RESUMEN
BACKGROUND: Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. METHODS: We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. RESULTS: Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. CONCLUSIONS: Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.
Asunto(s)
Anafilatoxinas/metabolismo , Células Endoteliales/metabolismo , Enfermedades Renales/patología , Riñón/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Daño por Reperfusión/complicaciones , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/complicaciones , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Riñón/metabolismo , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , PorcinosRESUMEN
Acute kidney injury (AKI) is emerging as a worldwide public health problem. Recent studies have focused on the possibility of using human adult renal stem/progenitor cells (ARPCs) to improve the repair of AKI. Here we studied the influence of ARPCs on the healing of cisplatin-injured renal proximal tubular epithelial cells. Tubular, but not glomerular, ARPCs provided a protective effect promoting proliferation of surviving tubular cells and inhibiting cisplatin-induced apoptosis. The recovery effect was specific to tubular ARPCs, occurred only after damage sensing, and was completely cancelled by TLR2 blockade on tubular ARPCs. Moreover, tubular, but not glomerular, ARPCs were resistant to the apoptotic effect of cisplatin. Tubular ARPCs operate mainly through the engagement of TLR2, the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs. These factors worked synergistically and were essential to the repair process. The involvement of tubular ARPC-secreted inhibin-A and decorin mRNA in the pathophysiology of AKI was also confirmed in transplant patients affected by delayed graft function. Hence, identification of this TLR2-driven recovery mechanism may shed light on new therapeutic strategies to promote the recovery capacity of the kidney in acute tubular damage. Use of these components, derived from ARPCs, avoids injecting stem cells.
Asunto(s)
Lesión Renal Aguda/terapia , Decorina/fisiología , Inhibinas/fisiología , Túbulos Renales Proximales/efectos de los fármacos , Riñón/citología , Trasplante de Células Madre , Receptor Toll-Like 2/fisiología , Adulto , Apoptosis , Proliferación Celular , Separación Celular , Cisplatino/toxicidad , Ciclina D1/fisiología , Decorina/genética , Humanos , Inhibinas/análisis , Túbulos Renales Proximales/patología , Regeneración , Receptor Toll-Like 2/antagonistas & inhibidoresRESUMEN
Adult renal progenitor cells (ARPCs) isolated from the human kidney may contribute to repair featuring acute kidney injury (AKI). Bone morphogenetic proteins (BMPs) regulate differentiation, modeling, and regeneration processes in several tissues. The aim of this study was to evaluate the biological actions of BMP-2 in ARPCs in vitro and in vivo. BMP-2 was expressed in ARPCs of normal adult human kidneys, and it was upregulated in vivo after delayed graft function (DGF) of renal transplantation, a condition of AKI. ARPCs expressed BMP receptors, suggesting their potential responsiveness to BMP-2. Incubation of ARPCs with this growth factor enhanced reactive oxygen species (ROS) production, NADPH oxidase activity, and Nox4 protein expression. In vivo, Nox4 was localized in BMP-2-expressing CD133+ cells at the tubular level after DGF. BMP-2 incubation induced α-smooth muscle actin (SMA), collagen I, and fibronectin protein expression in ARPCs. Moreover, α-SMA colocalized with CD133 in vivo after DGF. The oxidative stimulus (H(2)O(2)) induced α-SMA expression in ARPCs, while the antioxidant N-acetyl-cysteine inhibited BMP-2-induced α-SMA expression. Nox4 silencing abolished BMP-2-induced NADPH oxidase activation and myofibroblastic induction. We showed that 1) ARPCs express BMP-2, 2) this expression is increased in a model of AKI; 3) BMP-2 may induce the commitment of ARPCs toward a myofibroblastic phenotype in vitro and in vivo; and 4) this profibrotic effect is mediated by Nox4 activation. Our findings suggest a novel mechanism linking AKI with progressive renal damage.
Asunto(s)
Lesión Renal Aguda/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/fisiología , Riñón/metabolismo , NADPH Oxidasas/metabolismo , Células Madre/metabolismo , Lesión Renal Aguda/patología , Proteína Morfogenética Ósea 2/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Células Cultivadas , Fibronectinas/metabolismo , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Riñón/patología , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre/patología , Regulación hacia ArribaRESUMEN
A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1-fractalkine axis in the exacerbation of gross hematuria.
