Candidate biomarkers in exosome-like vesicles purified from rat urine samples
Ontology highlight
ABSTRACT:
INSTRUMENT(S): QToF-Ultima
ORGANISM(S): Rattus Norvegicus (rat)
SUBMITTER: 
Juan Casado-Vela
LAB HEAD: Juan Casado-Vela
PROVIDER: PRD000812 | Pride | 2009-12-01
REPOSITORIES: Pride
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Project description:There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment.We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease.The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified.Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury.
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Project description:Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.
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Project description:Frailty is a geriatric syndrome associated with progressive physical decline and significantly increases risk for falls, disability, hospitalizations, and death. However, much remains unknown regarding the biological mechanisms that contribute to aging and frailty, and to date, there are no clinically used prognostic or diagnostic molecular biomarkers. The present study profiled exosome-derived microRNAs isolated from the plasma of young, robust older, and frail older individuals and identified eight miRNAs that are uniquely enriched in frailty: miR-10a-3p, miR-92a-3p, miR-185-3p, miR-194-5p, miR-326, miR-532-5p, miR-576-5p, and miR-760. Furthermore, since exosomes can deliver miRNAs to alter cellular activity and behavior, these miRNAs may also provide insights into the biological mechanisms underlying frailty; KEGG analysis of their target genes revealed multiple pathways implicated in aging and age-related processes. Although further validation and research studies are warranted, our study identified eight novel candidate biomarkers of frailty that may help to elucidate the multifactorial pathogenesis of frailty.
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Project description:Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.
| S-EPMC4449362 | biostudies-literature
Project description:IntroductionData are sparse about the potential health risks of chronic low-dose contamination of humans by uranium (natural or anthropogenic) in drinking water. Previous studies report some molecular imbalances but no clinical signs due to uranium intake.ObjectivesIn a proof-of-principle study, we reported that metabolomics is an appropriate method for addressing this chronic low-dose exposure in a rat model (uranium dose: 40 mg L-1; duration: 9 months, n = 10). In the present study, our aim was to investigate the dose-effect pattern and identify additional potential biomarkers in urine samples.MethodsCompared to our previous protocol, we doubled the number of rats per group (n = 20), added additional sampling time points (3 and 6 months) and included several lower doses of natural uranium (doses used: 40, 1.5, 0.15 and 0.015 mg L-1). LC-MS metabolomics was performed on urine samples and statistical analyses were made with SIMCA-P+ and R packages.ResultsThe data confirmed our previous results and showed that discrimination was both dose and time related. Uranium exposure was revealed in rats contaminated for 9 months at a dose as low as 0.15 mg L-1. Eleven features, including the confidently identified N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide and 4-hydroxyphenylacetylglycine, discriminated control from contaminated rats with a specificity and a sensitivity ranging from 83 to 96 %, when combined into a composite score.ConclusionThese findings show promise for the elucidation of underlying radiotoxicologic mechanisms and the design of a diagnostic test to assess exposure in urine, in a dose range experimentally estimated to be above a threshold between 0.015 and 0.15 mg L-1.
| S-EPMC5025510 | biostudies-literature
Project description:BackgroundObesity is a rising risk factor for various diseases including cardiovascular diseases and Cancer. The limitations of targeted obesity-treatment approaches employed in the clinic presently underscore the importance of developing integrative management strategies for identification of specific biomarkers of obesity.ObjectivesGiven the specificity of exosome/extracellular vesicle (EV) biomarkers, we aimed here to identify the EV biomarkers of Ayurveda treatment - Lekhana Basti - for Obesity.MethodologyA total of eighteen 24-h urine samples from 6 participants with BMI>30 kg/m2 were used in this study, collected over 3 time-points during the Lekhana basti (medicated enema for obesity) treatment. Urine EV were isolated using Polyethylene Glycol (PEG). The proteins were resolved by 1-d gel electrophoresis and identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantified by label-free methods. Significant Protein-Protein Interactions, KEGG pathway analysis and enrichment, functional gene ontology (GO) annotation were identified and shortlisted in comparison to Obesity reference genes from DisGeNET.ResultsWith UniProt as a reference subsequent to LC-MS/MS-identification, a total of 210 exosome proteins were identified. Seventy-three proteins were overexpressed in pathway enrichment analysis. Further, GO functional annotation identified 15 common proteins involved. Finally, the 8 hub proteins associated with obesity were identified and their differential expression profile compared between three different time-points during Lekhana Basti treatment. Six protein markers overexpressed during obesity were downregulated post Lekhana Basti treatment, while 2 markers increased in abundance post-treatment.ConclusionTo our knowledge, this is the first study to isolate and identify urine EV protein abundance profiles from obese female participants of India. The study results indicate significant changes in the differential expression profile of 8 hub proteins involved in obesity, after Lekhana Basti treatment. The biomarker signature of the pilot study indicates the role of Ayurveda treatment and the possible pathways involved in the treatment of Obesity. Further, this study underlines the specificity of urine exosomes/EV as diagnostic markers as well as the potential of Ayurveda treatment in effective management of obesity.
