Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

中文翻译：

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尿液小细胞外囊泡衍生的 CCL21 mRNA 作为与糖尿病肾病发病机制相关的生物标志物

糖尿病肾病（DN）是肾功能衰竭的主要原因，而有效的早期诊断生物标志物仍然缺乏。在包括 4 名健康对照 (HC)、4 名糖尿病 (DM) 和 4 名活检证实的 DN 患者在内的筛查队列中，在尿细胞外囊泡 (EV) 中检测到 14 种细胞因子和趋化因子 mRNA，并在另外 16 名 HC 和 15 名 DM 患者中得到验证和 28 名 DN 患者。在候选生物标志物和临床参数以及肾脏组织学变化之间进行相关分析。在单次注射 STZ 的 DN 大鼠模型中也证实了这一发现。与 DM 患者和健康对照组相比，DN 患者尿液中分泌的小 EV 数量增加，表达 AQP1（近端小管标志物）和 AQP2（远端/集合小管标志物）。小 EV 衍生的 CCL21 mRNA 在 DN 患者中显着增加，并与蛋白尿和 eGFR 水平相关。有趣的是，在肾功能正常的 DN 患者中观察到来自尿液小 EV 的 CCL21 mRNA 升高，与 eGFR 和蛋白尿相比，可以更有效地将早期 DN 患者与 DM 区分开来。CCL21 在区分早期和明显 DN 方面也显示出准确的诊断能力。组织学上，CCL21 mRNA 表达随着肾小管间质炎症的恶化而逐渐增加，并且在 DN 患者的结节硬化组（III 类）中表现出最高水平。在 CCL21 高表达的 DN 患者中观察到 CD3 阳性 T 细胞包括 CD4 和 CD8 阳性 T 细胞群的显着浸润。除了，CD3 阳性 T 细胞的积累与 DN 患者肾小管间质中源自 CCL21 并与 CCL21 共定位的尿小 EV 水平相关。最后，在 STZ 诱导的 DN 大鼠模型中证实了肾皮质和泌尿小 EV 中 CCL21 表达的相关性。尿液小 EV 衍生的 CCL21 mRNA 可作为识别与发病机制相关的 DN 的早期生物标志物。CCL21 mRNA介导的T细胞浸润可能构成DN慢性炎症的关键机制。