Asunto(s)
Quimiocina CX3CL1/metabolismo , Glomerulonefritis por IGA/inmunología , Hematuria/inmunología , Inmunidad Innata , Inmunidad Mucosa , Leucocitos Mononucleares/inmunología , Receptores de Quimiocina/metabolismo , Adulto , Receptor 1 de Quimiocinas CX3C , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CX3CL1/orina , Femenino , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Glomerulonefritis por IGA/complicaciones , Glomerulonefritis por IGA/genética , Hematuria/genética , Humanos , Italia , Glomérulos Renales/inmunología , Ligandos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Quimiocina/genética , Recurrencia , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Regulación hacia Arriba , Adulto JovenRESUMEN
Dendritic cells (DCs) have a pivotal role in the autoimmune response of systemic lupus erythematosus. Plasmacytoid DCs infiltrate the kidney of patients with lupus nephritis, but factors regulating their recruitment to the kidney are unknown. Chemerin is the recently identified natural ligand of ChemR23, a receptor highly expressed by plasmacytoid DCs. We performed immunohistochemical and immunofluorescence analysis to study the ChemR23/Chemerin axis in renal biopsies from patients with lupus nephritis. We found ChemR23-positive DCs had infiltrated the kidney tubulointerstitium in patients with severe lupus nephritis. Chemerin association with tubular epithelial cells and renal lymphatic endothelial cells was found in patients with lupus nephritis but not in normal kidneys. Proximal tubular epithelial cells produced Chemerin in vitro, which was significantly down-modulated by added tumor necrosis factor (TNF)-α and interferon-γ as measured by quantitative PCR and enzyme-linked immunosorbent assay. Interestingly, TNF-α was capable of inducing a functionally active form of renal Chemerin, resulting in an efficient transendothelial migration of plasmacytoid DCs measured in transwell systems. Thus, the ChemR23/Chemerin axis may have a role in the recruitment of DCs within the kidney in patients affected by lupus nephritis.
Asunto(s)
Quimiocinas/metabolismo , Quimiotaxis , Células Dendríticas/inmunología , Riñón/inmunología , Nefritis Lúpica/inmunología , Receptores de Quimiocina/metabolismo , Transducción de Señal , Migración Transendotelial y Transepitelial , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Quimiocinas/genética , Técnicas de Cocultivo , Células Endoteliales/inmunología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Células Epiteliales/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/metabolismo , Riñón/patología , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/patología , Nefritis Lúpica/patología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Enfermedades Renales/patología , Lectinas/química , Daño por Reperfusión/metabolismo , Animales , Proteína Inhibidora del Complemento C1/biosíntesis , Complemento C1q/metabolismo , Complemento C4b/metabolismo , Modelos Animales de Enfermedad , Femenino , Supervivencia de Injerto , Humanos , Inmunohistoquímica/métodos , Isquemia/patología , Enfermedades Renales/metabolismo , Fragmentos de Péptidos/metabolismo , PorcinosRESUMEN
In the past few years, adult renal progenitor/stem cells (ARPCs) have been identified in human kidneys, and particularly in Bowman's capsule and proximal tubules. They may play an important role in the kidney regenerative processes and might prospectively be the ideal cell type for the treatment of both acute and chronic renal injury. In this study, microarray analysis identified 6 gene clusters that discriminated normal human glomerular and tubular ARPCs from renal proximal tubular epithelial cells and mesenchymal stem cells. The top-scored pathway in the ARPC gene expression profile contained growth factor receptors and immune system-related genes, including toll-like receptor 2 (TLR2). Stimulation of TLR2 by ligands that mime inflammatory mediators or damage associated molecular pattern molecules induced secretion of elevated amounts of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, and C3 via NF-kappaB activation. TLR2 stimulation also increased the ARPC proliferation rate, suggesting a role for TLR2 in ARPC activation via autocrine signaling. Moreover, TLR2 stimulation improved ARPC differentiation into renal epithelial cells and was responsible of ARPC branching morphogenesis and tubule-like structures formation. For the first time, this study provides a genomic characterization of renal multipotent progenitor cells and shows that TLR2 found on ARPCs might be responsible for their activation in the kidney, orchestrating the activation of crucial signaling networks necessary for renal repair.
