特发性膜性肾病尿液外泌体表达及临床意义

Expression of urinary exosomes in patients with idiopathic membranous nephropathy and its clinical significance

目的探讨特发性膜性肾病(IMN)患者尿液外泌体的表达及临床意义。方法对2016年9月至2018年1月盛京医院肾病科住院的49例IMN患者(IMN组)及同期21例健康志愿者(对照组)通过差速离心方法提取尿液外泌体,通过Westernblot检测外泌体标记蛋白(Alix,CD63和TSG101)的表达,将其与24h尿蛋白定量,估计肾小球滤过率和肾组织病变等临床病理指标做统计分析。结果 IMN患者尿液外泌体较对照组显著增加[Alix(t=12.74,P <0.0001),CD63(t=13.98,P <0.0001),TSG101(t=15.68,P<0.0001)],外泌体标记蛋白与蛋白尿呈正相关[Alix(r=0.451,P=0.002),CD6(r=0.408,P=0.002),TSG101(r=0.417,P<0.001)];尿液外泌体蛋白浓度与蛋白尿呈正相关(r=0.635,P<0.001)。肾组织补体C3和PLA2R荧光染色增强组较染色较弱组尿液外泌体表达均显著增加[分别为Alix(t=3.071,P=0.004;t=2.07,P=0.046),CD63(t=2.69,P=0.01;t=3.43,P=0.002),TSG101(t=3.493,P=0.001;t=2.202,P=0.036)];尿外泌体标记蛋白(Alix,CD63和TSG101)诊断IMN的ROC曲线下面积分别是0.922[95%CI(0.843~1.002),P<0.001],0.936[95%CI(0.868~1.003),P<0.001]和0.944[95%CI(0.893~0.996),P<0.001]。随访研究表明尿外泌体在保守治疗无效组以及免疫制剂有效或无效组中表达均增加。结论尿液外泌体可反映IMN活动性肾组织病理改变,有希望成为IMN疾病诊断、病情评估和预后判断的非侵入性生物标志物。

Objective To observes the expression level and clinical significance of urinary exosomes in IMN patients.Methods Totally 49 IMN patients(IMN group)hospitalized in Shengjing Hospital from Sep.2016 to Jan.2018 and 21 healthy volunteers(control group)were included in the study.Urinary exosomes were extracted by differential centrifugation.The expression of exosomal marker proteins(Alix,CD63,and TSG101)was detected by Westernblot,and their expression was analyzed,along with 24-hour urine protein quantification,estimated glomerular filtration rate and renal tissue lesion parameters.Results Urinary exosomes were significantly increased in IMN patients[Alix(t=12.74,P<0.0001),CD63(t=13.98,P<0.0001),TSG101(t=15.68,P<0.0001)],and the exosomal marker proteins were positively correlated with proteinuria[Alix(r=0.451,P=0.002),CD6(r=0.408,P=0.002),TSG101(r=0.417,P<0.001)];urinary exosomal protein concentration was positively correlated with proteinuria(r=0.635,P<0.001).The expression of urinary exosomes was increased in patients whose renal tissue complement C3 and PLA2R immunofluorescence intensity was increased[Alix(t=3.071,P=0.004;t=2.07,P=0.046),CD63(t=2.69,P=0.01;t=3.43,P=0.002),TSG101(t=3.493,P=0.001;t=2.202,P=0.036)].The area under the ROC curve of urinary exosome marker protein(Alix,CD63 and TSG101)diagnosis IMN was 0.922[95%CI(0.843-1.002),P<0.001],0.936[95%CI(0.868-1.003),P<0.001]and 0.944[95%CI(0.893-0.996),P<0.001].Follow-up study showed that the expression of urinary exosomes was increased in the conservative-treatment-ineffective group and the immune-agent-effective or-ineffective group.Conclusion Urinary exosomes can reflect the active pathological changes of renal tissue of IMN,and may be a non-invasive biomarker for the diagnosis,assessment and prognosis of IMN disease.