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Kidney Week

Abstract: TH-PO882

Extracellular Vesicle Cargo Derived from Kidney Cell Lines Is Affected in Insulin-Resistant and Highly Insulin-Sensitive Conditions

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Barreiro, Karina A., Institute for Molecular Medicine Finland FIMM, University of Helsinki,, Helsinki, Finland
  • Lay, Abigail Charlotte, University of Bristol, Bristol, United Kingdom
  • Coward, Richard, University of Bristol, Bristol, United Kingdom
  • Leparc, German G., Boehringer-Ingelheim, Biberach, Germany
  • Delic, Denis, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
  • Huber, Tobias B., University Medical Center Hamburg, Hamburg, Germany
  • Holthofer, Harry B., University of Helsinki, Finnish Institute of Molecular Medicine, Helsinki, Finland
Background

Kidney disease (DKD) is common, costly and the most feared complication of long standing diabetes. It is now clear that cellular insulin resistance is a major driver of this disease. Here we explored the extracellular vesicle (EV) secretion pattern by key DKD target cellsin the glomerulus (podocytes, endothelial and mesangial cells) and proximal tubular cells by harvesting the entire secreted EV repertoire in insulin sensitive and insulin resistant cells using Hydrostatic Filtration Dialysis (HFD).

Methods

EVs were isolated from 50ml of cell culture mediafrom conditionally immortalized human cells. Cells were rendered hyperinsulin-sensitive by stable insulin receptor expression, and insulin resistant by culturing in a diabetic environment as recently published.
Quality of the EV yield was verified with negative staining Electron Microscopy (EM) and Western blotting (WB). Vesicle concentration and size was determined by Nanoparticle Tracking Analysis (NTA). EV RNAs were profiled with Bioanalyzer Pico kit and subjected to miRNAseq, and mRNAseq. EV proteins were analyzed using tandem mass tag labeling.

Results

The isolated EVs appeared typical at EM and were positive for the EV-marker TSG101 in WB. RNA quantity and quality proved appropriate for miRNA analysis. 96 EV miRNAs and 109 mRNAs could characteristically discriminate between the cell types. Some EV miRNAs, mRNA and proteins showed treatment effects. KEGG and Gene Ontology gene enrichment analysis showed pathways associated with DKD.

Conclusion

EV analysis provides a novel approach to reveal valuable new details of the kidney secretome in the setting of insulin-resistant diabetic conditions. It is noteworthy that the characteristic changes can be found from culture medium of DKD target cell. Changes in the EV miRNAs, mRNA and proteomics may thus give valuable insight into mechanisms and targets to insulin resistance in DKD.