Raimondo Francesca, Chinello Clizia, Porcaro Luigi, Magni Fulvio, Pitto Marina
School of Medicine and Surgery, University Milan Bicocca, 20900 Monza, Italy.
Laboratory of Medical Genetics, Fondazione IRCCS Ca' Granda Hospital, 20162 Milan, Italy.
Proteomes. 2020 May 9;8(2):9. doi: 10.3390/proteomes8020009.
Renal tubular cells release urinary extracellular vesicles (uEV) that are considered a promising source of molecular markers for renal dysfunction and injury. We investigated uEV proteomes of patients with hereditary salt-losing tubulopathies (SLTs), focusing on those caused by Gitelman and Bartter (BS) syndromes, to provide potential markers for differential diagnosis. Second morning urine was collected from patients with genetically proven SLTs and uEV were isolated by the ultracentrifugation-based protocol. The uEV proteome was run through a diagonal bidimensional electrophoresis (16BAC/SDS-PAGE), to improve hydrophobic protein resolution. Sixteen differential spots from the proteome of two variants (BS2 and BS3) were analysed by nLC-ESI-MS/MS after in-gel tryptic digestion. A total of 167 protein species were identified from 7 BS2 spots and 9 BS3 spot. Most of these proteins were membrane-associated proteins, in particular transmembrane proteins, and were related to typical renal functions. The differential content of some uEV was then validated by immunoblotting. Our work suggests that uEV proteomics represents a promising strategy for the identification of differential SLT proteins. This could play a role in understanding the pathophysiological disease mechanisms and may support the recognition of different syndromes.
肾小管细胞释放尿细胞外囊泡（uEV），其被认为是肾功能不全和损伤分子标志物的一个有前景的来源。我们研究了遗传性失盐性肾小管病（SLT）患者的uEV蛋白质组，重点关注由吉特曼综合征和巴特综合征（BS）引起的疾病，以提供鉴别诊断的潜在标志物。从基因确诊的SLT患者中收集第二次晨尿，并通过基于超速离心的方案分离uEV。uEV蛋白质组通过对角线二维电泳（16BAC/SDS-PAGE）进行分析，以提高疏水蛋白的分辨率。在凝胶内胰蛋白酶消化后，通过nLC-ESI-MS/MS分析来自两个变体（BS2和BS3）蛋白质组的16个差异点。从7个BS2斑点和9个BS3斑点中共鉴定出167种蛋白质。这些蛋白质大多数是膜相关蛋白，特别是跨膜蛋白，并且与典型肾功能相关。然后通过免疫印迹验证了一些uEV的差异含量。我们的工作表明，uEV蛋白质组学是鉴定SLT差异蛋白的一种有前景的策略。这可能有助于理解疾病的病理生理机制，并可能有助于识别不同的综合征。
