尿胞外体在糖尿病肾病中的作用研究
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摘要
背景
糖尿病肾病(diabetic kidney disease,DKD)是糖尿病主要的微血管并发症之一,并且是引起终末期肾病(end stage of renal diseases,ESRD)最常见的原因。糖尿病肾病的临床分期如下：Ⅰ期：正常蛋白尿期,尿白蛋白/肌酐比率(urinary albumin/creatinine ratio,UACR)<30mg/g肌酐；Ⅱ期：微量蛋白尿期,UACR在30-300mg/g肌酐；Ⅲ期大量蛋白尿期,UACR≧300mg/g肌酐或者伴随持续蛋白尿伴血肌酐浓度<1.2mg/dl；Ⅳ期：慢性肾功能衰竭,血肌酐浓度至1.2mg/dl伴蛋白尿；V期：慢性透析治疗。
以往有人认为糖尿病肾病是一个单方向的过程,一旦发生微量白蛋白尿就直接发展为终末期肾病。但最近的研究表明,有些已被诊断为糖尿病。肾病的患者又恢复到正常蛋白尿状态,即使在微量蛋白尿阶段,有三分之一的糖尿病肾病患者失去了肾功能。因此,在诊断糖尿病肾病时,需要更灵敏更有特异性的标志物。
尿液对寻找诊断性标志物的研究是最有用的实验材料。因为它易于从患者处获得,收集过程十分方便并且为非侵入性。除了可溶性的血浆蛋白,尿液中的微囊泡,如胞外体和微粒,最近成为了尿液蛋白分析的靶点。尿液胞外体(exosome)是一种由小管细胞分泌的直径为40-100nm的膜性囊泡,而微粒是一种无膜结构的大小在100-1000nm的微泡。胞外体和微粒均以同样的脂质双层结构为特点,而大部分从尿液中分离的微囊泡被认为是胞外体。有报道证实了水通道蛋白-2仅仅存在于尿液胞外体中,而其他来源的胞外体中没有。这些胞外体可能为肾脏结构和功能损伤提供理想的生物标志物。
已有报道表明二肽基肽酶-4(dipeptidyl peptidase-4,DPP4,即CD26)与糖尿病有密切的关系。DPP4是一种膜相关蛋白,在所有组织中广泛的表达,其在肾脏异位聚集,主要位于皮质、刷状缘和微绒毛碎片中。DPP4可降解活性胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1), GLP-1是餐后由小肠L细胞分泌的肠降血糖素,可以通过葡萄糖依赖的途径增加胰岛素分泌。DPP4抑制剂沙格列汀和西格列汀已被批准用于2型糖尿病患者饮食和运动控制不佳的治疗。我们推测DPP4是尿液胞外体的组成成分,尿液中的胞外体浓度也许反映肾小管上皮细胞的损伤。我们用胞外体特异性特异性抗体(AD-1)和酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测尿液胞外体中的DPP4。 AD-1固定在板上后,这种抗体可以特异性捕获及纯化尿液中完整的胞外体,同时胞外体中DPP4的活性可以被测定。通过这种方法,我们测定了2型糖尿病患者尿液中的胞外体结合的DPP4活性,并且与年龄和性别匹配的正常个体进行对比。我们推测不同个体尿液样本的水合状态也许会导致尿液水/盐含量的不同,从而留取了24h尿,然后将其中的一部分用于我们的分析。我们评价胞外体结合的DPP4用作糖尿病肾病早期的生物标志物的效果。
目的
1.证实糖尿病肾病患者血液及尿液中存在胞外体。
2.尿胞外体结合的DPP4在正常人、糖尿病患者、糖尿病肾病微量白蛋白尿患者、糖尿病肾病大量白蛋白尿患者中的变化。
方法
1.选取127例2型糖尿病患者,根据24h尿白蛋白肌酐比分为糖尿病正常白蛋白尿组(DM,43例),微量白蛋白尿组(DN1,50例),大量白蛋白尿组(DN2,34例)。健康对照组34例。
2.采用特异性的单克隆抗体提纯胞外体,并验证抗体特异性,使用电镜对胞外体进行形态学鉴定。
3.酶联免疫吸附法检测尿液exosome-DPP4的水平及血exosome-DPP4水平,滤过离心的方法检测尿游离DPP4水平,使用Western Blot方法对胞外体进行分子标记物鉴定。
4.同时检测糖化血红蛋白(液相色谱法)、血胆固醇(化学修饰法)、肌酐(苦味酸法)、尿素氮(速率法),尿白蛋白的测定由芬兰Quickread101分析仪完成,结果采用UACR报告。
5.应用逐步多元回归方法分析DPP4与糖化血红蛋白、血胆固醇、肌酐、尿素氮、UACR相关性。
结果
1.血和尿中均存在胞外体,其形态是脂质双层膜结构。
2.血、尿的DPP4同源,尿exosome-DPP4是尿中DPP4的主要存在形式。
3.DM.DN1、DN2组尿exosome-DPP4水平均显著高于正常组(均P<0.01),尿exosome-DPP4与糖化血红蛋白、血胆固醇、肌酐、尿素氮、UACR具有显著相关性。逐步多元回归分析显示,UACR.糖化血红蛋白是尿exosome-DPP
4的独立危险因素(均P<0.001)
结论
尿exosome-DPP4水平与糖尿病肾病严重程度密切相关,对于糖尿病肾病诊断及预后的估计具有一定的临床价值。
背景
目前研究认为不仅1型糖尿病是一种自身免疫性疾病,2型糖尿病的发病也与机体的免疫功能密切相关。2型糖尿病是在遗传基础上由多因素促成的,患者有免疫机制的异常,其胰岛细胞抗体(Islet cell antibody, ICA)阳性者比ICA阴性者有更严重的β细胞损害。糖尿病肾病是其最常见的慢性并发症,因其发病机制不明,故其早期诊断与治疗已成为临床工作面临的重要课题。T淋巴细胞在调节炎症反应中起到了重要的作用,其中特别是辅助性T淋巴细胞(Thelper cell, Th)更为受到关注。IFN-γ可介导β细胞的直接毒性作用；刺激巨噬细胞、淋巴细胞,对胰岛β细胞进行直接杀伤。IL-4因子主要由2型辅助性T淋巴细胞(Th2)产生,介导体液免疫,调节抗体生成。干扰素-γ(Interferon-gamma, IFN-γ)/白介素-4(Interleukin-4, IL-4)的比值代表1型辅助性T细胞(Th1)和Th2的水平变化,目前研究认为Th亚群的激活以及Th1/Th2比例的失衡,参与了动脉粥样硬化、支气管哮喘等多种炎症反应相关疾病的病理过程,但其在2型糖尿病发病机制中的作用尚不完全清楚。
目的
探讨2型糖尿病患者尿液胞外体Th1/Th2的水平变化及在糖尿病肾病发生发展过程中的意义。
方法
选取120例2型糖尿病患者及健康对照组30例。采用特异性的单克隆抗体提纯尿胞外体,酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)检测尿液胞外体(Exosome)-IFN-γ、exosome-IL-4的水平。应用逐步多元回归方法分析Th1/Th2与糖化血红蛋白、胆固醇、尿白蛋白肌酐比、肌酐、尿素氮相关性。
结果
尿exosome-Th1/Th2与尿白蛋白肌酐比(P=0.015)、尿素氮(P=0.001)相关,进一步进行逐步多元回归分析显示尿素氮是exosome-Th1/Th2的独立危险因素(P=0.006)。
结论
尿exosome-Th1/Th2漂移与糖尿病肾病的早期诊断有密切的关系,具有一定的临床价值。
