ANCA-associated vasculitis (AAV) is a rare, but potentially severe autoimmune disease, even nowadays displaying increased mortality and morbidity. Finding early biomarkers of activity and prognosis is thus very important. Small extracellular vesicles (EVs) isolated from urine can be considered as a non-invasive source of biomarkers. We evaluated several protocols for urinary EV isolation. To eliminate contaminating non-vesicular proteins due to AAV associated proteinuria we used proteinase K treatment. We investigated the differences in proteomes of small EVs of patients with AAV compared to healthy controls by label-free LC–MS/MS. In parallel, we performed an analogous proteomic analysis of urine samples from identical patients. The study results showed significant differences and similarities in both EV and urine proteome, the latter one being highly affected by proteinuria. Using bioinformatics tools we explored differentially changed proteins and their related pathways with a focus on the pathophysiology of AAV. Our findings indicate significant regulation of Golgi enzymes, such as MAN1A1, which can be involved in T cell activation by N-glycans glycosylation and may thus play a key role in pathogenesis and diagnosis of AAV.

Significance

The present study explores for the first time the changes in proteomes of small extracellular vesicles and urine of patients with renal ANCA-associated vasculitis compared to healthy controls by label-free LC–MS/MS. Isolation of vesicles from proteinuric urine samples has been modified to minimize contamination by plasma proteins and to reduce co-isolation of extraluminal proteins. Differentially changed proteins and their related pathways with a role in the pathophysiology of AAV were described and discussed. The results could be helpful for the research of potential biomarkers in renal vasculitis associated with ANCA.

中文翻译：

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与总尿量比较，基于质谱的蛋白质组学探索ANCA相关血管炎中的小尿液细胞外囊泡

ANCA相关的血管炎（AAV）是一种罕见的但可能是严重的自身免疫性疾病，即使在如今，其死亡率和发病率也有所增加。因此，寻找活动和预后的早期生物标记非常重要。从尿液中分离出的小细胞外囊泡（EVs）可被视为生物标志物的非侵入性来源。我们评估了几种隔离尿液EV的协议。为了消除由于AAV相关蛋白尿污染的非囊泡蛋白，我们使用了蛋白酶K处理。我们通过无标记的LC-MS / MS研究了AAV患者的小型EV的蛋白质组与健康对照相比的差异。同时，我们对来自相同患者的尿液样品进行了类似的蛋白质组学分析。研究结果表明，EV和尿液蛋白质组存在显着差异和相似性，后者受到蛋白尿的严重影响。使用生物信息学工具，我们重点研究了AAV的病理生理学，探讨了差异变化的蛋白质及其相关途径。我们的发现表明，高尔基体酶（例如MAN1A1）的显着调节，其可能通过N-聚糖糖基化参与T细胞活化，因此可能在AAV的发病机理和诊断中起关键作用。

意义

本研究首次通过无标记LC-MS / MS探索了与健康对照相比，肾ANCA相关血管炎患者的小细胞外囊泡和尿液蛋白质组的变化。从蛋白尿尿样中分离囊泡的方法已进行了修改，以最大程度地减少血浆蛋白的污染并减少腔外蛋白的共分离。描述和讨论了差异变化的蛋白质及其在AAV的病理生理中起作用的相关途径。该结果可能有助于研究ANCA相关的肾血管炎的潜在生物标志物。