Proteomic Characterization of Urinary Extracellular Vesicles from Kidney-Transplanted Patients Treated with Calcineurin Inhibitors.

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.

Proteomic Research in Peritoneal Dialysis.

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.

Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs.

Tolerance in Kidney Transplantation: What Is on the B Side?

Regulatory B cells (Breg) are in the spotlight for their role in immune homeostasis maintenance and tolerance achievement as in the last years the correlation with functional and increased Breg numbers in autoimmune diseases and transplantation has been extensively proven. Their study is, however, in its infancy with still little knowledge and consensus on their origin, phenotype, and mechanism of action. All this hampers the pursuit of an effective Breg induction method for therapeutic purposes. In this review we aim to summarize the studies on human Breg and their implication in kidney transplantation and to further discuss the issues surrounding therapeutic applications of this cell subset.

Mesenchymal stromal cells induced regulatory B cells are enriched in extracellular matrix genes and IL-10 independent modulators.

Regulatory B cells (Breg) are essential players in tolerance and immune homeostasis. However, lack of specific Breg markers limit their potential in clinical settings. Mesenchymal stromal cells (MSC) modulate B cell responses and are described to induce Breg in vitro. The aim of this work was to characterize MSC induced Breg (iBreg) and identify specific Breg biomarkers by RNAseq. After 7-day coculture with adipose tissue-derived MSC, B cells were enriched in transitional B cell populations, with increased expression and secretion of IL-10 and no TNFα. In addition, iBreg showed potential to modulate T cell proliferation at 2 to 1 cell ratios and their phenotype remained stable for 72h. RNAseq analysis of sorted IL-10 positive and negative iBreg populations identified over 1500 differentially expressed genes (DEG) among both populations. Analysis of biological processes of DEG highlighted an enrichment of immune regulation and extracellular matrix genes in IL-10- iBreg populations, while IL-10+ iBreg DEG were mostly associated with cell activation. This was supported by T cells modulation assays performed in the presence of anti-IL-10 neutralizing antibodies showing the non-essential role of IL-10 in the immunomodulatory capacity of iBregs on T cells. However, based on RNAseq results we explored the role of TGF-ß and found out that it plays a major role on iBreg induction and iBreg immunomodulatory properties. Therefore, we report that MSC induce B cell populations characterized by the generation of extracellular matrix and immune modulation independently of IL-10.

Urinary vitronectin identifies patients with high levels of fibrosis in kidney grafts.

BACKGROUND: In kidney transplantation, fibrosis represents the final and irreversible consequence of the pathogenic mechanisms that lead to graft failure, and in the late stages it irremediably precedes the loss of renal function. The invasiveness of kidney biopsy prevents this condition from being frequently monitored, while clinical data are rather unspecific. The objective of this study was to find noninvasive biomarkers of kidney rejection. METHODS: We carried out proteomic analysis of the urinary Extracellular Vesicles (uEVs) from a cohort of kidney transplant recipients (n = 23) classified according to their biopsy-based diagnosis and clinical parameters as interstitial fibrosis and tubular atrophy (IFTA), acute cellular rejection (ACR), calcineurin inhibitors toxicity (CNIT) and normal kidney function (NKF). RESULTS: Shotgun mass spectrometry of uEV-proteins identified differential expression of several proteins among these different groups. Up to 23 of these proteins were re-evaluated using targeted proteomics in a new independent cohort of patients (n = 41) classified in the same diagnostic groups. Among other results, we found a differential expression of vitronectin (VTN) in patients displaying chronic interstitial and tubular lesions (ci and ct mean > 2 according to Banff criteria). These results were further confirmed by a pilot study using enzyme-linked immunosorbent assay (ELISA). CONCLUSION: Urinary vitronectin levels are a potential stand-alone biomarker to monitor fibrotic changes in kidney transplant recipients in a non-invasive fashion.

Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted Extracellular Vesicles.

Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell communication and typically reflect the phenotype of the cell of origin. MSC-EVs could resemble MSC functions, and are being contemplated as an improved alternative to the MSC-based immunomodulatory therapy. In the present work, we focused on the factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were isolated from MSC-conditioned medium (CM) using size-exclusion chromatography (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell proliferation was compared to that of the whole MSCs. Co-culture with MSC or unfractionated CM induced naïve and CD24hiCD38hi, IL-10 producing (Breg) phenotypes on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, inducing a naïve phenotype, and even though they did not induce the shift toward a CD24hiCD38hi population, MSC-PF fostered IL-10 production by B cells. Conversely, MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs induced CD24hiCD38hi B cells to a similar extent of that of MSC, but not bona fide Bregs since they did not produce IL-10. Our results show that B cell modulation by MSC is partially mediated by soluble factors other than EVs.

Corrigendum: Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted Extracellular Vesicles.

[This corrects the article DOI: 10.3389/fimmu.2019.01288.].

Extracellular-Vesicle Isolation from Different Biological Fluids by Size-Exclusion Chromatography.

This unit describes how to isolate extracellular vesicles (EVs) from different biological fluids using size-exclusion chromatography (SEC) and how to prepare your starting sample and the EV product for downstream applications. EVs are membrane nanovesicles with specific content that reflects the phenotype and functions of the cell of origin, including protected proteins, lipids, metabolites, and nucleic acids. EVs are thus an excellent resource for noninvasive biomarker discovery in a number of pathological situations and are a promising nanotherapeutic tool to overcome the disadvantages associated with cellular therapy. However, there are still no standardized methods to isolate pure EV preparations, as many approaches do not guarantee proper EV purification, free of contaminating non-EV molecules. Currently, SEC is one of the most promising approaches to purify EVs from any biological fluid, as it avoids co-isolation of contaminants and is user friendly and scalable. © 2019 by John Wiley & Sons, Inc.

Proteomic profiling of peritoneal dialysis effluent-derived extracellular vesicles: a longitudinal study.

BACKGROUND: Peritoneal dialysis (PD) is an optimal renal replacement therapy for patients while waiting for kidney transplantation, but functional failure of the peritoneal membrane (PM), mainly induced by exposure to PD solutions, force many patients to early abandon PD therapy. PM function is evaluated by the peritoneal equilibration test (PET), a tedious technique only detecting alterations in extensively damaged PM. In a previous study, we showed that peritoneal dialysis effluent contained extracellular vesicles (PDE-EV), and that their proteome was significantly different between newly enrolled and long-term PD patients. Here, we report the results of a longitudinal study and compare PDE-EV proteome changes with PET results. METHODS: PDE was collected from 11 patients every 6 months (coincident with PET controls) from 0 months up to 24 months on PD. PDE-EV were isolated by size-exclusion chromatography and the proteome was analyzed by mass spectrometry (LC-MS/MS). Bioinformatic analyses were conducted to evaluate differences between groups. RESULTS: At follow-up endpoint, patients were classified as Stable (n = 7) or Unstable (n = 4) according to PET evolution. Strikingly, PDE-EV from the Stable group showed a significantly higher protein expression compared to Unstable patients already at 6 months on PD, when PET alterations had not been detected yet. CONCLUSIONS: PDE-EV proteome show alterations much earlier than PET monitoring, thus unveiling the potential of PDE-EV proteins as feasible biomarkers of PM alteration in PD patients.

Characterization and proteomic profile of extracellular vesicles from peritoneal dialysis efflux.

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.

Inflammatory Conditions Dictate the Effect of Mesenchymal Stem or Stromal Cells on B Cell Function.

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 this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (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, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ 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-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ 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 pretreatment of MSC.

Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents.

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.