Urinary vesicles are tiny packages of potential for biomedical researchers. They offer a non-invasive and simple method for obtaining clinical samples representative of kidney and urogenital tract disease. As such, they offer scope for long-term studies but also present additional problems for biobanking.
The vesicles include exosomes, microvesicles and apoptotic blebs, among other extracellular components actively released into urine by epithelial lining cells. Unfortunately, collecting these vesicles requires lengthy sample preparation to concentrate large volumes of urine and remove the hyper-abundant Tamm–Horsfall protein (THP), which can interfere with analyte recovery. Musante et al. (2014) present a simplified collection method that maximizes recovery of representative vesicle populations without requiring biobanking large volumes of urine.1
Previously, sample preparation comprised lengthy sequential ultracentrifugation steps, requiring skilled laboratory technicians and expensive (potentially hazardous even) equipment. Musante et al. developed a faster method based around hydrostatic dialysis, which they suggest may eventually be simple enough for the urine donor to implement at point of collection.
In this method, sample preparation starts with simple centrifugation at 2,000 g to remove cellular debris and THP macropolymers from urine collected as the first morning void from four laboratory volunteers. In their study, the researchers took the supernatant and applied it to dialysis tubes. Using the solution’s own properties, they dialyzed the sample under gravity through a dialysis membrane with a 1,000 kDa cut-off. Taking the fluid retained above the membrane, they then concentrated the vesicles with further centrifugation at 40,000 g and 200,000 g.
Following this hydrostatic filtration dialysis (HFDa), the team checked each stage of sample preparation for urinary vesicle content using immunodetection. They separated each fraction using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), then checked for the presence of the exosomal marker TSG101 in each lane. The scientists found that they obtained vesicles in the fractions they predicted, that is, those collected by HFDa preparation. They noted with interest that the fluid flow-through from the initial 1,000 kDa dialysis step did not contain vesicles. However, the pellet obtained by the initial 2,000 g centrifugation showed a minor amount compared with the maximal recovery in the HFDa fractions.
The team further analyzed the HFDa fractions using transmission electron microscopy and TRPS (Tunable Resistive Pore Sensing) analysis to visualize the vesicle populations obtained. This confirmed that the new method obtained a representative mixed population of urinary vesicles without bias for onward analysis. Furthermore, the method also showed that the team recovered good quality miRNA, as quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific).
The team also examined the methodology with different starting volumes. They assessed recovery and population variability, recovering vesicles from urine in volumes of 15 ml, 50 ml, 100 ml and 200 ml. They replicated initial results but noted that the process was slightly less efficient when starting from an initial volume of 200 ml.
Compared with the traditional method, the researchers note that their HFDa technique results in faster and better recovery of urinary vesicles for onward biobanking. Furthermore, their initial centrifugation step removes all but 4% of interfering THP complexes from the sample without removing excess vesicles. In addition to the improvements in speed, they also note that the method is cost-effective, requiring neither specialized laboratory equipment nor highly trained staff.
1. Musante, L. et al. (2014) “A simplified method to recover urinary vesicles for clinical applications, and sample banking,” Scientific Reports 4, Article number 7532, doi:10.1038/srep07532.
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