Urinary extracellular vesicles. A promising shortcut to novel biomarker discoveries

Proteomic and genomic techniques have reached full maturity and are providing unforeseen details for the comprehensive understanding of disease pathologies at a fraction of previous costs. However, for kidney diseases, many gaps in such information remain to inhibit major advances in the prevention, treatment and diagnostics of these devastating diseases, which have enormous global impact. The discovery of ubiquitous extracellular vesicles (EV) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate with distant locations in processes of cancer spread, immunological regulation, barrier functions and general modulation of cellular activity. In this review, we describe some of the most prominent research streams and findings utilizing urinary extracellular vesicles as highly versatile and dynamic tools with their extraordinary protein and small regulatory RNA species. While being a highly promising approach, the relatively young field of EV research suffers from a lack of adherence to strict standardization and carefully scrutinized methods for obtaining fully reproducible results. With the appropriate guidelines and standardization achieved, urine is foreseen as forming a unique, robust and easy route for determining accurate and personalized disease signatures and as providing highly useful early biomarkers of the disease pathology of the kidney and beyond.Peer reviewe

From genetics to personalized nephrology : kidney research at a tipping point

Non peer reviewe

Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.Peer reviewe

Diabetes with kidney injury may change the abundance and cargo of urinary extracellular vesicles

BackgroundUrinary extracellular vesicles (uEVs) are derived from epithelia facing the renal tubule lumen in the kidney and urogenital tract; they may carry protein biomarkers of renal dysfunction and structural injury. However, there are scarce studies focusing on uEVs in diabetes with kidney injury.Materials and methodsA community-based epidemiological survey was performed, and the participants were randomly selected for our study. uEVs were enriched by dehydrated dialysis method, quantified by Coomassie Bradford protein assay, and adjusted by urinary creatinine (UCr). Then, they identified by transmission electron microscopy (TEM), nanoparticle track analysis (NTA), and western blot of tumor susceptibility gene 101.ResultsDecent uEVs with a homogeneous distribution were finally obtained, presenting a membrane-encapsulated structure like cup-shaped or roundish under TEM, having active Brownian motion, and presenting the main peak between 55 and 110 nm under NTA. The Bradford protein assay showed that the protein concentrations of uEVs were 0.02 ± 0.02, 0.04 ± 0.05, 0.05 ± 0.04, 0.07 ± 0.08, and 0.11 ± 0.15 μg/mg UCr, respectively, in normal controls and in prediabetes, diabetes with normal proteinuria, diabetes with microalbuminuria, and diabetes with macroproteinuria groups after adjusting the protein concentration with UCr by calculating the vesicles-to-creatinine ratio.ConclusionThe protein concentration of uEVs in diabetes with kidney injury increased significantly than the normal controls before and after adjusting the UCr. Therefore, diabetes with kidney injury may change the abundance and cargo of uEVs, which may be involved in the physiological and pathological changes of diabetes

Management of Tamm-Horsfall Protein for Reliable Urinary Analytics

Purpose 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. Experimental design The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS. Results Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap. Conclusions and clinical relevance The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.Peer reviewe

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.Peer reviewe

Protein and molecular characterization of a clinically compliant amniotic fluid stem cell derived extracellular vesicle fraction capable of accelerating muscle regeneration through the enhancement of angiogenesis

The secretome of human amniotic fluid stem cells (AFSC) has great potential as a therapeutic agent in regenerative medicine. However it must be produced in a clinically compliant manner before it can be used in humans. Here we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EV) that harbour much but not all the biological activity of the whole secretome. Proteomic characterisation of the EV and free secretome fraction show the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly we show that the EVs only contain miRNA and not mRNA. This suggests tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than the imposition of new, signalling pathways. The EVs harbour significant anti-inflammatory activity as well as promoting angiogenesis; the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury

Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins

Abstract Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation