Urinary Extracellular Vesicles Research Articles

Proteomic profiling of urinary extracellular vesicles differentiates breast cancer patients from healthy women.

Urinary extracellular vesicles (uEVs) reflect the biological conditions of the producing cells. The protein profiling of uEVs allow us to better understand cancer progression in several cancers such as bladder cancer, prostate cancer and kidney cancer but has not been reported in breast cancer. We have, herein, aimed at quantifying the concentration and at generating the proteomic profile of uEVs in patients with breast cancer (BC) as compared to that of healthy controls (CT). Urine samples were collected from 29 CT and 47 patients with BC. uEVs were isolated by using differential ultracentrifugation, and were then characterized by Western blotting and transmission electron microscopy. Moreover, a nanoparticle tracking analysis was used in order to measure the concentration and the size distribution of urine particles and uEVs. The proteomic profiling of the uEVs was facilitated through LC-MS/MS. The uEV concentration was not significantly different between the assessed groups. The undertaken proteomic analysis revealed 15,473 and 11,278 proteins in the BC patients' group and the CT group, respectively. Furthermore, a heat map analysis revealed a differential protein expression, while a principal component analysis highlighted two clusters. The volcano plot indicated 259 differentially expressed proteins (DEPs; 155 up- and 104 down-regulated proteins) in patients with BC compared with CT. The up-regulated proteins from BC-derived uEVs were enriched in pathways related to cancer progression (i.e., cell proliferation, cell survival, cell cycle, cell migration, carbohydrate metabolism, and angiogenesis). Moreover, we verified the expression of the upregulated DEPs using UALCAN for web-based validation. Remarkably, the results indicated that 6 of 155 up-regulated proteins (POSTN, ATAD2, BCAS4, GSK3β, HK1, and Ki-67) were overexpressed in BC compared with normal samples. Since these six proteins often act as markers of cell proliferation and progression, they may be potential biomarkers for BC screening and diagnosis. However, this requires validation in larger cohorts.

Renal Microvascular Inflammation in Human Obesity Detected by Urinary Extracellular Vesicles

Renal Microvascular Inflammation in Human Obesity Detected by Urinary Extracellular Vesicles

Mitochondrial DNA in Urinary Large Extracellular Vesicles as a Marker of Relapse in Children with Nephrotic Syndrome

Mitochondrial DNA in Urinary Large Extracellular Vesicles as a Marker of Relapse in Children with Nephrotic Syndrome

Identification of RNAs in Urinary Extracellular Vesicles for Detecting Renal Nrf2 Activation

Identification of RNAs in Urinary Extracellular Vesicles for Detecting Renal Nrf2 Activation

Urinary Extracellular Vesicles Reveal Distinct Biological Effects of Voclosporin in the Treatment of Lupus Nephritis

Urinary Extracellular Vesicles Reveal Distinct Biological Effects of Voclosporin in the Treatment of Lupus Nephritis

Podocyte-specific proteins in urinary extracellular vesicles of patients with IgA nephropathy: Vasorin and ceruloplasmin

Introduction: Urinary extracellular vesicles (uEVs) can be considered biomarkers of kidney diseases. EVs derived from podocytes may reflect podocyte damage in different glomerular diseases. IgA nephropathy (IgAN) is one of the most common forms of glomerulonephritis (GN) characterized by proteinuria and hematuria. This study aimed to analyze the uEVs of IgAN patients to understand the pathophysiological processes of the disease at the protein level. Methods: Patients with GN [biopsy-proven IgAN (n = 16) and membranous glomerulonephritis (MGN, n = 16)], and healthy controls (n = 16) were included in this study. The uEVs were extracted, characterized, and analyzed to evaluate the protein levels of candidate markers of IgAN, including vasorin precursor, aminopeptidase N, and ceruloplasmin by western-blot analysis. Results: Higher levels of both podocytes and EVs-related proteins were observed in the pooled urine samples of GN patients compared to the healthy controls. In IgAN patients, uEV-protein levels of vasorin were statistically lower while levels of ceruloplasmin were significantly higher compared to MGN (P = 0.002, P = 0.06) and healthy controls, respectively (P = 0.020, P= 0.001). Conclusion: Different levels of the studied proteins in uEVs may indicate podocyte injury and represent a direct association with the pathology of IgAN and MGN.

Hands-Free Proteomic Profiling of Urinary Extracellular Vesicles with a High-Throughput Automated Workflow.

Extracellular vesicles (EVs) have emerged as a promising source of disease biomarkers for noninvasive early stage diagnoses, but a bottleneck in EV sample processing restricts their immense potential in clinical applications. Existing methods are limited by a low EV yield and integrity, slow processing speeds, low sample capacity, and poor recovery efficiency. We aimed to address these issues with a high-throughput automated workflow for EV isolation, EV lysis, protein extraction, and protein denaturation. The automation can process clinical urine samples in parallel, resulting in protein-covered beads ready for various analytical methods, including immunoassays, protein quantitation assays, and mass spectrometry. Compared to the standard manual lysis method for contamination levels, efficiency, and consistency of EV isolation, the automated protocol shows reproducible and robust proteomic quantitation with less than a 10% median coefficient of variation. When we applied the method to clinical samples, we identified a total 3,793 unique proteins and 40,380 unique peptides, with 992 significantly upregulated proteins in kidney cancer patients versus healthy controls. These upregulated proteins were found to be involved in several important kidney cancer metabolic pathways also identified with a manual control. This hands-free workflow represents a practical EV extraction and profiling approach that can benefit both clinical and research applications, streamlining biomarker discovery, tumor monitoring, and early cancer diagnoses.

FRI070 The Urinary Extracellular Vesicles Small RNA Profile Of Obesity Displays Distinct Patterns By RNA Subclass

Abstract Disclosure: D. Choi: None. S. Kim: None. S. Kwon: None. Introduction: Urinary extracellular vesicles (EVs) small RNAs are thought to function as potential biomarkers for various physiological states and various diseases. Since urine contains a relatively small amount of microRNA compared to other biofluids, analyzing the distinct pattern of each subclass of small RNAs may provide additional information about gene expression. We attempted to analyze urinary EVs small RNAs by subclass in obesity before and after weight loss. Methods: Urinary extracellular vesicles small RNA sequencing was performed on 6 obese patients before and after their weight loss, which was achieved through bariatric surgery, and the results were compared with those of 6 healthy volunteers. Each distinct subclass of urinary small EVs RNAs was analyzed using a multi-dimensional scaling (MDS) plot. Results: RNA sequencing revealed total number of 5205 urinary EVs small RNAs. In the MDS plot comparing distinct subclass of urinary EVs small RNAs, there were different patterns observed between obesity and healthy volunteers, however, no significant patterns were found between obesity before and after weight loss. More distinct difference patterns between obesity and healthy volunteers were observed in miRNA, tRNA, snoRNA, and snRNA subclasses of urinary EVs small RNAs, whereas piRNA and YRNA subclasses showed indistinct difference patterns (Figure). Presentation: Friday, June 16, 2023

P17 - Urinary extracellular vesicles isolation: Improving the differential ultracentrifugation method for biomarker research

P17 - Urinary extracellular vesicles isolation: Improving the differential ultracentrifugation method for biomarker research

Identification of urinary extracellular vesicles differentially expressed RNAs in diabetic nephropathy via whole-transcriptome integrated analysis

