Quantification of human urinary exosomes by nanoparticle tracking analysis

Key points •  Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively. •  Current methods for the identification and quantification of urinary exosomes are time consuming and...

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Published in:The Journal of physiology 2013-12, Vol.591 (23), p.5833-5842
Main Authors: Oosthuyzen, Wilna, Sime, Nicole E. L., Ivy, Jessica R., Turtle, Emma J., Street, Jonathan M., Pound, John, Bath, Louise E., Webb, David J., Gregory, Christopher D., Bailey, Matthew A., Dear, James W.
Format: Article
Language:English
Subjects:
Adolescent
Adult
Animals
Aquaporin 2 - metabolism
Biomarkers - urine
Cell Line
Deamino Arginine Vasopressin - pharmacology
Diabetes Insipidus - urine
Exosomes
Female
Humans
Male
Mice
Mice, Inbred C57BL
Nanoparticles - analysis
Nanoparticles - chemistry
Particle Size
Techniques for Physiology
Urinalysis
Young Adult
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Summary:Key points •  Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively. •  Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. •  In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins. •  Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus. •  With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non‐invasive tool for investigating human renal physiology.   Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24‐ and AQP2‐positive particles of characteristic exosomal size. Extensive pre‐NTA processing of urine was not necessary. However, the intra‐assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at −80°C with the
ISSN:0022-3751
1469-7793
DOI:10.1113/jphysiol.2013.264069