Davide Medica

Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells

Endothelial progenitor cells are known to reverse acute kidney injury by paracrine mechanisms. We previously found that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. Here, we tested whether these microvesicles prevent acute kidney injury in a rat model of ischemia-reperfusion injury. The RNA content of microvesicles was enriched in microRNAs (miRNAs) that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from acute kidney injury by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. Microvesicles also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. The renoprotective effect of microvesicles was lost after treatment with RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells, or depletion of pro-angiogenic miR-126 and miR-296 by transfection with specific miR-antagomirs. Thus, microvesicles derived from endothelial progenitor cells protect the kidney from ischemic acute injury by delivering their RNA content, the miRNA cargo of which contributes to reprogramming hypoxic resident renal cells to a regenerative program.

Microvesicles derived from endothelial progenitor cells enhance neoangiogenesis of human pancreatic islets.

The efficacy of islet transplantation is limited by poor graft vascularization. We herein demonstrated that microvesicles (MVs) released from endothelial progenitor cells (EPCs) enhanced human islet vascularization. After incorporation into islet endothelium and β-cells, EPC-derived MVs favored insulin secretion, survival, and revascularization of islets transplanted in SCID mice. MVs induced in vitro islet endothelial cell proliferation, migration, resistance to apoptosis, and organization in vessel-like structures. Moreover, MVs partially overcame the antiangiogenic effect of rapamycin and inhibited endothelial–leukocyte interaction via L-selectin and CD40. MVs were previously shown to contain defined patterns of mRNAs. Here we demonstrated that MVs carried the proangiogenic miR-126 and miR-296 microRNAs (miRNAs). MVs pretreated with RNase or derived from Dicer knocked-down EPCs showed a reduced angiogenic effect. In addition, MVs overcame the antiangiogenic effect of the specific antagomiRs of miR-126 and miR-296, suggesting a relevant contribution of miRNAs delivered by MVs to islet endothelium. Microarray analysis of MV-stimulated islet endothelium indicated the upregulation of mRNAs coding for factors involved in endothelial proliferation, differentiation, and angiogenesis. In addition, MVs induced the activation of the PI3K-Akt and eNOS signaling pathways in islet endothelium. These results suggest that MVs activate an angiogenic program in islet endothelium that may sustain revascularization and β-cell function.

Endothelial progenitor cell-derived extracellular vesicles protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis

BACKGROUND Endothelial progenitor cells (EPCs) are known to induce tissue repair by paracrine mechanisms including the release of growth factors and extracellular vesicles (EVs), nanoparticles able to carry proteins and genetic information to target cells. The aim of this study was to evaluate whether EVs derived from EPCs may protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis. METHODS EVs were isolated by serial ultracentrifugation from supernatants of cultured human EPCs and characterized for their protein and RNA content. In vivo, EVs were injected i.v. in the experimental rat model of mesangiolytic anti-Thy1.1 glomerulonephritis evaluating renal function, proteinuria, complement activity and histological lesions. In vitro, the biological effects of EPC-derived EVs were studied in cultured rat mesangial cells incubated with anti-Thy1.1 antibody and rat or human serum as complement source. RESULTS After i.v. injection in Thy1.1-treated rats, EVs localized within injured glomeruli and inhibited mesangial cell activation, leucocyte infiltration and apoptosis, decreased proteinuria, increased serum complement haemolytic activity (CH50) and ameliorated renal function. EV treatment decreased intraglomerular deposition of the membrane attack complex (MAC or C5b-9) and expression of smooth muscle cell actin and preserved the endothelial antigen RECA-1 and the podocyte marker synaptopodin. The protective effect of EVs was significantly reduced by pre-treatment with a high dose of RNase (1 U/mL), suggesting a key role for EV-carried RNAs in these mechanisms. Indeed, EPC-derived EVs contained different mRNAs coding for several anti-apoptotic molecules and for the complement inhibitors Factor H, CD55 and CD59 and the related proteins. The in vitro experiments aimed to investigate the mechanisms of EV protection indicated that EVs transferred to mesangial cell mRNAs coding for Factor H, CD55 and CD59 and inhibited anti-Thy1.1 antibody/complement-induced apoptosis and C5b-9/C3 mesangial cell deposition. CONCLUSIONS EVs derived from EPCs exert a protective effect in Thy1.1 glomerulonephritis by inhibition of antibody- and complement-mediated injury of mesangial cells.

Caffeic acid, a phenol found in white wine, modulates endothelial nitric oxide production and protects from oxidative stress-associated endothelial cell injury.