背景
慢性’肾脏病(Chronic kidney disease,CKD)是最常见的严重危害人们身心健康的一类肾病,近年来其发病率呈稳步上升的趋势,平均每年的发病率增加约5%-8%,已经成为全球性重要的公共卫生问题。据2007年2月20日国际肾脏病学会的公告,全球每10人中就有1人患有肾脏疾病,每年死于与慢性肾脏病相关的心血管疾病患者超过10万人。慢性肾功能不全临床症状隐匿,可以完全没有症状或症状不明显,而且肾脏的代偿功能极其强大,即使肾脏功能已经损失50%以上的肾功能不全患者仍可能没有任何症状。常见的慢性肾病有糖尿病肾病、高血压肾病、药物性肾病、1gA肾病和狼疮性肾病。慢性肾病如不及时治疗,将发展成为慢性肾功能衰竭(Chronic renal failure, CRF)和尿毒症。对于肾衰期和尿毒症期患者,目前除血液净化、腹膜透析和肾移植外,尚无理想的治疗方法。我们通过研究慢性肾病患者尿胞外体结合型酶及自由型酶的含量变化,来发现与慢性肾病有关的特异性强的标志物。
目的
探讨慢性肾病肾功能不全患者尿液胞外体结合型及自由型碱性磷酸酶(Exosome-alkaline phosphatase, EXO-ALP)、γ-谷氨酰转肽酶(Exosome-Gamma-glutamyl transferase, EXO-y-GT)及亮氨酸氨基肽酶(Exosome-Leucinea minopeptidase, EXO-LAP)的水平变化。
方法
选取10例2型糖尿病肾病、10例狼疮性肾病、10例止痛剂肾病,符合慢性肾病第1V期标准,健康对照组14例。采用特异性的单克隆抗体(AD-1)提纯尿胞外体,利用胞外体携带的碱性磷酸酶作为标记物,鉴定抗体对胞外体的特异性。用琼脂糖凝胶方法分离碱性磷酸酶同工酶,通过免疫层析方法检测尿液EXO-ALP、EXO-y-GT、EXO-LAP的水平及自由型ALP、γ-GT及LAP水平。
结果
1AD-1抗体分别与不同的自由型ALP和EXO-ALP反应,仅仅EXO-ALP可以与抗体结合。
2把EXO-ALP与AD-1反应,然后把反应物进行琼脂糖电泳,可见EXO-ALP与抗体结合后,电泳迁移率降低。
3免疫层析结果表明正常人尿胞外体结合型ALP、LAP和γ-GT的含量高于自由型的ALP、LAP和γ-GT,3种慢性肾病肾功不全的病人尿胞外体结合型ALP、LAP和γ-GT及尿自由型γ-GT的含量明显下降,而自由型ALP、LAP酶水平升高。
结论
慢性肾病肾功能不全患者尿胞外体结合型的三种酶水平均较正常人降低,机制还需进一步研究。
Background
Diabetic kidney disease (DKD) is one of the major microvascular complications of diabetes and the most common cause of end stage of renal diseases (ESRD). The clinical stages of diabetic nephropathy are classified as follows:stage1, normoalbuminuria (urinary albumin/creatinine ratio (ACR<30mg/g creatinine); stage2, microalbuminuria (ACR from30-300mg/g creatinine); stage3, macroalbuminuria (ACR≧300mg/g creatinine and/or persistent proteinuria with serum concentration of creatinine<1.2mg/dl); stage4, chronic renal failure (serum concentration of creatinine≧1.2mg/dl with proteinuria); stage5, under chronic dialysis therapy.
Previously, DKD was thought to be a unidirectional process that started with microalbuminuria and lead to end-stage renal failure. However, recent studies have shown that a large proportion of patients diagnosed with DKD may reverse back to normoalbuminuria. Therefore, the more sensitive and reliable markers for early detection of DKD are needed.
Urine is one of the most useful resources for such a study and its collection is simple and non-invasive. In addition to soluble plasma proteins, urinary microvesicles, such as exosomes and microparticles, have recently been the targets of urine proteomic analysis. Urinary exosomes are membrane vesicles secreted by tubular cells with a diameter of40-100nm, while microparticles are membrane-shed vesicles with a size range between100and1000nm. Both the exosomes and microparticles are characterized by the same lipid bilayer, but the majority of microvesicles isolated from urine are thought to be exosomes. A previous report has shown that aquaporin-2was only identified in urinary exosomes and not in exosomes from other sources, thereby revealing a link between these exosomes and their urogenital tract origin. These exosomes may carry novel biomarkers for renal dysfunction and structural injury.