BackgroundDiabetic nephropathy (DN) is a common systemic microvascular complication of diabetes and a leading cause of chronic kidney disease worldwide. Urinary extracellular vesicles (uEVs), which are natural nanoscale vesicles that protect RNA from degradation, have the potential to serve as an invasive diagnostic biomarker for DN. MethodsWe enrolled 24 participants, including twelve with renal biopsy-proven T2DN and twelve with T2DM, and isolated uEVs using ultracentrifugation. We performed microarrays for mRNAs, lncRNAs, and circRNAs in parallel, and Next-Generation Sequencing for miRNAs. Differentially expressed RNAs (DE-RNAs) were subjected to CIBERSORTx, ssGSEA analysis, GO enrichment, PPI network analysis, and construction of the lncRNA/circRNA-miRNA-mRNA regulatory network. Candidate genes and potential biomarker RNAs were validated using databases and machine learning models. ResultsA total of 1684 mRNAs, 126 lncRNAs, 123 circRNAs and 66 miRNAs were found in uEVs in T2DN samples compared with T2DM. CIBERSORTx revealed the involvement of uEVs in immune activity and ssGSEA explored possible cell or tissue sources of uEVs. A ceRNA co-expression and regulation relationship network was constructed. Candidate genes MYO1C and SP100 mRNA were confirmed to be expressed in the kidney using Nephroseq database, scRNA-seq dataset, and Human Protein Atlas database. We further selected 2 circRNAs, 2 miRNAs, and 2 lncRNAs from WGCNAs and ceRNAs and demonstrated their efficacy as potential diagnostic biomarkers for T2DN using machine learning algorithms. ConclusionsThis study reported, for the first time, the whole-transcriptome genetic resources found in urine extracellular vesicles of T2DN patients. The results provide additional support for the possible interactions, and regulators between RNAs from uEVs themselves and as potential biomarkers in DN.

One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles.

Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.

Extracellular Vesicles as Source of Biomarkers in Glomerulonephritis.

Kidney disease is a global health and healthcare burden. Glomerulonephritis (Gn), both primary and secondary, is generally characterized by an inflammatory glomerular injury and may lead to end-stage renal disease. Kidney biopsy is fundamental to the diagnosis; however, kidney biopsy presents some concerns that may partly hamper the clinical process. Therefore, more accurate diagnostic tools are needed. Extracellular vesicles (EVs) are membranous vesicles released by cells and found in bodily fluids, including urine. EVs mediate intercellular signaling both in health and disease. EVs can have both harmful and cytoprotective effects in kidney diseases, especially Gn. Previous findings reported that the specific cargo of urinary EV contains an aerobic metabolic ability that may either restore the recipient cell metabolism or cause oxidative stress production. Here, we provide an overview of the most recent proteomic findings on the role of EVs in several aspects of glomerulopathies, with a focus on this metabolic and redox potential. Future studies may elucidate how the ability of EVs to interfere with aerobic metabolism and redox status can shed light on aspects of Gn etiology which have remained elusive so far.

Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis.

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

One-pot preparation of bi-functional POSS-based hybrid monolith via photo-initiated polymerization for isolation of extracellular vesicles

Extracellular vesicles (EVs) are important participants in numerous pathophysiological processes, and could be used as valuable biomarkers to detect and monitor various diseases. However, facile EV isolation methods are the essential and preliminary issue for their downstream analysis and function investigation. In this work, a polyhedral oligomeric silsesquioxanes (POSS) based hybrid monolith combined metal affinity chromatography (MAC) and distearoyl phospholipid ethanolamine (DSPE) function was developed via photo-initiated thiol-ene polymerization. This synthesis process was facile, simple and convenient, and the obtained hybrid monolith could be applied to efficiently isolate EVs from bio-samples by taking advantages of the specific bond of Ti4+ and phosphate groups on the phospholipid membrane of EVs and the synergistic effect of DSPE insertion. Meanwhile, the eluted EVs could maintain their structural integrity and biological activity, suggesting they could be used for downstream application. Furthermore, 75 up-regulated proteins and 56 down-regulated proteins were identified by comparing the urinary EVs of colorectal cancer (CRC) patients and healthy donors, and these proteins might be used as potential biomarkers for early screening of CRC. These results demonstrated that this hybrid monolith could be used as a simple and convenient tool for isolating EVs from bio-samples and for wider applications in biomarker discovery.

Total and Extracellular Vesicle cAMP Contents in Urine Are Associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD) Progression.