Asunto(s)
Túbulos Renales Proximales/citología , Células Madre/fisiología , Receptor Toll-Like 2/fisiología , Antígeno AC133 , Adulto , Antígenos CD/metabolismo , Antígeno CD24/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Células Clonales/fisiología , Complemento C3/metabolismo , Perfilación de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Glomérulos Renales/fisiología , Túbulos Renales Proximales/fisiología , FN-kappa B/metabolismo , Péptidos/metabolismo , Receptor Toll-Like 2/agonistasRESUMEN
Ischemia-reperfusion injury (IRI) in kidney transplantation is the major cause of delayed graft function (DGF), an event associated with an increased risk of acute rejection. The aim of this study was to evaluate T helper (Th) cell phenotype in renal transplants with DGF. T-bet (Th1), GATA-3 (Th2) and IL-17 (Th17) protein expression was investigated in pretransplant biopsies, DGF and acute tubular damage (ATD) caused by calcineurin-inhibitor toxicity. Intracytofluorimetric analysis of IFN-γ, IL-4 and IL-17 was performed to analyze Th1, Th2 and Th17 responses in peripheral blood mononuclear cells of recipients with early graft function (EGF) and DGF, before (T0) and 24 h after transplantation (T24). In pretransplant biopsies, T-bet(+) , GATA-3(+) and IL-17(+) cells were barely detectable. In DGF, T-bet(+) and IL-17(+) cells were significantly increased compared with pretransplant and ATD. More than 90% of T-bet(+) and less then 5% of IL-17(+) cells were CD4(+) . GATA-3(+) cells were increased to a lower extent. T-bet(+) /GATA-3(+) cell ratio was significantly higher in DGF. Peripheral CD4(+) IFN-γ/IL-4 ratio was significantly decreased in DGF, while CD4(+) /IL-17(+) cells did not differ between T0 and T24 in DGF. Our data suggest that DGF is characterized by a prevalent Th1 phenotype within the graft. This event might represent a link between DGF and acute rejection.
Asunto(s)
Funcionamiento Retardado del Injerto/patología , Trasplante de Riñón/inmunología , Linfocitos T Colaboradores-Inductores/patología , Células TH1/patología , Células Th2/patología , Adulto , Animales , Isquemia Fría/efectos adversos , Funcionamiento Retardado del Injerto/inmunología , Rechazo de Injerto/fisiopatología , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Persona de Mediana Edad , Daño por Reperfusión/patologíaRESUMEN
BACKGROUND: The term chronic allograft nephropathy (CAN) was deleted in the Eighth Banff Classification and two new categories were introduced: chronic T-cell-mediated rejection (CTMR) and chronic active humoral rejection (CAHR). The aim of this study was to revise our CAN cases diagnosed in the last 4 years, analyse allograft survival rates and identify types of infiltrating cells in the different settings. METHODS: Seventy-nine patients with biopsy-proven CAN were examined and classified into four groups according to the Banff 2005 criteria: CTMR, CAHR, chronic calcineurin inhibitor toxicity (CNITOX) and interstitial fibrosis and tubular atrophy not otherwise specified (NOS). CD4, CD8, CD20, CD68, CD103, Foxp3 and IL-17 protein expression and C4d deposits were investigated. RESULTS: We diagnosed 20 CTMR, 13 CAHR, 28 CNITOX, and 18 NOS. Death-censored graft survival at 4 years from renal biopsy was worse in CAHR compared with the other types of chronic injury. Glomerular CD8(+) cells were increased in CTMR vs CNITOX and NOS. Interstitial CD4(+) and CD8(+) cells were increased in CTMR vs CNITOX. CD68(+) cells in glomerular and peritubular capillaries were higher in CAHR vs CNITOX, CTMR and NOS. CD103(+) cells were higher in cases with tubulitis than in those without. T regulatory and T helper 17 cells were rarely observed in the different settings. CONCLUSIONS: Graft survival was worse in patients with CAHR. The presence of any grade transplant glomerulopathy and chronic allograft vasculopathy are poorer prognostic factors. Infiltrating CD8(+), CD103(+) and CD4(+) cells may help to differentiate CTMR from other types of chronic injury, thus improving diagnostic/prognostic features of biopsy in patients with chronic allograft dysfunction.
