Microfiltration isolation of human urinary exosomes for characterization by MS

Merchant, Michael L.; Powell, David W.; Wilkey, Daniel W.; Cummins, Timothy D.; Deegens, Jeroen K. J.; Rood, Ilse M.; McAfee, K. Jill; Fleischer, Cornelia; Klein, Elias; Klein, Jon B.

doi:10.1002/prca.200800093

Cited by 175 publications

(153 citation statements)

References 48 publications

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“…Although many of the innate immune proteins identified in our analysis had been previously identified in urinary exosomal analyses (Supplemental Table 1), 12,13,[16][17][18][19][20][21] enrichment for this protein grouping has not previously been reported. However, enrichment analysis relies on the reliability of the background proteome.…”

Section: Discussionmentioning

confidence: 84%

“…However, most studies on urine exosomes to date have focused on biomarker discovery, resulting in the publication of several urine exosome protein compendia. 12,13,[16][17][18][19][20][21] Published reports of the urinary exosomal proteome have limited value in illuminating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates, 22,23 and previous reports are not free of these limitations.…”

mentioning

confidence: 99%

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Human Urinary Exosomes as Innate Immune Effectors

Hiemstra¹,

Charles

Gracia

et al. 2014

Journal of the American Society of Nephrology

133

120

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Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein.Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

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Section: Discussionmentioning

confidence: 84%

mentioning

confidence: 99%

Human Urinary Exosomes as Innate Immune Effectors

Hiemstra¹,

Charles

Gracia

et al. 2014

Journal of the American Society of Nephrology

133

120

View full text Add to dashboard Cite

show abstract

“…UF can rapidly isolate EVs from body fluids [66,67], but the yield is low because of the loss of small size EVs, which have a similar size to protein aggregates, necessary to guarantee the purity [68]. …”

Section: Microfluidics-based Ev Isolationmentioning

confidence: 99%

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

Guo

Tao

Dawn

2018

J of Extracellular Vesicle

133

106

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Extracellular vesicles (EVs), which can be found in almost all body fluids, consist of a lipid bilayer enclosing proteins and nucleic acids from their cells of origin. EVs can transport their cargo to target cells and have therefore emerged as key players in intercellular communication. Their potential as either diagnostic and prognostic biomarkers or therapeutic drug delivery systems (DDSs) has generated considerable interest in recent years. However, conventional methods used to study EVs still have significant limitations including the time-consuming and low throughput techniques required, while at the same time the demand for better research tools is getting stronger and stronger. In the past few years, microfluidics-based technologies have gradually emerged and have come to play an essential role in the isolation, detection and analysis of EVs. Such technologies have several advantages, including low cost, low sample volumes, high throughput and precision. This review summarizes recent advances in microfluidics-based technologies, compares conventional and microfluidics-based technologies, and includes a brief survey of recent progress towards integrated “on-a-chip” systems. In addition, this review also discusses the potential clinical applications of “on-a-chip” systems, including both “liquid biopsies” for personalized medicine and DDS devices for precision medicine, and then anticipates the possible future participation of cloud-based portable disease diagnosis and monitoring systems, possibly with the participation of artificial intelligence (AI).

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“…They are suitable for most laboratories and easy to be utility, however, they are costly, especially if a large number of biological samples need to be processed. Besides that, various techniques including filtration [20][21][22], microfluidic-based method [23,24], immuno-affinity and peptide-based isolation [25], size-exclusion chromatography [26][27][28], and so on, have also been proposed for enrichment of exosomes. They have advantages and disadvantages, can be selected according to the experimental command.…”

Section: Introductionmentioning

confidence: 99%

Effective enrichment of urinary exosomes by polyethylene glycol for RNA detection

Lv¹,

Yang²

2018

biomedicalresearch

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Urinary exosome is a kind of extracellular vesicles secreted by the cells of kidney and urinary tract. It is an ideal non-invasive biomarker for obtaining information about the state of renal cells. There are few exosomes in urine which are affected by many factors. Enriching exosomes in urine with inexpensive and efficient method is a major challenge. Because exosomes are virus-sized minuscule membrane vesicles or sacs and people used to enrich virus by Polyethylene Glycol (PEG), here we use PEG to enrich the exosomes in urine of 17 volunteers. To confirm the harvest of exosomes, the morphology of exosomes were observed by Transmission Electron Microscopy (TEM), the common exosomal protein markers (CD63, CD9, tumor susceptibility gene 101, Flotillin-1 and β-actin) were detected by Western blot, and the size distribution was analysed by Malvin Nano-ZS. Next, the exosomal housekeeping gene of miRNA (SnRNA-U6) and mRNA (β-actin) were tested by qRT-PCR to prove that this method could be used in subsequent experiments.

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Microfiltration isolation of human urinary exosomes for characterization by MS

Cited by 175 publications

References 48 publications

Human Urinary Exosomes as Innate Immune Effectors

Human Urinary Exosomes as Innate Immune Effectors

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

Effective enrichment of urinary exosomes by polyethylene glycol for RNA detection

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