| Diabetic nephropathy(DN) is one common chronic microvascular complications of diabetes, seriously affecting the quality of life. The incidence of diabetic nephropathy increased year by year, according to estimates, in2030the number of DM patients in the world will increase from171000000in2000to366000000, including30%-40%T2DM will evolve into DNIn Europe and the United States, DN is the most common cause of the end stage renal disease (ESRD),accounting for about44%of all ESRD cases.In some domestic developed regions diabetic nephropathy is the first cause of ESRD hemodialysis patients.Its exact pathogenesis remains unclear. Glucose toxicity, renal hemodynamics changes, lipid metabolic disorder, hypertension and smoking,genetic, race, gender, environment and so on were risk factors for diabetic nephropathy, and at the same time with genetic polymorphism.The early diagnosis of DN and interfere with the development have gained more and more attention by the medical profession. As insidious onset, DN are difficulty to diagnose at its early stage. Renal biopsy is a trauma, T2DM patients are difficult to accept it as a routine examination. While urine contains disease information,the urine sample can be collected simple, non-invasive, repeatable.At present, the clinical commonly used urinary albumin as DN early diagnosis index, but many studies found that the specificity and accuracy of MALB diagnosis of DN is not so good.With the development of proteomics, urine protein group combined with the fluid technology provides technical support for the early screening markers in the diagnosis of DN. Using proteomics technology may find some new DN early diagnosis marker.Through the study of weak cation (WCX) beads combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) are observe urine specimens of patients with type2diabetes mellitus and animal model in comparative proteomics analysis, and intervention research on animal mode.The content of this paper is divided into three parts as follows.Part one:Urinary polypeptide analysis in the type2diabetic nephropathy with magnetic bead separation and MALDI-TOF MS.Part two:Establishment of Rat Model of Type2Diabetic Nephropathy and Rat Diabetic Nephropathy’s early-stage Urinary Polypeptide Analysis.Part three:Protein expression profile of the urine peptides on early stage of type2diabetic nephropathy rats treated by irbesartan.Part one:Urinary polypeptide analysis in the type2diabetic nephropathy with magnetic bead separation and MALDI-TOF MSObjective:To detect urinary polypeptide patterns of the type2diabetic nephropathy (T2DN)’s every-stage in order to screen and to find out the potential biomarkers. Methods: A total of68urine samples from type2diabetic patients with normoalbuminuria(A,n=24),microalbuminuria(B,n=22)and macroalbuminuria(C,n=22),and healthy controls(D,n=20) were analyzed by MALDI-TOF-MS (the matrix auxiliary laser analytic ionization-flight time mass spectrometry) and ClinProtTM software was used to profile and screen the polypeptide patterns in urine of the patients.Results:When the relative molecular mass<12000, A compared to D groups, there are15difference statistically significant protein peak (P<0.01). B compared to D groups1difference statistically significant low expression of the protein peak (P=0.0298). C compared to D groups, there are10difference statistically significant protein peak (P<0.01). Conclusion: WCX magnetic beads separation coupled to MALDI-TOF-MS is a fast, convenient and high throughput analyzing method capable of screening some relative specific, potential biomarkers from the urine of DN subgroups thus it possesses better clinical value. Part two:Establishment of Rat Model of Type2Diabetic Nephropathy and Rat Diabetic Nephropathy’s early-stage Urinary Polypeptide AnalysisObjective:To monitor and determine the urinary polypeptides of type2diabetic rat models with early stages of diabetic nephropathy which were induced by high-sugar and high-fat feeding combined with low doses of Streptozocin.Methods:Male SD rats at four weeks of age were randomly divided into model group (A group,n=30),which were intraperitoneally injected low doses of streptozotocin after having the sucrose and fat enriched diets for6weeks,and control group (B group, n=10) with conventional diet. Body weight and urine samples from A and B groups were collected for urime albumin excretin every two weeks.At-2、2、12weeks kidney samples were collected for pathological diagnosis and blood samples were collected for serum insulin, cholesterol, and triglyceride levels. Urine samples were divided into four groups:A1group (-2、 weeks urine samples of A group,10samples), A2group (12weeks urine samples of A group,10samples) and B group (12weeks urine samples of B group,10samples). These urinary polypeptides were enriched by weak cations (WCX) magnetic beads purification kits, then combined with Matrix auxiliary laser analytic ionization-flight time mass spectrometry(MALDI-TOF-MS) and ClinProTools software were used to profile and screen the polypeptide patterns in urine. Results:①The rate of modeling in rats fed with high-sugar and high-fat diet for6weeks and received STZ injection was87%.Blood glucose of rats in A group increased significantly than those in B group(P<0.05),body weight of rats in DM group decreased significantly than those in B group (P<0.05).