Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR and their total protein content, NTA and Cryo-electron microscopy profiles and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs.

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is however unknown how MSC interact with B cells under inflammatory conditions. In the present study, adipose tissue-derived MSC were pre-treated with 50 ng/ml IFN-γ for 96h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43-selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40 and IL-2 and after 7 days B cell proliferation, phenotype, IgG and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38hi CD24hi B cells (Bregs) and IL-10 production, while MSC-IFN-y significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-y required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase (IDO) expression was highly induced in MSC-IFN-y and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions MSC stimulate Breg induction, whereas under inflammatory conditions MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pre-treatment of MSC.

Peritoneal Dialysis (PD) is considered the best option for a cost-effective mid-term dialysis in patients with Chronic Renal Failure. However, functional failure of the peritoneal membrane (PM) force many patients to stop PD treatment and start haemodialysis. Currently, PM functionality is monitored by the peritoneal equilibration test, a tedious technique that often show changes when the membrane damage is advanced. As in other pathologies, the identification and characterization of extracellular vesicles (EVs) in the peritoneal dialysis efflux (PDE) may represent a non-invasive alternative to identify biomarkers of membrane failure. Using size-exclusion chromatography, we isolated EVs from PDE in a group of patients. Vesicles were characterized by the presence of tetraspanin markers, nanoparticle tracking analysis profile, cryo-electron microscopy and mass spectrometry. Here, we report the isolation and characterization of PDE-EVs. Based on mass spectrometry, we have found a set of well-conserved proteins among patients. Interestingly, the peptide profile also revealed remarkable changes between newly enrolled and longer-treated PD patients. These results are the first step to the identification of PDE-EVs based new markers of PM damage, which could support clinicians in their decision-making in a non-invasive manner.

Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs.

Peritoneal dialysis (PD) is an established home care, cost-effective renal replacement therapy (RRT), which offers several advantages over the most used dialysis modality, hemodialysis. Despite its potential benefits, however, PD is an under-prescribed method of treating uremic patients. Infectious complications (primarily peritonitis) and bio-incompatibility of PD solutions are the main contributors to PD drop-out, due to their potential for altering the functional and anatomical integrity of the peritoneal membrane. To improve the clinical outcome of PD, there is a need for biomarkers to identify patients at risk of PD-related complications and to guide personalized interventions. Several recent studies have shown that proteomic investigation may be a powerful tool in the prediction, early diagnosis, prognostic assessment, and therapeutic monitoring of patients on PD. Indeed, analysis of the proteome present in PD effluent has uncovered several proteins involved in inflammation and pro-fibrotic insult, in encapsulating peritoneal sclerosis, or even in detecting early changes before any measurable modifications occur in the traditional clinical parameters used to evaluate PD efficacy. We here review the proteomic studies conducted thus far, addressing the potential use of such omics methodology in identifying potential new biomarkers of the peritoneal membrane welfare in relation to dialytic prescription and adequacy.

Use of immunosuppressive drugs is still unavoidable in kidney-transplanted patients. Since their discovery, calcineurin inhibitors (CNI) have been considered the first-line immunosuppressive agents, in spite of their known nephrotoxicity. Chronic CNI toxicity (CNIT) may lead to kidney fibrosis, a threatening scenario for graft survival. However, there is still controversy regarding CNIT diagnosis, monitoring and therapeutic management, and their specific effects at the molecular level are not fully known. Aiming to better characterize CNIT patients, in the present study, we collected urine from kidney-transplanted patients treated with CNI who (i) had a normal kidney function, (ii) suffered CNIT, or (iii) presented interstitial fibrosis and tubular atrophy (IFTA). Urinary extracellular vesicles (uEV) were enriched and the proteome was analyzed to get insight into changes happening during CNI. Members of the uroplakin and plakin families were significantly upregulated in the CNIT group, suggesting an important role in CNIT processes. Although biomarkers cannot be asserted from this single pilot study, our results evidence the potential of uEV as a source of non-invasive protein biomarkers for a better detection and monitoring of this renal alteration in kidney-transplanted patients.