[Immunosuppressive treatment after kidney transplant: the frontier of chronic antibody-mediated rejection]. / Terapia immunosoppressiva nel trapianto renale: la frontiera del rigetto cronico anticorpo-mediato.

The recognition of antibody-mediated rejection as an important factor in the reduction of long-term renal graft survival represents a new challenge to the immunosuppressive strategies of recent years, which have been quite successful in reducing the acute rejection rates as well as the side effects of pharmacological immunosuppression. The search for an effective treatment of chronic anti-donor antibody disease has been pursued mostly through limited single-center experiences and therefore in a dispersed fashion, without leading to the definition of a consolidated approach. The most frequently used pharmacological approaches stem from the experience of antibody-mediated acute rejection. In this review we will critically analyze the results reported so far of various intervention strategies and we will discuss future pharmacological novelties targeting the humoral immune response.

Protective effect of resin adsorption on septic plasma-induced tubular injury.

INTRODUCTION: A pro-apoptotic effect of circulating mediators on renal tubular epithelial cells has been involved in the pathogenesis of sepsis-associated acute kidney injury (AKI). Adsorption techniques have been showed to efficiently remove inflammatory cytokines from plasma. The aim of this study was to evaluate the efficiency of the hydrophobic resin Amberchrom CG161 M to adsorb from septic plasma soluble mediators involved in tubular injury. METHODS: We enrolled in the study 10 critically ill patients with sepsis-associated AKI and we evaluated the effects of their plasma on granulocyte adhesion, apoptosis and functional alterations of cultured human kidney tubular epithelial cells. We established an in vitro model of plasma adsorption and we studied the protective effect of unselective removal of soluble mediators by the Amberchrom CG161 M resin on septic plasma-induced tubular cell injury. RESULTS: Plasma from septic patients induced granulocyte adhesion, apoptosis and altered polarity in tubular cells. Plasma adsorption significantly decreased these effects and abated the concentrations of several soluble mediators. The inhibition of granulocyte adhesion to tubular cells was associated with the down-regulation of ICAM-1 and CD40. Resin adsorption inhibited tubular cell apoptosis induced by septic plasma by down-regulating the activation of caspase-3, 8, 9 and of Fas/death receptor-mediated signalling pathways. The alteration of cell polarity, morphogenesis, protein reabsorption and the down-regulation of the tight junction molecule ZO-1, of the sodium transporter NHE3, of the glucose transporter GLUT-2 and of the endocytic receptor megalin all induced by septic plasma were significantly reduced by resin adsorption. CONCLUSIONS: Septic plasma induced a direct injury of tubular cells by favouring granulocyte adhesion, by inducing cell apoptosis and by altering cell polarity and function. All these biological effects are related to the presence of circulating inflammatory mediators that can be efficiently removed by resin adsorption with a consequent limitation of tubular cell injury.

Macrophage stimulating protein may promote tubular regeneration after acute injury.

Macrophage-stimulating protein (MSP) exerts proliferative and antiapoptotic effects, suggesting that it may play a role in tubular regeneration after acute kidney injury. In this study, elevated plasma levels of MSP were found both in critically ill patients with acute renal failure and in recipients of renal allografts during the first week after transplantation. In addition, MSP and its receptor, RON, were markedly upregulated in the regenerative phase after glycerol-induced tubular injury in mice. In vitro, MSP stimulated tubular epithelial cell proliferation and conferred resistance to cisplatin-induced apoptosis by inhibiting caspase activation and modulating Fas, mitochondrial proteins, Akt, and extracellular signal-regulated kinase. MSP also enhanced migration, scattering, branching morphogenesis, tubulogenesis, and mesenchymal de-differentiation of surviving tubular cells. In addition, MSP induced an embryonic phenotype characterized by Pax-2 expression. In conclusion, MSP is upregulated during the regeneration of injured tubular cells, and it exerts multiple biologic effects that may aid recovery from acute kidney injury.

Platelet-activating factor synthesis and response on pancreatic islet endothelial cells: relevance for islet transplantation.

BACKGROUND: Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. METHODS: We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. RESULTS: HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. CONCLUSIONS: Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.

Relationship among C1q-fixing de novo donor specific antibodies, C4d deposition and renal outcome in transplant glomerulopathy.

