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.

OBJECTIVE: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications. DESIGN: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays. RESULTS: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjö gren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling. CONCLUSIONS: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies. Oral Diseases (2010) 16, 34-38

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 aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

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.

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.

Star RA. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury.

The remnant kidney model in C57BL6 mice does not develop progressive chronic kidney disease (CKD). In this study we modified the model to mimic features of human CKD and to define accelerants of disease progression using three strains of mice. Following the procedure there was a progressive increase in albuminuria, progressive loss in renal function, severe glomerulosclerosis and interstitial fibrosis, hypertension, cardiac fibrosis, and anemia by 4 weeks in CD-1 mice and by 12 weeks in 129S3 mice. In contrast, even after 16 weeks, the C57BL/6 mice with a remnant kidney had modestly increased albuminuria without increased blood pressure and without developing CKD or cardiac fibrosis. The baseline blood pressure, determined by radiotelemetry in conscious animals, correlated with CKD progression rates in each strain. Administering angiotensin II overcame the resistance of C57BL/6 mice to CKD following renal mass reduction displaying high blood pressure and albuminuria, severe glomerulosclerosis, and loss of renal function by 4 weeks. Decreasing blood pressure with olmesartan but not hydralazine in CD-1 mice with a remnant kidney reduced CKD progression and cardiac fibrosis. C57BL/6 mice with a remnant kidney and DOCA-salt hypertension developed modest CKD. Each strain had similar degrees of interstitial fibrosis in three different normotensive models of renal fibrosis. Thus reducing renal mass in CD-1 or 129S3 mice mimics many features of human CKD. Angiotensin II can convert the C57BL/6 strain from CKD-resistant to susceptible in this disease model.

Acute kidney injury (AKI) occurs in about half of patients in septic shock and the mortality of AKI with sepsis is extremely high. An effective therapeutic intervention is urgently required. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that also have pleiotropic actions. They have been reported to increase the survival of septic or infectious patients. But the effect of simvastatin, a widely used statin, on sepsis-induced AKI is unknown. The effects of simvastatin and tumor necrosis factor (TNF)-alpha neutralizing antibody were studied in a clinically relevant model of sepsis-induced AKI using cecal ligation and puncture (CLP) in elderly mice. Simvastatin significantly improved CLP-induced mortality and AKI. Simvastatin attenuated CLP-induced tubular damage and reversed CLP-induced reduction of intrarenal microvascular perfusion and renal tubular hypoxia at 24 h. Simvastatin also restored towards normal CLP-induced renal vascular protein leak and serum TNF-alpha. Neither delayed simvastatin therapy nor TNF-alpha neutralizing antibody improved CLP-induced AKI. Simvastatin improved sepsis-induced AKI by direct effects on the renal vasculature, reversal of tubular hypoxia, and had a systemic anti-inflammatory effect.