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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.

SUBMITTER: Cheruvanky A

PROVIDER: S-EPMC2271070 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

Cheruvanky Anita A Zhou Hua H Pisitkun Trairak T Kopp Jeffrey B JB Knepper Mark A MA Yuen Peter S T PS Star Robert A RA

American journal of physiology. Renal physiology 20070116 5

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, aqu ...[more]

PMID: 17229675

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Project description:Background and study aims Bowel cancer is diagnosed in 40,000 people each year in the UK. Early detection and treatment of bowel cancer is important as it improves survival. This has led to a “fast-track” system to speed up waiting times and access to hospital tests. Deciding who to test is a hard for GPs and hospital doctors because bowel symptoms are common and are not usually due to cancer. In addition, not all people with a bowel cancer develop symptoms. Therefore, at present the vast majority of patients are required to undergo testing. An internal camera examination of the large bowel (colonoscopy) is the best way to look at the large bowel. This is invasive, can be uncomfortable and needs strong laxatives to prepare the bowel. There is a risk of unplanned admission to hospital and surgery. This test is also expensive and the demand is increasing in the NHS. In the UK, 300,000 people a year are referred through the fast-track system for suspected bowel cancer. Over 90% of these have normal results, meaning they have undergone unpleasant testing that did not help them. It is now possible to look at natural chemicals, produced by normal bacteria that live in everybody’s bowel, to help diagnose bowel conditions. Bowel cancer alters the chemical “signature”. The aim of this study is to test the urine of 600 people referred by their GP to the fast-track pathway for these chemicals to see how good they are at predicting bowel cancer. Who can participate? Patients aged 18 and over, referred by their GP for fast-track assessment due to symptoms of colorectal cancer What does the study involve? Participants provide a urine sample when they attend the hospital for colonoscopy or CT scanning. Participants are also asked to complete a short questionnaire collecting demographic information and some data about lifestyle including smoking status, alcohol use and dietary habit (meat eating, vegetarian or vegan diet). From that point on their clinical pathway is unaffected by participation in the study and the results of the volatile compound testing are not be available to the clinical team. At the end of their investigations (maximum of 63 days), a member of the research team accesses patient notes to collect disease outcome data coded as cancer, polyp or normal. The participants receive treatment as normal judged on the clinical data available to the clinicians caring for them. The urine samples are sent for analysis at the University of the West of England. They are analysed using three separate machines to identify which of these machines is the best for use in a clinical setting, to take forward into a larger study. Data from the testing are then analysed along with the data about disease status in order to assess the sensitivity and reliability of the testing for the identification of patients at high risk of having colon cancer.

Project description:BackgroundBrain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples.MethodsThe principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers.ResultsUsing comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases.ConclusionThe MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

Dataset Information

Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

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Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.

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