Gu Dongfeng, Chen Yunying, Masucci Monica, Xiong Chongxiang, Zou Hequn, Holthofer Harry
Clin Nephrol. 2020 Supplement-Jan;93(1):129-133. doi: 10.5414/CNP92S123.
Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), and the most frequent cause of end-stage renal disease (ESRD) in many countries. Urinary extracellular vesicles (UEVs) are considered a rich non-invasive source of markers for renal diseases. In this study, UEV enrichment and analysis in diabetic nephropathy (DN) was performed in a community epidemiological survey supported through the ISN CKHDP program.
Patients were divided into five groups according to severity of kidney damage. A hydrostatic dialysis method was used for UEV enrichment followed by quantitation using Coomassie protein assays and subsequent adjustment using urinary creatinine levels. UEVs were then characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting of tumor susceptibility gene product TSG101. Two-dimensional DIGE (2D-DIGE) was used to analyze differential protein expression in the UEVs. Mass spectrometry (MS) was conducted and MASCOT search engine was used to identify potential biomarkers.
Bradford protein assay showed that protein concentration of UEVs in diabetics with kidney injury increased significantly as compared to normal controls. UEVs present a round, cup-shaped, membrane-encapsulated structure under TEM, and the main peak of UEVs show 55 - 110 nm nanoparticles with NTA. MS and MASCOT identified 22 differential proteins, and MASP2, CALB1, S100A8, and S100A9 were selected as potential biomarkers of early DN based on bioinformatic analysis.
Our results show UEV proteome changes in different stages of DN. The results of this study show four unique proteins that undergo changes in early DN. These promising discoveries may prompt a new field of research focused on improving the diagnosis of DN.
糖尿病肾病（DN）是糖尿病（DM）的主要并发症，也是许多国家终末期肾病（ESRD）最常见的病因。尿细胞外囊泡（UEVs）被认为是肾脏疾病标志物丰富的非侵入性来源。在本研究中，通过国际肾脏病学会慢性肾脏病和透析项目（ISN CKHDP）支持的社区流行病学调查，对糖尿病肾病（DN）患者进行了尿细胞外囊泡（UEV）的富集和分析。
根据肾脏损伤严重程度将患者分为五组。采用静水压透析法富集UEV，然后用考马斯亮蓝蛋白分析法进行定量，并随后用尿肌酐水平进行校正。然后通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）以及肿瘤易感基因产物TSG101的蛋白质免疫印迹法对UEV进行表征。使用二维差异凝胶电泳（2D-DIGE）分析UEV中的差异蛋白表达。进行质谱分析（MS），并使用MASCOT搜索引擎鉴定潜在的生物标志物。
Bradford蛋白分析法显示，与正常对照组相比，肾损伤糖尿病患者的UEV蛋白浓度显著增加。在透射电子显微镜下，UEV呈现圆形、杯状、膜包裹结构，通过纳米颗粒跟踪分析显示UEV的主峰为55 - 110纳米的纳米颗粒。质谱分析和MASCOT鉴定出22种差异蛋白，基于生物信息学分析，选择MASP2、CALB1、S100A8和S100A9作为早期DN的潜在生物标志物。
我们的结果显示了DN不同阶段的UEV蛋白质组变化。本研究结果显示有四种独特的蛋白质在早期DN中发生变化。这些有前景的发现可能会促使一个专注于改善DN诊断的新研究领域的出现。