Previous reports have indicated that a high level of plasma dipeptidyl peptidase-4(DPP4), also known as CD26, is positively correlated with Diabetes Mellitus (DM). DPP4degrades active glucagon-like peptide-1(GLP-1), an incretin released from L cells in the intestine after meal intake that enhances insulin secretion in a glucose-dependent manner. DPP4inhibitor showed blood glucose-lowering effects in both animal models of diabetes and patients with type2diabetes. Currently, the DPP4inhibitors such as sitagliptin, vidagliptin have been widely used as an adjunct to diet and exercise to improve glycemic control in adults with type2diabetes. DPP4is a membrane-associated peptidase and is widely expressed in all tissues. In the kidney, where the enzyme is exceptionally concentrated, it is located primarily in the cortex and found in the brush-border and microvillus fractions. On the basis of these findings, we hypothesized that DPP4might be a protein component of urinary exosome, and its activity might represent the concentration of exosome, secreted by tubular epithelial cells in the urine. To test this hypothesis, we have developed an ELISA method for DPP4on urinary exosome using a specific monoclonal antibody, AD-1.After it is immobilized on the plate, the antibody can specifically capture and purify the intact exosome from urine samples, and the DPP4activity in the exosome can be determined. With this assay, urinary exosome-bound DPP4activity in patients with type2diabetes mellitus was tested and compared with age-and sex-matched normal individuals. We hypothesized that the variable hydration state of individuals providing urine specimens may lead to some differences in water/salt content of urine, so we pooled all24-hour urine samples from an individual, then used a portion of that for our analysis. The applicability of exosome-bound DPP4as an early biomarker for determining the status of DKD was evaluated.
Objectives
The present study was designed to identify the changes in exosome-DPP4levels in human urine and serum, and to determine whether there were correlations with the severity of DKD.
Methods
127patients with type2diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/creatinine ratio (UACR):microalbuminuria group (n=50); macroalbuminuria group (n=34) and normoalbuminuria group (n=43).34age-and sex-matched non-diabetic healthy subjects were selected as controls. Exosome-bound DPP4and free urinary DPP4were separated by a filtra-centrifugation method. The total exosome was captured by a specific monoclonal antibody, AD-1. DPP4activity was determined by measuring the cleavage of chromogenic free4-nitroaniline from Gly-Pro-p-nitroanilide at405nm with an ELISA plate reader. DPP4protein levels were determined by ELISA and Western blot. Simultaneous detections of glycated hemoglobin (liquid chromatography), blood cholesterol (chemical modification), creatinine (picric acid method), blood urea nitrogen (rate method) were used. Determination of urinary albumin was finished by Quickread101analyzer of Finland,and the results using the UACR report. Multivariate stepwise regression method was for statistical analysis.
Results
1. The exosomes exsisted in urine and serum,and its structure was lipid bilayer membrane.
2. Comparison of the urinary and serum immuno-precipitates in the blot revealed that DPP4protein from human urine was consistent with its serum origin. Results showed that the exosome-bound type was the major form of DPP4in urine.
3. The urinary exosome-DPP4of T2DM group was significantly higher compared to the controls. The urinary exosome-DPP4was positively correlated with UACR in T2DM patients. These findings suggested that the urinary level of exosome-bound DPP4was associated with the severity of DKD.
Conclusions
These findings indicate that urinary exosome-bound DPP4is a specific marker for DKD. The measurement of urinary exosome-bound DPP4may be a useful method for the screening and diagnosis of DKD in T2DM patients.
Background
Not only type1diabetes is considerd as an autoimmune disease, the incidence of type2diabetes is closely related to immune system. Type2diabetes is on the genetic basis by a number of factors contributed to the patients with immune abnormalities.The patients with positive islet cell antibodies (ICA) have more severe P-cell damage than that with ICA negative.Diabetic kidney disease(DKD) is the most common chronic complication, but its pathogenesis is unknown.The early diagnosis and treatment of clinical work have become an important issue. T lymphocytes especially the helper T lymphocytes (Th) play an important role in the regulation of inflammatory responses. Interferon-gamma (IFN-y) direct toxic effects may be mediated by beta cells,and it stimulates macrophages and lymphocytes to destroy pancreatic β cells.Interleukin-4(IL-4) produced by T helper2cell (Th2) mediates humoral immunity, and regulates the production of antibody.The ratio of IFN-γ/IL-4is on behalf of T helper1cell (Th1) and Th2levels.
The previous study has suggested that the activation of Th subsets and the imbalance of Th1/Th2involved in atherosclerosis, bronchial asthma and other inflammatory-related diseases in the pathological process, however,,it is not fully understood in the pathogenesis of type2diabetes.
Objective
To investigate the imbalance of urinary exosomal Th1/Th2and its correlation with diabetic kidney disease.
Method
Thirty healthy volunteers and120patients with type2diabetes mellitus(T2DM) were included. The healthy volunteers served as control. Urinary exosome-IFN-y and exosome-IL-4levels were determined by Enzyme-linked immunosorbent assay(ELISA).Multiple stepwise linear regression was used to analyze the relationship of exosome-Th1/Th2with glycated hemoglobin (HbA1c), cholesterol (CH), urinary albumin/creatinine ratio (UACR), creatinine (CR) and urea nitrogen (BUN).
Results
Correlation analysis showed that urinary exosome-Th1/Th2was positively correlated with UACR (P=0.015) and BUN(P=0.001). Multiple stepwise linear regression analysis showed that BUN was the independent determinant for exosome-Th1/Th2(P=0.006).
Conclusions
Urinary exosome-Th1/Th2correlates with early diagnosis evaluation of diabetic kidney disease.
Background
Chronic kidney disease has become an important global public health problem. It is reported that about1in every10people was suffered from kidney disease, and each year more than100,000people have died from cardiovascular disease associated with chronic kidney disease according to the announcement of the International Society of Nephrology on February20,2007. Chronic renal insufficiency caused by chronic kidney disease is a kind of common and frequently-occurring disease. The clinical symptom of chronic renal insufficiency may be hidden, the patients can have no symptom or obvious symptoms.The compensatory function of the kidney is extremely powerful, even when the kidney function has already lost more than50%,the patients may still have not any symptoms.
Diabetic kidney disease, hypertension kidney disease, drug-induced nephropathy,1gA nephropathy and lupus nephritis are common chronic kidney diseases.If not treated,it will develop into chronic renal failure (CRF) and uremia. In addition to blood purification, peritoneal dialysis and kidney transplantation, there is no ideal treatment for the renal failure and uremia patients.
ALP is widely distributed in the material exchange active cell membrane of human tissues, such as intestinal epithelium, liver and bile capillary membrane, and the arterioles and capillaries artery department of endothelium, et al.When cells were subjected to injury, degeneration and necrosis, intracellular enzymes may go into the urine probably due to cells damaged or organelle function changes inducing the obstacles of the synthesis or the decreased tubular cells reabsorption. Leucine aminopeptidase (LAP) is a kind of protease widespread in human tissues especially in intrahepatic. Unlike other liver enzymes, LAP can be detected not only in blood samples, but also in urine. LAP increased generally in acute nephritis, tubular injury, renal failure and toxic kidney damage.Urinary LAP activity changes in response to the injury of the proximal tubule. Glutamyl endopeptidase mainly distributed in kidney, brain, prostate and liver tissue,highest in the kidney.
In the study, urinary exosome binding enzymes and free enzymes were detected in patients with chronic kidney disease. We look forward to finding specific markers associated with chronic kidney disease, so we can reach early prevention of end-stage kidney failure.
Objective
To explore the urinary exosome binding alkaline phosphatase (EXO-ALP), gamma-glutamylacyl transpeptidase (EXO-γ-GT) and leucine aminopeptidase (EXO-LAP) levels and free type of the above three enzymes in patients with stage Ⅳ chronic kidney disease.
Methods
Ten patients with stage Ⅳ type2diabetic kidney disease, ten cases of stage Ⅳ lupus nephritis, and ten cases of stage Ⅳ analgesic nephropathy were included. The14healthy volunteers served as control. Specific monoclonal antibody (AD-1) purified urinary exosome. The alkaline phosphatase was as the marker to identify the specificity of the antibody against exosome. Alkaline phosphatase isoenzyme separated by agarose gel method. The levels of EXO-ALP, EXO-γ-GT, EXO-LAP and free types of ALP, γ-GT and LAP in urine were measured by immunochromatography method.
Results
1AD-1antibody reacted with the free type of ALP and EXO-ALP respectively,and only the EXO-ALP can bind with the antibody.
2The reactants that EXO-ALP reacted with AD-1was done with agarose gel electrophoresis.The result showed that after the EXO-ALP binding with the antibody, the electrophoretic migration rate decreased.
3Immunochromatographic result showed that urinary EXO-ALP, EXO-y-GT, EXO-LAP are higher than the free type of ALP, LAP and y-GT in normal people. Urinary EXO-ALP, EXO-y-GT, EXO-LAP and urinary free y-GT levels decreased,and the free type of ALP, LAP levels increased inversely in the patients with stage IV chronic kidney disease.
Conclusion
The urinary exosome-bound enzymes in stage IV chronic kidney disease decreased, and the mechanism needs further study.
糖尿病肾病(diabetic kidney disease,DKD)是糖尿病主要的微血管并发症之一,并且是引起终末期肾病(end stage of renal diseases,ESRD)最常见的原因。糖尿病肾病的临床分期如下：Ⅰ期：正常蛋白尿期,尿白蛋白/肌酐比率(urinary albumin/creatinine ratio,UACR)<30mg/g肌酐；Ⅱ期：微量蛋白尿期,UACR在30-300mg/g肌酐；Ⅲ期大量蛋白尿期,UACR≧300mg/g肌酐或者伴随持续蛋白尿伴血肌酐浓度<1.2mg/dl；Ⅳ期：慢性肾功能衰竭,血肌酐浓度至1.2mg/dl伴蛋白尿；V期：慢性透析治疗。
