A Comprehensive Proteomics Analysis of Urinary Extracellular Vesicles Identifies a Specific Kinase Protein Profile as a Novel Hallmark of Medullary Sponge Kidney Disease.
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ABSTRACT:
SUBMITTER: Bruschi M
PROVIDER: S-EPMC9171619 | biostudies-literature | 2022 Jun
REPOSITORIES: biostudies-literature
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A Comprehensive Proteomics Analysis of Urinary Extracellular Vesicles Identifies a Specific Kinase Protein Profile as a Novel Hallmark of Medullary Sponge Kidney Disease.
Bruschi Maurizio M Granata Simona S Petretto Andrea A Verlato Alberto A Ghiggeri Gian Marco GM Stallone Giovanni G Candiano Giovanni G Zaza Gianluigi G
Kidney international reports 20220302 6
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Project description:Medullary sponge kidney (MSK) disease is a rare and neglected kidney condition often associated with nephrocalcinosis/nephrolithiasis and cystic anomalies in the precalyceal ducts. Little is known about the pathogenesis of this disease, so we addressed the knowledge gap using a proteomics approach. The protein content of microvesicles/exosomes isolated from urine of 15 MSK and 15 idiopathic calcium nephrolithiasis (ICN) patients was investigated by mass spectrometry, followed by weighted gene coexpression network analysis, support vector machine (SVM) learning, and partial least squares discriminant analysis (PLS-DA) to select the most discriminative proteins. Proteomic data were verified by ELISA. We identified 2998 proteins in total, 1764 (58.9%) of which were present in both vesicle types in both diseases. Among the MSK samples, only 65 (2.2%) and 137 (4.6%) proteins were exclusively found in the microvesicles and exosomes, respectively. Similarly, among the ICN samples, only 75 (2.5%) and 94 (3.1%) proteins were exclusively found in the microvesicles and exosomes, respectively. SVM learning and PLS-DA revealed a core panel of 20 proteins that distinguished extracellular vesicles representing each clinical condition with an accuracy of 100%. Among them, three exosome proteins involved in the lectin complement pathway maximized the discrimination between MSK and ICN: Ficolin 1, Mannan-binding lectin serine protease 2, and Complement component 4-binding protein β. ELISA confirmed the proteomic results. Our data show that the complement pathway is involved in the MSK, revealing a new range of potential therapeutic targets and early diagnostic biomarkers.
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Project description:Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.
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Project description:There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers.
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Project description:Medullary sponge kidney (MSK) disease is a rare and neglected kidney condition often associated with nephrocalcinosis/nephrolithiasis and cystic anomalies in the precalyceal ducts. Little is known about the pathogenesis of this disease, so we addressed the knowledge gap using a proteomics approach. The protein content of microvesicles/ exosomes isolated from urine of 15 MSK and 15 idiopathic calcium nephrolithiasis (ICN) patients was investigated by mass spectrometry, followed by weighted gene coexpression network analysis, support vector machine (SVM) learning, and partial least squares discriminant analysis (PLS-DA) to select the most discriminative proteins. Proteomic data were verified by ELISA. We identified 2998 proteins in total, 1764 (58.9%) of which were present in both vesicle types in both diseases. Among the MSK samples, only 65 (2.2%) and 137 (4.6%) proteins were exclusively found in the microvesicles and exosomes, respectively. Similarly, among the ICN samples, only 75 (2.5%) and 94 (3.1%) proteins were exclusively found in the microvesicles and exosomes, respectively. SVM learning and PLS- DA revealed a core panel of 20 proteins that distinguished extracellular vesicles representing each clinical condition with an accuracy of 100%. Among them, three exosome proteins involved in the lectin complement pathway maximized the discrimination between MSK and ICN: Ficolin 1, Mannan-binding lectin serine protease 2 and Complement component 4-binding protein β. ELISA confirmed proteomic results. Our data show that the complement pathway is involved in MSK, revealing a new range of potential therapeutic targets and early diagnostic biomarkers.
