RESUMO
Chronic kidney disease involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in Irp1+/+ and Irp1-/- mice, we asked if iron regulatory proteins (IRPs), the central regulators of cellular iron metabolism and suppressors of EPO production, contribute to the etiology of anemia in kidney failure. We identified a significant reduction in IRP protein level and RNA binding activity that associates with a loss of the iron uptake protein transferrin receptor 1 (TfR1), increased expression of the iron storage protein subunits H- and L-ferritin, and a low but overall variable level of stainable iron in the obstructed kidney. This reduction in IRP RNA binding activity and ferritin RNA levels suggests the concomitant rise in ferritin expression and iron content in kidney failure is IRP dependent. In contrast, the reduction in the Epo mRNA level in the obstructed kidney was not rescued by genetic ablation of IRP1, suggesting disruption of normal hypoxia-inducible factor (HIF)-2α regulation. Furthermore, reduced expression of some HIF-α target genes in UUO occurred in the face of increased expression of HIF-α proteins and prolyl hydroxylases 2 and 1, the latter of which is not known to be HIF-α mediated. Our results suggest that the IRP system drives changes in cellular iron metabolism that are associated with kidney failure in UUO but that the impact of IRPs on EPO production is overridden by disrupted hypoxia signaling.NEW & NOTEWORTHY This study demonstrates that iron metabolism and hypoxia signaling are dysregulated in unilateral obstructive nephropathy. Expression of iron regulatory proteins (IRPs), central regulators of cellular iron metabolism, and the iron uptake (transferrin receptor 1) and storage (ferritins) proteins they target is strongly altered. This suggests a role of IRPs in previously observed changes in iron metabolism in progressive renal disease. Hypoxia signaling is disrupted and appeared to dominate the action of IRP1 in controlling erythropoietin expression.
Assuntos
Anemia/etiologia , Ferro/metabolismo , Rim/metabolismo , Insuficiência Renal/etiologia , Obstrução Ureteral/complicações , Anemia/metabolismo , Anemia/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Modelos Animais de Doenças , Eritropoetina/genética , Eritropoetina/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Fibrose , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Hyperpolarized 13 C magnetic resonance imaging (MRI) may be used to non-invasively image the transport and chemical conversion of 13 C-labeled compounds in vivo. In this study, we utilize hyperpolarized 13 C MRI to evaluate metabolic markers in the kidneys longitudinally in a mouse model of partial unilateral ureteral obstruction (pUUO). Partial obstruction was surgically induced in the left ureter of nine adult mice, leaving the right ureter as a control. 1 H and hyperpolarized [1-13 C]pyruvate MRI of the kidneys was performed 2 days prior to surgery (baseline) and at 3, 7 and 14 days post-surgery. Images were evaluated for changes in renal pelvis volume, pyruvate, lactate and the lactate to pyruvate ratio. After 14 days, mice were sacrificed and immunohistological staining of both kidneys for collagen fibrosis (picrosirius red) and macrophage infiltration (F4/80) was performed. Statistical analysis was performed using a linear mixed effects model. Significant kidney × time interaction effects were observed for both lactate and pyruvate, indicating that these markers changed differently between time points for the obstructed and unobstructed kidneys. Both kidneys showed an increase in the lactate to pyruvate ratio after obstruction, suggesting a shift towards glycolytic metabolism. These changes were accompanied by marked hydronephrosis, fibrosis and macrophage infiltration in the obstructed kidney, but not in the unobstructed kidney. Our results show that pUUO is associated with increased pyruvate to lactate metabolism in both kidneys, with injury and inflammation specific to the obstructed kidney. The work also demonstrates the feasibility of the use of hyperpolarized 13 C MRI to study metabolism in renal disease.
Assuntos
Isótopos de Carbono/metabolismo , Rim/metabolismo , Imageamento por Ressonância Magnética , Obstrução Ureteral/metabolismo , Animais , Biomarcadores/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Camundongos , Espectroscopia de Prótons por Ressonância Magnética , Tamanho da AmostraRESUMO
Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function.
