA-small interfering RNA (siRNA), which could inhibit RhoA expression. RhoA, ROCK, FN, CTGF, and TNF-alpha expression was detected by real-time reverse transcription-PCR (RT-PCR) and ELISA. High glucose up-regulated RhoA and downstream ROCK-I expression (P< 0.05). RhoA, CTGF, and TNF-alpha of HMCs cultured under high glucose were expressed significantly more than those of HMCs in the normal glucose group (P< 0.05); this was dependent on ROCK signaling. FN up-regulation by high glucose, shown to be mediated by CTGF and TNF-alpha, was prevented by Y-27632 or fasudil (P< 0.05). RhoA siRNA was also markedly attenuated by ROCK-I, FN, CTGF, and TNF-alpha up-regulation by high glucose (P< 0.05). The present study demonstrates that the Rho/ROCK signaling pathway is involved in the up-regulation of TNF-alpha CTGF, and FN in DKD. The ROCK inhibitors Y-27632 and fasudil, in addition to RhoA siRNA, could effectively reverse the high glucose-induced expression of ROCK-I, FN, CTGF, and TNF-alpha and reduce glomerular fibrosis and inflammation. We conclude that suppression of the Rho/ROCK signaling pathway could provide a new intervention target for preventing and treating DKD.