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Am J Physiol Renal Physiol (January 16, 2007). doi:10.1152/ajprenal.00434.2006
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Submitted on October 31, 2006
Accepted on January 5, 2007

Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator

Anita Cheruvanky1, Hua Zhou1, Trairak Pisitkun2, Jeffrey B Kopp3, Mark A Knepper4, Peter S.T. Yuen1, and Robert A. Star5*

1 NIDDK, NIH, Bethesda, Maryland, United States
2 NHLBI, NIH, Bethesda, Maryland, United States
3 Bethesda, Maryland, United States; NIDDK, NIH, Bethesda, Maryland, United States
4 Bethesda, Maryland, United States; NHLBI, NIH, Bethesda, Maryland, United States
5 Bethesda, Maryland, United States; NIDDK, NIH, Bethesda, Maryland, United States; Renal Diagnosis and Therapeutics Unit, 10 Center Drive, Room 3N108, Bethesda, Maryland, 20892-1268, United States

* To whom correspondence should be addressed. E-mail: robert_star{at}nih.gov.

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 3000 x g for 10-30 min. Urinary exosomal markers, TSG101, AQP2, NES, Annexin V, ACE, 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 5 male patients with focal segmental glomerulosclerosis (FSGS) and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; 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.




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