Abstract: TH-PO302
Minimal Volume of Urine for Analysis of Urinary Extracellular Vesicles Recovered at Low Relative Centrifugation Force
Session Information
- AKI Basic: Oxidative Injury and Nephrotoxins
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Acute Kidney Injury
- 001 AKI: Basic
Authors
- Musante, Luca, University of Virginia, Charlottesville, Virginia, United States
- Bontha, Sai Vineela, University of Virginia, Charlottesville, Virginia, United States
- Rudy, Christine, University of Virginia, Charlottesville, Virginia, United States
- La salvia, Sabrina, University of Virginia, Charlottesville, Virginia, United States
- Lannigan, Joanne, University of Virginia, Charlottesville, Virginia, United States
- Mas, Valeria, University of Virginia, Charlottesville, Virginia, United States
- Erdbruegger, Uta, University of Virginia Health System , Charlottesville, Virginia, United States
Background
Urinary extracellular vesicles (uEVs) provide a relative novel source of valuable biomarkers for kidney and urogenital diseases. Although the bulk of the research has focused mainly on exosomes as the primary source of extracellular vesicles (EVs). Only recently have uEVs recovered at low relative centrifugation force (RCF) been regarded as an additional important fraction of EVs carrying biomarkers. The number of MVs released by podocytes has shown to be higher in the urine of patients with diabetes mellitus type 1 without any kidney complications. This study aims to investigate what is the minimal amount of urine which enables the detection and characterization uEVs in the low RCF pellet.
Methods
First morning urine was centrifuged at RCF of 3,200 g. The supernatant was split in 0.5, 1.0, 1.5, 3.0, 4.5, 9.0 and 13.5 ml fractions to enrich uEVs by centrifugation at RCF of 21,000g. Tunable Resistive Pulse Sensing (TRPS), imaging flow cytometry, Cryo-Transmission Electron Microscopy and quantitative real time PCR were employed to establish the minimal volume of urine to provide uEVs for analysis.
Results
uEVs could be detected by TRSP and imaging flow cytometry, and it was possible to quantify selected miRNA starting from 0.5 ml of urine. Cryo-Transmission Electron Microscopy provided adequate images starting from a minimal volume of 1.5 ml of urine, showing uEVs of different size (60-500 nm) and morphology. Finally, western blot detection of uEVs proteins listed in Vesiclepedia like TSG101, TMEM27, IGFB-7 and TIMP-2 could be detected starting from 3.0-4.5 ml of urine. Interestingly, the detection of IGFB-7 and TIMP-2, approved early biomarkers of acute kidney injury (AKI), underlines the important role of uEVs in the detection and assessment of urinary biomarkers.
Conclusion
Depending on the sensitivity of the technique in use, a minimal volume of 0.5 ml urine is sufficient for a single analysis; however for multi analysis the volume of urine depends on the limit of detection of the techniques in use.
Funding
- Other NIH Support