Assuntos
Anticorpos/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Transição Epitelial-Mesenquimal , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Transplante de Rim , Imunologia de Transplantes , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat-shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction, supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies, we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and ß-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-ß-catenin complex.
Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Animais , Caderinas/metabolismo , Membrana Celular/metabolismo , Colágeno Tipo III/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fibrose , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , beta Catenina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: Mouse kidney transplantation is a challenging technique for novice microsurgeons. Factors that affect transplant outcomes for a clinical surgeon starting microsurgery have not yet been investigated. MATERIALS AND METHODS: 110 consecutive mouse kidney transplants were performed over a 9-month period. Data were recorded, and surgical results and complication were analyzed. RESULTS: Three and thirty day survival rates improved from 0 (0/6) to 92.3 % (12/13) between months 1 and 9. Bleeding, arterial thrombosis, kidney failure and hydronephrosis were the most common causes of transplant failure. From month 1 to month 7, using the same surgical technique, practice significantly decreased the incidence of bleeding and increased the 3-day survival rate; however, it didn't significantly decrease the incidence of thrombosis, kidney failure, but improved the 30-day survival rate. From month 8, when surgical technique used on artery anastomosis switched from continuous suture to interrupted suture, surgical survival rate at 3 and 30 days improved significantly. Interestingly, ischemia time was not a significant factor determining the success of transplantation in this study. CONCLUSIONS: Practice is essential for novice microsurgeons, and the choice of surgical techniques significantly affects surgical results. The use of interrupted arterial sutures can significantly improve mouse kidney transplantation outcomes compared with continuous sutures. Ischemic time was not a factor in determining successful of kidney transplantation in mice in this study.
Assuntos
Modelos Animais de Doenças , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Microcirurgia/métodos , Animais , Competência Clínica , Curva de Aprendizado , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Duração da Cirurgia , Fatores de Tempo , Falha de Tratamento , Resultado do Tratamento , Isquemia QuenteRESUMO
BACKGROUND: Transplant glomerulopathy (TG) is a pathological feature of chronic active antibody-mediated rejection (cAMR) and is associated with renal allograft failure. The specific role of B cells in the pathogenesis of TG is unclear. METHODS: We used a minor mismatched rat kidney transplant model with B cell-deficient recipients, generated by clustered regularly interspaced short palindromic repeats/Cas9 technology, to investigate the impact of B-cell depletion on the pathogenesis of TG. We hypothesized that B-cell deficiency would prevent TG in the rat kidney transplant model of cAMR. Treatment groups included syngeneic, allogeneic, sensitized allogeneic, and B cell-deficient allogeneic transplant recipients. RESULTS: B cell-deficient recipients demonstrated reduced TG lesions, decreased microvascular inflammation, reduced allograft infiltrating macrophages, and reduced interferon gamma transcripts within the allograft. Allograft transcript levels of interferon gamma, monocyte chemoattractant protein-1, and interleukin-1ß correlated with numbers of intragraft macrophages. B cell-deficient recipients lacked circulating donor-specific antibodies and had an increased splenic regulatory T-cell population. CONCLUSIONS: In this model of cAMR, B-cell depletion attenuated the development of TG with effects on T cell and innate immunity.
Assuntos
Linfócitos B/imunologia , Glomerulonefrite/prevenção & controle , Rejeição de Enxerto/prevenção & controle , Isoanticorpos/sangue , Rim/imunologia , Animais , Linfócitos B/metabolismo , Proliferação de Células , Células Cultivadas , Doença Crônica , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Imunidade Celular , Imunidade Inata , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Rim/patologia , Ativação Linfocitária , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Ratos Transgênicos , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Background: Extracellular ATP binds to purinergic receptors and promotes inflammatory responses. We tested whether oxidized ATP (oATP), P2X7 receptor antagonist can attenuate acute kidney allograft rejection. Methods: Brown Norway kidney allografts were transplanted into Lewis recipients. Three groups were defined: oATP (n=8), cyclosporine A (n=6), and no treatment (n=8). On day 7, we assessed kidney allograft survival, function, and rejection characteristics. We further determined T-cell, B-cell, and macrophage response to oATP in vivo and in vitro and examined intragraft inflammatory gene transcripts. Results: Kaplan-Meier survival analyses demonstrated significantly better graft survival rates in oATP and CsA groups compared with no treatment (P<0.05). Similarly, serum creatinine (Scr) and BUN levels were significantly lower in oATP and CsA groups (P<0.05). oATP reduced both T cell-mediated rejection and antibody-mediated rejection, inhibited B-cell and T-cell activation, and downregulated intragraft IL-6 mRNA levels (P<0.0001). In vitro, oATP prevented proliferation in mixed lymphocyte reaction assays, and inhibited macrophage P2X7R activity in a dose-dependent manner. Conclusions: Our findings suggest that oATP mitigates kidney allograft rejection by inhibiting T-cell, B-cell, and macrophage activity and indicate a potential role for the purinergic system and oATP in solid organ transplantation.
