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Urine Exosome Isolation and Characterization

  • Jonathan M. Street
  • Erik H. Koritzinsky
  • Deonna M. Glispie
  • Peter S. T. YuenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1641)

Abstract

Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research. Although faster and simpler methods have been developed, ultracentrifugation remains a mainstay for purification. A single ultracentrifugation step provides an enriched preparation suitable for biomarker discovery, and a second ultracentrifugation on a sucrose/D2O cushion provides the purest exosome preparation currently available and may be preferred for bioactivity assays. The concentration of exosomes can be determined using Nanosight Nanoparticle Tracking Analysis and their contents studied with a variety of approaches including western blots for proteins and RT-qPCR for microRNAs.

Key words

Exosomes Biomarker Urine TSG101 Ultracentrifugation Nanosight Nephrotoxicity 

Notes

Acknowledgments

This study was supported by the Intramural Research Program of the NIH, the National Institute of Diabetes, and Digestive and Kidney Diseases (NIDDK).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jonathan M. Street
    • 1
  • Erik H. Koritzinsky
    • 1
  • Deonna M. Glispie
    • 1
  • Peter S. T. Yuen
    • 1
    Email author
  1. 1.Renal Diagnostics and Therapeutics UnitNIDDK, National Institutes of HealthBethesdaUSA

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