Effect Of Benazepril On ILK And α-SMA Activity In Glomerular Mesangial Cells Exposed To High Glucose

Objective: To investigate the expression of ILK andα-SMA in the glomerular mesangial cells induced by high-glucose and observe the effect of Benazepril on the the expression of ILK andα-SMA. Recently diabetic nephropathy (DN) has become the most common cause of end-stage renal disease (ESRD), which is one of the most serious complications of diabetes mellitus in the United States and Europe. But, up to now, the mechanisms of the onset of DN and its preventive measures in early stage are not clear. The thickening of glomerular basement membrane(GBM), the proliferation of the glomerular mesangial cells (GMC) and the accumulation of the mesangial matrix are the main pathological changes in DN which led to glomerulosclerosis. Glomerular mesangial cells are the most active cells in the glomerular, which play an important role for the normal histio-structure and physiologic function in the kidney. So it is very important to explore and clarify the mechanisms of physiological and pathological changes in Glomerular mesangial cells for the clinical prevention and finding DN in early time.Intergrin-linked Kinase is a kind of serine / threonine protein kinase in the cytoplasm, which plays a role in regulating cell adhesion, maintaining cell survival, morphology and regulating gene expression through many kinds of signal transduction pathways including integrin, growth factor and Wnt. Recently, some researches show that there is nothing or little ILK in the GMC of normal kidney tissues. But the increasing expression of ILK induce the cell proliferation and the secretion of extracellular matrix (ECM). A-smooth muscle actin (α-SMA) is a special cytoskeleton protein of vascular smooth muscle. It is the symbol of the vascular smooth muscle, which has no expression in normal maturation GMC. Butα-SMA can be expressed in the pathological GMC because of its transformed phenotype. The increasing expression ofα-SMA in GMC are related to the proliferation of GMC and the accumulation of extracellular matrix,which are also lead to the continuous decline of renal function. Because the proliferation of GMC and the accumulation of extracellular matrix are the early pathological changes in DN, the relations among ILK,α-SMA, the GMC proliferation and the secretion of ECM will help us to clarify the mechanism of the the pathogenesis of DN more deeply.Benazepril is one of the new generation of angiotensin -converting enzyme inhibitors(ACEIs), which can decrease angiotensinⅡ(AngⅡ) in the circulation and organisms by inhibiting the angiotensin-converting enzyme. It can also weaken the stimulus of AngⅡand prevent AngⅡfrom combineing with its receptors. Some experiments have proved that the GMC can autocrine AngⅡby the stimulus of high glucose. And then it will combine with AT1 receptors in GMC, which led to activate the signal transduction pathyways PKC. So some other cell growth factor genes and proteins will be expressed, which lead to the proliferation and differentiation of GMC. In this experiment we use the effects of benazepril on ILK andα-SMA to observe their expressions in the cultured GMC exposed by high glucose and the protective effects of the drug, which may provide us experimental theory in clinical DN.Methods: The mesangial cells of SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups: normal glucose( D-glucose 5.5mmol/L, group NG ) ,Mannitol-treated normal glucose group (D-glucose 5.5mmol/L+ mannitol 20mmol/L,group MG), high glucose (D-glucose 30mmol/L,group HG), Benazepril-treated high glucose group(D-glucose 30mmol/L+ Benazepril 10μmol/L, group HG+Benazepril). NG, MG, HG ,HG+Benazepril were all collected on 3hour, 6hour, 12hour, 24 hour, 48 hour and 72 hour respectively. The mRNA of ILK andα-SMA expressions of the four groups were detected by reverse transcriptase- polymerase chain reaction (RT-PCR) . The protein of ILK andα-SMA were detected by western blot.Results:1.The shapes of the cultured cells showed as fusiform, irregular stellar, trianglar or branch-shaped by phase contrast microscopy. When the cells growed overlap-intensively, their endochylemas had some ecphymas and there are a lot of microfilaments in it. Their nucleus were in the center, which were round or oval. 2.The expressions of ILK mRNA and protein in HG group were significantly increased on 3h compared to those in NG group (P <0.05). The increasing expression of ILK in HG group were time-dependent and the expression reached the peak on 48h (P <0.05), while decreased on 72h(P <0.05). The expression of ILK in HG+Benazepril group were lower than that in HG group (P <0.01), but failed to drop to the same level as that in NG(P<0.01). There were no significant differences of ILK expressions between MG group and NG group at the same time(P> 0.05). 3.The expressions ofα-SMA mRNA and protein in HG were higher than that in NG at anytime(P <0.05). The increasing expression ofα-SMA was also time-dependent and it reached the peak on 72h(P<0.05). The expression ofα-SMA in HG+Benazepril was lower than that in HG(P<0.01), while failed to drop to the same level as that in NG(P <0.01). The expressions ofα-SMA mRNA and protein in MG were higher than that in NG (P <0.05),so we couldn't eliminate the factor of high osmotic pressure which may cause the increasing ofα-SMA.Conclusion: 1.High glucose induced ILK expression in the GMC in a time-dependent manner. 2.High glucose could also inducedα-SMA expression in the GMC in a time-dependent manner and mediated the phenotypic transformation of GMC. 3.Benazepril could inhibit the processes that were mentioned above in GMC. 4.The abnormal expressions ofα-SMA suggested that there was phenotypic transformation of GMC in glucose. The increasing ofα-SMA may be dependent on high osmotic pressure.