Asunto(s)
Rechazo de Injerto/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Trasplante de Riñón/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Adulto , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto/metabolismo , Rechazo de Injerto/fisiopatología , Supervivencia de Injerto/fisiología , Humanos , Inmunohistoquímica , Cadenas alfa de Integrinas/metabolismo , Interleucina-17/metabolismo , Glomérulos Renales/fisiopatología , Trasplante de Riñón/fisiología , Túbulos Renales/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Trasplante HomólogoRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease involving several organs. SLE patients developing lupus nephritis (LN) frequently have the worst outcome. Recent data have shown that dendritic cells (DCs) may have a central role in SLE pathogenesis directing the immune response against auto-antigens. In this study we describe a reduction in circulating BDCA1+ and BDCA3+ myeloid DCs, and BDCA2+ plasmacytoid DCs in patients with active LN compared to those in the remission state. Analysis of LN biopsies revealed a strong tubulo-interstitial infiltrate of BDCA1+, BDCA3+ and BDCA4+ DCs which were negative for DC-LAMP, a specific marker of mature DCs. The extent of the DCs infiltrate was higher in class III/IV LN than in normal kidney. These results show for the first time that three DCs subsets, decreased at circulating levels, are recruited within the kidney, indicating that DCs might play a pathogenic role in SLE patients with nephritis.
Asunto(s)
Células Dendríticas/citología , Túbulos Renales/patología , Nefritis Lúpica/patología , Células Mieloides/citología , Adulto , Antígenos CD1 , Antígenos de Superficie/metabolismo , Femenino , Glicoproteínas , Humanos , Masculino , TrombomodulinaRESUMEN
BACKGROUND: Delayed graft function (DGF) is associated with acute tubular necrosis. In this setting, surviving tubular cells may proliferate and replace injured cells. CD133Pax-2cells may play a role in the regeneration of tubular damage. The aim of this study was to demonstrate the presence of these cells in human kidneys before transplantation and in grafts with DGF. METHODS: Ten normal kidneys (group 1) and pretransplant biopsy of 25 deceased donors (group 2) were examined. The latter group included 10 kidneys with early graft function (2A) and 15 with DGF (2B). Group 2B patients received a second biopsy during DGF (2C). CD133, Pax-2, and Ki-67 protein expression was investigated by confocal microscopy. RESULTS: CD133Pax-2 and CD133Pax-2cells were present within the Bowman's capsule and proximal tubules in all groups except group 2B. Number of CD133Pax-2 and CD133Pax-2cells at tubular level was similar in groups 1 and 2A. Within group 2B we observed a striking reduction in both cell types. There was a significant increase of both cell populations within group 2C, compared with group 2B. CD133Pax-2 and CD133Pax-2cell number in group 2 correlated inversely with cold ischemia time. Pax-2Ki-67cells were absent from group 1 and 2B samples, and increased significantly in groups 2A and 2C. Proliferating CD133 cells increased significantly in group 2C. CONCLUSIONS: Our data suggest that regenerative response in posttransplant acute tubular necrosis, underlying DGF, is characterized by an increase in proliferating renal progenitor/stem cells CD133Pax-2 and CD133Pax-2 cells involved in repairing tubular damage.
Asunto(s)
Funcionamiento Retardado del Injerto/patología , Trasplante de Riñón/efectos adversos , Necrosis Tubular Aguda/patología , Riñón/patología , Células Madre/patología , Antígeno AC133 , Adulto , Antígenos CD/análisis , Proliferación Celular , Femenino , Glicoproteínas/análisis , Humanos , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , Factor de Transcripción PAX2/análisis , Péptidos/análisisRESUMEN
BACKGROUND: Chronic allograft nephropathy (CAN) is characterized by deposition of extracellular matrix (ECM) in all renal compartments. PAI-1 seems to play a pivotal role in ECM turnover in CAN. Rapamycin has been shown to improve long-term graft survival in patients with CAN. The aim of the study was to evaluate the molecular mechanisms underlying the beneficial effects of rapamycin on CAN progression at glomerular and tubulointerstitial level. METHODS: After a biopsy-proven CAN diagnosis (T0), 18 patients on calcineurin inhibitors (CNI) were randomly assigned in a 2:1 ratio to continue CNI (6 patients) or to receive rapamycin (RAPA; 12 patients). After 2 years of treatment (T24), all patients underwent a second renal biopsy. Morphometric analysis was conducted at T0 and at T24. PAI-1 expression was evaluated at T0 and T24 by immunohistochemistry. We evaluated the effect of rapamycin on PAI-1 gene expression in cultured proximal tubular cells incubated with CD40L or thrombin, two potential CAN pathogenic mediators. RESULTS: The RAPA group showed a significant regression of glomerulosclerotic lesions and only a 26% increase in interstitial fibrosis after 2 years compared to baseline, whereas the CNI group showed progression of glomerulosclerosis and 112% increase in fibrosis. Glomerular and tubulointerstitial PAI-1 expression was reduced compared to the baseline in the RAPA group, while they were unchanged in the CNI group. In vitro data showed that rapamycin significantly reduced PAI-1 gene expression induced by both CD40L and thrombin in proximal tubular epithelial cells. CONCLUSIONS: These data suggest that rapamycin may modulate ECM deposition in CAN reducing PAI-1 expression.
