Urinary Extracellular Vesicles Research Articles

Uromodulin mRNA from Urinary Extracellular Vesicles Correlate to Kidney Function Decline in Type 2 Diabetes Mellitus

Background: Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline. Methods: A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline. Results: We observe that mild and severe DKD groups show a significant 3.2- and ­4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and ­non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function. Conclusion: Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity.

Metabolic alterations in urine extracellular vesicles are associated to prostate cancer pathogenesis and progression

ABSTRACTUrine contains extracellular vesicles (EVs) that concentrate molecules and protect them from degradation. Thus, isolation and characterisation of urinary EVs could increase the efficiency of biomarker discovery. We have previously identified proteins and RNAs with differential abundance in urinary EVs from prostate cancer (PCa) patients compared to benign prostate hyperplasia (BPH). Here, we focused on the analysis of the metabolites contained in urinary EVs collected from patients with PCa and BPH. Targeted metabolomics analysis of EVs was performed by ultra-high-performance liquid chromatography–mass spectrometry. The correlation between metabolites and clinical parameters was studied, and metabolites with differential abundance in PCa urinary EVs were detected and mapped into cellular pathways. We detected 248 metabolites belonging to different chemical families including amino acids and various lipid species. Among these metabolites, 76 exhibited significant differential abundance between PCa and BPH. Interestingly, urine EVs recapitulated many of the metabolic alterations reported in PCa, including phosphathidylcholines, acyl carnitines, citrate and kynurenine. Importantly, we found elevated levels of the steroid hormone, 3beta-hydroxyandros-5-en-17-one-3-sulphate (dehydroepiandrosterone sulphate) in PCa urinary EVs, in line with the potential elevation of androgen synthesis in this type of cancer. This work supports urinary EVs as a non-invasive source to infer metabolic changes in PCa.

Increased Urinary Extracellular Vesicle Sodium Transporters in Cushing Syndrome With Hypertension.

Increased renal sodium reabsorption contributes to hypertension in Cushing syndrome (CS). Renal sodium transporters can be analyzed noninvasively in urinary extracellular vesicles (uEVs). To analyze renal sodium transporters in uEVs of patients with CS and hypertension. Observational study. University hospital. The uEVs were isolated by ultracentrifugation and analyzed by immunoblotting in 10 patients with CS and 7 age-matched healthy participants. In 7 patients with CS, uEVs were analyzed before and after treatment. Abundance of protein in uEVs. The 10 patients with CS were divided in those with suppressed and nonsuppressed renin-angiotensin-aldosterone system (RAAS; n = 5 per group). Patients with CS with suppressed RAAS had similar blood pressure but significantly lower serum potassium than patients with CS with nonsuppressed RAAS. Compared with healthy participants, only patients with suppressed RAAS had higher phosphorylated Na+-K+-Cl- cotransporter type 2 (pNKCC2) and higher total and phosphorylated Na+-Cl- cotransporter (pNCC) in uEVs. Serum potassium but not urinary free cortisol correlated with pNKCC2, pNCC, and Na+-Cl- cotransporter (NCC) in uEVs. Treatment of CS reversed the increases in pNKCC2, NCC, and pNCC. CS increases renal sodium transporter abundance in uEVs in patients with hypertension and suppressed RAAS. Potassium has recently been identified as an important driver of NCC activity, and low serum potassium may also contribute to increased renal sodium reabsorption and hypertension in CS. These results may also be relevant for hypertension induced by exogenous glucocorticoids.

Urinary extracellular vesicle biomarkers in urological cancers: From discovery towards clinical implementation.

Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect an individual's metabolic and pathophysiologic state. Despite intensive research into the discovery of urinary biomarkers to facilitate early diagnosis, accurate prognosis and prediction of therapy response in urological cancers, none of these markers has reached widespread use. Their implementation into daily clinical practice is hampered by a substantial degree of heterogeneity in performance characteristics and uncertainty about reliability, clinical utility and cost-effectiveness, in addition to several technical limitations. Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery because of their role in intercellular communication and the resemblance of their molecular content to that of the releasing cells. We review currently used urinary biomarkers in the clinic and attempts that have been made to identify EV-derived biomarkers for urological cancers. In addition, we discuss technical and methodological considerations towards their clinical implementation.

FRI-164 - Serum and urine extracellular vesicles contain mRNA biomarkers for primary sclerosing cholangitis (PSC) and cholangiocarcinoma (CCA)

FRI-164 - Serum and urine extracellular vesicles contain mRNA biomarkers for primary sclerosing cholangitis (PSC) and cholangiocarcinoma (CCA)

Representation Analysis of miRNA in Urine Microvesicles and Cell-Free Urine in Prostate Diseases

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesicles-associated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urinary extracellular vesicles and demonstrated diagnostic potential of their miRNAs. In this study, we have performed a comparative analysis of 84 miRNA in paired samples of urine microvesicles and urine supernatant from healthy men, patients with benign prostate hyperplasia, and prostate cancer patients using miRCURY LNA miRNA qPCR Panels. In all groups of patients, miRNA subsets characterized by different distribution between the urinary fractions have been found. In this context, two groups of miRNAs have been identified, which are involved in several signaling pathways including those associated with prostate cancer development.

Altered Proteome of Extracellular Vesicles Derived from Bladder Cancer Patients Urine.

