The Expression Of RANTES And CCR5 In Renal Tubular Epithelial Cells Under High Glucose Condition And The Interventional Effect Of Simvastatin On The Cells

Objective: To observe the expression of RANTES, CCR5 and TGF-β1 in human proximal tubular epithelial cells(HK-2) cultivated under the condition of high glucose, to investigate the effects of these factors in the tubulointerstitial injury of diabetic nephropathy(DN), and to evaluate the influence of simvastatin on the cytokines, to discuss statin's protective mechanism in DN independently on lipid-lowering effect.Methods: HK-2 cells were cultivated with deferent concentrations of simvastatin for 24h, 48h and 72h in vitro, the cells proliferation was detected by MTT colorimetry to determine the suitable interventional dose of the drug on the cells. Then HK-2 cells were divided into four groups in different medium:①NG(glucose concentration: 5.5mmol/l);②HG(glucose concentration: 25mmol/l);③HG+Sim(2) (HG+simvastatin 2μmol/l);④HG+Sim(4) (HG+simvastatin 4μmol/l). Listed below was measured at 24h, 48h and 72h: the expression of RANTESmRNA in the cells by RT-PCR, the protein concentration of RANTES in supernatant fluid by ELISA, and the distributions and contents of CCR5, TGF-β1 in cells by immunohistochemistry.Results: 1. The secretion of RANTES in HK-2 increased under high glucose condition. At 24h the expression of RANTESmRNA increased in HG in a time-dependent manner, which is significantly higher than that in NG(P<0.05); the protein content of RNATES in HG markedly increased at 48h(P<0.05). The enhancement of both mRNA and protein was more obvious at 72h(P<0.01). 2. Immunohistochemistry analysis showed the expression of CCR5 and TGF-β1 in HK-2 cells was few in NG.. They both increased under high glucose at 24h(P<0.01, P<0.05) and positive expression was buffy in the plasma of the cells. They enhanced in a time-dependent manner, having more obvious statistical difference at 72h(P<0.01). 3. The expression of RANTESmRNA and protein content in HK-2 cells under high glucose condition both decreased distinctly after treated with two concentrations of simvastatin(P<0.05). Positive cells of CCR5 and TGF-β1 also decreased significantly compared with them in HG after the intervention by simvastatin(P<0.05). It showed a concentration-dependent manner between the groups of simvastatin 2μmol/l and 4μmol/l. The correlation analysis revealed that RANTES, CCR5 and TGF-β1 were positively correlated(P<0.01).Conclusions: 1. High glucose stimulated the expression of RANTES in HK-2 cells in a time-dependent manner, which could induce the infiltration and activation of inflammatory cells, and this may be an important pathway in DN. 2. High glucose induced expression of CCR5 in HK-2 cells, which could be a reflection of the tubulointerstitial inflammation and it might interact with RANTES or other cytokines, participating the inflammatory process. 3. High glucose enhanced expression of TGF-β1, which is a key factor resulting in fibrosis. And it was found there were positive correlations among TGF-β1, RANTES and CCR5, which revealed that all these factors may inter-communicate and cooperate, leading to renal tubulointerstitial disease. 4. Simvastatin could suppress the overexpression of the cytokines and cytokine receptor under high glucose, and the effect was in a dose-dependent manner, which showed statin might inhibit inflammation and fibrosis of tubulointerstitium in DN through down-regulating expression of those factors, playing a protective role on DN.