Asunto(s)
Inmunosupresores/farmacología , Enfermedades Renales/inmunología , Glomérulos Renales/patología , Trasplante de Riñón/efectos adversos , Túbulos Renales/patología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Sirolimus/farmacología , Adulto , Biopsia , Ligando de CD40/farmacología , Línea Celular , Células Cultivadas , Progresión de la Enfermedad , Fibrosis/metabolismo , Fibrosis/patología , Fibrosis/prevención & control , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/fisiopatología , Trasplante de Riñón/patología , Túbulos Renales/metabolismo , Túbulos Renales/fisiopatología , Persona de Mediana Edad , Esclerosis/metabolismo , Esclerosis/patología , Esclerosis/prevención & control , Trombina/farmacología , Trasplante HomólogoRESUMEN
BACKGROUND: Arteriovenous fistula (AVF) stenosis is the major cause of vascular access failure in hemodialysis. Adventitial remodeling has been suggested to play a role in the pathogenesis of AVF stenosis. This study aimed to evaluate adventitial fibrosis in stenotic AVF and investigate the underlying molecular mechanisms. METHODS: Forty-four patients undergoing surgery for AVF creation were examined; ten presented AVF failure, with histological-proven AVF stenosis. RESULTS: In stenotic AVF we observed a significant increase of adventitia extracellular matrix deposition and alpha-smooth muscle actin (α-SMA)(+) cell numbers; most of these cells were myofibroblast (α-SMA(+)/vimentin(+)). Phosphorylated platelet-derived growth factor ß receptor (p-PDGFRß) was significantly increased within the adventitia of stenotic compared to native AVF, along with a marked increase in the phosphorylation of Akt and ERK, two key kinases in PDGFRß signalling. Myofibroblasts were the main cell type associated with the activation of p-PDGFRß. At the same time, we observed a significant adventitial vessels rarefaction in stenotic AVF, as demonstrated by a reduced CD34 expression. This event was associated with a marked reduction in the expression of KDR/fetal liver kinase-1, the main vascular endothelial growth factor receptor. The degree of adventitial fibrosis was directly correlated with the extent of adventitial α-SMA and inversely associated with adventitial CD34 expression. Finally, we observed an increase in CD34(+)/α-SMA(+) cells within the adventitia of failed AVF. CONCLUSION: This study suggests that AVF failure is associated with an increased adventitial fibrosis, myofibroblast activation and capillary rarefaction, potentially linked with endothelial-to-mesenchymal transition. In this scenario, our data suggest that PDGF may play a pathogenic role.