BACKGROUND: The C1q-binding properties of donor specific antibodies (DSA) may be related to antibody-mediated rejection and poor outcome. METHODS: We retrospectively studied 35 kidney transplant recipients with transplant glomerulopathy (TG) and de novo DSA (dnDSA). C1q dnDSA were measured in the serum stored at renal biopsy and the association among C1q-fixing dnDSA, C4d deposition and graft loss was examined. RESULTS: Of the 35 patients with dnDSA and TG, 15 (42.9%) had C1q-positive dnDSA and 20 (57.1%) had C1q-negative dnDSA. Ten out of 15 patients with C1q-positive dnDSA (66.6%) and 5 with C1q-negative dnDSA (25%) had C4d positive staining renal biopsies (P=0.02), being the C1q-negative dnDSA/C4d-negative TG 42.9% of the total. The C1q-positive dnDSA group has significantly higher IgG DSA Class II MFI than the C1q-negative dnDSA group (P=0.004). Patients with C4d deposits have significantly higher IgG DSA MFI for both Class I and Class II than those without C4d deposits (P=0.02). We found a trend toward higher graft loss in the C1q-positive dnDSA group (60%) versus the C1q-negative dnDSA group (40%) without a statistical significance (P=0.31). CONCLUSION: Our study provides further characterization of TG associated with dnDSA. The major part of dnDSA-associated TG was C1q-negative and the presence of C1q-fixing dnDSA did not significantly correlate with graft outcome.

Isolation, characterization and potential role in beta cell-endothelium cross-talk of extracellular vesicles released from human pancreatic islets.

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.

Different regulatory and cytotoxic CD4+ T lymphocyte profiles in renal transplants with antibody-mediated chronic rejection or long-term good graft function.

Comparative analysis of the different subsets of CD4(+) T-lymphocytes may provide hints on the immunologic mechanisms operating in the long-term fate of a kidney transplant. We analyzed peripheral regulatory CD4(+) T cells (Tregs) and CD4(+) cytotoxic T lymphocytes (CTLs) in antibody-mediated chronic rejection (AMCR), in middle-term kidney transplants (2-4 years, MTKT) with good graft function and rejection-free history, in long-term kidney transplants (>15 years, LTKT) and in normal healthy subjects (NHS). Transplant groups with good prognosis (MTKT and LTKT) displayed a significant lower amount of CD4(+)CD25(high) T lymphocytes than NHS, with a trend of a higher percentage in AMCR than in MTKT and LTKT. However, CD4(+)CD25(high) Foxp3(+) cells were significantly higher in LTKT and MTKT than AMCR. Characterization of CD4(+)CD25(high) T cells showed a marked increase of intracellular CTLA-4 in the AMCR group in respect to the other transplant groups, while the expression of the surface molecule seemed to follow a reverse trend. In addition, CD27, a costimulatory receptor involved in long-term T cell survival and prevention of immune tolerance, is significantly reduced in CD4(+)CD25(high) and CD4(+)Foxp3(+) T cells in the LTKT in respect to the other transplant groups. CD4(+)CD25(high)CD45RO(+) and CD4(+)Foxp3(+)CD45RO(+) regulatory T cells with memory function were increased in LTKT compared to NHS and for the latter also in AMCR group. Finally, CD4(+)CTLs that were quantified on the basis of granzyme A expression, were more represented in AMCR patients in comparison to the other groups. Strikingly, CD27 in the CD4(+)CTLs was suppressed in LTKT and MTKT and markedly expressed in AMCR group. No significant differences in the expression of CD28 were observed among different groups. In conclusion, different profiles of Tregs and CD4(+)CTL populations correlate with different long-term conditions of kidney-transplanted patients, suggesting their role in the development of immunologic events in kidney transplantation.

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.

Magnetic resonance imaging of gadolinium-labeled pancreatic islets for experimental transplantation.

New imaging techniques that couple anatomical resolution to sensitivity may greatly contribute to improving islet transplantation. In the present work, a report is given of the direct detection of islets by magnetic resonance imaging (MRI) after ex vivo cell labeling with the MRI T(1) contrast agent GdHPDO3A. Experiments on mouse and human islets demonstrated well-tolerated uptake of GdHPDO3A, based on morphology, viability, glucose-dependent insulin response and apoptosis/toxicity gene array profile. GdHPDO3A loading was sufficient for in vitro MRI cell detection. In vivo isotransplanted mouse islets into the kidney capsule and xenotransplanted human islets within the mouse liver were detected. Imaging specificity was supported by the absence of signal in unlabeled islet transplants, its persistence upon using fat-suppression MRI protocols and the colocalization with the transplanted islets. In conclusion, direct islet imaging with high spatial and contrast resolution after labeling with GdHPDO3A is demonstrated, allowing visualization of kidney subcapsular mouse islet grafts and intrahepatic human islet xenografts.