Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients.

Diabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney's physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-ß1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.

Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques.

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs' identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.

Acute and chronic glomerular damage is associated with reduced CD133 expression in urinary extracellular vesicles.

Extracellular vesicles released into urine (uEVs) can represent interesting biomarkers of renal cell damage. CD133, a stem/progenitor cell marker expressed by renal progenitor cells, is highly expressed in uEVs of healthy individuals. In the present study, we evaluated the level of CD133 in the uEVs of patients with acute and chronic glomerular damage by cytofluorimetric analysis. The level of CD133+ uEVs was significantly decreased in pediatric patients with acute glomerulonephritis during the acute phase of renal damage, while it was restored after the subsequent recovery. A similar decrease was also observed in patients with chronic glomerulonephritis. Moreover, CD133+ uEVs significantly declined in patients with type 2 diabetes, used as validation group, with the lowest levels in patients with albuminuria with diabetic nephropathy. Indeed, receiver-operating characteristic curve analysis indicates the ability of CD133+ uEV values to discriminate the health condition from that of glomerular disease. In parallel, a significant decrease of CD133 in renal progenitor cells and in their derived EVs was observed in vitro after cell treatment with a combination of glucose and albumin overload, mimicking the diabetic condition. These data indicate that the level of CD133+ uEVs may represent an easily accessible marker of renal normal physiology and could provide information on the "reservoir" of regenerating cells within tubules.

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model.

Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho.

The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury.

BACKGROUND: Mesenchymal stromal cells (MSCs) and renal stem/progenitors improve the recovery of acute kidney injury (AKI) mainly through the release of paracrine mediators including the extracellular vesicles (EVs). Several studies have reported the existence of a resident population of MSCs within the glomeruli (Gl-MSCs). However, their contribution towards kidney repair still remains to be elucidated. The aim of the present study was to evaluate whether Gl-MSCs and Gl-MSC-EVs promote the recovery of AKI induced by ischemia-reperfusion injury (IRI) in SCID mice. Moreover, the effects of Gl-MSCs and Gl-MSC-EVs were compared with those of CD133+ progenitor cells isolated from human tubules of the renal cortical tissue (T-CD133+ cells) and their EVs (T-CD133+-EVs). METHODS: IRI was performed in mice by clamping the left renal pedicle for 35 minutes together with a right nephrectomy. Immediately after reperfusion, the animals were divided in different groups to be treated with: Gl-MSCs, T-CD133+ cells, Gl-MSC-EVs, T-CD133+-EVs or vehicle. To assess the role of vesicular RNA, EVs were either isolated by floating to avoid contamination of non-vesicles-associated RNA or treated with a high dose of RNase. Mice were sacrificed 48 hours after surgery. RESULTS: Gl-MSCs, and Gl-MSC-EVs both ameliorate kidney function and reduce the ischemic damage post IRI by activating tubular epithelial cell proliferation. Furthermore, T-CD133+ cells, but not their EVs, also significantly contributed to the renal recovery after IRI compared to the controls. Floating EVs were effective while RNase-inactivated EVs were ineffective. Analysis of the EV miRnome revealed that Gl-MSC-EVs selectively expressed a group of miRNAs, compared to EVs derived from fibroblasts, which were biologically ineffective in IRI. CONCLUSIONS: In this study, we demonstrate that Gl-MSCs may contribute in the recovery of mice with AKI induced by IRI primarily through the release of EVs.

Extracellular vesicles in the urine: markers and mediators of tissue damage and regeneration.

As in several body fluids, urine is a rich reservoir of extracellular vesicles (EVs) directly originating from cells facing the urinary lumen, including differentiated tubular cells, progenitor cells and infiltrating inflammatory cells. Several markers of glomerular and tubular damage, such as WT-1, ATF3 and NGAL, as well as of renal regeneration, such as CD133, have been identified representing an incredible source of information for diagnostic purposes. In addition, urinary extracellular vesicles (uEVs) appear to be involved in the cell-to-cell communication along the nephron, although this aspect needs further elucidation. Finally, uEVs emerge as potential amplifying or limiting factors in renal damage. Vesicles from injured cells may favour fibrosis and disease progression whereas those from cells with regenerative potential appear to promote cell survival. Here, we will discuss the most recent findings of the literature, on the light of the role of EVs in diagnosis and therapy for damage and repair of the renal tissue.

Urinary CD133+ extracellular vesicles are decreased in kidney transplanted patients with slow graft function and vascular damage.

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133+ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133(+) EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133(+) EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133(+) EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133(+) EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.

Analysis of secreted proteins for the study of bladder cancer cell aggressiveness.