Asunto(s)
Adventicia/patología , Derivación Arteriovenosa Quirúrgica/efectos adversos , Oclusión de Injerto Vascular/patología , Fallo Renal Crónico/terapia , Diálisis Renal , Remodelación Vascular , Venas/patología , Adulto , Adventicia/química , Anciano , Biomarcadores/análisis , Biopsia , Constricción Patológica , Células Endoteliales/química , Células Endoteliales/patología , Femenino , Fibrosis , Oclusión de Injerto Vascular/etiología , Oclusión de Injerto Vascular/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Miofibroblastos/química , Miofibroblastos/metabolismo , Estudios Prospectivos , Transducción de Señal , Insuficiencia del Tratamiento , Venas/químicaRESUMEN
In this paper we studied the in vivo neoadjuvant Androgen Deprivation Therapy (ADT) effect on the expression of TGF-ß1 and its receptor Tß-RII. Mechanisms of androgen dependence are critical to understanding prostate cancer progression to androgen independence associated with disease mortality, and TGF-ß is thought to support prostatic apoptosis as its expression coincides with androgen ablation in benign and cancer tissues. Increase of both mRNA and protein level were shown for the first time only in the patients who underwent neoadjuvant ADT for 1-month. This transient increase of TGF-ß expression after androgen ablation suggested cooperation of the pathways in prostate regression. Since no alteration was observed in the gene transcriptional activity, the molecular mechanism of this cooperation, probably act at the post-transcriptional level. TGF-ß1 and Tß-RII specific signals were co-localized within the neoplastic prostate epithelium. Our results suggests that the androgens deprivation by means of ADT for 1-month, involves a shift of the TGF-ß1 mechanism in prostate cancer, suggesting that the TGF-ß1 promotes prostate epithelial cell proliferation and inhibits apoptosis in a autocrine way.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Anciano , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
AIMS: This study investigated on (i) the role of gp91(phox)/NOX2 in reactive oxygen species (ROS) generation in hemodialysis (HD) patients, and (ii) the link between clotting activation and ROS production in this setting. RESULTS: The study was performed on peripheral blood mononuclear cells (PBMCs) isolated from HD patients randomized to polysulphon/polyamide (S-group, n=30) or ethylene-vinyl-alcohol (EVAL) membrane (E-group, n=30) treatment and from healthy subjects (control group, n=15). ROS generation was increased in PBMCs of HD patients compared with healthy subjects. S-group showed higher levels of intracellular ROS generation than control, whereas E-group did not. In addition, S-group displayed an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity compared with E-group and healthy subjects. A further increase in NADPH activity shortly after HD treatment was observed only in S-group. The plasma levels of the prothrombin fragment F1+2, a marker of in vivo clotting activation, were significantly higher in S-group than in E-group. Moreover, a heightened thrombin generation was recorded in the plasma of S-group. Intracellular ROS production correlated with NADPH oxidase activity and coagulation priming in HD patients. The in vitro validation study demonstrated that incubation of PBMCs with activated FX induced a significant increase in intracellular ROS production, superoxide generation, and gp91(phox)/NOX2 expression. INNOVATION: The pivotal role of NADPH oxidase in the upregulation of ROS in HD patients makes this enzyme a potential target for therapeutic intervention in the treatment of HD-related oxidative stress. CONCLUSION: The EVAL membrane, by reducing clotting activation, inhibits gp91(phox)/NOX2-related ROS production in HD patients.
Asunto(s)
NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Diálisis Renal , Adulto , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Membranas Artificiales , NADPH Oxidasas/antagonistas & inhibidores , Nylons/química , Nylons/farmacología , Polivinilos/química , Polivinilos/farmacología , Sulfonas/química , Sulfonas/farmacología , Propiedades de SuperficieRESUMEN
BACKGROUND: The mechanisms underlying the development of proteinuria in renal-transplant recipients converted from calcineurin inhibitors to sirolimus are still unknown. METHODS: This is a single-center cohort study. One hundred ten kidney transplant recipients converted from calcineurin inhibitors to sirolimus in the period from September 2000 to December 2005 were included in the study. All patients underwent a graft biopsy before conversion (T0) and a second protocol biopsy 2 years thereafter (T2), according to our standard clinical protocol. On the basis of the changes observed in proteinuria between T0 and T2 (median 70%), the patients were divided into two groups: group I (<70%) and group II (>70%). The authors blinded the sirolimus blood trough levels. We investigated in vivo the effects of sirolimus on nephrin, podocin, CD2ap, and actin protein expression. Slit diaphragm (SD)-associated protein expressions were evaluated in T0 and T2 biopsies. The same analysis was performed in cultured human podocytes treated with different doses of sirolimus (5, 10, 20, and 50 ng/mL). RESULTS: The SD protein expression in group II T2 biopsies was significantly reduced compared with the T0 biopsies and with T2 group I biopsies. In addition, sirolimus blood trough levels directly and significantly correlated with the SD protein expression at T2 graft biopsies. Group II patients presented significantly higher sirolimus blood levels than group I. In vitro study confirmed that sirolimus effect on podocytes was dose dependent. CONCLUSIONS: Our data suggest that sirolimus-induced proteinuria may be a dose-dependent effect of the drug on key podocyte structures.
