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. 2023 Feb;12(2):e12304.
doi: 10.1002/jev2.12304.

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles

Karina Barreiro  1 , Abigail C Lay  2 , German Leparc  3 , Van Du T Tran  4 , Marcel Rosler  3 , Lusyan Dayalan  2 , Frederic Burdet  4 , Mark Ibberson  4 , Richard J M Coward  2 , Tobias B Huber  5 , Bernhard K Krämer  6 , Denis Delic  3   6 , Harry Holthofer  1   5
Affiliations

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles

Karina Barreiro et al. J Extracell Vesicles. 2023 Feb.

Abstract

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.

Keywords: RNA; exosomes; extracellular vesicles; glomerular endothelial cells; kidney; mesangial cells; miRNA; podocytes; proteomics; proximal tubule cells.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Experimental workflow: EV were isolated from 50 ml of cell free culture media by hydrostatic filtration dialysis. Vesicles were characterised by TEM, WB and NTA. Isolated total RNA was subjected to miRNA‐ and RNA‐sequencing (n = 5 per cell type). Isolated proteins were subjected to quantitative proteomics (TMT approach). Omics dataset were used to define cell‐type‐enriched EV features. Lists of enriched features were tested in human uEV datasets. EV, extracellular vesicles; miRNA‐seq, miRNA sequencing; NTA, nanoparticle tracking analysis; RNA‐seq, RNA sequencing; TMT, tandem mass tag; TEM, transmission electron microscopy; uEV, urinary extracellular vesicles; WB, Western blotting
FIGURE 2
FIGURE 2
EV preparations characterisation and quality control. (a) Electron microscopy micrographs of EV samples isolated by HFD. (b) Representative NTA profiles of uEV total RNA. (c) Diameter mean per cell type EV evaluated by NTA. (d) Particle yield per ml of cell free culture media as measured by NTA. (e) Western blotting. Immunodetection of EV markers (TSG101, CD63 and CD81). (f) Representative RNA electropherograms obtained by Agilent RNA 6000 Pico Kit. Black arrows: extracellular vesicles. GEC, glomerular endothelial cells; EV, extracellular vesicles; MC, mesangial cells; NTA, nanoparticle tracking analysis; POD, podocytes; PTC, proximal tubule cells
FIGURE 3
FIGURE 3
Principal component analysis (PCA) on miRNA‐seq, RNA‐seq and proteomics data from the four extracellular vesicles (EV) isolates. (a) PCA including GEC, (b) PCA excluding GEC in order to avoid GEC bias. GEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
FIGURE 4
FIGURE 4
Gene ontology biological process enrichment analysis with multi‐omics data. Heatmap depicts selected enriched biological processes per cell and molecule type. Analysis was done using cell‐type‐enriched EV features (POD‐EV, PTC‐EV, MC‐EV and GEC‐EV). GGEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
FIGURE 5
FIGURE 5
Gene ontology molecular function enrichment analysis with multi‐omics data. Heatmap depicts selected enriched molecular functions per cell and molecule type. Analysis was done using cell‐type‐enriched EV features (POD‐EV, PTC‐EV, MC‐EV and GEC‐EV). GEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
FIGURE 6
FIGURE 6
Identification of cell‐type‐enriched extracellular vesicles (EV) miRNAs in urinary extracellular vesicles (uEV). Venn diagrams depict overlapping between cell‐type‐enriched EV miRNAs (POD‐EV, PTC‐EV, MC‐EV and GEC‐EV miRNAs) and human uEV miRNAs. GEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
FIGURE 7
FIGURE 7
Identification of cell‐type‐enriched extracellular vesicles (EV) genes in urinary extracellular vesicles (uEV). Venn diagrams depict overlapping between cell‐type‐enriched EV mRNAs (POD‐EV, PTC‐EV, MC‐EV and GEC‐EV mRNAs) and human uEV mRNAs. Top 15 mRNA (by expression in uEV) are listed for each cell type. GEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
FIGURE 8
FIGURE 8
Identification of cell‐type‐enriched extracellular vesicles (EV) proteins in urinary extracellular vesicles (uEV). Venn diagrams depict overlapping between cell‐type‐enriched EV proteins (POD‐EV, PTC‐EV, MC‐EV and GEC‐EV proteins) and human uEV proteins. GEC, glomerular endothelial cells; MC, mesangial cells; POD, podocytes; PTC, proximal tubule cells
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References

    1. Abashev, T. M. , Metzler, M. A. , Wright, D. M. , & Sandell, L. L. (2017). Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium. Developmental Dynamics, 246(2), 135–147. 10.1002/dvdy.24476 - DOI - PMC - PubMed
    1. Aguado‐Fraile, E. , Ramos, E. , Conde, E. , Rodríguez, M. , Martín‐Gómez, L. , Lietor, A. , Candela, Á. , Ponte, B. , Liaño, F. , & García‐Bermejo, M. L. (2015). A pilot study identifying a set of microRNAs as precise diagnostic biomarkers of acute kidney injury. PLoS ONE, 10(6), e0127175. 10.1371/journal.pone.0127175 - DOI - PMC - PubMed
    1. Ahluwalia, T. S. , Lindholm, E. , & Groop, L. C. (2011). Common variants in CNDP1 and CNDP2, and risk of nephropathy in type 2 diabetes. Diabetologia, 54(9), 2295–2302. 10.1007/s00125-011-2178-5 - DOI - PubMed
    1. Ai, K. , Zhu, X. , Kang, Y. , Li, H. , & Zhang, L. (2020). miR‐130a‐3p inhibition protects against renal fibrosis in vitro via the TGF‐β1/Smad pathway by targeting SnoN. Experimental and Molecular Pathology, 112, 104358. 10.1016/j.yexmp.2019.104358 - DOI - PubMed
    1. Akaishi, J. , Onda, M. , Okamoto, J. , Miyamoto, S. , Nagahama, M. , Ito, K. , Yoshida, A. , & Shimizu, K. (2006). Down‐regulation of transcription elogation factor A (SII) like 4 (TCEAL4) in anaplastic thyroid cancer. BMC cancer, 6, 260. 10.1186/1471-2407-6-260 - DOI - PMC - PubMed

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