Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.
Bristol Renal, Bristol Medical School, Faculty of Health Sciences, University of Bristol, Bristol, UK.
J Extracell Vesicles. 2023 Feb;12(2):e12304. doi: 10.1002/jev2.12304.
Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.
细胞外囊泡（EV）是所有细胞分泌的膜性颗粒，存在于体液中。已确定的 EV 内容物包括多种 RNA 种类、蛋白质、脂质和代谢物，这些物质被认为反映了其亲本细胞的生理状态。然而，迄今为止，对于复杂 EV 混合物中富含细胞类型的 EV 货物，特别是在尿液中的情况，了解甚少。为了测试培养的不同人类肾脏细胞分泌的 EV 是否存在差异，并且可以再现正常尿液中的发现，我们全面分析了条件永生化的人类肾脏细胞系（足细胞、肾小球内皮细胞、系膜细胞和近端肾小管细胞）中 EV 的成分（特别是 miRNA、长 RNA 和蛋白质），并与尿液中分泌的 EV 进行了比较。通过静水过滤透析（HFD）从细胞培养物中分离出细胞培养衍生的 EV，并通过电子显微镜（EM）、纳米颗粒跟踪分析（NTA）和 Western blot（WB）进行表征。从 EV 中分离出 RNA，并进行 miRNA 和 RNA 测序，通过串联质量标签蛋白质组学对蛋白质进行分析。在每种细胞类型中都检测到了代表性的 EV miRNA、RNA 和蛋白质，并与人类尿源性 EV 分离物（uEV）、EV 货物数据库、肾脏活检批量 RNA 测序和蛋白质组学以及单细胞转录组学进行了比较。这表明，体外 EV 特征的很大一部分也存在于体内数据集。因此，突出了我们体外模型的稳健性，并表明这种方法能够在生物流体中剖析细胞类型特异性 EV 货物，并有可能鉴定肾脏疾病的细胞类型特异性 EV 生物标志物。
