Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator

Abstract

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.

Figure 1. The isolation of urinary exosomes by nanomembrane concentrator

Urinary exosomes were isolated from 10 ml, 1.5 ml or 0.5 ml urine samples by the nanomembrane concentrator as described in methods.

Figure 2. Efficiency of urinary exosome isolation by nanomembrane concentrator

Ten ml of normal human fresh urine samples processed by nanomembrane concentrator. An equal proportion was loaded in each lane, and western blots were performed for TSG101, AQP-2, NSE, Annexin V, and PODXL. Lane 1: 200,000 × g pellets as exosome positive controls; lane 2: combined retentate (CR); lane 3: nanomembrane flowthrough was ultracentrifuged (200,000 × g 1 hr); lane 4: retentate (R); lane 5: proteins remaining on the nanomembrane after retentate was removed and washed with heated SDS-containing wash (HSW) buffer.

Figure 3. Electron microscopy of urinary vesicles

Urinary vesicles obtained by nanomembrane concentrator (A) and differential ultracentrifugation (B). Black bar denotes 100 nm.

Figure 4. Isolation of exosomal proteins from a small urine volume by nanomembrane concentrator

Fresh urine samples (0.5 or 1.5 ml) were processed by nanomembrane, proteins were recovered (CR, R, and HSW indicated in Figure 1), and western blots were performed for TSG101, AQP2, and NSE. Lane 1–3: 0.5 ml of urine and lane 4–6:1.5 ml of urine.

Figure 5. Effect of storage on urinary exosomal proteins isolated by nanomembrane concentrator

Urine sample from one healthy volunteer was processed in three storage conditions, within 1 hr (lane 1–3); stored at −80°C (lane 4–6); refrigerated at 4°C for 24 hr and then stored at −80°C (lane 7–9) after collection. Urinary proteins (CR, R, and HSW indicated in Figure 1) were analyzed by western blotting of TSG 101, AQP2, ACE, and PODXL. High molecular weight (HMW) and low molecular weight (LMW) isoforms of ACE are shown.

Figure 6. PODXL in patients with FSGS isolated by nanomembrane concentrator

Urine samples were obtained from 9 patients with FSGS and 8 healthy normal volunteers. Urinary PODXL by western blotting in the urinary proteins from the combined retentate. The amount of proteins was normalized by urine creatinine before loading onto the gels.