Effects Of Megsin Gene On The EMMPRIN And MMP-2 In The Mouse Glomerular Mesangial Cells Exposed To High Glucose

Objective: Diabetic nephropathy (DN), as a serious chronic microvascular implication of diabeties, has become one of the main etiological factors resulting in end stage of renal failure with the increasing incidence of diabetics worldwide. So there is a great need to clarify the pathogenesis as well as to seek the methords of preventing and treating DN for nephrologist nowadays. The pathogenesis of DN is affected by many factors. Its main pathological changes are glomerular sclerosis and renal interstitial fibrosis, which results from the imbalance and remodeling of synthesis and degradation of extracellular matrix(ECM).And the abnormal expression and activation of catabolic enzymes regulating ECM metabolism plays an important role during this pathological condition.Megsin,a novel member of the serine protease inhibitor superfamily, predominantly expressed in mesangial cells utilizing a 3,-directed regional cDNA library from cultured human mesangial cells. In the pathophysiology of DN, proliferation of mesangial cells and accumulation of tracellular mesangial matrix are primary events leading the progression of DN. Megsin gene and protein are up-regulated in DN,but the precise mechanism has not been illuminated.Matrix metalloproteinases(matrixmetalloproteinases,MMP) is an important regulation of renal ECM metabolism degradation systems,Including gelatinase,interstitial collagenase,matrixlysin,secretion of elastase and membrane-type MMP and so on,their activeness relies on the zinc ion. A recent study found that megsin potentially inhibits total enzymatic activities of MMP-2,MMP-9 and plasmin in the mouse glomerular mesangial cells exposed to high glucose. A specific monoclonal anti-megsin neutralizing antibodies can enhance the activity of the above-mentioned cytokines. The mechanism of the inhibition of MMP activities by megsin is independent of TGF-β.Therefore, discussing the role of megsin in the extracellular matrix plays an great significance of revealing the mechanism of DN.The study was undertaken by cultured mouse mesangial cells exposed to high glucose in vitro and interfered in the expression of megsin gene by transfection technology in order to investigate the expression of megsin, EMMPRIN,MMP-2 and type IV collagen and discuss the role of megsin in the development of extracellular matrix metabolism in DN.To further clarify the pathogenesis of DN and provide experimental theoretic basis for the prevention and treatment of DN in the early stage.Methods: Cultured glomerular mesangial cells were randomly divided into five groups:normal glucose group (group A,D-glucose 5.5mmol/L),high glucose group (group B,D-glucose 30mmol/L),high glucose + empty vector plasmid group (group C,D-glucose 30mmol/L),high glucose + megsin plasmid group (group D,D-glucose 30mmol/L),high glucose + megsin siRNA plasmid group (group E,D-glucose 30mmol/L).Large collection of constructed empty vector,megsin expression plasmid and megsin siRNA expression plasmid,transfecting transiently with the corresponding plasmid of mouse GMC by Lipofectamine2000 according to experimental design(the efficiency of transfection is determined by the protein expression of megsin in each group detecting by western blot ) and giving the corresponding group of corresponding stimulus are needed.These five groups were harvested at 12hours, 24hours and 48hours.The expression of Megsin, EMMPRIN and MMP-2 was measured by immunocytochemistry and western blot. Radioimmunoassay was conducted for the content of typeⅣcollagen in the supernatants of each group.(the results of total protein with correction).Result: 1 Immunocytochemistry results: under normal glucose condition,megsin expressed in the cytoplasm of MC, weak expression, light brown yellow. In all timepoints, protein expression of group B was higher than the group A in a time-dependent manner. There has no significant difference between group C and group B at each time point (P>0.05);Compared with group C of the same period, the protein expression of megsin in group D increased significantly, dark brown yellow. But group E reduced, the difference was significant (p < 0.01).Under normal glucose condition, EMMPRIN and MMP-2 expressed in the cytoplasm of MC, dark brown yellow. The protein expression of EMMPRIN and MMP-2 in group B was lower than group A in a time-dependent manner. There has no significant difference between group C and group B at each time point (P>0.05). Compared with group C of the same period, the protein expression of EMMPRIN and MMP-2 in group D reduced significantly, weak expression, light brown yellow. But, group E increased, the difference was significant (p<0.01).2 Western blotting results: a small amount of megsin expressed in normal glucose group. The protein expression of group B was higher than the group A in a time-dependent manner.There has no significant difference between group C and group B at each time point (P>0.05);Compared with group C of the same period, the protein expression of megsin in group D increased and group E reduced significantly, the difference was significant (p<0.01). A large number of EMMPRIN and MMP-2 expressed in normal glucose group .The protein expression of group B was lower than group A in a time-dependent manner. There has no significant difference between group C and group B at each time point (P>0.05). Compared with group C of the same period, the protein expression of EMMPRIN and MMP-2 in group D reduced and group E increased, the difference was significant (p<0.01).3 Radioimmunoassay results : from the beginning of 12 hours,the concentrations of type IV collagen (cell total protein correction) in supernatant of MC became higher in group B than that of group A,peaked at 48hours(P<0.05).There was no significant difference between group C and group B(P>0.05).Compared with group C of the same period, the concentrations of type IV collagen increased in group D, But group E reduced, the difference was significant (p<0.01).Conclusions: 1 In cultured mesangial cells, high glucose could promote the expression of megsin and decrease EMMPRIN and MMP-2.This suggestes that megsin, EMMPRIN and MMP-2 may be take part in the pathogenesis of DN.2 In the condition of high glucose, the expression of EMMPRIN and MMP-2 could decrease in the mesangial cells of overexpressed megsin, but typeⅣcollagen increase. After mesangial cells transfected megsin siRNA, the expression of megsin reduce, EMMPRIN and MMP-2 increase and typeⅣcollagen decrease.Megsin may affect the expression of MMP-2 through EMMPRIN and suppress degradation of typeⅣcollagen,resulting in progressive accumulation of extracellular matrix.3 The application of megsin RNA interference technology may alleviate the pathological process of extracellular matrix accumulation and provide a new idea in the prevention and treatment of early DN.