②WCX magnetic beads separation coupled to MALDI-TOF-MS is capable of profiling the polypeptide patterns of the four groups’urine samples. ClinProTools software was used to analysis these polypeptide patterns. There was no significant differences between A1group and B group (P>0.05). While4peaks were up-regulated, while3peaks were down-regulated in A2group compared to B group. Conclusion:①A rat model of type2diabetic nephropathy was developed successfully by combination of dietary-induced insulin resistance and low-doses of STZ-induced hyperglycemia.②WCX magnetic beads separation coupled to MALDI-TOF-MS was capable of profiling the polypeptide patterns of the urine collected from the T2DM rats and the control group, as well as screening some relative specific, potential biomarkers from the urine samples. Part three:Protein expression profile of the urine peptides on early stage of type2diabetic nephropathy rats treated by irbesartanObjective:Observe the protein expression profile of the urinary polypeptides of type2diabetic rat models with early stages of diabetic nephropathy treated by losartan.Methods:①30Male SD rats at four weeks of age were randomly divided into three groups:A group, control group;B group,diabetis group; C group,irbesartan treat group.B and C group which were intraperitoneally injected low doses of streptozotocin after having the sucrose and fat enriched diets for6weeks, and control group with conventional diet.②After the success of modeling the C group is fed irbesartan50mg/(kg-d),B group with the same volume of distilled water.③Body weight, blood pressure and tail vein blood glucose from three groups were collected every two weeks.At-2、12weeks urine samples for urime albumin excretin, kidney samples for pathological diagnosis and blood samples for serum insulin, cholesterol, and triglyceride levels were collected.④Urine samples were divided into four groups:A1group (-2weeks urine samples of A group,8samples), A2group (12weeks urine samples of A group,8samples)、B group (12weeks urine samples of B group,8samples) and C group (12weeks urine samples of C group,8samples). These urinary polypeptides were enriched by weak cations (WCX) magnetic beads purification kits, then combined with Matrix auxiliary laser analytic ionization-flight time mass spectrometry(MALDI-TOF-MS) and ClinProTools software were used to profile and screen the polypeptide patterns in urine.Results:①The rate of modeling in rats fed with high-sugar and high-fat diet for6weeks and received STZ injection was87%.Blood glucose of rats in A group increased significantly than those in B group(P<0.05), body weight of rats in B and C group decreased significantly than those in A group (P<0.05).②WCX magnetic beads separation coupled to MALDI-TOF-MS is capable of profiling the polvpeptide patterns of the four groups’urine samples. ClinProTools software was used to analysis these polypeptide patterns. There was no significant differences between A1group and A2group (P>0.05). While4peaks were up-regulated and3peaks were down-regulated in B group compared to A2group(P<0.05). There were3peaks were up-regulated and2peaks were down-regulated in C group compared to A2group(P<0.05). There were1peaks were up-regulated and1peaks were down-regulated in C group compared to B group(P<0.05). Conclusion:①A rat model of type2diabetic nephropathy was developed successfully by combination of dietary-induced insulin resistance and low-doses of STZ-induced hyperglycemia.(2)WCX magnetic beads separation coupled to MALDI-TOF-MS was capable of profiling the polypeptide patterns of the urine collected from the T2DM rats and the control group, as well as screening some relative specific, potential biomarkers from the urine samples.③The early use of irbesartan can significantly reduce the spectrum of urine polypeptide abnormalities in rat with diabetic nephropathy.④The early use of irbesartan can delay or prevent the process of renal pathological changes in rats with diabetic nephropathy. |
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