Clin Nephrol. 2021;96(1):107-113. doi: 10.5414/CNP96S19.
Long-term kidney allograft survival requires a personalized approach to allograft injury recognition in a timely and reliable manner. Kidney biopsy is invasive and unsuitable for continuous function assessment. Alternatively, in urine, we find extracellular vesicles (uEVs), stable carriers of kidney pathology signals. Analysis of uEVs and their cargo could allow for more frequent and non-invasive assessment of allograft function. We aimed to optimize the uEVs isolation method applicable for kidney allograft injury biomarker studies.
To this end, we optimized several steps of size-exclusion chromatography (SEC)-based method for uEVs isolation from second morning urine of kidney allograft recipients. uEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western analysis, and quantitative PCR.
According to TEM and NTA, SEC isolated high concentrations (8.64 × 10 EVs/mL of urine) of EVs that showed typical morphology and mean size (171 nm), but addition of EDTA and filtration step were needed to remove impurities. Additionally, typical EV proteins Hsc70, CD63, flotillin, tubulin, GAPDH, and miR hsa-let-7i were detected in isolated uEVs, further confirming their identity.
Optimized method based on SEC was effective and adequate in isolating pure EVs from urine of kidney allograft recipients and could be used in future biomarker studies.
长期的肾移植体存活需要及时可靠地采用个性化方法来识别移植物损伤。肾活检具有侵袭性，不适合连续功能评估。相比之下，在尿液中，我们发现了细胞外囊泡（uEVs），这是肾脏病理信号的稳定载体。对 uEVs 及其货物的分析可以更频繁、更无创地评估移植物功能。我们旨在优化适用于肾移植体损伤生物标志物研究的 uEVs 分离方法。
为此，我们优化了基于大小排阻色谱（SEC）的方法中的几个步骤，以从肾移植受者第二天早晨的尿液中分离 uEVs。通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）、western 分析和定量 PCR 对 uEVs 进行了表征。
根据 TEM 和 NTA，SEC 分离出高浓度（尿液中 8.64×10 EVs/mL）的 EVs，其具有典型的形态和平均大小（171nm），但需要添加 EDTA 和过滤步骤来去除杂质。此外，在分离的 uEVs 中还检测到了典型的 EV 蛋白 Hsc70、CD63、flotillin、微管蛋白、GAPDH 和 miR hsa-let-7i，进一步证实了其身份。
基于 SEC 的优化方法在从肾移植受者的尿液中分离纯净的 EVs 方面是有效且充分的，可用于未来的生物标志物研究。
