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Human urinary extracellular vesicles protein LC-MS/MS

ABSTRACT: Urinary extracellular vesicles (uEV) are a precious new source of biomarkers reflecting the physiological state of the upstream parent cells and organ systems. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes heavily with all vesicle isolation attempts, precipitates with normal urinary proteins thus representing an unwanted “contaminant” in urinary assays. Here we developed a simple method to manage THP efficiently. Accordingly, uEV were isolated by hydrostatic filtration dialysis (HFD) and treated with a solution of urea to release most uEV. Presence of uEV was then confirmed by transmission electron microscopy, Western blot and proteomic profiling in MS, all showing that urea treatment did not affect integrity of uEV.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

SUBMITTER: Xiaomeng Xu

LAB HEAD: Harry Holthofer

PROVIDER: PXD013429 | Pride | 2019-09-30

REPOSITORIES: Pride

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Publications

Management of Tamm-Horsfall Protein for Reliable Urinary Analytics.

Xu Xiaomeng X Barreiro Karina K Musante Luca L Kretz Oliver O Lin Hanfei H Zou Hequn H Huber Tobias B TB Holthofer Harry H

Proteomics. Clinical applications 20190908 6

<h4>Purpose</h4>Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted "contaminant" in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently.<h4>Experimental design</h4>The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of ...[more]

PMID: 31424164

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Transcriptomic profiling of urine extracellular vesicles reveals alterations of CDH3 in prostate cancer

Project description:One of the challenges of current research in prostate cancer is to improve the differential non-invasive diagnosis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Extracellular vesicles (EV) are emerging structures with promising properties for intercellular communication. In addition, the characterization of EV in biofluids is an attractive source of non-invasive diagnostic, prognostic and predictive biomarkers. Here we show that urinary EV (uEV) from prostate cancer patients exhibit genuine and differential physical and biological properties. Importantly, transcriptomics characterization of uEVs led us to define the decreased abundance of Cadherin 3, type 1 (CDH3) transcript in uEV from PCa patients. Tissue and cell line analysis strongly suggested that the status of CDH3 in uEVs is a distal reflection of changes in the expression of this cadherin in the prostate tumor. Our results reveal that uEVs could represent a non-invasive tool to inform about the molecular alterations in prostate cancer. RNA extraction was performed in 10 ultracentrifuge-isolated-uEVs samples (4 BPH, 6 Pca)

2016-01-13 | E-GEOD-75441 | biostudies-arrayexpress

Neat urine proteome during menstrual cycle

Project description:Progress and advancement in assisted reproductive technologies (ART) and its outcomes are limited by the importance of research of endometrial receptivity being overlooked. Due to endometrial biopsy being invasive and in vitro studies lacking reproducibility in vivo, urine is an appealing alternative biofluid source for biomarker research as it can be collected in large quantities non-invasively. The discovery of extracellular vesicles (EVs) in urine (uEVs), has also opened a new avenue in this biomarker research, with these EVs harbouring thousands of proteins that hold promise for biomarker development. In this study urine was collected from human female volunteers and samples representing the different phases of the menstrual cycle were subjected to EV isolation via differential centrifugation and size exclusion chromatography. The resulting uEVs were analysed via Nanoparticle tracking analysis (NTA) to examine the different concentration and size of particles and proteomic analysis performed using shotgun label-free mass spectrometry on the uEV samples and neat urine samples. Our results showed that uEVs were found in numbers depending on the menstrual cycle phase but uEV size was not statistically altered during different stages of the menstrual cycle. Proteomics showed 50% of proteins detected in the neat urine were also present in the uEV samples with 813 proteins were unique in the uEV samples. Proteomics analysis also showed that the menstrual cycle phase affect the uEVs proteomic profile, with some proteins shown to be significantly upregulated and downregulated during the window of implantation phase of the cycle compared to the other non-receptive periods. This data highlights that uEVs characteristics are altered depending on the menstrual cycle phase suggesting the potential of uEVs being used as biomarkers for improving fertility.In this dataset we have the neat urine results.