ADPKD is the most common genetic renal disease, characterized by the presence of multiple cysts which, through slow and gradual growth, lead to glomerular filtration rate (GFR) decline and end-stage renal disease. Cystic growth is associated with increased intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP). Extracellular vesicles (EVs) are proposed to participate in "remote sensing" by transporting different cargoes, but their relevance to ADPKD progression is poorly understood. This study aimed to determine whether cAMP is contained in urinary EVs and, if so, how total and/or EV cAMP contents participate in disease progression. Fourteen ADPKD patients, naïve for V2 receptor antagonism treatment, and seven controls were studied. Progression was evaluated by estimating GFR (eGFR) and height-adjusted total kidney volume (htTKV). Fresh morning urine was collected to determine cAMP by the competitive radioligand assay. Urine EVs were isolated using an adapted centrifugation method and characterized by electron microscopy, dynamic light scanning, flow cytometry with FITC CD63 labeling, protein and RNA content, and AQP2 and GAPDH mRNA detection. Total and EV cAMP was measurable in both control and patient urine samples. Total cAMP was significantly correlated with eGFR and its annual change but inversely correlated with htTKV. The cAMP-EVs showed a bimodal pattern with htTKV, increasing to ~1 L/m and falling at larger sizes. Our results demonstrate that urine cAMP correlates with ADPKD progression markers, and that its extracellular delivery by EVs could reflect the architectural disturbances of the organ.

Extracellular Vesicles: Investigating the Pathophysiology of Diabetes-Associated Hypertension and Diabetic Nephropathy.

Extracellular vesicles (EVs) include exosomes, microvesicles, and apoptotic bodies. EVs are released by all cell types and are found in biological fluids including plasma and urine. Urinary extracellular vesicles (uEVs) are a mixed population of EVs that comprise small EVs that are filtered and excreted, EVs secreted by tubular epithelial cells, and EVs released from the bladder, urethra, and prostate. The packaged cargo within uEVs includes bioactive molecules such as metabolites, lipids, proteins, mRNAs, and miRNAs. These molecules are involved in intercellular communication, elicit changes in intracellular signaling pathways, and play a role in the pathogenesis of various diseases including diabetes-associated hypertension and diabetic nephropathy. uEVs represent a rich source of biomarkers, prognosis markers, and can be loaded with small-molecule drugs as a vehicle for delivery.

Polyethylene glycol-based isolation of urinary extracellular vesicles, an easily adoptable protocol

Urine is a highly advantageous biological specimen for biomarker research and is a non-invasive source. Most of the urinary biomarkers are non-specific, volatile and need extensive validation before clinical adoption. Extracellular vesicles are secreted by almost all cells and are involved in homoeostasis, intercellular communication, and cellular processes in healthy and pathophysiological states. Urinary extracellular vesicles (UEVs) are released from the urogenital system and mirror the molecular processes of physiological and pathological states of their source cells. Therefore, UEVs serve as a valuable source of biomarkers for the non-invasive diagnosis of various pathologies. They hold a promising source of multiplex biomarkers suitable for prognosis, diagnosis, and therapy monitoring. UEVs are easily accessible, non-invasive, and suited for longitudinal sampling. Although various techniques are available for isolating UEVs, there is yet to be a consensus on a standard and ideal protocol. We have optimized an efficient, reliable, and easily adoptable polyethylene glycol (PEG) based UEV isolation technique following MISEV guidelines. The method is suitable for various downstream applications of UEVs. This could be a cost-effective, consistent, and accessible procedure for many clinical labs and is most suited for longitudinal analysis. Adopting the protocol will pave the way for establishing UEVs as the ideal biomarker source. •Urine can be collected non-invasively and repeatedly, hence a very useful specimen for biomarker discovery. Urinary EVs (UEVs), derived from urine, offer a stable diagnostic tool, but standardised isolation and analysis approaches are warranted.•To have enough UEVs for any study, large volumes of urine sample are necessary, which limits different isolation methods by cost, yield, and time.•The protocol developed could help researchers by offering a cost-effective and dependable UEV isolation method and may lay the foundation for UEVs adoption in clinical space.

