Primary Studies On The Relationship Between Advanced Glycation End Products,Glucose Fluctuation And Diabetic Nephropathy

Hyperglycemia is a symbol of diabetes and also a cause of nephropathy complications.Constant hyperglycemia results in non-enzyme glycation of a variety of proteins in kidneys and the formation of Advanced Glycation End Products(AGEs).So there are more and more importance attached to the role of hyperglycemia and AGEs in diabetic nephropathy.Though there are many animal holistic models for the study of DN,and also experiments about the lesion of kidney cells only by AGEs or GLU,there is no article published about combined damage of hyperglycemia and AGEs.The hazard of blood sugar fluctuation to diabetics in clinical study attracts more attention,however there is no report about pathogenesis of diabetic nephropathy caused by blood sugar fluctuation.To study the pathogenesis of diabetic nephropathy,the thesis focuses on the effect of hyperglycemia and AGEs and the variation of glucose levels on kidney cells and the expression of Receptor for Advanced Glycation End Products(RAGE) on cell level through the study of combined effect of hyperglycemia and AGEs on kidney cells and the establishment of the cell model treated with the medium whose glucose concentration is varying.And the experiment was launched mainly on three levels.Firstly the injury of AGEs and hyperglycation to Rat Glomerular Mesangial Cell(RGMC) and Mouse Kidney Cell(MKC);Secondly,the damage effect of glucose fluctuation on RGMC and MKC;Thirdly,the relationship between glucose variation and the expression of RAGE of RGMC cells.1.The discussion of the pathogenesis of DN through the study of combined injury of AGEs and hyperglycemia to RGMC and MKC.RGMC were arranged into six groups , 0mg/mlAGEs+5.5mmol/LGLU(contro;l) , 2mg/mlAGEs , 12mg/mlAGEs, 30mmol/LGLU,2mg/ml AGEs+30mmol/LGLU, 12mg/mlAGEs+30mmol/LGLU,and--cultured for 24 hrs.Then MKC were transferred into 24 wells plates,4 wells repeated per group;the final concentration of AGEs is 0,2,4,5,10,20,25mg/ml,the finalconcentration of GLU is 0,5,10,30,50,100,150mmol/L when single damage;while combined damage,the groups were 0,4,10,25mg/mlAGEs,5mg/mlAGEs+30mmol/L~ GLU and 10mg/mlAGEs+30mmol/L.The results suggested that with the concentration of AGEs and hyperglycemia increasing and treated time prolonging,the leak rate of LDH and leak capacity of NAG of kidney cells rose,but the level of GSH and activity of SOD dropped. And co-existence of AGEs and hyperglycemia prick up the damage to cells.2.The discuss of the pathogenesis of DN through the study of the effect of the variation of GLU concentration on kidney cells by unceasing change of the GLU concentration in the medium for RGMC and MKC ,and simulation of blood sugar fluctuation in diabetics.Cells were grouped into control group (GLU5mmol/L), hyperglycemia group(30mmol/L,GLU),hypoglycemia group(1.5mmol/LGLU)and fluctuation group(1.5mmol/L←→30mmol/LGLU).Fluctuation scheme l:In one cycle, cells were cultured in hyperglycemic medium for 3 hrs,then hypoglycemic medium for 2 hrs.Fluctuation scheme 2:In one cycle,cells were cultured in hypoglycemic medium for 2 hrs,then hyperglycemic medium for 3 hrs.Fluctuation scheme 3:Cells were cultured in hypoglycemic medium for 6 hrs and then in hyperglycemic medium for 18 hrs.Fluctuation scheme 4:Cells were cultured in hypoglycemic medium for 12 hrs and then in hyperglycemic medium for 12 hrs.The results suggested that cells proliferated to some degree in GLU fluctuation group,which validate that the early proliferation of GMC results in glomeruloscerosis; and the damage degree was more serious than hyperglycemic and hypoglycemic groups and it showed that LDH leak rate and extracellular NAG activity increased markedly, GSH level and SOD activity fell noticeably within cells.During the change of the sugar concentration,the longer cells were treated with hypoglycemia,the more seribus lesion of cells.3.Analysis and treatment of the data with BIOED software suggested that the expression of RAGE-mRNA increased in turn in control group,hyperglycemia group,-hypoglycemia group and fluctuation group(fluctuation scheme2).And...