Assuntos
Rejeição de Enxerto , Linfócitos T , Trifosfato de Adenosina/metabolismo , Aloenxertos/metabolismo , Rejeição de Enxerto/prevenção & controle , Rim/metabolismo , Macrófagos/metabolismo , Linfócitos T/metabolismoRESUMO
Chronic active antibody-mediated rejection is a major cause of allograft failure in kidney transplantation. Microvascular inflammation and transplant glomerulopathy are defining pathologic features of chronic active antibody-mediated rejection and are associated with allograft failure. However, the mechanisms of leukocyte infiltration and glomerular endothelial cell injury remain unclear. We hypothesized MHC class II ligation on glomerular endothelial cells (GEnC) would result in upregulation of adhesion molecules and production of chemoattractants. A model of endothelial cell activation in the presence of antibodies to MHC classes I and II was used to determine the expression of adhesion molecules and chemokines. Murine GEnC were activated with IFNγ, which upregulated gene expression of ß2-microglobulin (MHC class I), ICAM1, VCAM1, CCL2, CCL5, and IL-6. IFNγ stimulation of GEnC increased surface expression of MHC class I, MHC class II, ICAM1, and VCAM1. Incubation with antibodies directed at MHC class I or class II did not further enhance adhesion molecule expression. Multispectral imaging flow cytometry and confocal microscopy demonstrated MHC molecules co-localized with the adhesion molecules ICAM1 and VCAM1 on the GEnC surface. GEnC secretion of chemoattractants, CCL2 and CCL5, was increased by IFNγ stimulation. CCL2 production was further enhanced by incubation with sensitized plasma. Endothelial activation induces de novo expression of MHC class II molecules and increases surface expression of MHC class I, ICAM1 and VCAM1, which are all co-localized together. Maintaining the integrity and functionality of the glomerular endothelium is necessary to ensure survival of the allograft. IFNγ stimulation of GEnC propagates an inflammatory response with production of chemokines and co-localization of MHC and adhesion molecules on the GEnC surface, contributing to endothelial cell function as antigen presenting cells and an active player in allograft injury.
Assuntos
Aloenxertos/imunologia , Moléculas de Adesão Celular/metabolismo , Células Endoteliais/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Glomérulos Renais/patologia , Animais , Apresentação de Antígeno , Células Cultivadas , Citometria de Fluxo , Isoanticorpos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Transporte Proteico , Regulação para CimaRESUMO
BACKGROUND: B-cell depletion is a common treatment of antibody-mediated rejection (ABMR). We sought to determine the specific immunopathologic effects of this therapeutic approach in kidney transplantation. METHODS: This was a prospective observational study of kidney transplant recipients diagnosed with late ABMR (>3 months after transplant). Patients received treatment with pulse steroids, IVIG, and rituximab. Donor specific HLA antibodies (DSA), kidney allograft pathology, renal function, immune cell phenotypes, and 47 circulating cytokines were assessed at baseline and at three months. RESULTS: We enrolled 23 patients in this study between April 2015 and March 2019. The majority of patients were male (74%) and Caucasian (78%) with an average age of 45.6±13.8 years. ABMR was diagnosed at 6.8±5.9 years (4 months-25 years) post-transplant. Treatment was associated with a significant decline in circulating HLA class I DSA (P=0.003) and class II DSA (P=0.002) and peritubular capillaritis (ptc, P=0.04) compared to baseline. Serum creatinine, BUN, eGFR, and proteinuria (UPC) remained stable. Circulating B-cells were depleted to barely detectable levels (P≤0.001), whereas BAFF (P=0.001), APRIL (P<0.001), and IL-10 (P=0.02), levels increased significantly post-treatment. Notably, there was a significant rise in circulating CD4+ (P=0.02) and CD8+ T-cells (P=0.003). We also noted a significant correlation between circulating cytotoxic CD8+ T-cells and BAFF (P=0.05), regulatory T-cells and IL10 (P=0.002), and HLA class I DSA (P=0.005). CONCLUSIONS: Short-term pulse steroids/IVIG/rituximab therapy was associated with inhibition of ABMR (DSA and ptc), stabilization of kidney function, and increased regulatory B-cell and T-cell survival cytokines. Additional studies are needed to understand the implications of B cell-depletion on the crosstalk between T-cells, B-cells, and humoral components that regulate ABMR.
