Effects Of Megsin Gene Transfection On Expression Of P38MAPK, MCP-1 And ICAM-1 In Renal Tissue Of Diabetic Mice

Objective:Diabetes and its complications have become a public health problem. One of the most important complications is diabetic nephropathy, which is nowadays the main cause of end-stage renal failure. In spite of our greater understanding of this complication, the intimate mechanisms leading to the development and progression of renal injury are not well understood. How to effectively prevent and treat early diabetic nephropathy is currently a very hot subject concerned by scholars at home and abroad.Recent studies demonstrated that inflammatory cytokines and proinflammatory cytokines such as acute phase protein, chemokines and adhesion molecule probably play a major part in the development of diabetic nephropathy. Megsin is an mesangium-predominant gene,located in 18q21.3, binding with the serine protease,and play a serine protease inhibitor (serpin) activity. Serpin family is composed of a large number of members,their functions relate to blood coagulation, fibrinolysis, inflammation,cell proliferation, apoptosis,signal transduction, digestive and other systems. We speculate that megsin, as a member of the serpin, may participates in certain disfunction of various glomerular disease .Discussing megsin′s roles in renal tissue of diabetic mice has great significance to reveal the mechanism of diabetic nephropathy.We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid,transfected them to STZ-induced diabetic mice, then investigate the relationship between megsin , phospho-p38MAPK,P38,MCP-1 and ICAM-1, to discuss the possible roles of megsin in inflammation of nephropathy, which may provide an important therapeutic targets that can be translated into clinical treatments for diabetic nephropathy. Methods:60 healthy male unilateral nephrectomy CD-1 mice were randomly divided to 4 groups.One of 4 groups was regarded as normal control group(A),then the others received a single intraperitoneal injection of STZ(dissolved in 0.1mol/L citrate buffer,pH 4.5)at a dose of 150mg/kg Wt.The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L randomly and urinary glucose(+++)～(++++) after 72 hours of STZ injection.Then megsin plasmid,megsin-siRNA plasmid and control plasmid via the tail vein of mice were severally injected to diabetic control group(B),megsin plasmid group(C)and megsin-siRNA group(D).All mice were allowed free access to food and water during the experiment. At the end of 2 week,4 week,12 week,collecting the blood,24-hour urine and renal tissue samples,testing serum creatinine;24h urinary protein excretion rate,creatinine;kidney weight/body weight ratio; staining renal tissue by HE,MASSON and periodic acid - Schiff (PAS) , observed the changes in glomerular pathology in ordinary light microscope.The expression of megsin, phospho-p38MAPK,P38,MCP-1 and ICAM-1 in renal cortex was measured with immunohistochemical method and Western-Blot.The results were semiquantitatively analyzed with an image-processing system. All the data were analysed by SPSS13.0 statistics software,P value<0.05 was considered to have statistical significance.Results:1. The mice of group B,C and D all presented polydipsia, polyuria and polyphagia after 4 or 5 days of STZ injection.There is no significant difference in three groups.Kidney mass/body mass ratio,UP and Scr were higher in group B and C at 12 weeks(P<0.01),group C in those were highest(P<0.01),group D is less than group B(P<0.01).2.light microscopy:at 2 and 4 weeks,the number of glomerular cells in group B was more than in group A (p<0.01),but less than group C(p<0.01),the number of glomerular cells decreased in group B at 12 weeks ,but still higher than in group A (p<0.01), less than group C(p<0.01) ;glomerular hypertrophy, thickening of GBM and accumulation of ECM in group B were significantly higher than those in group A and group D,group C is the most serious.3. Immunohistochemistry and renal tissue protein Westen-bolt: The results of immunohistochemistry and Western blot showed that the expression of megsin, phospho-p38MAPK, MCP-1 and ICAM-1,at the end of 2 weeks, was obviously increased in group B and C than group A and D( (p<0.05,p<0.01). Group D is less than the same period of group B and C,there was a significant difference (p<0.05,p < 0.01). Changes of above-mentioned indicators were more significantly different among these groups at 4 weeks (p<0.05,p<0.01).At 12 weeks the expression level of phospho-p38MAPK decreased in group B,but still higher than in groupA and group D(p<0.05,p<0.01),other indicators's expression such as megsin, MCP-1 and ICAM-1 further enhanced , there was significant difference (p<0.05,p<0.01).Conclusion:1 The expression of megsin enhanced in diabetic mice,that consisting with thickening of GBM,accumulation of ECM,inflammatory cell infiltration, mesangial cells hyperplasia,renal hypertrophy and renal dysfunction,suggesting that megsin may play a role in the development and progression of diabetic nephropathy.2 Over-expressed megsin may increase the expression levels of ICAM-1,MCP-1 and phospho-p38MAPK in renal tissue which may help explain the synergistic effect of megsin with other factors in promotes early diabetic nephropathy.However the specific ways have to be further studied.3 Megsin siRNA plasmid can decrease the expression level of megsin, ICAM-1,MCP-1 and phospho-p38MAPK,reduce 24-hr excretion of urinary protein,lower the level of creatinine,lessen the glomerular enlargement,mesangial cell proliferation and renal dysfunction that can be translated into clinical treatments for diabetic nephropathy.Megsin may be a new target for the therapy of diabetic nephropathy.