Dataset Information

Identification and proteomic profiling of exosomes in human urine.

ABSTRACT: Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

SUBMITTER: Pisitkun T

PROVIDER: S-EPMC516573 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification and proteomic profiling of exosomes in human urine.

Pisitkun Trairak T Shen Rong-Fong RF Knepper Mark A MA

Proceedings of the National Academy of Sciences of the United States of America 20040823 36

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-dens ...[more]

PMID: 15326289

Similar Datasets

Project description:Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals.CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR).Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples.Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools.

Dataset Information

Identification and proteomic profiling of exosomes in human urine.

Publications

Identification and proteomic profiling of exosomes in human urine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets