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.

Figure 1. The isolation of three urinary fractions

Urinary sediment (17,000 × g pellet), exosome fraction (200,000 × g pellet) and acetone insoluble supernatant (17,000 × g supernatant precipitated by acetone) were isolated as described in methods.

Figure 2. The effect of protease inhibitors

Eight fresh urine samples were collected without (A) and with (B) protease inhibitors, and exosome fractions were prepared, and evaluated by western blotting for NKCC2. Sample loading was normalized by urine creatinine. B1-B8 and D1-D8 represent 2 sets of different volunteers.

Figure 3. The effect of storage and vortexing

Samples were pooled from 3 individuals. Amount of urinary exosome-associated protein (200,000 × g pellet) (A), Coomassie blue-stained gel of equal fraction volume of protein (B), Western blot (10 :l aliquot) for NHE3, TSG101, ALIX and AQP2 (C).

Figure 4. Effect of long-term storage

(A) Western blot of NHE3, TSG101, ALIX and AQP2 abundance in exosome fraction normalized by urine creatinine from 10 ml freshly-collected urine samples (lane 1-3) or after long-term storage (-80°C for 7 months; lane 4-6) from 3 different individuals. Lane 1 and 4, lane 2 and 5, lane 3 and 6 are from the same volunteer. (B) Western blot of cytosolic exosome-associated proteins (NSE and MDH) normalized by urine creatinine in fresh (lane 1-3) and long-term stored (-80°C for 7 months; lane 4-6) urine samples from 3 different individuals. Lanes as in A.

Figure 5. The amount of total protein in three different urinary fractions of human first and second morning urine

The amount of total protein in first (open bar) and second (closed bar) morning urine samples in different urinary fractions from three volunteers (a, b and c), 1: morning first urine, 2: morning second urine. Protein content in the exosome fraction (after 200,000 × g 1hr spin, A), urinary sediment proteins (after 17,000 × g 15 min spin, B), and supernatant proteins (after 17,000 × g 15 min spin, C).

Figure 6. Protein electrophoretic patterns of three different urinary fractions of human first and second morning urine

Coomassie blue-stained gels of urinary proteins in exosome fraction after 200,000 × g 1hr spin (A and B), urinary sediment proteins after 17,000 × g 15 min spin (C and D) and supernatant proteins after 17,000 × g 15 min spin (E and F) normalized by urinary creatinine (A, C, E) or urine flow rate (B, D, F). Legend: a, b and c represent different volunteers, 1: first morning urine, 2: second morning urine.

Figure 7. Specific exosome-associated proteins in first and second morning urine

Abundance of NHE3, TSG101, ALIX and AQP2 by western blotting in the urinary exosome fraction normalized by urinary creatinine (A) or urine flow rate (B). Legend: a, b and c represent different volunteers, 1: first morning urine, 2: second morning urine.