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Project description:ObjectiveOsteoporotic fracture is one of the most common health risks and aggravates the quality of life among postmenopausal women worldwide. In this study, osteoporosis-associated protein biomarkers were identified from urine of osteoporotic female Sprague-Dawley rats developed by ovariectomy.MethodFour months after the operation, the bone mineral density of the femur of ovariectomized rats was significantly lowered in comparison with that of the sham operated rats. The protein profiles of the urine samples collected from the sham, ovariectomized (OVX) and 2 month-old non-operated (Young) rats were compared by 2-D gel and MS spectrometry.ResultsProteins consistently expressed between Young and sham but differentially expressed in OVX rats were selected and identified. One down-regulated 21 kDa protein, superoxide dismutase (SOD), and 1 up-regulated 53-54 kDa protein, alph-1-antitrypsin (A1AT), were selected from urine of the ovariectomized rats by 2-D gel analysis. Further, a total of 30 with 19 up-regulated and 11-down-regulated proteins were selected by LC-MS analysis with more than 2-fold differences in spectral counts. The fact that SOD and A1AT are also listed in the 30 differential proteins suggests that our biomarker isolation procedure suitably represents osteoporosis-associated proteins in urine.ConclusionSupporting the facts, the differential expressions of SOD and A1AT in urine could be validated by Western blotting. These urinary osteoporosis-associated proteins have high potentials to become candidates for non-invasive diagnosis of osteoporosis from urine.
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Project description:Background A molecular diagnostic test that could directly evaluate Treponema pallidum subspecies pallidum antigens in urine would greatly facilitate the prompt diagnosis of syphilis. Methods A multi-platform high resolution shotgun proteomics approach was used to investigate the presence of T. pallidum proteins in male urine samples collected from individuals with and without syphilis. Study participants were included in an observational cohort study conducted at the Institute of Tropical Medicine, Antwerp, Belgium from 2014 to 2017. In total, N = 60 individuals were included in this sub-study, including n = 36 with early symptomatic stage (primary/secondary), n = 13 early latent and n = 5 late latent stage and n = 6 controls. Almost all participants were HIV-positive (56/60; 93 %) and identified as men who have sex with men (MSM) (59/60; 98 %). Sixteen (27 %) individuals were serum PCR positive for T. pallidum at the time of diagnosis. Ten pools containing urine from six participants (±1.5 mL per individual) were created based on clinical staging. Urine protein was extracted from the pools by a two-step molecular weight cut-off centrifugal filtration to remove large proteins and non-protein material. Samples were digested, subjected to multi-dimensional liquid chromatographic separation based on hydrophobicity and were analysed with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or liquid chromatography- electrospray ionization quadruple ion mobility time-of-flight high-definition mass spectrometry (LC/ESI-Q-IM-TOF) in data dependent (MALDI-TOF/TOF) and data independent HDMSE (ESI-IM-Q-TOF) acquisition modes. Results In total, 26 peptides corresponding to four unique T. pallidum proteins were uncovered in four different pooled urine samples, from n = 3 symptomatic (primary/secondary stage; PSS) and n = 1 latent stage pool. Peptides from GTPase Obg (Tp0742) were detected in two separate PSS pooled samples. Protein designations are listed as in the currently annotated T. pallidum proteome available at the UniProt database (Proteome ID: UP000014259) which is related to the genome assembly and annotation ID GCA_000410535.2 listed on the European Nucleotide Archive. Eleven peptides from an uncharacterized protein (Tp0369) and 8 peptides from a protein annotated as the Borrelia-like p83/100 protein (Tp0486) were detected in two different PSS pooled samples. A protein annotated as an ABC superfamily transport protein (Tp0804) was uncovered in a latent stage pooled sample. BLAST analyses revealed low peptide sequence similarity with other pathogenic and commensal prokaryotes and human proteins. Conclusions High resolution mass spectrometric analyses of protein extracts from pooled urine samples resulted in the detection of four T. pallidum proteins. This is the first successful account of applying proteomics approaches to detect treponemal antigens in clinical samples. These proteins could represent potential biomarkers for downstream diagnostic applications.