2021-12-09 | PXD025547 | Pride
Project description:Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 proteins as present in all OC-EVs samples. The functional enrichment analysis using Gene Ontology terms and an Ingenuity Pathway Analysis revealed that the identified proteins were involved in several cellular metabolic processes, including translation, synthesis, expression, and protein metabolism. Notably, the proteins were also involved in critical canonical pathways with essential functions in oocyte and embryo development, such as ERK/MAPK, EIF2, PI3K/AKT, and mTOR signaling. These data would be an important resource for studying canine reproductive physiology and establishing a successful in vitro embryo production system in dogs.
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Project description:Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.
| S-EPMC7956503 | biostudies-literature
Project description:BackgroundCancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells' EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard.MethodsEVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach.ResultsBoth TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs' cargo identified candidate proteins (including uPAR and PDGFRβ) that may be involved during this process.ConclusionsTNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.
| S-EPMC9520807 | biostudies-literature
Project description:Background: Renal cell carcinoma (RCC) accounts for about 2% of all cancers. Renal biopsy is the gold standard among the diagnostic tools, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for ccRCC detection are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, seems to be a general characteristic of malignant transformation. Consistently, cancer-derived EVs are enriched in the blood, urine and various malignant effusions of cancer patients. Therefore, urinary samples can be a non-invasive approach for discovering diagnostic biomarkers. Methods: We enrolled 33 clear-cell RCC (ccRCC) patients and 22 healthy subjects (HS), age and sex-matched, for urine collection and extracellular vesicles isolation by differential centrifugation. Transcriptional profiles of urinary EVs from 12 patients with ccRCC and 11 HS were generated using the Illumina HumanHT-12 v4 BeadChip oligonucleotide arrays. Microarray analysis led to the identification of RNA that were then validated using RT-qPCR. Results: We showed for the first time that urinary exosomal shuttle RNA (esRNA) was significantly different in ccRCC patients compared to HS and we identified three EVs esRNA involved in the tumor biology that are potentially suitable as non-invasive biomarkers. GSTA1, CEBPA and PCBD1 RNA levels decreased in urinary EVs of patients compared to HS. After 1 month post-operation, the levels of RNA increased to reach the normal level. Conclusions: This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC. Total RNA obtained from urinary extracellular vesicles isolated from ccRCC patients and healthy subjects.
2016-09-01 | E-GEOD-72922 | biostudies-arrayexpress
Project description:IntroductionVarious kidney diseases reportedly show different urinary extracellular vesicle (EV) RNA profiles. Although obesity is one of the main causes of chronic kidney disease, the expression pattern of urinary EV RNA in obesity is uncertain. Our aim was to sequence the small RNA profiles of urinary EVs in obese patients before and after weight reduction and compare them to those of healthy volunteers (HVs).MethodsWe recruited age-sex-matched obese patients and HVs. The small RNA profiles of urinary EVs were analyzed using RNA sequencing. To evaluate the effect of weight reduction, small RNA profiles of urinary EVs 6 months after bariatric surgery were also analyzed.ResultsThe proportion of urinary EVs transfer RNA and microRNA of obese patients differed from that of HVs. Obese patients showed differential expression of 1,343 small RNAs in urinary EVs compared to HVs (fold change ≥2 and p value <0.05). Among those, 61 small RNAs were upregulated in obese patients and downregulated after weight reduction, whereas 167 small RNAs were downregulated in obese patients and upregulated after weight reduction. RNA sequencing revealed the correlation between the specific urinary EV small RNAs and clinical parameters including body weight, low-density lipoprotein cholesterol, triglyceride, high-density lipoprotein cholesterol, serum glucose, estimated glomerular filtration rate, and albuminuria.ConclusionObese patients showed distinct urinary EV small RNA profiles compared to HVs. Weight reduction altered urinary EV small-RNA profiles in obese patients.
| S-EPMC9021654 | biostudies-literature
Project description:BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.
| S-EPMC6921509 | biostudies-literature