以往有人认为糖尿病肾病是一个单方向的过程,一旦发生微量白蛋白尿就直接发展为终末期肾病。但最近的研究表明,有些已被诊断为糖尿病。肾病的患者又恢复到正常蛋白尿状态,即使在微量蛋白尿阶段,有三分之一的糖尿病肾病患者失去了肾功能。因此,在诊断糖尿病肾病时,需要更灵敏更有特异性的标志物。
尿液对寻找诊断性标志物的研究是最有用的实验材料。因为它易于从患者处获得,收集过程十分方便并且为非侵入性。除了可溶性的血浆蛋白,尿液中的微囊泡,如胞外体和微粒,最近成为了尿液蛋白分析的靶点。尿液胞外体(exosome)是一种由小管细胞分泌的直径为40-100nm的膜性囊泡,而微粒是一种无膜结构的大小在100-1000nm的微泡。胞外体和微粒均以同样的脂质双层结构为特点,而大部分从尿液中分离的微囊泡被认为是胞外体。有报道证实了水通道蛋白-2仅仅存在于尿液胞外体中,而其他来源的胞外体中没有。这些胞外体可能为肾脏结构和功能损伤提供理想的生物标志物。
已有报道表明二肽基肽酶-4(dipeptidyl peptidase-4,DPP4,即CD26)与糖尿病有密切的关系。DPP4是一种膜相关蛋白,在所有组织中广泛的表达,其在肾脏异位聚集,主要位于皮质、刷状缘和微绒毛碎片中。DPP4可降解活性胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1), GLP-1是餐后由小肠L细胞分泌的肠降血糖素,可以通过葡萄糖依赖的途径增加胰岛素分泌。DPP4抑制剂沙格列汀和西格列汀已被批准用于2型糖尿病患者饮食和运动控制不佳的治疗。我们推测DPP4是尿液胞外体的组成成分,尿液中的胞外体浓度也许反映肾小管上皮细胞的损伤。我们用胞外体特异性特异性抗体(AD-1)和酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测尿液胞外体中的DPP4。 AD-1固定在板上后,这种抗体可以特异性捕获及纯化尿液中完整的胞外体,同时胞外体中DPP4的活性可以被测定。通过这种方法,我们测定了2型糖尿病患者尿液中的胞外体结合的DPP4活性,并且与年龄和性别匹配的正常个体进行对比。我们推测不同个体尿液样本的水合状态也许会导致尿液水/盐含量的不同,从而留取了24h尿,然后将其中的一部分用于我们的分析。我们评价胞外体结合的DPP4用作糖尿病肾病早期的生物标志物的效果。
目的
1.证实糖尿病肾病患者血液及尿液中存在胞外体。
2.尿胞外体结合的DPP4在正常人、糖尿病患者、糖尿病肾病微量白蛋白尿患者、糖尿病肾病大量白蛋白尿患者中的变化。
方法
1.选取127例2型糖尿病患者,根据24h尿白蛋白肌酐比分为糖尿病正常白蛋白尿组(DM,43例),微量白蛋白尿组(DN1,50例),大量白蛋白尿组(DN2,34例)。健康对照组34例。
2.采用特异性的单克隆抗体提纯胞外体,并验证抗体特异性,使用电镜对胞外体进行形态学鉴定。
3.酶联免疫吸附法检测尿液exosome-DPP4的水平及血exosome-DPP4水平,滤过离心的方法检测尿游离DPP4水平,使用Western Blot方法对胞外体进行分子标记物鉴定。
4.同时检测糖化血红蛋白(液相色谱法)、血胆固醇(化学修饰法)、肌酐(苦味酸法)、尿素氮(速率法),尿白蛋白的测定由芬兰Quickread101分析仪完成,结果采用UACR报告。
5.应用逐步多元回归方法分析DPP4与糖化血红蛋白、血胆固醇、肌酐、尿素氮、UACR相关性。
结果
1.血和尿中均存在胞外体,其形态是脂质双层膜结构。
2.血、尿的DPP4同源,尿exosome-DPP4是尿中DPP4的主要存在形式。
3.DM.DN1、DN2组尿exosome-DPP4水平均显著高于正常组(均P<0.01),尿exosome-DPP4与糖化血红蛋白、血胆固醇、肌酐、尿素氮、UACR具有显著相关性。逐步多元回归分析显示,UACR.糖化血红蛋白是尿exosome-DPP
4的独立危险因素(均P<0.001)
结论
尿exosome-DPP4水平与糖尿病肾病严重程度密切相关,对于糖尿病肾病诊断及预后的估计具有一定的临床价值。
背景
目前研究认为不仅1型糖尿病是一种自身免疫性疾病,2型糖尿病的发病也与机体的免疫功能密切相关。2型糖尿病是在遗传基础上由多因素促成的,患者有免疫机制的异常,其胰岛细胞抗体(Islet cell antibody, ICA)阳性者比ICA阴性者有更严重的β细胞损害。糖尿病肾病是其最常见的慢性并发症,因其发病机制不明,故其早期诊断与治疗已成为临床工作面临的重要课题。T淋巴细胞在调节炎症反应中起到了重要的作用,其中特别是辅助性T淋巴细胞(Thelper cell, Th)更为受到关注。IFN-γ可介导β细胞的直接毒性作用；刺激巨噬细胞、淋巴细胞,对胰岛β细胞进行直接杀伤。IL-4因子主要由2型辅助性T淋巴细胞(Th2)产生,介导体液免疫,调节抗体生成。干扰素-γ(Interferon-gamma, IFN-γ)/白介素-4(Interleukin-4, IL-4)的比值代表1型辅助性T细胞(Th1)和Th2的水平变化,目前研究认为Th亚群的激活以及Th1/Th2比例的失衡,参与了动脉粥样硬化、支气管哮喘等多种炎症反应相关疾病的病理过程,但其在2型糖尿病发病机制中的作用尚不完全清楚。
目的
探讨2型糖尿病患者尿液胞外体Th1/Th2的水平变化及在糖尿病肾病发生发展过程中的意义。
方法
选取120例2型糖尿病患者及健康对照组30例。采用特异性的单克隆抗体提纯尿胞外体,酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)检测尿液胞外体(Exosome)-IFN-γ、exosome-IL-4的水平。应用逐步多元回归方法分析Th1/Th2与糖化血红蛋白、胆固醇、尿白蛋白肌酐比、肌酐、尿素氮相关性。
结果
尿exosome-Th1/Th2与尿白蛋白肌酐比(P=0.015)、尿素氮(P=0.001)相关,进一步进行逐步多元回归分析显示尿素氮是exosome-Th1/Th2的独立危险因素(P=0.006)。
结论
尿exosome-Th1/Th2漂移与糖尿病肾病的早期诊断有密切的关系,具有一定的临床价值。
背景
慢性’肾脏病(Chronic kidney disease,CKD)是最常见的严重危害人们身心健康的一类肾病,近年来其发病率呈稳步上升的趋势,平均每年的发病率增加约5%-8%,已经成为全球性重要的公共卫生问题。据2007年2月20日国际肾脏病学会的公告,全球每10人中就有1人患有肾脏疾病,每年死于与慢性肾脏病相关的心血管疾病患者超过10万人。慢性肾功能不全临床症状隐匿,可以完全没有症状或症状不明显,而且肾脏的代偿功能极其强大,即使肾脏功能已经损失50%以上的肾功能不全患者仍可能没有任何症状。常见的慢性肾病有糖尿病肾病、高血压肾病、药物性肾病、1gA肾病和狼疮性肾病。慢性肾病如不及时治疗,将发展成为慢性肾功能衰竭(Chronic renal failure, CRF)和尿毒症。对于肾衰期和尿毒症期患者,目前除血液净化、腹膜透析和肾移植外,尚无理想的治疗方法。我们通过研究慢性肾病患者尿胞外体结合型酶及自由型酶的含量变化,来发现与慢性肾病有关的特异性强的标志物。
目的
探讨慢性肾病肾功能不全患者尿液胞外体结合型及自由型碱性磷酸酶(Exosome-alkaline phosphatase, EXO-ALP)、γ-谷氨酰转肽酶(Exosome-Gamma-glutamyl transferase, EXO-y-GT)及亮氨酸氨基肽酶(Exosome-Leucinea minopeptidase, EXO-LAP)的水平变化。
方法
选取10例2型糖尿病肾病、10例狼疮性肾病、10例止痛剂肾病,符合慢性肾病第1V期标准,健康对照组14例。采用特异性的单克隆抗体(AD-1)提纯尿胞外体,利用胞外体携带的碱性磷酸酶作为标记物,鉴定抗体对胞外体的特异性。用琼脂糖凝胶方法分离碱性磷酸酶同工酶,通过免疫层析方法检测尿液EXO-ALP、EXO-y-GT、EXO-LAP的水平及自由型ALP、γ-GT及LAP水平。
结果
1AD-1抗体分别与不同的自由型ALP和EXO-ALP反应,仅仅EXO-ALP可以与抗体结合。
2把EXO-ALP与AD-1反应,然后把反应物进行琼脂糖电泳,可见EXO-ALP与抗体结合后,电泳迁移率降低。
3免疫层析结果表明正常人尿胞外体结合型ALP、LAP和γ-GT的含量高于自由型的ALP、LAP和γ-GT,3种慢性肾病肾功不全的病人尿胞外体结合型ALP、LAP和γ-GT及尿自由型γ-GT的含量明显下降,而自由型ALP、LAP酶水平升高。
结论
慢性肾病肾功能不全患者尿胞外体结合型的三种酶水平均较正常人降低,机制还需进一步研究。
Background
Diabetic kidney disease (DKD) is one of the major microvascular complications of diabetes and the most common cause of end stage of renal diseases (ESRD). The clinical stages of diabetic nephropathy are classified as follows:stage1, normoalbuminuria (urinary albumin/creatinine ratio (ACR<30mg/g creatinine); stage2, microalbuminuria (ACR from30-300mg/g creatinine); stage3, macroalbuminuria (ACR≧300mg/g creatinine and/or persistent proteinuria with serum concentration of creatinine<1.2mg/dl); stage4, chronic renal failure (serum concentration of creatinine≧1.2mg/dl with proteinuria); stage5, under chronic dialysis therapy.
