Street Jonathan M, Koritzinsky Erik H, Glispie Deonna M, Yuen Peter S T
Renal Diagnostics and Therapeutics Unit, NIDDK, National Institutes of Health, 10 Center Drive, Bldg 10, Room 3N108, Bethesda, MD, 20892, USA.
Methods Mol Biol. 2017;1641:413-423. doi: 10.1007/978-1-4939-7172-5_23.
Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research. Although faster and simpler methods have been developed, ultracentrifugation remains a mainstay for purification. A single ultracentrifugation step provides an enriched preparation suitable for biomarker discovery, and a second ultracentrifugation on a sucrose/DO cushion provides the purest exosome preparation currently available and may be preferred for bioactivity assays. The concentration of exosomes can be determined using Nanosight Nanoparticle Tracking Analysis and their contents studied with a variety of approaches including western blots for proteins and RT-qPCR for microRNAs.
外泌体是纳米级的、被膜包裹的囊泡，有可能用于检测肾毒性，并揭示肾脏随后的反应。每个肾单位节段的上皮细胞都可对外泌体尿群有贡献，尿外泌体富含潜在生物标志物，包括转运蛋白和受体等膜蛋白、转录因子和微小RNA。这些外泌体生物标志物在接触肾毒性物质后可能会上调或下调。外泌体分离是一个正在研究的领域。尽管已经开发出更快、更简单的方法，但超速离心仍然是纯化的主要方法。单次超速离心步骤可提供适合生物标志物发现的富集制剂，在蔗糖/二辛基磺酸钠垫层上进行的第二次超速离心可提供目前可得的最纯外泌体制剂，可能更适合用于生物活性测定。外泌体的浓度可以使用纳米可视纳米颗粒追踪分析来确定，其内容物可以通过多种方法进行研究，包括蛋白质的蛋白质免疫印迹法和微小RNA的逆转录定量聚合酶链反应法。
