Urine Exosome Isolation and Characterization

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.

Keywords: Biomarker; Exosomes; Nanosight; Nephrotoxicity; TSG101; Ultracentrifugation; Urine.

Figure 1.

Flow chart outlining the ultracentrifugation protocol for urinary exosome isolation. A single ultracentrifugation is typical for biomarker discovery studies (left). If a higher level of purity is required, such as for bioactivity assays, an additional ultracentrifugation step using a Sucrose/D₂O cushion may be added (right).

Figure 2.

Sample image from NTA 3.1 illustrating a proper concentration of particles present in the field of view (20-100 particles).

Figure 3.

Sample images from NTA 3.1 illustrating how to choose a proper detection threshold setting for analysis. When the detection threshold is set properly (A), there should be red crosses on each particle in the field of view and no crosses on other parts of the field. The number of blue crosses present should also be minimized (less than five). When the detection threshold is set too low (B), there will be crosses on non-particle background as well as an abundance of blue crosses visible. When the detection threshold is set too high (C), there will be particles visible but without red crosses on them.

Figure 4.

Sample size-concentration distribution of urine exosomes isolated from a healthy patient obtained using Nanosight NTA. Exosomes were resuspended in 1X PBS. The sample was diluted in order for the measured concentration to fall within the dynamic range of the instrument.