磁珠结合质谱分析2型糖尿病肾病早期尿液多肽及药物干预研究
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摘要
糖尿病肾病(Diabetic Nephropathy,DN)是2型糖尿病(Type2diabetes mellitus, T2DM)患者常见的、严重影响生活质量的慢性并发症,发病率逐年升高,据估测,2030年全世界T2DM患者数量将从2000年的1.71亿增加到3.66亿,其中30%-40%的T2DM将发展为DN,在欧美国家DN是终未期肾病(End-stage renal disease,ESRD)的最常见病因,约占全部ESRD病例的44%。早期诊断DN并干预其发展越来越受到医学界的重视,但DN起病隐袭,早期缺乏明显的临床症状。目前临床上早期诊断DN存在较大困难。。肾活检是早期诊断最可靠的方法,然而,很多患者不愿意接受这种具有创伤性的侵入检查,很多接受检查的患者肾脏组织病理活检并不能找到特征性的病理改变。肾穿刺活检术不能作为T2DM患者检查的常规项目。而尿液富含肾脏病变信息,取材简单、无创伤,可重复取样,目前被广泛认可的糖尿病肾病的早期实验室诊断标准是持续性微量白蛋白尿,已有许多研究发现尿微量白蛋白(Microalbuminuria, MALB)作为DN早期诊断指标的准确性和特异性不高,2型糖尿病患者大多年龄较大,常伴有高血压、冠心病等合并症,微量白蛋白尿也可因这些并发症引起。且在MALB出现时,DN已发展到第Ⅲ期,因而只依靠监测MALB并不能及时准确地对DN做出诊断,会带来较高的误诊和漏诊。因此2型糖尿病肾病的早期诊断迫切需要寻找新的特异性标志物以判断病情,指导治疗,评价疗效。随着蛋白质组学技术的发展,将其与体液技术相结合的尿液蛋白质组学技术为DN早期诊断标志物的筛选提供了技术支持,利用尿液蛋白质组学技术可能发现一些新的DN早期诊断标志物并作为药物疗效评价的指标。本研究通过弱阳离子(weak cation exchange, WCX)磁珠分离系统联合基质辅助激光解吸电离-飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry, MALDI-TOF-MS)技术对2型糖尿病患者及模型动物的尿液标本进行比较蛋白组学分析,并对模型动物进行干预研究。本课题研究内容共分为以下三部分。
第一部分：2型糖尿病肾病患者尿小分子多肽差异分析；
第二部分：2型糖尿病肾病大鼠模型的建立及肾病早期尿小分子多肽差异分析；
第三部分：厄贝沙坦干预对糖尿病肾病大鼠尿小分子多肽影响。
第一部分：2型糖尿病肾病患者尿小分子多肽差异分析
目的：利用弱阳离子磁珠分离系统结合MALDI-TOF-MS的方法获取糖尿病肾病患者的尿小分子多肽谱。
方法：按Mogensen标准,68例2型糖尿病患者经测三天平均微量白蛋白尿排泄率(Urinary Albamin Excretion Rate, UAER)后分组,其中正常蛋白尿(A)组24例,微量白蛋白(B)组22例,大量蛋白(C)组22例；健康对照(D)组20例。A及B组肾小球率过滤(glomerular filtration rate,GFR)均>90ml/(min·1.73m2),C组GFR60～89ml/(min·1.73m2)。采用WCX磁珠纯化试剂盒富集尿多肽,再经MALDI-TOF-MS技术,采集尿液多肽谱,应用ClinProtTM软件进行生物学比较分析。
结果：质荷比<12000时,A组与D组相比,有15个蛋白质峰差异有统计学意义(P<0.01)。B组与D组相比,有1个蛋白质峰差异有统计学意义(P=0.0298)。C组与D组相比,有10个蛋白质峰差异有统计学意义(P<0.01)。
结论：弱阳离子磁珠分离系统联合MALDI-TOF-MS蛋白质检测技术,能够获得健康人及DN患者尿液小分子多肽谱,为进一步从中寻找早期筛查及评价干预效果的标志物提供基础。
第二部分：2型糖尿病肾病大鼠模型的建立及肾病早期尿小分子多肽差异分析
目的：探寻高糖高脂饮食联合小剂量链脲佐菌素(Streptozocin, STZ)诱导致2型糖尿病大鼠糖尿病肾病早期尿液小分子差异多肽。