Secreted proteins play a key role in cell signaling, communication, and migration. We recently described the development of an aggressive variant (T24M) of the bladder cancer cell line T24. Using this cell line model, the objective of our work was the identification of secreted proteins involved in the acquisition of the aggressive phenotype. Using in vitro assays, we demonstrate that conditioned media of the T24M cells promote motility of the parental less aggressive T24 cells. Proteomic analysis of cell culture conditioned media by the use of 2-dimensional gel electrophoresis coupled to MALDI TOF MS and LC-MS approaches resulted in enrichment and detection of multiple classical extracellular and secreted proteins such as fibronectin, cystatin, fibrillin, fibulin, interleukin 6, etc. Comparison of the secretome of the T24 and T24M cells indicated differences in proteins with potential involvement in the mechanisms of cell aggressiveness including SPARC, tPA, and clusterin. These findings were further confirmed by Western blot analysis. In the case of SPARC, further studies involving transwell assays indicated that blockage of the protein in the presence of SPARC-specific Abs results in decreased cell motility. Collectively, our study provides a 2DE-based comprehensive analysis of bladder cancer cell secretome. The results indicate various secreted proteins with potential involvement in bladder cancer cell aggressiveness and more specifically provide initial evidence for special role of SPARC in bladder cancer cell motility and invasiveness.

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.

2D-electrophoresis and the urine proteome map: where do we stand?

The discovery of urinary biomarkers is a main topic in clinical medicine. The development of proteomics has rapidly changed the knowledge on urine protein composition and probably will modify it again. Two-dimensional electrophoresis (2D-PAGE) coupled with mass spectrometry has represented for years the technique of choice for the analysis of urine proteins and it is time to draw some conclusions. This review will focus on major methodological aspects related to urine sample collection, storage and analysis by 2D-PAGE and attempt to define an advanced normal urine protein map. Overall, 1118 spots were reproducibly found in normal urine samples but only 275 were characterized as isoforms of 82 proteins. One-hundred height spots belonging to 30 proteins were also detected in plasma and corresponded to typical plasma components. The identity of most of the proteins found in normal urine by 2D-PAGE remains to be determined, the majority being low-molecular weight proteins (<30 kDa). Equalization procedures would also enhance sensitivity of the analysis and allow low abundance proteins to be characterized. Therefore, we are still on the way to define the normal urine composition. Technology advancements in concentrating procedure will improve sensitivity and give the possibility to purify proteins for mass spectrometry.

Serum albumin fragmentation in end-stage renal disease patients--a pilot study.

BACKGROUND: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. METHODS: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. RESULTS: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M(r) of 12.5-29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. CONCLUSIONS: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood.

Combinatorial peptide ligand libraries for urine proteome analysis: investigation of different elution systems.

Proteome treatments with peptide libraries in view of reducing high-abundance proteins and increasing the concentration of rare species involve the adsorption on solid-phase material. Subsequent elution of captured proteins may not be fully effective except when sequences of eluting agents are used. The standard way utilized up to the present has been a three- to four-step, sequential elution system consisting of various agents mixed together such as urea, thiourea, CHAPS, sodium chloride, citric or acetic acid and some polar solvents such as ACN and isopropanol. Elution sequences produce distinct fractions adding to the burden of having to analyze all of them. An alternative, highly effective, single elution to reduce the workload is here reported for the first time, namely elution in boiling 10% SDS added with 3% DTE. This single step elutes almost quantitatively the adsorbed proteins, thus ensuring, for all practical purposes, a full recovery. This high efficiency is believed to be due to the fact that the SDS micelles bury the polypeptide chains within their hydrophobic core, thus shielding them from the surroundings and impeding accidental adsorption to surfaces. Suggestions for selecting the best method to eliminate the excess of SDS for further protein analysis are also evaluated. The merits and limits of this novel system are assessed and discussed.

The oxido-redox potential of albumin methodological approach and relevance to human diseases.

In humans, an increased synthesis of reactive oxygen species (ROS) may be a relevant cause of amplification of physiologic processes resulting in inflammatory organ damage or neoplasia. Efficient anti-oxidative systems targeting oxidative stress are thus essential to prevent tissue damage. In plasma, proteins proved to be the first line of defence against ROS and albumin, the highest concentration plasma protein, has a key role in this antioxidant function. Recent studies have clearly documented that albumin oxido-redox potential changes upon oxidation by different oxidants thus becoming a deputy biomarker of this process. ROS react primarily with the free (34)Cysteine ((34)Cys) residue of albumin to form two reversible intermediate derivatives, sulfenic-(SOH-alb) and sulfinic acid (SO(2)H-alb), resulting in sulfonic acid (SO(3)H-alb), the final stable product of the reaction. Upon stable oxidation (SO(3)H-alb), albumin properties are altered: the protein becomes more susceptible to trypsin digestion and is degraded faster compared to the non-oxidized counterpart. The present review focuses on the characterization of albumin chemical changes induced by ROS, their relevance in human pathology and the most recent advances in the approach to oxidation adduct analysis.