Asunto(s)
Inmunosupresores/administración & dosificación , Trasplante de Riñón/efectos adversos , Proteinuria/etiología , Sirolimus/administración & dosificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Línea Celular , Estudios de Cohortes , Proteínas del Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/sangre , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Trasplante de Riñón/patología , Trasplante de Riñón/fisiología , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Neprilisina/metabolismo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/patología , Proteinuria/fisiopatología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus/efectos adversos , Sirolimus/sangre , Factores de Tiempo , Proteínas WT1/metabolismoRESUMEN
OBJECTIVE: Pentraxin-3 (PTX3) has been suggested to play a role in the development of vascular pathology. Stenosis of arteriovenous fistula (AVF) leading to its failure is the major cause of morbidity in hemodialysis patients. To date, little is known on the pathogenesis of AVF stenosis. The aim of the present study was to investigate the potential role of PTX3 in this setting. METHODS AND RESULTS: A sample of venous wall was collected at the time of AVF formation in 44 patients with end stage renal disease. Ten patients developed AVF stenosis and from these patients a second portion of the venous wall was obtained during surgical revision of the AVF. Confocal laser scanning microscopy demonstrated that PTX3 immunostaining, hardly detectable in native AVF, was significantly increased in failed AVF, showing a specific co-localization with endothelial cell markers. Circulating mononuclear cells isolated at the time of AVF revision presented a significantly higher PTX3 mRNA expression than those collected during AVF creation. Interestingly, a significant deposition of C5b-9 on endothelial cells, co-localizing with PTX3, was observed in stenotic AVF. CONCLUSION: The present study demonstrates for the first time a close association between PTX3 deposition and complement activation at the endothelial cell level in failed AVF and suggests a role for PTX3 in modulating innate immunity in the pathogenesis of AVF stenosis.
Asunto(s)
Anastomosis Arteriovenosa/inmunología , Proteína C-Reactiva/metabolismo , Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Constricción Patológica/inmunología , Fallo Renal Crónico/terapia , Diálisis Renal , Componente Amiloide P Sérico/metabolismo , Adulto , Anciano , Biomarcadores/metabolismo , Proteína C-Reactiva/genética , Endotelio Vascular/inmunología , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Componente Amiloide P Sérico/genética , Insuficiencia del TratamientoRESUMEN
Chronic allograft nephropathy (CAN) represents the main cause of renal allograft loss after 1 yr of transplantation. Calcineurin inhibitor (CNI) use is associated with increased graft expression of profibrotic cytokines, whereas rapamycin inhibits fibroblast proliferation. The aim of this randomized, prospective, open-label, single-center study was to evaluate the histologic and clinical effect of rapamycin on biopsy-proven CAN. Eighty-four consecutive patients who had biopsy-proven CAN and received a transplant were randomized to receive either a 40% CNI reduction plus mycophenolate mofetil (group 1; 50 patients) or immediate CNI withdrawal and rapamycin introduction with a loading dose of 0.1 mg/kg per d and a maintaining dose aiming at through levels of 6 to 10 ng/ml (group 2; 34 patients). The follow-up period was 24 mo. At the end of follow-up, 25 patients (group 1, 10 patients; group 2, 15 patients) underwent a second biopsy. CAN lesions were graded according to Banff criteria. alpha-Smooth muscle actin (alpha-SMA) protein expression was evaluated in all biopsies as a marker of fibroblast activation. Graft function and Banff grading were superimposable at randomization. Graft survival was significantly better in group 2 (P = 0.0376, chi2 = 4.323). CAN grading worsened significantly in group 1, whereas it remained stable in group 2. After 24 mo, all group 1 biopsies showed an increase of alpha-SMA expression at the interstitial and vascular levels (P < 0.001); on the contrary, alpha-SMA expression was dramatically reduced in group 2 biopsies (P = 0.005). This study demonstrates that rapamycin introduction/CNI withdrawal improves graft survival and reduces interstitial and vascular alpha-SMA expression, slowing down the progression of allograft injury in patients with CAN.
Asunto(s)
Glomerulonefritis Membranosa/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/patología , Sirolimus/uso terapéutico , Biopsia con Aguja , Enfermedad Crónica , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Glomerulonefritis Membranosa/patología , Rechazo de Injerto , Supervivencia de Injerto , Humanos , Inmunohistoquímica , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/cirugía , Masculino , Probabilidad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Método Simple Ciego , Inmunología del Trasplante/fisiología , Trasplante HomólogoRESUMEN
CHF2819 is a novel orally active acetylcholinesterase inhibitor (AChEI) developed for the treatment of Alzheimer's disease (AD). CHF2819 is a selective inhibitor of AChE, it is 115 times more potent against this enzyme than against butyrylcholinesterase (BuChE). Moreover, CHF2819 is more selective for inhibition of central (brain) AChE than peripheral (heart) AChE. In vivo CHF2819, 0.5, 1.5, and 4.5 mg/kg p.o., significantly and in dose-dependent manner increased acetylcholine (ACh) levels in hippocampus of young adult rats. Moreover, aging animals, with lower basal ACh levels than young adult rats, also exhibit a marked increase in hippocampal levels of this neurotransmitter after administration of CHF2819. At 1.5 mg/kg p.o. CHF2819 attenuated scopolamine-induced amnesia in a passive avoidance task. Furthermore, it decreased dopamine (DA) levels and increased extracellular levels of 5-hydroxytryptamine (5-HT) in the hippocampus, without modifying norepinephrine (NE) levels. By oral administration to young adult rats CHF2819 did not affect extracellular hippocampal levels of glutamate (Glu), aspartate (Asp), gamma-aminobutyric acid (GABA), taurine (Tau), arginine (Arg) or citrulline (Cit). Functional observational battery (FOB) screening demonstrated that CHF2819 (1.5 and 4.5 mg/kg p.o.) does not affect activity, excitability, autonomic, neuromuscular, and sensorimotor domains, as well as physiological endpoints (body weight and temperature). CHF2819 induced, however, involuntary motor movements (ranging from mild tremors to myoclonic jerks) in a dose-dependent manner. The neurochemical and behavioral profiles of CHF2819 suggest that this orally active novel AChEI could be of clinical interest for the treatment of Alzheimer-type dementia associated with multiple neurotransmitter abnormalities in the brain. In particular, CHF2819 might be a useful therapeutic drug for AD patients with cognitive impairment accompanied by depression.
Asunto(s)
Carbamatos/farmacología , Inhibidores de la Colinesterasa/farmacología , Óxidos N-Cíclicos/farmacología , Fenilcarbamatos , Acetilcolina/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Aminoácidos/metabolismo , Animales , Monoaminas Biogénicas/metabolismo , Carbamatos/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Óxidos N-Cíclicos/uso terapéutico , Hipocampo/metabolismo , RatasRESUMEN
Ischemia-reperfusion (I-R) injury in transplanted kidney, a key pathogenic event of delayed graft function (DGF), is characterized by tubular cell apoptosis and interstitial inflammation. Akt-mammalian target of rapamycin-S6k and NF-kappaB-inducing kinase (NIK)-NF-kappaB axis are the two main signaling pathways regulating cell survival and inflammation. Rapamycin, an immunosuppressive drug inhibiting the Akt axis, is associated with a prolonged DGF. The aim of this study was to evaluate Akt and NF-kappaB axis activation in patients who had DGF and received or not rapamycin and in a pig model of I-R and the role of coagulation priming in this setting. In graft biopsies from patients who were not receiving rapamycin, phosphorylated Akt increased in proximal tubular, interstitial, and mesangial cells with a clear nuclear translocation. The same pattern of activation was observed for S6k and NIK. However, in rapamycin-treated patients, a significant reduction of S6k but not Akt and NIK activation was observed. A time-dependent activation of phosphatidylinositol 3-kinase, Akt, S6k, and NIK was observed in the experimental model with the same pattern reported for transplant recipients who did not receive rapamycin. Extensive interstitial and glomerular fibrin deposition was observed both in pig kidneys upon reperfusion and in DGF human biopsies. It is interesting that the activation of both Akt and NIK-NF-kappaB pathways was induced by thrombin in cultured proximal tubular cells. In conclusion, the data suggest that (1) coagulation may play a pathogenic role in I-R injury; (2) the Akt axis is activated after I-R, and its inhibition may explain the prolonged DGF observed in rapamycin-treated patients; and (3) NIK activation in I-R and DGF represents a proinflammatory, rapamycin-insensitive signal, potentially leading to progressive graft injury.