Previously, DKD was thought to be a unidirectional process that started with microalbuminuria and lead to end-stage renal failure. However, recent studies have shown that a large proportion of patients diagnosed with DKD may reverse back to normoalbuminuria. Therefore, the more sensitive and reliable markers for early detection of DKD are needed.
Urine is one of the most useful resources for such a study and its collection is simple and non-invasive. In addition to soluble plasma proteins, urinary microvesicles, such as exosomes and microparticles, have recently been the targets of urine proteomic analysis. Urinary exosomes are membrane vesicles secreted by tubular cells with a diameter of40-100nm, while microparticles are membrane-shed vesicles with a size range between100and1000nm. Both the exosomes and microparticles are characterized by the same lipid bilayer, but the majority of microvesicles isolated from urine are thought to be exosomes. A previous report has shown that aquaporin-2was only identified in urinary exosomes and not in exosomes from other sources, thereby revealing a link between these exosomes and their urogenital tract origin. These exosomes may carry novel biomarkers for renal dysfunction and structural injury.
Previous reports have indicated that a high level of plasma dipeptidyl peptidase-4(DPP4), also known as CD26, is positively correlated with Diabetes Mellitus (DM). DPP4degrades active glucagon-like peptide-1(GLP-1), an incretin released from L cells in the intestine after meal intake that enhances insulin secretion in a glucose-dependent manner. DPP4inhibitor showed blood glucose-lowering effects in both animal models of diabetes and patients with type2diabetes. Currently, the DPP4inhibitors such as sitagliptin, vidagliptin have been widely used as an adjunct to diet and exercise to improve glycemic control in adults with type2diabetes. DPP4is a membrane-associated peptidase and is widely expressed in all tissues. In the kidney, where the enzyme is exceptionally concentrated, it is located primarily in the cortex and found in the brush-border and microvillus fractions. On the basis of these findings, we hypothesized that DPP4might be a protein component of urinary exosome, and its activity might represent the concentration of exosome, secreted by tubular epithelial cells in the urine. To test this hypothesis, we have developed an ELISA method for DPP4on urinary exosome using a specific monoclonal antibody, AD-1.After it is immobilized on the plate, the antibody can specifically capture and purify the intact exosome from urine samples, and the DPP4activity in the exosome can be determined. With this assay, urinary exosome-bound DPP4activity in patients with type2diabetes mellitus was tested and compared with age-and sex-matched normal individuals. We hypothesized that the variable hydration state of individuals providing urine specimens may lead to some differences in water/salt content of urine, so we pooled all24-hour urine samples from an individual, then used a portion of that for our analysis. The applicability of exosome-bound DPP4as an early biomarker for determining the status of DKD was evaluated.
Objectives
The present study was designed to identify the changes in exosome-DPP4levels in human urine and serum, and to determine whether there were correlations with the severity of DKD.
Methods
127patients with type2diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/creatinine ratio (UACR):microalbuminuria group (n=50); macroalbuminuria group (n=34) and normoalbuminuria group (n=43).34age-and sex-matched non-diabetic healthy subjects were selected as controls. Exosome-bound DPP4and free urinary DPP4were separated by a filtra-centrifugation method. The total exosome was captured by a specific monoclonal antibody, AD-1. DPP4activity was determined by measuring the cleavage of chromogenic free4-nitroaniline from Gly-Pro-p-nitroanilide at405nm with an ELISA plate reader. DPP4protein levels were determined by ELISA and Western blot. Simultaneous detections of glycated hemoglobin (liquid chromatography), blood cholesterol (chemical modification), creatinine (picric acid method), blood urea nitrogen (rate method) were used. Determination of urinary albumin was finished by Quickread101analyzer of Finland,and the results using the UACR report. Multivariate stepwise regression method was for statistical analysis.