Assuntos
Rejeição de Enxerto , Isoanticorpos , Aloenxertos , Feminino , Rejeição de Enxerto/tratamento farmacológico , Sobrevivência de Enxerto , Antígenos HLA , Humanos , Imunoglobulinas Intravenosas/farmacologia , Rim/fisiologia , Masculino , Rituximab/uso terapêutico , Esteroides/farmacologiaRESUMO
Alloantibody represents a significant barrier in kidney transplant through the sensitization of patients prior to transplant through antibody mediated rejection (ABMR). APRIL BLyS are critical survival factors for mature B lymphocytes plasma cells, the primary source of alloantibody. We examined the effect of APRIL/BLyS blockade via TACI-Ig (Transmembrane activator calcium modulator cyclophilin lig interactor-Immunoglobulin) in a preclinical rodent model as treatment for both desensitization ABMR. Lewis rats were sensitized with Brown Norway (BN) blood for 21 days. Following sensitization, animals were then sacrificed or romized into kidney transplant (G4, sensitized transplant control); desensitization with TACI-Ig followed by kidney transplant (G5, sensitized + pre-transplant TACI-Ig); kidney transplant with post-transplant TACI-Ig for 21 days (G6, sensitized + post-transplant TACI-Ig); desensitization with TACI-Ig followed by kidney transplant post-transplant TACI-Ig for 21 days (G7, sensitized + pre- post-transplant TACI-Ig). Animals were sacrificed on day 21 post-transplant tissues were analyzed using flow cytometry, IHC, ELISPOT, RT-PCR. Sensitized animals treated with APRIL/BLyS blockade demonstrated a significant decrease in marginal zone non-switched B lymphocyte populations (p<0.01). Antibody secreting cells were also significantly reduced in the sensitized APRIL/BLyS blockade treated group. Post-transplant APRIL/BLyS blockade treated animals were found to have significantly less C4d deposition less ABMR as defined by Banff classification when compared to groups receiving APRIL/BLyS blockade before transplant or both before after transplant (p<0.0001). The finding of worse ABMR in groups receiving APRIL/BLyS blockade before both before after transplant may indicate that B lymphocyte depletion in this setting also resulted in regulatory lymphocyte depletion resulting in a worse rejection. Data presented here demonstrates that the targeting of APRIL BLyS can significantly deplete mature B lymphocytes, antibody secreting cells, effectively decrease ABMR when given post-transplant in a sensitized animal model.