2018-10-18 | PXD009707 | Pride
Project description:BackgroundExosomes are involved in cell-to-cell communication, neovascularization, cancer metastasis, and drug resistance, which all play an important role in the occurrence and progression of hepatocellular carcinoma (HCC). Because there are few mechanistic studies about the function of exosomes in HCC, the goals of this study were to identify exosome-related genes in HCC, to establish a reliable prognostic model for HCC, and to explore underlying mechanisms.MethodsThe exoRBase and The Cancer Genome Atlas (TCGA) databases were used to analyze differentially expressed genes (DEGs). Cox regression and least absolute shrinkage and selection operator analyses were used to identify DEGs closely related to the overall survival of patients with HCC. An exosome-related prognostic model was then constructed in TCGA and validated in the International Cancer Genome Consortium database. A nomogram was developed to predict survival. CIBERSORT was used to estimate the abundance of different types of immune cells. Immunotherapy-related DEGs were used to predict the effect of immunotherapy.ResultsForty-eight exosome-related DEGs were obtained; of them, five [exportin 1 (XPO1), lysosomal thiol reductase (IFI30), F-box protein 16 (FBXO16), calmodulin 1 (CALM1), MORC family CW-type zinc finger 3 (MORC3)] were selected to construct a predictive model. Patients with HCC were then divided into low- and high-risk groups using the best cut-off value, as determined by the X-tile software. Prognosis was significantly poorer in the high-risk than in the low-risk group (P=0.009; hazard ratio =2.65). Features related to exosomes were found to positively regulate immune response. Further analysis showed a higher risk score was associated with higher expression of immune checkpoint molecules, including programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), T cell Ig and ITIM domain (TIGIT), and indoleamine-2,3-dioxygenase 1 (IDO1).ConclusionsThis study has identified a novel signature based on exosome-related genes that has potential as a prognostic biomarker for HCC. Our research provides an immunological perspective for the development of precision treatment for HCC.
| S-EPMC9403937 | biostudies-literature
Project description:BackgroundBladder cancer (BC), a common cancer of the urinary system, has a low mortality but an extremely high recurrence rate. Patients who have undergone initial surgical treatment often undergo frequent prognostic examinations with a substantial burden of discomfort and costs. Urine samples can reflect early disease processes in the urinary system and may be an excellent source of biomarkers.MethodsIn the present study, we used the liquid chromatography with tandem mass spectrometry (LC-MS/MS) to perform proteomic analysis of pre- and postoperative urine samples from patients with stage III BC to identify biomarkers of cancer prognosis. Candidate biomarkers from proteomic analysis were simultaneously validated using western blotting in an independent cohort and immunohistochemical (IHC) staining, combined with gene expression data of BC samples in The Cancer Genome Atlas (TCGA).ResultsThe comparison of pre- and postoperative urine samples from the same patients led to the discovery of several significantly differentially expressed proteins, whose functions could be closely related to the occurrence and development of BC. We confirmed a representative group of candidate biomarker molecules, such as cadherin-related family member 2 (CDHR2), heat shock protein beta-1 (HSP27), and heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1).ConclusionsThe candidate biomarker molecules can distinguish between pre- and postoperative urine samples, and alterations in their expression levels are significantly associated with recurrence rates in patients with BC. Therefore, these molecules may become useful biomarkers for the monitoring and prognosis of BC.
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