Results
1. The exosomes exsisted in urine and serum,and its structure was lipid bilayer membrane.
2. Comparison of the urinary and serum immuno-precipitates in the blot revealed that DPP4protein from human urine was consistent with its serum origin. Results showed that the exosome-bound type was the major form of DPP4in urine.
3. The urinary exosome-DPP4of T2DM group was significantly higher compared to the controls. The urinary exosome-DPP4was positively correlated with UACR in T2DM patients. These findings suggested that the urinary level of exosome-bound DPP4was associated with the severity of DKD.
Conclusions
These findings indicate that urinary exosome-bound DPP4is a specific marker for DKD. The measurement of urinary exosome-bound DPP4may be a useful method for the screening and diagnosis of DKD in T2DM patients.
Background
Not only type1diabetes is considerd as an autoimmune disease, the incidence of type2diabetes is closely related to immune system. Type2diabetes is on the genetic basis by a number of factors contributed to the patients with immune abnormalities.The patients with positive islet cell antibodies (ICA) have more severe P-cell damage than that with ICA negative.Diabetic kidney disease(DKD) is the most common chronic complication, but its pathogenesis is unknown.The early diagnosis and treatment of clinical work have become an important issue. T lymphocytes especially the helper T lymphocytes (Th) play an important role in the regulation of inflammatory responses. Interferon-gamma (IFN-y) direct toxic effects may be mediated by beta cells,and it stimulates macrophages and lymphocytes to destroy pancreatic β cells.Interleukin-4(IL-4) produced by T helper2cell (Th2) mediates humoral immunity, and regulates the production of antibody.The ratio of IFN-γ/IL-4is on behalf of T helper1cell (Th1) and Th2levels.
The previous study has suggested that the activation of Th subsets and the imbalance of Th1/Th2involved in atherosclerosis, bronchial asthma and other inflammatory-related diseases in the pathological process, however,,it is not fully understood in the pathogenesis of type2diabetes.
Objective
To investigate the imbalance of urinary exosomal Th1/Th2and its correlation with diabetic kidney disease.
Method
Thirty healthy volunteers and120patients with type2diabetes mellitus(T2DM) were included. The healthy volunteers served as control. Urinary exosome-IFN-y and exosome-IL-4levels were determined by Enzyme-linked immunosorbent assay(ELISA).Multiple stepwise linear regression was used to analyze the relationship of exosome-Th1/Th2with glycated hemoglobin (HbA1c), cholesterol (CH), urinary albumin/creatinine ratio (UACR), creatinine (CR) and urea nitrogen (BUN).
Results
Correlation analysis showed that urinary exosome-Th1/Th2was positively correlated with UACR (P=0.015) and BUN(P=0.001). Multiple stepwise linear regression analysis showed that BUN was the independent determinant for exosome-Th1/Th2(P=0.006).
Conclusions
Urinary exosome-Th1/Th2correlates with early diagnosis evaluation of diabetic kidney disease.
Background
Chronic kidney disease has become an important global public health problem. It is reported that about1in every10people was suffered from kidney disease, and each year more than100,000people have died from cardiovascular disease associated with chronic kidney disease according to the announcement of the International Society of Nephrology on February20,2007. Chronic renal insufficiency caused by chronic kidney disease is a kind of common and frequently-occurring disease. The clinical symptom of chronic renal insufficiency may be hidden, the patients can have no symptom or obvious symptoms.The compensatory function of the kidney is extremely powerful, even when the kidney function has already lost more than50%,the patients may still have not any symptoms.
Diabetic kidney disease, hypertension kidney disease, drug-induced nephropathy,1gA nephropathy and lupus nephritis are common chronic kidney diseases.If not treated,it will develop into chronic renal failure (CRF) and uremia. In addition to blood purification, peritoneal dialysis and kidney transplantation, there is no ideal treatment for the renal failure and uremia patients.
ALP is widely distributed in the material exchange active cell membrane of human tissues, such as intestinal epithelium, liver and bile capillary membrane, and the arterioles and capillaries artery department of endothelium, et al.When cells were subjected to injury, degeneration and necrosis, intracellular enzymes may go into the urine probably due to cells damaged or organelle function changes inducing the obstacles of the synthesis or the decreased tubular cells reabsorption. Leucine aminopeptidase (LAP) is a kind of protease widespread in human tissues especially in intrahepatic. Unlike other liver enzymes, LAP can be detected not only in blood samples, but also in urine. LAP increased generally in acute nephritis, tubular injury, renal failure and toxic kidney damage.Urinary LAP activity changes in response to the injury of the proximal tubule. Glutamyl endopeptidase mainly distributed in kidney, brain, prostate and liver tissue,highest in the kidney.
In the study, urinary exosome binding enzymes and free enzymes were detected in patients with chronic kidney disease. We look forward to finding specific markers associated with chronic kidney disease, so we can reach early prevention of end-stage kidney failure.
Objective
To explore the urinary exosome binding alkaline phosphatase (EXO-ALP), gamma-glutamylacyl transpeptidase (EXO-γ-GT) and leucine aminopeptidase (EXO-LAP) levels and free type of the above three enzymes in patients with stage Ⅳ chronic kidney disease.
Methods
Ten patients with stage Ⅳ type2diabetic kidney disease, ten cases of stage Ⅳ lupus nephritis, and ten cases of stage Ⅳ analgesic nephropathy were included. The14healthy volunteers served as control. Specific monoclonal antibody (AD-1) purified urinary exosome. The alkaline phosphatase was as the marker to identify the specificity of the antibody against exosome. Alkaline phosphatase isoenzyme separated by agarose gel method. The levels of EXO-ALP, EXO-γ-GT, EXO-LAP and free types of ALP, γ-GT and LAP in urine were measured by immunochromatography method.
