Model Establishment Of Diabetic Nephropathy Combined With Membranous Nephropathy And Research On Its Mechanism

Diabetic nephropathy is a major reason for end-stage renal failure.In recent years, As the prevalence of diabetes in developing countries (including China) increases rapidly, DN will also be the main reason for ESRD in China in the near future. Clinical practice guidelines of KDOQI which is released in 2007 proposed the concept of diabetic kidney disease for the first time.DKD is a clinical performance with kidney damage of diabetes patients, including not only the traditional concept disease of the DN, but also the non-diabetic renal disease in diabetic patients, such as primary glomerular diseases with diabetes, kidney damage in hypertension and so on. In 2007, International Diabetes Federation (IDF) estimates that the number of diabetes in China was about 39.8 million, so the number of NDRD patients was not small. the reasons for proteinuria in diabetic patients were complex,especially the mechanism of proteinuria in membranous nephropathy with diabetic nephropathy and renal pathological changes play a vital role in the choice of treatment.Objective:This experiment wanted to eatablish a rat model of membranous nephropathy combined with diabetic nephropathy.In this study,we used CD4+CD25+ regulatory T cells and p27kip1 as targets for the study.By observing the relationship between diabetic nephropathy with membranous nephropathy and pathological changes and clarifying the mechanism for occurrence of urine protein and the disease pathogenesis, we provided a new way of thinking for the prevention and treatment of NDRD.Methods:Male Wistar rats were randomly divided into four groups:normal group (n=10), diabetic nephropathy (n=10), membranous nephropathy group (n=15), diabetic nephropathy+membranous nephropathy group (n=20).The rat model of diabetic nephropathy was established by single intraperitoneal injection of STZ (55 mg/kg), we could confirme the success of the model of diabetic nephropathy by three consecutive blood glucose measurements which were higher than 16.7 mmol/L as well as urine protein. We dealed with the diabetic rat model according to the modeling approach membranous nephropathy,which was emulsified C-BSA with incomplete Freund's adjuvant, and multi-point injected subcutaneously in rats for pre-immunization. Pre-immunization 1 week later, we began formal immunization, each rat was intraperitoneally injected C-BSA 16mg/kg each time(preparation of a concentration of 10mg/ml),3 times per week,4 weeks in all.After 2 weeks,4 weeks, 6 weeks,8 weeks of the start of formal immunization and before 24 hours of the rats were killed,we collected 24-hour urinary protein.8 weeks later, the rats were killed, we collected the heart blood before execution, collected specimens from the thymus, spleen, kidney tissue, we observed renal pathological changes by renal tissue. We detected the ratio of CD4+CD25+Treg in rat peripheral blood PBMC cell by the way of FCM. P27KIP1mRNA levels were measured by RT-PCR and P27KIP1 protein expression were measured by. Western blotting.Results:we successfully constructed a rat model of diabetic nephropathy, membranous nephropathy model,diabetic nephropathy combined with membranous nephropathy model.The general condition, body weight, kidney weight/body weight, urine protein, blood glucose of every disease model group appears specific features variation. The pathology results showed that:By the way of microscope,the kidney of DN rats showed increased glomerular volume, diffuse mesangial area widened, extracellular matrix diffuse increased,part of the visible capillary basement membrane thickening and capillary cavity occlusion. MN Group glomerular capillary wall was significantly thicker, rigid, tubular epithelial cells has diffuse granular degenerated, focal degeneration, protein tube can be seen in the small tube, glomerular basement membrane were also thickening, rigid, even nail formation and subepithelial material-Stenotrophomonas fuchsin existed; DN+ MN Group:glomerular volume increased, renal mesangial proliferation, matrix increased, diffuse basement membrane thickening,The model group PBMC of peripheral blood CD4+CD25+Treg cells in CD4+T cell ratio:Compared with the control group, CD4+CD25+Treg in peripheral blood of every models were significantly decreased (P<0.05). The renal p27 kip1 mRNA and protein expression: p27 levels had no changes in the transcription, p27 expression of membranous nephropathy group compared with normal were not different (P> 0.05); p27 expression of diabetic nephropathy and membranous nephropathy combined with diabetic nephropathy group were significantly increased (P<0.05).Conclusion:1, we successfully prepared diabetic nephropathy, membranous nephropathy animal models,for the first time we established an animal model of diabetic nephropathy with glomerulonephritis; 2, CD4+CD25+regulatory T cells of peripheral blood in diabetic nephropathy,membranous nephropathy, diabetic nephropathy with membranous nephropathy were significantly reduced, but in the spleen and thymus has no significant difference, CD4+CD25+regulatory T cells played an important role in the study of immune mechanism of diabetic nephropathy with membranous nephropathy.3, In diabetic nephropathy, membranous nephropathy, membranous nephropathy in diabetes mellitus, p27 levels had no changes in the transcription, but protein expression is different, p27 expression in diabetic nephropathy and membranous nephropathy combined with diabetic nephropathy group are significantly increased.