Introduction Several studies demonstrated that endothelium dependent vasodilatation is impaired in cardiovascular and chronic kidney diseases because of oxidant stress-induced nitric oxide availability reduction. The Mediterranean diet, which is characterized by food containing phenols, was correlated with a reduced incidence of cardiovascular diseases and delayed progression toward end stage chronic renal failure. Previous studies demonstrated that both red and white wine exert cardioprotective effects. In particular, wine contains Caffeic acid (CAF), an active component with known antioxidant activities. Aim of the study The aim of the present study was to investigate the protective effect of low doses of CAF on oxidative stress-induced endothelial injury. Results CAF increased basal as well as acetylcholine—induced NO release by a mechanism independent from eNOS expression and phosphorylation. In addition, low doses of CAF (100 nM and 1 μM) increased proliferation and angiogenesis and inhibited leukocyte adhesion and endothelial cell apoptosis induced by hypoxia or by the uremic toxins ADMA, p-cresyl sulfate and indoxyl sulfate. The biological effects exerted by CAF on endothelial cells may be at least in part ascribed to modulation of NO release and by decreased ROS production. In an experimental model of kidney ischemia-reperfusion injury in mice, CAF significantly decreased tubular cell apoptosis, intraluminal cast deposition and leukocyte infiltration. Conclusion The results of the present study suggest that CAF, at very low dosages similar to those observed after moderate white wine consumption, may exert a protective effect on endothelial cell function by modulating NO release independently from eNOS expression and phosphorylation. CAF-induced NO modulation may limit cardiovascular and kidney disease progression associated with oxidative stress-mediated endothelial injury.

Neutrophil Gelatinase Associated Lipocalin Is an Early and Accurate Biomarker of Graft Function and Tissue Regeneration in Kidney Transplantation from Extended Criteria Donors

Background Delayed graft function (DGF) is an early complication of kidney transplantation (KT) associated with increased risk of early loss of graft function. DGF increases using kidneys from extended criteria donors (ECD). NGAL is a 25KDa protein proposed as biomarker of acute kidney injury. The aim of this study was to investigate the role of NGAL as an early and accurate indicator of DGF and Tacrolimus (Tac) toxicity and as a mediator of tissue regeneration in KT from ECD. Methods We evaluated plasma levels of NGAL in 50 KT patients from ECD in the first 4 days after surgery or after Tac introduction. Results Plasma levels of NGAL at day 1 were significantly higher in DGF group. In the non DGF group, NGAL discriminated between slow or immediate graft function and decreased more rapidly than serum creatinine. NGAL increased after Tac introduction, suggesting a role as marker of drug toxicity. In vitro, hypoxia and Tac induced NGAL release from tubular epithelial cells (TEC) favoring an autocrine loop that sustains proliferation and inhibits apoptosis (decrease of caspases and Bax/Bcl-2 ratio). Conclusions NGAL is an early and accurate biomarker of graft function in KT from ECD favoring TEC regeneration after ischemic and nephrotoxic injury.

Erratum to: The exciting “bench to bedside” journey of cell therapies for acute kidney injury and renal transplantation

Acute kidney injury (AKI) is characterized by an increasing incidence and poor outcomes in both developed and undeveloped countries. AKI is also acquiring importance in the setting of kidney transplantation (KT): besides all the classical forms of AKI that KT patients may undergo, several transplant-specific injuries can also lead to the loss of graft function. The mechanisms of tissue damage in native and grafted kidneys share several common pathogenic elements. Since appropriate therapeutic treatments are still lacking—probably due to the disease complexity—clinicians are forced to provide only supportive care. In this composite scenario, cell therapies represent an evolving frontier for AKI treatment in native and transplanted kidneys: ex-vivo manipulated stem or immune cells are able to counteract renal dysfunction by a wide range of biological mechanisms. In this review, we will discuss the potential applications of cell therapies in AKI and KT by analyzing the available clinical data and the most promising experimental prospects from a “bench to bedside” perspective.

Endothelial Progenitor Cell-Derived Extracellular Vesicles Inhibit Kidney Ischemia-Reperfusion Injury through the transfer of Specific Micrornoa and Mrna Coding for the Transcription Factor NRF2: Relevance for Delayed Kidney Graft Function