方法：①40只4周龄SD (Sprague-Dawley)大鼠随机分为糖尿病模型(A)组30只和对照(B)组10只,A组应用高糖高脂饮食6周联合STZ注射(以注射时计为0),诱导致2型糖尿病大鼠模型,B组正常饮食；②每2周测体质量,尾静脉取血检测血糖；-2、2、12周周末收集各组大鼠24小时尿液、血样本和肾脏标本。尿液测24小时白蛋白排泄率,血样本测胰岛素、胆固醇、甘油三脂等指标,肾脏标本HE染色光镜下观察。③将大鼠尿液分为四组：A1组(-2周末时A组尿液,10个标本)、A2组(12周末时A组尿液,10个标本)；B组(12周末时B组尿液,10个标本)。采用弱阳离子磁珠联合MALDI-TOF-MS技术对各组尿液样本进行尿液多肽谱的采集,应用ClinProTools软件进行生物学比较分析。
结果：①高糖高脂饮食6周联合25mg/kg STZ腹腔注射诱导2型糖尿病肾病大鼠模型,成模率87%,成模大鼠具有高血糖、高血脂、胰岛素抵抗等特点,并出现DN相应的形态及功能改变。②弱阳离子磁珠联合MALDI-TOF-MS技术检测三组尿液。ClinProTools软件分析后发现A1和B组间无明显差异。A2组和B组间多肽谱比较发现有7个差异多肽峰具有统计学意义,在A2组表达高表达有4个,低表达的有3个。
结论：①利用高糖高脂饮食6周联合小剂量STZ可以成功诱导2型糖尿病肾病大鼠模型。②弱阳离子磁珠联合MALDI-TOF-MS技术,能获得2型糖尿病大鼠糖尿病肾病早期和正常对照大鼠尿液多肽谱,并有效地鉴定出峰度较高的差异峰。
第三部分：厄贝沙坦干预对糖尿病肾病大鼠尿小分子多肽影响
目的探讨血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)1型受体拮抗剂(Angiotensin receptor blocker, ARB)厄贝沙坦对2型糖尿病大鼠尿小分子多肽的影响。方法①将30只实验大鼠随机分成正常对照组(A组)、糖尿病组(B组)、厄贝沙坦治疗组(C组)。高糖高脂饮食联合小剂量STZ诱导致2型糖尿病大鼠糖尿病肾病早期,以STZ注射时计为0；②C组在造模成功后3天即开始喂服厄贝沙坦[50mg/(kg·d),杭州赛诺菲安万特民生制药有限公司)],B组则以等体积蒸馏水灌胃。每2周末测体质量、血压,尾静脉取血检测血糖；-2、12周末收集各组大鼠24小时尿液、血样本和肾脏标本。尿液测24小时白蛋白排泄率,血样本测胰岛素、胆固醇、甘油三脂等指标。肾脏标本测肾重后制作病理切片。③将大鼠尿液分为四组：A1组(-2周时A组尿液,8个标本)、A2组(12周时A组尿液,8个标本)、B组(12周时B组尿液,8个标本)和C组(12周时C组尿液,8个标本)。采用弱阳离子磁珠联合MALDI-TOF-MS技术对各组尿液样本进行尿液多肽谱的采集,应用ClinProTools软件进行生物学比较分析。
结果：①弱阳离子磁珠联合MALDI-TOF-MS技术检测四组尿液。ClinProTools软件分析后发现A1和A2组间无明显差异。A2组和B组间多肽谱比较发现有7个差异多肽峰具有统计学意义,在B组表达高表达有4个,低表达的有3个。C组和A2组间多肽谱比较发现有5个差异多肽峰具有统计学意义,在C组表达高表达有3个,低表达的有2个。C组和B组间多肽谱比较发现有2个差异多肽峰具有统计学意义,在C组表达高表达有1个,低表达的有1个。②C组12周时肾脏病理结构与同时期B组比较改变轻微。
结论：①弱阳离子磁珠联合MALDI-TOF-MS技术,能获得2型糖尿病大鼠糖尿病肾病早期和正常对照大鼠尿液多肽谱,并有效地鉴定出峰度较高的差异峰。②厄贝沙坦早期干预可以明显改善糖尿病肾病大鼠尿液多肽谱的异常。③厄贝沙坦早期干预可以延缓或阻止糖尿病肾病大鼠病理改变进程。
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 MS
Objective: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 Analysis
Objective: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 irbesartan
Objective: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.
