| ObjectiveGlycosylation is an important post-translational modification of proteins,the structure and function of glycosylated proteins may change, protein glycosylation is closely related to diabetes mellitus and diabetic complications, the AGE-modified proteins in renal tissues of diabetic rats are analyzed and identified in this study, which will explore novel molecular markers for the early diagnosis and treatment of patients with diabetic nephropathy.Methods1.50 healthy SD rats were randomly divided into three groups:diabetic rats for lmonth and 3months. healthy control group.2. The diabetic rats were induced by intraperitoneal injection streptozotocin with a dose of 55mg/kg,the control group injected with citrate buffer,the blood glucose concentration of tail vein in third and sixth days after injection was measured, when which were higher than 16.7mmol/L, diabetic rats forl month and 3 months were obtained.3. The morphological changes in renal tissues of diabetic rats for 1 month and 3 months were observed with light and electron microscopes.4. Preparation of homogenate of rats, the concentrations of AGE-modified proteins in kidneys of normal and diabetic rats were measured by ELISA kit,the correlation between blood glucose concentration and concentration of AGE-modified proteins in renal tissues of diabetic rats were analyzed by pearson correlation analysis.5. The proteins in renal tissues were extracted and concentration was measured,the AGE-modified proteins were separated by 1-DE and western blot,the AGE-modified protein bands of differentially expressed were analyzed through quantity-one 4.6.2software.6. Proteins bands expressed differentially were cut from gels,then proteins were digested by enzyme and generated peptides were identified by Liquid Chromatograph Mass Spectrometer/ Mass Spectrometer (LC-MS/MS),the results were obtained by Mascot datebase search.Results1. The pathlolgical morphological changes of renal tissues(1) Structure changes in light microscopes:the glomerular mesangial area of focal segmental of diabetic rats for 1 month expanded,mainly an increase in mesangial matrix,with or without mesangial cell proliferatin compared with normal control rats;the changes of mesangial area with diabetic rats for 3 months were more obvious,and focal segmental capillary basement member thickening appeared in diabetic rats for 3 months.(2) Structure changes in electron microscopes:the mesangial matrix of diabetic rats for 1 month increased, chromatin of endothelial cells and mesangial cells were not agglutinated compared with normal control group,the changes of the mesangial matrix of diabetic rats for 3 months were more obvious, chromatin of endothelial cells and mesangial cells were agglutinated,presenting morphological changes of apoptotic cells,thickening of basement membrane appeared uneven,partial foot lossed and isolated from basement membrane.2. Elisa analysis showed the concentrations of AGEs in renal tissues of 1 month and 3 months diabetic rats[(420.9±53.0) ug/ml and (618.8±86.1)ug/ml, respectively] were significantly (p<0.05, p<0.001 respectively) higher than which of normal rats[(332.3±51.4)ug/ml], the concentrations of AGEs of 3 months diabetic rats[(618.8±86.1)ug/ml] were significantly (p<0.001) higher than which of 1 month[(420.9±53.0)ug/ml]. Pearson correlation analysis showed the concentrations (ug/ml) of AGE-modified proteins in renal tissues of diabetic rats were positively correlated with blood glucose concentration.3.1-DE/Western-blot, quantity-one4.6.2 analysis showed four AGE-modified proteins in diabetic rats were upregulated compared with the control group, in addition,the expression of these four proteins in diabetic ratsfor 3 months were significantly higher than which of 1 month.4. Q Exactive mass spectrometry analysis showed the AGE-modified proteins which were upregulated in renal tissues of diabetic rats were:actin, serotransferrin, catalase, serum Albumin.Conclusions:The concentration and levels of AGE-modified proteins in renal tissues of diabetic rats were increased,which were related with the duration of diabetes mellitus,four AGE-modified proteins were identified:actin, serotransferrin, catalase, aerum albumin.glycosylation may affect the structure and function of these proteins and lead to diabetic kidney damage,the specific mechanism for which needs to be further studied. |
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