Assuntos
Fator Ativador de Células B/imunologia , Dessensibilização Imunológica/métodos , Rejeição de Enxerto/prevenção & controle , Transplante de Rim , Proteínas Recombinantes de Fusão/farmacologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Fator Ativador de Células B/antagonistas & inibidores , Fator Ativador de Células B/genética , Complemento C4b/antagonistas & inibidores , Complemento C4b/biossíntese , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imunização/métodos , Imunofenotipagem , Isoanticorpos/biossíntese , Masculino , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/patologia , Ratos , Ratos Endogâmicos Lew , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
BACKGROUND: Increasingly, it is being appreciated that B cells have broad roles beyond the humoral response and are able to contribute to and regulate inflammation. The specific role of B cells in the pathogenesis of early allograft inflammation remains unclear. METHODS: To address this question, we generated B cell-deficient (B) Lewis rats via clustered regularly interspaced short palindromic repeats (CRISPR) technology. In a full mismatch transplant model, kidneys from Brown Norway donors were transplanted into B Lewis recipients or wild type Lewis recipients. T cell-mediated rejection was attenuated with cyclosporine. RESULTS: Renal inflammation was reduced at 1 week after transplant (Banff scores for interstitial inflammation, microvascular inflammation, glomerulitis, and C4d) in allografts from B recipients. The reduction in interstitial inflammation was predominantly due to a decline in graft infiltrating macrophages. Intragraft T-cell numbers remained unchanged. In addition, B-cell deficiency was associated with increased T regulatory cells and reduced splenic T follicular helper cells at baseline; and significantly increased intragraft and splenic IL-10 mRNA levels after transplant. In vitro, B and wild type splenic T cells produced similar levels of IFN-γ in response to T cell-specific activation. CONCLUSIONS: B-cell deficiency in this model produced an anti-inflammatory phenotype with a shift toward regulatory T-cell populations, production of anti-inflammatory cytokines (IL-10), and a reduction in allograft inflammation. These findings define a role for B cells to influence the cell populations and mediators involved in the pathogenesis of early allograft inflammation.
Assuntos
Linfócitos B/fisiologia , Inflamação/prevenção & controle , Transplante de Rim , Macrófagos/fisiologia , Aloenxertos , Animais , Interferon gama/biossíntese , Interleucina-10/genética , Ativação Linfocitária , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Linfócitos T/imunologiaRESUMO
BACKGROUND: We hypothesized that immunomodulatory properties of mesenchymal stromal cells (MSC) may be considered for desensitization. METHODS: Autologous or allogeneic bone marrow derived MSC were infused via tail vein at 0.5 M (0.5 × 106), 1 M, or 2 M cells/dose on days -2, 3, 6, 9, 12 (prevention) or 14, 17, 20, 23, 26 (treatment) relative to transfusion in a Brown Norway to Lewis rat model (10 groups total, n = 6 per group). RESULTS: At 4 weeks, pooled analyses demonstrated that autologous and allogeneic MSC were equally effective in reducing IgG1 and IgG2a de novo donor-specific antibody (dnDSA, P < 0.001). Dose-response studies indicated that moderate-dose MSC (5 M total) was most effective in reducing IgG1, IgG2a, and IgG2c dnDSA (P ≤ 0.01). Time course studies determined that preventive and treatment strategies were equally effective in reducing IgG1 and IgG2a dnDSA (P ≤ 0.01). However, individual group analyses determined that moderate-dose (5 M) treatment with autologous MSC was most effective in reducing IgG1, IgG2a, and IgG2c dnDSA (P ≤ 0.01). In this group, dnDSA decreased after 1 week of treatment; regulatory B cells increased in the spleen and peripheral blood mononuclear cells; and transitional B cells increased in the spleen, peripheral blood mononuclear cells, and bone marrow (P < 0.05 for all). CONCLUSIONS: Our findings indicate that autologous MSC prevent transfusion-elicited sensitization and upregulate transitional, and regulatory B cells. Additional studies are needed to determine the biological relevance of these changes after kidney transplantation.