第一部分：2型糖尿病肾病患者尿小分子多肽差异分析；
第二部分：2型糖尿病肾病大鼠模型的建立及肾病早期尿小分子多肽差异分析；
第三部分：厄贝沙坦干预对糖尿病肾病大鼠尿小分子多肽影响。
第一部分：2型糖尿病肾病患者尿小分子多肽差异分析
目的：利用弱阳离子磁珠分离系统结合MALDI-TOF-MS的方法获取糖尿病肾病患者的尿小分子多肽谱。
方法：按Mogensen标准,68例2型糖尿病患者经测三天平均微量白蛋白尿排泄率(Urinary Albamin Excretion Rate, UAER)后分组,其中正常蛋白尿(A)组24例,微量白蛋白(B)组22例,大量蛋白(C)组22例；健康对照(D)组20例。A及B组肾小球率过滤(glomerular filtration rate,GFR)均>90ml/(min·1.73m2),C组GFR60～89ml/(min·1.73m2)。采用WCX磁珠纯化试剂盒富集尿多肽,再经MALDI-TOF-MS技术,采集尿液多肽谱,应用ClinProtTM软件进行生物学比较分析。
结果：质荷比<12000时,A组与D组相比,有15个蛋白质峰差异有统计学意义(P<0.01)。B组与D组相比,有1个蛋白质峰差异有统计学意义(P=0.0298)。C组与D组相比,有10个蛋白质峰差异有统计学意义(P<0.01)。
结论：弱阳离子磁珠分离系统联合MALDI-TOF-MS蛋白质检测技术,能够获得健康人及DN患者尿液小分子多肽谱,为进一步从中寻找早期筛查及评价干预效果的标志物提供基础。
第二部分：2型糖尿病肾病大鼠模型的建立及肾病早期尿小分子多肽差异分析
目的：探寻高糖高脂饮食联合小剂量链脲佐菌素(Streptozocin, STZ)诱导致2型糖尿病大鼠糖尿病肾病早期尿液小分子差异多肽。
方法：①40只4周龄SD (Sprague-Dawley)大鼠随机分为糖尿病模型(A)组30只和对照(B)组10只,A组应用高糖高脂饮食6周联合STZ注射(以注射时计为0),诱导致2型糖尿病大鼠模型,B组正常饮食；②每2周测体质量,尾静脉取血检测血糖；-2、2、12周周末收集各组大鼠24小时尿液、血样本和肾脏标本。尿液测24小时白蛋白排泄率,血样本测胰岛素、胆固醇、甘油三脂等指标,肾脏标本HE染色光镜下观察。③将大鼠尿液分为四组：A1组(-2周末时A组尿液,10个标本)、A2组(12周末时A组尿液,10个标本)；B组(12周末时B组尿液,10个标本)。采用弱阳离子磁珠联合MALDI-TOF-MS技术对各组尿液样本进行尿液多肽谱的采集,应用ClinProTools软件进行生物学比较分析。
结果：①高糖高脂饮食6周联合25mg/kg STZ腹腔注射诱导2型糖尿病肾病大鼠模型,成模率87%,成模大鼠具有高血糖、高血脂、胰岛素抵抗等特点,并出现DN相应的形态及功能改变。②弱阳离子磁珠联合MALDI-TOF-MS技术检测三组尿液。ClinProTools软件分析后发现A1和B组间无明显差异。A2组和B组间多肽谱比较发现有7个差异多肽峰具有统计学意义,在A2组表达高表达有4个,低表达的有3个。
结论：①利用高糖高脂饮食6周联合小剂量STZ可以成功诱导2型糖尿病肾病大鼠模型。②弱阳离子磁珠联合MALDI-TOF-MS技术,能获得2型糖尿病大鼠糖尿病肾病早期和正常对照大鼠尿液多肽谱,并有效地鉴定出峰度较高的差异峰。
第三部分：厄贝沙坦干预对糖尿病肾病大鼠尿小分子多肽影响
目的探讨血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)1型受体拮抗剂(Angiotensin receptor blocker, ARB)厄贝沙坦对2型糖尿病大鼠尿小分子多肽的影响。方法①将30只实验大鼠随机分成正常对照组(A组)、糖尿病组(B组)、厄贝沙坦治疗组(C组)。高糖高脂饮食联合小剂量STZ诱导致2型糖尿病大鼠糖尿病肾病早期,以STZ注射时计为0；②C组在造模成功后3天即开始喂服厄贝沙坦[50mg/(kg·d),杭州赛诺菲安万特民生制药有限公司)],B组则以等体积蒸馏水灌胃。每2周末测体质量、血压,尾静脉取血检测血糖；-2、12周末收集各组大鼠24小时尿液、血样本和肾脏标本。尿液测24小时白蛋白排泄率,血样本测胰岛素、胆固醇、甘油三脂等指标。肾脏标本测肾重后制作病理切片。③将大鼠尿液分为四组：A1组(-2周时A组尿液,8个标本)、A2组(12周时A组尿液,8个标本)、B组(12周时B组尿液,8个标本)和C组(12周时C组尿液,8个标本)。采用弱阳离子磁珠联合MALDI-TOF-MS技术对各组尿液样本进行尿液多肽谱的采集,应用ClinProTools软件进行生物学比较分析。
结果：①弱阳离子磁珠联合MALDI-TOF-MS技术检测四组尿液。ClinProTools软件分析后发现A1和A2组间无明显差异。A2组和B组间多肽谱比较发现有7个差异多肽峰具有统计学意义,在B组表达高表达有4个,低表达的有3个。C组和A2组间多肽谱比较发现有5个差异多肽峰具有统计学意义,在C组表达高表达有3个,低表达的有2个。C组和B组间多肽谱比较发现有2个差异多肽峰具有统计学意义,在C组表达高表达有1个,低表达的有1个。②C组12周时肾脏病理结构与同时期B组比较改变轻微。
结论：①弱阳离子磁珠联合MALDI-TOF-MS技术,能获得2型糖尿病大鼠糖尿病肾病早期和正常对照大鼠尿液多肽谱,并有效地鉴定出峰度较高的差异峰。②厄贝沙坦早期干预可以明显改善糖尿病肾病大鼠尿液多肽谱的异常。③厄贝沙坦早期干预可以延缓或阻止糖尿病肾病大鼠病理改变进程。
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 MS
Objective: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 Analysis
Objective: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 irbesartan
Objective: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.
引文
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