Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Abstract

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

“…They are a promising source of diagnostic biomarkers in oncology as well as other diseases [2,3]. EVs from urine can reveal urological diseases or tumours and their progression [2,4,5].…”

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

“…Urine EVs were prepared from healthy subjects by an ultracentrifugation method [10]. In brief, urine samples were centrifuged at 3000×g for 15 min at 25 °C to remove sediments, cells, and cell debris.…”

A qualitative proteome investigation of the sediment portion of human urine: Implications in the biomarker discovery process

“…Despite the analytical diversity, aspects of these protocols are universally accepted. For example, to preserve the proteome over extended periods, urine is frozen shortly after collection [8,9]. Upon thawing, sediments from the sample are typically removed by filtration or centrifugation, a necessary step in proteome visualization by gel electrophoresis [10].…”

“…The identities of the so-called lost proteins were not discussed in this report. Furthermore, Zhou et al [9] determined that the sediment phase of urine accounts for approximately 50% of the total protein content by mass and is primarily comprised of uromodulin. Thus, as a simple prefractionation technique, removal of the sediment phase can be considered beneficial for the analysis of low-abundant protein components in urine supernatants [12].…”

“…However, Zhou et al clearly demonstrated (through SDS-PAGE images) that several other proteins contribute to the sediment phase of urine; a comprehensive study to identify these proteins has yet to be conducted. Most recently, it has been shown that vortexing a urine sample aids in the solubilization of proteins from urinary exosomes [9]. Interestingly, although urinary sediments are routinely analyzed through microscopy as a means of diagnosing physiological conditions [13], current proteome investigations focus only on the solubilized portion of the sample.…”