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°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°C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°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°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.

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°C or refrigerated overnight at 4°C then stored at −80°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.

Although diagnosis and staging of acute kidney injury uses serum creatinine, acute changes in creatinine lag behind both renal injury and recovery. The risk for mortality increases when acute kidney injury accompanies sepsis; therefore, we sought to explore the limitations of serum creatinine in this setting. In mice, induction of sepsis by cecal ligation and puncture in bilaterally nephrectomized mice increased markers of nonrenal organ injury and serum TNF-α. Serum creatinine, however, was significantly lower in septic animals than in animals subjected to bilateral nephrectomy and sham cecal ligation and puncture. Under these conditions treatment with chloroquine decreased nonrenal organ injury markers but paradoxically increased serum creatinine. Sepsis dramatically decreased production of creatinine in nephrectomized mice, without changes in body weight, hematocrit, or extracellular fluid volume. In conclusion, sepsis reduces production of creatinine, which blunts the increase in serum creatinine after sepsis, potentially limiting the early detection of acute kidney injury. This may partially explain why small absolute increases in serum creatinine levels are associated with poor clinical outcomes. These data support the need for new biomarkers that provide better measures of renal injury, especially in patients with sepsis.

Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte injury models (puromycin-treated rats or podocin-Vpr transgenic mice) and from patients with focal segmental glomerulosclerosis, acute kidney injury and matched controls. Exosomes were isolated by differential centrifugation and found to contain activating transcription factor 3 (ATF3) and Wilms Tumor 1 (WT-1) proteins detected by Western blot. These factors were found in the concentrated exosomal fraction, but not in whole urine. ATF3 was continuously present in urine exosomes of the rat models following acute injury at times earlier than the increase in serum creatinine. ATF3 was found in exosomes isolated from patients with acute kidney injury but not from patients with chronic kidney disease or controls. Urinary WT-1 was present in animal models before significant glomerular sclerosis and in 9/10 patients with focal segmental glomerulosclerosis but not in 8 controls. Our findings suggest that transcription factor ATF3 may provide a novel renal tubular cell biomarker for acute kidney injury while WT-1 may detect early podocyte injury. Measurement of urinary exosomal transcription factors may offer insight into cellular regulatory pathways.