Quantification of human urinary exosomes by nanoparticle tracking analysis

Abstract

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 addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

Figure 1. Nanoparticle tracking analysis (NTA) of human urine samples

A, screen shot from 1:1000 diluted whole urine sample revealing a range of particle sizes. B, example NTA trace depicting the nanoparticle distribution profile for an individual subject. C, concentration of urinary particles (0–300 nm diameter) in urine samples from five study participants. The concentration is expressed as number of particles per mmol urinary creatinine.

Figure 2. Exosome marker specific fluorescent labelling

A, CD24, flotillin-1 and TSG101 localize to a density range of 1.12–1.16 g cm⁻³ on a sucrose density gradient following isopycnic centrifugation. The positive control was human urinary exosomes unseparated by isopycnic centrifugation. B, human urine samples labelled with CD24–quantum dots (Qdots) in light scatter mode (dashed line, all particles) and with the fluorescent filter in place (continuous line, CD24-labelled particles). Results from three study participants are presented. Particle concentration is expressed per mmol urinary creatinine. C, human urine samples labelled with aquaporin 2 (AQP2)–Qdots in light scatter mode (dashed line, all particles) and with the fluorescent filter in place (continuous line, AQP2-labelled particles). Results from three study participants are presented. Particle concentration is expressed per mmol urinary creatinine. D, human urine labelled with mouse IgG conjugated to Qdot as an isotype control in light scatter mode (dashed line) and with the fluorescent filter in place (continuous line). Note the absence of a ‘peak’ in the particle size range 0–100 nm with the fluorescent filter in place.

Figure 3. Comparison of different exosome isolation methods

A, human urinary exosomes concentrated by ultracentrifugation conjugated with CD24–Qdots in light scatter mode (dashed line, all particles) and with the fluorescent filter in place (continuous line, CD24-labelled particles). Results from three study participants are presented. Particle concentration is expressed per mmol urinary creatinine. B, human urinary exosomes concentrated by Exoquick™ reagent conjugated with CD24–Qdots in light scatter mode (dashed line, all particles) and with the fluorescent filter in place (continuous line, CD24-labelled particles). Results from three study participants are presented. Particle concentration is expressed per millimolar urinary creatinine.

Figure 4. Changes in AQP2-positive exosomes following desmopressin stimulation

A, the difference in exosomal concentration in the cell culture media is expressed as the area under the curve (AUC) for particles sized 20–100 nm that labelled with an antibody to AQP2. The cells were stimulated with desmopressin (3.16 ng ml⁻¹) for 48 or 96 h. *P < 0.05 T-TEST. B, difference in exosomal concentration in the urine samples from desmopressin-treated (n= 6) or control mice (n= 5), expressed as the AUC for particles sized 20–100 nm that labelled with an antibody to AQP2. Particle concentration is expressed per mmol urinary creatinine. *P < 0.05 T-TEST.

Figure 5. Nanoparticle tracking analysis tracked changes in AQP2-positive exosome concentration following desmopressin treatment of a patient with central diabetes insipidous

For A and B, urine aliquots were collected over 2 separate days. The AQP2-exosome concentration in the urine samples is expressed as the AUC for particles sized 20–100 nm that labelled with an antibody to AQP2. Particle concentration is expressed per mmol urinary creatinine. The time of administration of desmopressin treatment is indicated by the dashed line.

Figure 6. Different storage protocols and urine particle concentration

Nanoparticle tracking analysis was used to measure the particle concentration between 20 and 100 nm in diameter (AUC₂₀₋₁₀₀) after storage at various temperatures for 2 h, 1 day or 1 week. The baseline was immediate measurement after sample collection. n= 5 per group. *P < 0.05 (ANOVA) for significant differences between protease inhibition and none. Abbreviations: RT, room temperature; and PI, protease inhibitors.