Proteomic analysis of extracellular vesicles (EVs) from biological fluid is a powerful approach to discover potential biomarkers for human diseases including cancers, as EV secreted to biological fluids are originated from the affected tissue. In order to investigate significant molecules related to the pathogenesis of bladder cancer, EVs were isolated from patient urine which was analyzed by mass spectrometry based proteomics. Comparison of the EV proteome to the whole urine proteome demonstrated an increased number of protein identification in EV. Comparative MS analyses of urinary EV from control subjects and bladder cancer patients identified a total of 1,222 proteins. Statistical analyses provided 56 proteins significantly increased in bladder cancer urine, including proteins for which expression levels varied by cancer stage (P-value < 0.05). While urine represents a valuable, noninvasive specimen for biomarker discovery in urologic cancers, there is a high degree of intra- and inter-individual variability in urine samples. The enrichment of urinary EV demonstrated its capability and applicability of providing a focused identification of biologically relevant proteins in urological diseases.

Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50–150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies.

Enhanced urinary extracellular vesicle isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Enhanced urinary extracellular vesicle isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Reproducible isolation of high-purity urinary extracellular vesicles for uro-oncological biomarker studies

Reproducible isolation of high-purity urinary extracellular vesicles for uro-oncological biomarker studies

Exosomal biomarkers for kidney disease.

Development of minimally invasive biomarkers is necessary for early detection, prognosis prediction, severity assessment and treatment monitoring in different kidney diseases. Recently, many studies have been conducted worldwide on marker proteins and micro RNA(miRNA)contained in urinary extracellular vesicles(EVs)including exosomes. This article reviews urinary EV-contained biomarkers, including proximal tubular endocytic receptor megalin, for kidney diseases such as diabetic nephropathy.

Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients.

Background Extracellular vesicles (EVs) are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs) in urinary EVs from HIV+ patients and compare them to HIV− samples. Methods Urine samples were collected from HIV+ (n = 35) and HIV− (n = 12) individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS), and nanoparticle tracking analysis (NTA). Western blots confirmed the presence of HIV proteins. Gene ontology (GO) analysis was performed using FunRich and HIV Human Interaction database (HHID). Results EVs from urine were 30–400 nm in size. More EVs were in HIV+ patients, P < 0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV−. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV− samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins. Conclusion Differences in the proteomic profile of EVs from HIV+ versus HIV− samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.

Representation analysis of miRNA from clarified urine and urine microvesicles in prostate malignancies and non-malignant neoplasms

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesiclesassociated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urine extracellular vesicles and demonstrated diagnostic potential of their miRNA cargo. In this study, we have performed a comparative analysis of the expression of 84 miRNA in paired samples of urine microvesicles and clarified urine from healthy men, patients with benign hyperplasia and cancer of the prostate using miRCURY LNA miRNA qPCR Panels. Subsets of miRNAs with differences in expression between the fractions of the urine were found in all three groups. Two groups of miRNA were identified based on the patterns of their differential expression. They regulate several key signaling pathways associated with prostate cancer development.

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires.

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment

ABSTRACTUrinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 1010/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

Genome-wide Profiling of Urinary Extracellular Vesicle microRNAs Associated With Diabetic Nephropathy in Type 1 Diabetes

IntroductionDiabetic nephropathy (DN) is a form of progressive kidney disease that often leads to end-stage renal disease (ESRD). It is initiated by microvascular complications due to diabetes. Although microalbuminuria (MA) is the earliest clinical indication of DN among patients with type 1 diabetes (T1D), it lacks the sensitivity and specificity to detect the early onset of DN. Recently, microRNAs (miRNAs) have emerged as critical regulators in diabetes as well as various forms of kidney disease, including renal fibrosis, acute kidney injury, and progressive kidney disease. Additionally, circulating extracellular miRNAs, especially miRNAs packaged in extracellular vesicles (EVs), have garnered significant attention as potential noninvasive biomarkers for various diseases and health conditions.MethodsAs part of the University of Pittsburgh Epidemiology of Diabetes Complications (EDC) study, urine was collected from individuals with T1D with various grades of DN or MA (normal, overt, intermittent, and persistent) over a decade at prespecified intervals. We isolated EVs from urine and analyzed the small-RNA using NextGen sequencing.ResultsWe identified a set of miRNAs that are enriched in urinary EVs compared with EV-depleted samples, and identified a number of miRNAs showing concentration changes associated with DN occurrence, MA status, and other variables, such as hemoglobin A1c levels.ConclusionMany of the miRNAs associated with DN occurrence or MA status directly target pathways associated with renal fibrosis (including transforming growth factor-β and phosphatase and tensin homolog), which is one of the major contributors to the pathology of DN. These miRNAs are potential biomarkers for DN and MA.

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

Urinary Biomarkers for Chronic Kidney Disease with a Focus on Gene Transcript.

Objective:In the upcoming era of precision medicine, searching for the early, noninvasive biomarkers has been the cornerstone and major challenge in the management of chronic kidney disease (CKD). Urine contains rich biological information which could be the ideal source for noninvasive biomarkers of CKD. This review will discuss the recent advance in urinary biomarker.Data Sources:This review was based on data in articles published in the PubMed databases up to June 20, 2017, with the following keywords: “Chronic kidney disease”, “Biomarker”, and “Urine”.Study Selection:Original articles and important reviews on urinary biomarker were selected for this review.Results:Urinary biomarker studies of CKD mainly focused on urine sediment, supernatant, and urinary extracellular vesicles. The gene transcript (microRNA [miRNA], messenger RNA [mRNA]) biomarkers have been recently shown with diagnostic potential for CKD reflecting kidney function and histological change. However, challenges regarding technique and data analysis need to be resolved before translation to clinic.Conclusions:Different fractions of urine contain rich information for biomarker discovery, among which urine (extracellular vesicles) mRNA, miRNA, might represent promising biomarker for CKD.

Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer.

The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89–90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation.