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Abstract: TH-PO873

Transcriptome of Extracellular Vesicles Derived from In-Vitro Podocytes, Proximal Tubule, and Mesangial Cells

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Holthofer, Harry B., University of Helsinki, Finnish Institute of Molecular Medicine, Helsinki, Finland
  • Barreiro, Karina A., Institute for Molecular Medicine Finland FIMM, University of Helsinki,, Helsinki, Finland
  • Lay, Abigail Charlotte, University of Bristol, Bristol, United Kingdom
  • Liang, Wei, Renmin Hospital of Wuhan University, Wuhan, China
  • Xiaomeng, Xu, University of Helsinki, Finnish Institute of Molecular Medicine, Helsinki, Finland
  • Luo, Sihui, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
  • Bork, Tillmann, University Hospital Freiburg, Freiburg, BW, Germany
  • Coward, Richard, University of Bristol, Bristol, United Kingdom
  • Delic, Denis, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
  • Huber, Tobias B., University Medical Center Hamburg, Hamburg, Germany
Background

Extracellular vesicles (EVs) are lipid-bilayer structures of variable sizes (30-1000nm) secreted by all cell types. They show a characteristic vesicle surface consisting of a variety of proteins and lipids while the intracellular content is a rich array of proteins and distinct RNA species. These reflect accurately the physiological state of their cells of origin. In diabetes, development of renal insulin resistance has a major role in end-organ complications like Diabetic Kidney Disease (DKD). Little is still known of the secreted EV repertoire of especially podocytes.
Here our objective was to characterize EVs secreted by DKD target cells, podocytes, proximal tubular cells and mesangial cells using the Hydrostatic Filtration Dialysis (HFD) for EV harvesting.

Methods

We used cell culture media from podocytes, proximal tubule and mesangial cells in four conditions; 1) Basal 2) Insulin resistant 3) Insulin receptor transfected 4) Insulin receptor transfected and insulin resistant. EVs were isolated from 50ml of cell culture media, respectively using the recently described HFD. Quality of EV yield was analyzed by negative-staining EM and by Western blotting. Vesicle concentration was determined by Nanoparticle Tracking Analysis (NTA). Isolated RNAs were profiled with Bioanalyzer Pico kit and subjected to RNAseq after cDNA library preparation using QIAseq miRNA Library Kit. RNAseq was performed using HiSeq 3000 (Illumina) Paired-end (2X150) protocol.

Results

The isolated EVs appeared typical at EM and were positive for the EV-marker TSG101 in Western blotting. RNA yield was of high quantity and quality and thus suitable for RNAseq. Different treatments had distinct effect on vesiculation of the investigated cells. Ninety-six EV miRNAs could clearly discriminate between cell types and engineered cells. There were distinct EV miRNAs to reflect treatment effect within each of the individual cell types studied.

Conclusion

EV analysis provides a novel approach to reveal valuable pathophysiology, pathway and signaling information of cultured target cells. Changes in EV miRNAs may give insight into insulin resistance on DKD target cells and on diabetic nephropathy.