Background and Aim Activation of the complement cascade and oxidative stress in tubular epithelial and peritubular endothelial cells are hallmarks of delayed kidney graft function (DGF) due to ischemia-reperfusion injury (IRI). Endothelial progenitor cells (EPCs) are bone marrow-derived precursors known to reverse IRI by paracrine mechanisms including the release of extracellular vesicles (EV), small particles playing a role in intercellular communication through the transfer of specific mRNA and microRNA. The aim of this study was to evaluate the regenerative role of EPC-derived EV in kidney IRI through the horizontal transfer of RNA involved in the inhibition of complement activation and of oxidative stress. Methods EPC were isolated from peripheral blood of healthy volouteers and EV obtained by supernatant ultracentrifugation were characterized for size, protein and RNA content. We evaluated the effects of EV in a rat model of kidney IRI and in vitro in human tubular epithelial and endothelial cells cultured in hypoxia in vitro. Results EPC-derived EV sized 60-130 nm and carried different subsets of mRNAs and microRNAs able to modulate cell proliferation, angiogenesis and apoptosis (eNOS, Akt, Bcl-XL, miR-126, miR-296). By RT-PCR, we also found within EV mRNAs coding for the complement inhibitors factor H, DAF (CD55), CD59 and for the anti-oxidant transription factgor nrf2. In experimental kidney IRI, EPC EV localized within peritubular capillaries and tubular cells exerting morphologic and functional protection from by reducing tubular cell apoptosis/senescence, endothelial-to-mesenchymal transition (EndoMT) and leukocyte infiltration. Moreover, EV administration reduced C5b9 deposition and enhanced the expression of factor H, DAF, CD59 and nrf2 in the ischemic kidney. In vitro, EV reduced hypoxia-induced apoptosis/senescence of tubular epithelial (caspase activation, Klotho expression) and endothelial cells (EndoMT) by up-regulating the expression of factor H, DAF, CD59 and nrf2, thus confirming the in vivo data. The role of specific mRNA transfer to hypoxic renal cells was confirmed by experiments using RNase-treated EV, EV released from EPC engineered to knock-down the complement inhibitors or nrf2 by specific siRNA, or EV produced by EPC transfected with siRNA Dicer, the intracellular enzyme essential for microRNA maturation. Conclusions EPC-derived EV protect the kidney from IRI by delivering pro-angiogenic, anti-apoptotic, complement inhibitors and anti-oxidant microRNAs and mRNAs coding for factor H, DAF, CD59 and nrf2 to injured tubular epithelial and endothelial cells. These results suggest the potential use of EPC-derived EVs as therapeutic tool to avoid or at least to limit IRI-associated DGF in kidney transplantation without the potential adverse effects of whole stem cell therapy including maldifferentiation and tumorigenesis.

Perfluorocarbon solutions limit tubular epithelial cell injury and promote CD133+ kidney progenitor differentiation: potential use in renal assist devices for sepsis-associated acute kidney injury and multiple organ failure

Background The renal assist device (RAD) is a blood purification system containing viable renal tubular epithelial cells (TECs) that has been proposed for the treatment of acute kidney injury (AKI) and multiple organ failure. Perfluorocarbons (PFCs) are oxygen carriers used for organ preservation in transplantation. The aim of this study was to investigate the effect of PFCs on hypoxia- and sepsis-induced TEC injury and on renal CD133+ progenitor differentiation in a microenvironment similar to the RAD. Methods TECs were seeded in a polysulphone hollow fibre under hypoxia or cultured with plasma from 10 patients with sepsis-associated AKI in the presence or absence of PFCs and were tested for cytotoxicity (XTT assay), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling assay, caspases, enzyme-linked immunosorbent assay, Fas/Fas Ligand pathway activation), mitochondrial activity, cell polarity [transepithelial electrical resistance (TEER)] and adenosine triphosphate production. The effect of PFCs on proliferation and differentiation of human CD133+ progenitors was also studied. Results In the presence of PFCs, TECs seeded into the polysulphone hollow fibre showed increased viability and expression of insulin-like growth factor 1, hepatocyte growth factor and macrophage-stimulating protein. Plasma from septic patients induced TEC apoptosis, disruption of oxidative metabolism, alteration of cell polarity and albumin uptake, down-regulation of the tight junction protein ZO-1 and the endocytic receptor megalin on the TEC surface. These detrimental effects were significantly reduced by PFCs. Moreover, PFCs induced CD133+ renal progenitor cell proliferation and differentiation towards an epithelial/tubular-like phenotype. Conclusions PFCs improved the viability and metabolic function of TECs seeded within a polysulphone hollow fibre and subjected to plasma from septic AKI patients. Additionally, PFCs promoted differentiation towards a tubular/epithelial phenotype of CD133+ renal progenitor cells.