Exploring urinary extracellular vesicles for organ transplant monitoring: A comprehensive study for detection of allograft dysfunction using immune-specific markers

BackgroundAllograft dysfunction (AGD) is a common complication following solid organ transplantation (SOT). This study leverages the potential of urinary extracellular vesicles (UEVs) for the non-invasive detection of AGD. AimWe aimed to assess the diagnostic value of T-cell and B-cell markers characteristic of T-cell-mediated and antibody-mediated rejection in UEV-mRNA using renal transplantation as a model. Materials and methodsUEVs were isolated from 123 participants, spanning healthy controls, functional transplant recipients, and biopsy-proven AGD patients. T-cell and B-cell marker mRNA expressions were evaluated using RT-qPCR. ResultsWe observed significant differences in marker expression between healthy controls and AGD patients. ROC analysis revealed an AUC of 0.80 for T-cell markers, 0.98 for B-cell markers, and 0.94 for combined markers. T-cell markers achieved 81.3 % sensitivity, 80 % specificity, and 80.4 % efficiency. A triad of T-cell markers (PRF1, OX40, and CD3e) increased sensitivity to 87.5 % and efficiency to 82.1 %. B-cell markers (CD20, CXCL3, CD46, and CF3) delivered 100 % sensitivity and 97.5 % specificity. The combined gene signature of T-cell and B-cell markers offered 93.8 % sensitivity and 95 % specificity. ConclusionOur findings underscore the diagnostic potential of UEV-derived mRNA markers for T-cells and B-cells in AGD, suggesting a promising non-invasive strategy for monitoring graft health.

The effect of surface micropatterns on the flow in a lab-on-a-chip with nanowires

Nanowire-anchored microfluidic devices have opened new opportunities for in situ extractions of microRNAs (miRNAs) from extracellular vesicles (EVs) in body fluids. The diagnosis precision of the devices largely depends on the EV extraction efficiency. In this article, we demonstrated a maskless photolithography process with direct covalent bonding for fabricating microfluidic devices embedded with zinc oxide (ZnO) nanowires as a lab-on-a-chip for extracting urinary EVs. The chips were designed with two distinct surface micropatterns: herringbone arrow and cylindrical micropillars. We investigated the effect of surface pattern, groove height-to-width ratio and nanowire orientation on the microflow. The computational fluid dynamics (CFD) simulations suggested that the herringbone pattern is more effective in reducing the flow velocity thus prolonging the mixing time. Furthermore, a critical inter-pattern groove width-to-height ratio was shown to induce circulating currents in the concave regions of the micropatterns. This design shows potential in increasing the fluid residency time within the device, thus increasing the probability of trapping EVs in circulating flows. In addition, the randomly oriented nanowires resulted in a significant amount of near-wall vorticity. Overall, the combination of a surface micropattern and the randomness of nanowire orientation suggests enhanced particulate mixing in the wall-adjacent zones and the potential to improve the EV extraction efficiency.

Nanoparticle Tracking Analysis of Urinary Extracellular Vesicle Proteins as a New Challenge in Laboratory Medicine.

Urinary extracellular vesicle (uEV) proteins may be used as specific markers of kidney damage in various pathophysiological conditions. The nanoparticle-tracking analysis (NTA) appears to be the most useful method for the analysis of uEVs due to its ability to analyze particles below 300 nm. The NTA method has been used to measure the size and concentration of uEVs and also allows for a deeper analysis of uEVs based on their protein composition using fluorescence measurements. However, despite much interest in the clinical application of uEVs, their analysis using the NTA method is poorly described and requires meticulous sample preparation, experimental adjustment of instrument settings, and above all, an understanding of the limitations of the method. In the present work, we demonstrate the usefulness of an NTA. We also present problems encountered during analysis with possible solutions: the choice of sample dilution, the method of the presentation and comparison of results, photobleaching, and the adjustment of instrument settings for a specific analysis. We show that the NTA method appears to be a promising method for the determination of uEVs. However, it is important to be aware of potential problems that may affect the results.