尿液外泌体miRNA-22在糖尿病肾病中的水平变化及其与足细胞损伤、病情严重程度的关系

Urinary Exosomes miRNA-22 Level Changes in Diabetic Nephropathy and Its Relationship with Podocyte Damage and Severity

目的 探讨尿液外泌体miRNA-22在糖尿病肾病(diabetic nephropathy, DN)中的水平变化及其与足细胞损伤、病情严重程度的关系。方法 纳入2017-01/2019-01月于作者医院就诊的100例2型糖尿病合并DN患者(DN组),以同期来作者医院进行健康体检的30例健康人群作为对照组。收集研究者尿液并提取外泌体,蛋白质印迹法(Western blot)检测尿液中外泌体标志物溶酶体颗粒糖蛋白(lysosome intact membrane protein, CD63),采用聚合酶链反应(polymerase chain reaction, PCR)检测外泌体miRNA-22水平,Western blot检测nephrin蛋白水平,鉴定尿液中外泌体,比较DN组与对照组外泌体miRNA-22及nephrin蛋白水平。根据尿微量白蛋白定量指标将患者分为微量白蛋白尿组(n=50)和大量白蛋白尿组(n=50),比较微量白蛋白尿组与大量白蛋白尿组外泌体miRNA-22及nephrin蛋白水平,Pearson相关性分析法评价外泌体miRNA-22与nephrin蛋白水平的相关性,Spearman相关分析评价外泌体miRNA-22与DN病情严重程度的相关性。结果 通过透射电子显微镜在尿液中观察到直径40~100 nm的圆形结构,并且DN组中外泌体表面标志蛋白CD63的表达显著高于对照组(P<0.05);与对照组比较,DN组外泌体miRNA-22相对表达量升高(P<0.05),nephrin蛋白水平降低(P<0.05)。与微量白蛋白尿组比较,大量白蛋白尿组外泌体miRNA-22相对表达量升高(P<0.05),nephrin蛋白水平降低(P<0.05)。Pearson相关性分析结果显示,外泌体miRNA-22与nephrin蛋白水平呈负相关(r=-0.769,P<0.05),Spearman相关性分析结果显示,外泌体miRNA-22与DN患者Mogensen分期呈正相关(r=0.815,P<0.05)。结论 miRNA-22可能参与了DN的发生发展,尿液外泌体miRNA-22与nephrin蛋白水平呈负相关,可反映足细胞损伤状态,且外泌体miRNA-22与DN患者Mogensen分期呈正相关,外泌体miRNA-22可反映病情严重程度。

Objective To investigate the level changes of urinary exosome miRNA-22 in diabetic nephropathy(DN) and its relationship with podocyte damage and severity of disease. Methods A total of 100 patients with type 2 diabetes mellitus combined with DN who visited author′s hospital from January 2017 to January 2019 were selected as DN group, and 30 healthy people who came to the hospital for health check-up during the same period were selected as the control group. The exosomes were collected and extracted from the urine, and the exosome marker lysosome intact membrane protein(CD63) was detected by immunoblotting(Western blot), the exosome miRNA-22 level was detected by polymerase chain reaction(PCR), and the nephrin protein level was detected by Western blot to identify the exosomes in the urine. Urine exosomes were identified and miRNA-22 and nephrin protein levels of exosomes in DN group and control group were compared. The patients were divided into microalbuminuria group(n=50) and massive albuminuria group(n=50) according to the quantitative index of urinary microalbumin, and the exosomal miRNA-22 and nephrin protein levels were compared between microalbuminuria group and massive albuminuria group, The correlation between exosomal miRNA-22 and nephrin protein level was evaluated by Pearson correlation analysis, and the correlation between exosomal miRNA-22 and DN severity were evaluated by Spearman correlation analysis. Results Circular structures with diameters of 40-100 nm were observed in urine by transmission electron microscopy, and the expression of exosomal surface marker protein CD63 was significantly higher in the DN group than in the control group(P<0.05). Compared with the control group, the relative expression of exosomal miRNA-22 was increased and nephrin protein level was decreased(P<0.05) in the DN group. Compared with microalbuminuria group, the relative expression of miRNA-22 in exosomes in the albuminuria group was increased(P<0.05), and the nephrin protein level was decreased(P<0.05). Pearson correlation analysis showed that exosomal miRNA-22 was negatively correlated with nephrin protein levels(r=-0.769, P<0.05), and Spearman correlation analysis showed that exosomal miRNA-22 was positively correlated with Mogensen stage in DN patients(r=0.815, P<0.05). Conclusion miRNA-22 may be involved in the development of DN. Urinary exosomal miRNA-22 is negatively correlated with nephrin protein level, which can reflect the status of podocyte damage, and exosomal miRNA-22 is positively correlated with Mogensen stage of DN patients, which can reflect the severity of the disease.