Plasma NGAL Is An Early Biomarker of Graft Function, Calcineurin Inhibitor Nephrotoxicity and Tubular Regeneration in Kidney Transplantation from Extended Criteria Donors: 902

Delayed graft function (DGF) is an early complication of kidney transplantation (KT) usually defined as the need for dialysis in the first week after the procedure. The main cause of DGF is ischemiareperfusion injury and the incidence of DGF has been increasing in the last years for the use of kidneys from extended criteria donors (ECD). Moreover, DGF is associated with an increased risk of acute rejection due to enhanced tissue immunogenicity and to a premature loss of kidney graft function. Plasma Neutrophil Gelatinase Associated Lipocalin (NGAL) is a 25 kDa protein belonging to the lipocalin family secreted by leukocytes and different epithelia including renal tubular epithelial cells. NGAL has been proposed as early biomarker of ischemic and toxic acute kidney injury and of DGF in kidney graft. The aims of this study were to evaluate: 1) plasma NGAL in 50 patients in the first 24h after KT from ECD; 2) the relationship between plasma NGAL and DGF, slow graft function (SGF) and immediate graft function (IGF); 3) the trend of serum creatinine (sCr) and plasma NGAL in the first 5 days after KT; 4) plasma NGAL before and after the introduction of calcineurin inhibitors (CNI, tacrolimus or cyclosporine); 5) the in vitro role of NGAL in tubular regeneration after ischemic injury. Fifty patients were enrolled in the study (immunosuppression with basiliximab, MMF and steroids: CNI introduced when sCr < 2.5 mg/dl). Patients were divided in 3 groups: DGF, SGF (sCr >3 mg/dl at day 6 after KT) and IGF (sCr < 3 mg/dl at day 6 after KT). Plasma NGAL levels were measured by a fluorimetric method (Alere, San Diego, CA). Protein and mRNA NGAL levels, proliferation and apoptosis were evaluated in isolated human tubular cells cultured under hypoxia or with therapeutic doses of tacrolimus/cyclosporine. Patients‘ demographics and characteristics were: male 67%, recipient age 57.65 yr, donor age 65 yr, cold ischemia time 16.8 h, HLA mismatches 3.46, recipient BMI 24.2, donor hypertension 64.4%, donor eGFR 88.66 ml/min. The incidence of DGF was 28%: in the 72% of patients without DGF, SGF occurred in 55%, IGF in 45%. Plasma NGAL levels (< 24h after KT) were significantly higher in DGF than in SGF and IGF groups (DGF 654.94 ng/ml; SGF 439.75 ng/ml; IGF 357.37 ng/ml). A decline of plasma NGAL but not of sCr was detectable at day 2 after KT with a further decrease at day 3, 4 and 5. By contrast, NGAL increased after 24 hr from CN

Perfluorocarbon (PFC) Protects Kidney Tubular Epithelial Cells (TEC) by Septic Plasma-Induced Apoptosis And Promotes CD133+ Stem Cell Differentiation: Relevance for Bioartifical Renal Assist Devices (RAD).

LIAL CELLS (TEC) BY SEPTIC PLASMA-INDUCED APOPTOSIS AND PROMOTES CD133 STEM CELL DIFFERENTIATION: RELEVANCE FOR BIOARTIFICAL RENAL ASSIST DEVICES (RAD) Vincenzo Cantaluppi, Federico Figliolini, Davide Medica, Sergio Dellepiane, Sylvia Giacalone, Alessandro D Quercia, Giuseppe P Segoloni, Giovanni Camussi. Nephrology, Dialysis and Renal Transplantation Unit, Department of Internal Medicine, University of Turin, San Giovanni Battista “Molinette” Hospital, Turin, Turin, Italy. Purpose of Study: Extracorporeal blood purification techniques including RAD have been proposed for sepsis-associated acute kidney injury (AKI). The aim of this study was to evaluate the effect of oxygen carrier PFC on septic plasma-induced TEC injury and on renal stem cell differentiation. Methods: Plasma was drawn by 10 patients with sepsis/ AKI (RIFLE). TEC were incubated with plasma in presence/absence of PFC evaluating: apoptosis (TUNEL, caspases), trans-epithelial electrical resistance (TER) and albumin uptake. We studied proliferation (BrdU) and differentiation of CD133 stem cells. Results: Septic plasma induced: 1) a cytotoxic effect on TEC through the activation of the death receptor/mitochondrial apoptotic pathways, 2) an alteration of cell polarity (TER) and albumin uptake; 3) down-regulation of the tight junction protein ZO-1 and of the endocytic receptor megalin. These detrimental effects were significantly reduced in presence of PFC. PFC induced CD133 progenitor proliferation and differentiation toward an epithelial phenotype (increase of TER and expression of markers of fully differentiated TEC). Summary: PFC protects TEC from septic plasmainduced injury and promotes CD133 stem cell differentiation. PFC may improve the results of RAD therapy in sepsis-associated AKI and find a potential application in AKI treatment.