High-volume hemofiltration does not protect human kidney endothelial and tubular epithelial cells from septic plasma-induced injury.

High volume hemofiltration (HVHF) could remove from plasma inflammatory mediators involved in sepsis-associated acute kidney injury (SA-AKI). The IVOIRE trial did not show improvements of outcome and organ dysfunction using HVHF. The aim of this study was to evaluate in vitro the biological effects of plasma of patients treated by HVHF or standard volume hemofiltration (SVHF). We evaluated leukocyte adhesion, apoptosis and functional alterations of endothelial cells (EC) and tubular epithelial cells (TEC). In vitro data were correlated with plasma levels of TNF-α, Fas-Ligand (FasL), CD40-Ligand (CD40L), von Willebrand Factor (vWF) and endothelial-derived microparticles. An experimental model of in vitro hemofiltration using LPS-activated blood was established to assess cytokine mass adsorption during HVHF or SVHF. Plasma concentrations of TNF-ɑ, FasL, CD40L and von Willebrand Factor (vWF) were elevated at the start (d1h0) of both HVHF and SVHF, significantly decreased after 6 h (d1h6), remained stable after 12 h (d1h12) and then newly increased at 48 h (d3h0). Plasma levels of all these molecules were similar between HVHF- and SVHF-treated patients at all time points considered. In addition, the levels of endothelial microparticles remained always elevated, suggesting the presence of a persistent microvascular injury. Plasma from septic patients induced leukocyte adhesion on EC and TEC through up-regulation of adhesion receptors. Moreover, on EC, septic plasma induced a cytotoxic and anti-angiogenic effect. On TEC, septic plasma exerted a direct pro-apoptotic effect via Fas up-regulation and caspase activation, loss of polarity, altered expression of megalin and tight junction molecules with an impaired ability to internalize albumin. The inhibition of plasma-induced cell injury was concomitant to the decrease of TNF-α, Fas-Ligand and CD40-Ligand levels. The protective effect of both HVHF and SVHF was time-limited, since a further increase of circulating mediators and plasma-induced cell injury was observed after 48 h (d3h0). No significant difference of EC/TEC damage were observed using HVHF- or SVHF-treated plasma. The in vitro hemofiltration model confirmed the absence of a significant modulation of cytokine adsorption between HVHF and SVHF. In comparison to SVHF, HVHF did not increase inflammatory cytokine clearance and did not reverse the detrimental effects of septic plasma-induced EC and TEC injury. Further studies using adsorptive membranes are needed to evaluate the potential role of high dose convective therapies in the limitation of the harmful activity of plasma soluble factors involved in SA-AKI.Trial registration IVOIRE randomized clinical trial; ClinicalTrials.gov (NCT00241228) (18/10/2005).

Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury.

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.

Citrate anion improves chronic dialysis efficacy, reduces systemic inflammation and prevents Chemerin-mediated microvascular injury.

Systemic inflammation and uremic toxins (UT) determine the increased cardiovascular mortality observed in chronic hemodialysis (HD) patients. Among UT, the adipokine Chemerin induces vascular dysfunction by targeting both endothelial and vascular smooth muscular cells (EC and VSMC). As Citrate anion modulates oxidative metabolism, systemic inflammation and vascular function, we evaluated whether citrate-buffered dialysis improves HD efficiency, inflammatory parameters and chemerin-mediated microvascular injury. 45 patients were treated in sequence with acetate, citrate and, again, acetate-buffered dialysis solution (3 months per interval). At study admission and after each treatment switch, we evaluated dialysis efficacy and circulating levels of chemerin and different inflammatory biomarkers. In vitro, we stimulated EC and VSMC with patients' plasma and we investigated the role of chemerin as UT. Citrate dialysis increased HD efficacy and reduced plasma levels of CRP, fibrinogen, IL6 and chemerin. In vitro, patients' plasma induced EC and VSMC dysfunction. These effects were reduced by citrate-buffered solutions and paralleled by the decrease of chemerin levels. Consistently, chemerin receptor knockdown reduced EC and VSMC dysfunction. In conclusion, Switching from acetate to citrate improved dialysis efficacy and inflammatory parameters; in vitro, chemerin-induced EC and VSMC injury were decreased by using citrate as dialysis buffer.

Acute Tubular Injury is Associated With Severe Traumatic Brain Injury: in Vitro Study on Human Tubular Epithelial Cells.

Acute kidney injury following traumatic brain injury is associated with poor outcome. We investigated in vitro the effects of plasma of brain injured patients with acute tubular kidney injury on kidney tubular epithelial cell function. we performed a prospective observational clinical study in ICU in a trauma centre of the University hospital in Italy including twenty-three ICU patients with traumatic brain injury consecutively enrolled. Demographic data were recorded on admission: age 39 ± 19, Glasgow Coma Score 5 (3-8). Neutrophil Gelatinase-Associated Lipocalin and inflammatory mediators were measured in plasma on admission and after 24, 48 and 72 hours; urine were collected for immunoelectrophoresis having healthy volunteers as controls. Human renal proximal tubular epithelial cells were stimulated with patients or controls plasma. Adhesion of freshly isolated human neutrophils and trans-epithelial electrical resistance were assessed; cell viability (XTT assay), apoptosis (TUNEL staining), Neutrophil Gelatinase-Associated Lipocalin and Megalin expression (quantitative real-time PCR) were measured. All patients with normal serum creatinine showed increased plasmatic Neutrophil Gelatinase-Associated Lipocalin and increased urinary Retinol Binding Protein and α1-microglobulin. Neutrophil Gelatinase-Associated Lipocalin was significantly correlated with both inflammatory mediators and markers of tubular damage. Patient' plasma incubated with tubular cells significantly increased adhesion of neutrophils, reduced trans-epithelial electrical resistance, exerted a cytotoxic effect and triggered apoptosis and down-regulated the endocytic receptor Megalin compared to control. Plasma of brain injured patients with increased markers of subclinical acute kidney induced a pro-inflammatory phenotype, cellular dysfunction and apoptotic death in tubular epithelial cells.

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.

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.

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.

[New mechanisms and recent insights in the pathogenesis of acute kidney injury (AKI)]. / Nuovi meccanismi e recenti acquisizioni nella patogenesi del danno renale acuto.

Acute kidney injury (AKI) is a frequent complication in hospitalized patients often associated with multiple organ failure, increased mortality and progression toward chronic kidney disease. The identification of new cellular and molecular targets involved in AKI may lead to an improvement of diagnostic and therapeutic approaches. In recent years, the pathogenetic mechanisms of AKI have been fully elucidated: tubular epithelial cells and endothelial cells present in the microvasculature have been identified as the main targets of ischemia and of nephrotoxic drugs. Indeed, endothelial cell injury is associated with an extension phase of AKI, whereas tubular cells are subjected to an alteration of cell polarity, mislocalization of tight junction proteins and membrane transporters, and finally to the development of necrosis or apotosis. Apoptosis, or programmed cell death, is also a key component of sepsis-associated AKI in which the mechanisms of tissue damage are associated not only with hypoperfusion but also with a direct detrimental effect of bacterial products and inflammatory mediators on resident kidney cells. Endothelial and tubular epithelial cells also represent the main targets in the immunological mechanisms of AKI in kidney transplantation during cell-mediated and antibody-mediated rejection. Recent studies evidenced new molecules as early biomarkers of AKI. Among these molecules, NGAL and KIM-1 play a possible role in the progression toward chronic kidney disease. Lastly, the new frontier of AKI therapy is represented by the use of bone marrow-derived mesenchymal stem cells able to induce a regenerative program in the damaged kidney.

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 ß-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 ß-cell function.