2023-12-04 | PXD047128 | Pride

The kidney releases in the urine and circulation a non-polymerizing form of Uromodulin that retains the external hydrophobic patch domain

Project description:Uromodulin (Tamm-Horsfall protein, THP) is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs (TAL) apically in the urine, and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supra-molecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the C terminus. However, THP in the circulation is not polymerized, and it remains unclear if non-aggregated forms of THP exist natively in the urine. We propose that an alternative processing path, which retains the EHP domain, can lead to a non-polymerizing form of THP. We generated an antibody that specifically recognizes THP with retained EHP (THP+EHP) and established its presence in the urine in a non-polymerized native state. Proteomic characterization of urinary THP+EHP revealed its C-terminus to end at F617. In the human kidney, THP+EHP was not only detected in TAL cells, but also diffusely in the renal parenchyma. Using C-terminus proteomic sequencing and immunoblotting, we then demonstrated that serum THP has also retained EHP. In a small cohort of patients at risk for acute kidney injury (AKI), admission urinary THP+EHP was significantly lower in patients who subsequently developed AKI during hospitalization. Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing, which could explain the release of non-polymerizing THP in the circulation. Larger studies as needed to establish the utility of urinary THP+EHP as a sensitive biomarker of kidney health and susceptibility to injury.

2022-11-08 | PXD027914 | Pride

Urine Extracellular Vesicles as a Non-Invasive Implantation Window Monitoring Method

2023-12-04 | PXD047127 | Pride

Detection of N‑glycoprotein associated with IgA nephropathy in urine as a potential diagnostic biomarker using glycosylated proteomic analysis

Project description:The present study aimed to clarify the potential diagnosis value of the urinary N-glycoproteins using mass spectrometry (MS) analysis in patients with IgA nephropathy (IgAN).

2023-08-13 | PXD041151 | Pride

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine

Project description:Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease, profiling the proteome of EV separated from prostate tumor interstitial fluid and matched uEV, and a comparative proteomic analysis with uEV from patients with bladder and renal cancer.

2020-04-09 | PXD015289 | Pride

Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

Project description:Comparison of the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine to matched diagnostic formalin-fixed paraffin embedded (FFPE) tumour DNAs for 23 well-characterised UBC patients.

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Transcriptional responses of uropathogenic E. coli to stresses caused by sodium chloride or urea.

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Matrix Metalloproteinase-7 in Urinary Extracellular Vesicles Identifies Rapid Disease Progression in Autosomal Dominant Polycystic Kidney Disease

Project description:Background: In autosomal dominant polycystic kidney disease (ADPKD) there is an unmet need for early markers of rapid disease progression to facilitate counseling and selection for kidney-protective therapy. Our aim was to identify markers for rapid disease progression in urinary extracellular vesicles (uEVs). Methods: uEVs were isolated at baseline in two independent cohorts including patients with rapid disease progression and stable disease; patients were matched by age, sex, total kidney volume and mutation. uEV biomarker candidates were identified by mass spectrometry and further analyzed in a third and fourth cohort using immunoblotting and an enzyme-linked immunosorbent assay (ELISA). Single-nucleotide RNA sequencing of healthy and ADPKD tissue was used to confirm findings and identify the cellular origin of the uEV biomarker. Results: In the first two cohorts matrix metalloproteinase-7 (MMP-7) was significantly higher in uEVs of patients with rapid disease progression compared to stable disease. In the third cohort, immunoblotting confirmed a >2-fold increase in uEV-MMP-7 in patients with rapid disease progression compared to stable disease. In the fourth cohort, whole urine rather than uEVs were analyzed with MMP-7 ELISA demonstrating a significant but weak correlation with kidney function decline. Single-nucleotide RNA sequencing showed higher MMP-7 expression in ADPKD kidney tissue than in healthy kidney tissue and localized MMP-7 to the proximal tubule and thick ascending limb. Conclusion: uEV-associated MMP-7 is elevated in ADPKD patients with rapid disease progression and should be further investigated as predictive biomarker. Our findings also suggests that MMP-7 performs better when analyzed in uEVs than in whole urine.

2023-12-18 | PXD042533 | Pride

Transcriptional responses of uropathogenic E. coli to stresses caused by sodium chloride or urea.

Project description:E. coli which cause urinary tract infections must respond to high osmolarity in the urinary tract as well as the presence of urea. We used microarrays to measure the differntial gene expression of uropathogenic strain CFT073 in conditions of high osmolarity of urea v. minimal media RNA was extracted from CFT073 in each growth condition and hybridized to an Affymetrix microarray. The differentially expressed genes were analyzed by expression pattern and function.

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