Results
1AD-1antibody reacted with the free type of ALP and EXO-ALP respectively,and only the EXO-ALP can bind with the antibody.
2The reactants that EXO-ALP reacted with AD-1was done with agarose gel electrophoresis.The result showed that after the EXO-ALP binding with the antibody, the electrophoretic migration rate decreased.
3Immunochromatographic result showed that urinary EXO-ALP, EXO-y-GT, EXO-LAP are higher than the free type of ALP, LAP and y-GT in normal people. Urinary EXO-ALP, EXO-y-GT, EXO-LAP and urinary free y-GT levels decreased,and the free type of ALP, LAP levels increased inversely in the patients with stage IV chronic kidney disease.
Conclusion
The urinary exosome-bound enzymes in stage IV chronic kidney disease decreased, and the mechanism needs further study.
引文
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41. Mitic B,Lazarevic G,Vlahovic P,et al. Diagnostic Value of the Aminopeptidase N, N-Acetyl-b-D-Glucosaminidase and Dipeptidylpeptidase Ⅳ in Evaluating Tubular Dysfunction in Patients with Glomerulopathies. Renal Failure, 2008, 30:896-903.
42. Mannucci E, Pala L, Ciani S,et al. Hyperglycaemia increases dipeptidyl peptidase IV activity in diabetes mellitus. Diabetologia,2005,48:1168-1172.
43. Ryskjaer J, Deacon CF, Carr RD, et al. Plasma dipeptidyl peptidase-IV activity in patients with type-2 diabetes mellitus correlates positively with HbAlc levels, but is not acutely affected by food intake. European Journal of Endocrinology, 2006,155:485-493.
44. Meneilly GS, Demuth HU, McIntosh CHS, et al. Effect of ageing and diabetes on glucose-dependent insulinotropic polypeptide and dipeptidyl peptidase Ⅳ responses to oral glucose. Diabetic Medicine,2000,17(5):346-350.
45. McKillop AM, Duffy NA, Lindsay JR,et al. Decreased dipeptidyl peptidase-Ⅳ activity and glucagon-like peptide-1 (7-36) amide degradation in type 2 diabetic subjects. Diabetes Research and Clinical Practice,2008,79:79-85.
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18. Deng JT, Parsons PG.Solid phase immunoassay for high molecular weight alkaline phosphatase in human sera using a specific monoclonal antibody. Clinica chimica acta; international journal of clinical chemistry,1988, 176(3):291-301.
19. Miranda, Daniel T. Bond, Mary McKee et al. Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.Kevin C. Kidney international,2010,78:191-199.
20. Trams EG, Lauter CJ, Salem Jr N, et al. Exfoliation of membrane ecto-enzymes in the form ofmicrovesicles. Biochim Biophys Acta,1981,645: 63-70.
21. Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling ofthe transferrin receptor in rat reticulocytes. J Cell Biol,1983,97: 329-339.
22. Pan BT, TengK Wu C, et al. Electron microscopic evidence for externalization ofthe transferrin receptor in vesicular form in sheepreticulocytes. J Cell Biol, 1985,101:942-948.
23. Lin XP, Almqvist N, Telemo E, Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production ofexosomes. Blood Cells Mol Dis,2005,35:122-128.
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29. Gonzales PA, Pisitkun T, Hoffert JD, et al. Large-scale proteomics and phosphoproteomics of urinary exosomes. J. Am. Soc. Nephrol,2009,20:363-379.
30. Conde-Vancells, J., Rodriguez-Suarez, E., Gonzalez, E.,et al. Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples.Proteomics Clin Appl,2010,4:416-425.
31. Cheruvanky A, Zhou H, Pisitkun T, et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol,2007,292:F1657-F1661.
32. Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scandinavian journal of immunology,2007, 66:159-165.
33. Hopsu-Havu VK,Glenner GG.A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-beta-naphthylamide[J]. Histochemie,1966,7:197-201.
34. Hopsu-Havu VK, Sarimo SR.Purification and characterization of an aminopeptidase hydrolyzing glysyl-proline-naphthylamide[J].Hoppe Seylers Z Physiol Chem,1967,348:1540-1550.
35. Hopsu-Havu VK, Rintola P, Glenner GG.A dog kidney aminopeptidase liberating N-terminal peptides. Partial purification and characteristics[J]. Acta Chem Scand,1968,22:299-308.
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39. Sato T, Yamochi T, Aytac U,et al. CD26 regulates p38 mitogen-activated protein kinase-dependent phosphorylation of integrin beta 1, adhesion to extracellular matrix, and tumorigenicity of T-anaplastic large cell lymphoma Karpas 299. Cancer Res,2005,65:6950-6956.
40. Yamochi T, Aytac U, Sato T, et al. Regulation of p38 phosphorylation and topoisomerase II alpha expression in the B-cell lymphoma line Jiyoye by CD26/dipeptidyl peptidase IV is associated with enhanced in vitro and in vivo sensitivity to doxorubicin. Cancer Res,2005,65:1973-1983.
41. Mitic B,Lazarevic G,Vlahovic P,et al. Diagnostic Value of the Aminopeptidase N, N-Acetyl-b-D-Glucosaminidase and Dipeptidylpeptidase Ⅳ in Evaluating Tubular Dysfunction in Patients with Glomerulopathies. Renal Failure, 2008, 30:896-903.
42. Mannucci E, Pala L, Ciani S,et al. Hyperglycaemia increases dipeptidyl peptidase IV activity in diabetes mellitus. Diabetologia,2005,48:1168-1172.
43. Ryskjaer J, Deacon CF, Carr RD, et al. Plasma dipeptidyl peptidase-IV activity in patients with type-2 diabetes mellitus correlates positively with HbAlc levels, but is not acutely affected by food intake. European Journal of Endocrinology, 2006,155:485-493.
44. Meneilly GS, Demuth HU, McIntosh CHS, et al. Effect of ageing and diabetes on glucose-dependent insulinotropic polypeptide and dipeptidyl peptidase Ⅳ responses to oral glucose. Diabetic Medicine,2000,17(5):346-350.
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