RESUMO
BACKGROUND: We hypothesized that nicotinamide adenosine diphosphate oxidase 2 (Nox2) plays an important role in cyclosporine A (CsA)-induced chronic hypoxia. METHODS: We tested this hypothesis in Fisher 344 rats, C57BL/6 J wild type and Nox2-/- mice, and in liver transplant recipients with chronic CsA nephrotoxicity. We used noninvasive molecular imaging (blood oxygen level-dependent magnetic resonance imaging and dynamic contrast-enhanced magnetic resonance imaging) and molecular diagnostic tools to assess intrarenal oxygenation and perfusion, and the molecular phenotype of CsA nephrotoxicity. RESULTS: We observed that chemical and genetic inhibition of Nox2 in rats and mice resulted in the prevention of CsA-induced hypoxia independent of regional perfusion (blood oxygen level-dependent magnetic resonance imaging and dynamic contrast-enhanced magnetic resonance imaging, pimonidazole, HIF-1α). Nicotinamide adenosine diphosphate oxidase 2 knockout was also associated with decreased oxidative stress (Nox2, HIF-1α, hydrogen peroxide, hydroxynonenal), and fibrogenesis (α-smooth muscle actin, picrosirius red, trichrome, vimentin). The molecular signature of chronic CsA nephrotoxicity using transcriptomic analyses demonstrated significant changes in 40 genes involved in injury repair, metabolism, and oxidative stress in Nox2-/- mice. Immunohistochemical analyses of kidney biopsies from liver transplant recipients with chronic CsA nephrotoxicity showed significantly greater Nox2, α-smooth muscle actin and picrosirius levels compared with controls. CONCLUSIONS: These studies suggest that Nox2 is a modulator of CsA-induced hypoxia upstream of HIF-1α and define the molecular characteristics that could be used for the diagnosis and monitoring of chronic calcineurin inhibitor nephrotoxicity.
Assuntos
Ciclosporina/efeitos adversos , Hipóxia/induzido quimicamente , Rim/efeitos dos fármacos , Rim/patologia , Transplante de Fígado , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Actinas/metabolismo , Animais , Compostos Azo/metabolismo , Biópsia , Inibidores de Calcineurina/química , Meios de Contraste/química , Peróxido de Hidrogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/patologia , Imageamento por Ressonância Magnética , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Perfusão , Fenótipo , Ratos , Ratos Endogâmicos F344 , Vimentina/metabolismoRESUMO
BACKGROUND: Heat shock protein (HSP) 27 plays a cytoprotective role through its antioxidant, antiapoptotic, and actin-stabilizing properties during cell stress. The authors hypothesized that HSP27 is involved in chronic allograft nephropathy (CAN), a chronic state of inflammation and stress. METHODS: The authors used the Fisher 344-to-Lewis model of CAN. Transplants were performed in 3-month-old recipient rats. HSP27 mRNA and protein levels were determined using semiquantitative polymerase chain reaction, microarray (stress-toxicity, GEArray) analyses, gene sequencing, immunoblotting, and immunohistochemical analyses at 10 days and 6 months posttransplant. P38 mitogen-activated protein kinase (MAPK), manganese (Mn) superoxide dismutase (SOD), copper-zinc (CuZn) SOD, FasL, Bax, hypoxia-inducible factor (HIF)-1alpha, and CD3 lymphocytes were studied in parallel as selective biomarkers of oxidative stress (OS), apoptosis, hypoxia, and graft-infiltrating immune cells. RESULTS: Six months after transplantation, kidney allografts displayed histologic and functional features of CAN, including tubular atrophy, interstitial fibrosis, glomerulosclerosis, and increased proteinuria and serum creatinine levels. HSP27 mRNA and protein levels in CAN were reduced by 50% and 85%, respectively (P=0.04). Immunohistochemical analyses revealed a "shift" in HSP27 from the medulla to the cortex in allografts with CAN. Bax, phosphorylated p38-MAPK, HIF-1alpha, and MnSOD followed a parallel relocation pattern. CD3 lymphocyte density and tubular FasL expression were also greater in the cortex of allografts with CAN. Time-course analyses revealed that most of these changes were present as early as 10 days posttransplant. CONCLUSIONS: The shift of HSP27 from the medulla to the cortex, combined with greater CD3, p38-MAPK, Bax, FasL, HIF-1alpha, and MnSOD immunoreactivity in this area of the kidney, likely represents an allograft-level response to CAN-related OS-hypoxia.
Assuntos
Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Transplante de Rim/patologia , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Transplante Homólogo/patologia , Animais , Sequência de Bases , Doença Crônica , Proteínas de Choque Térmico HSP27 , Córtex Renal/citologia , Córtex Renal/patologia , Medula Renal/citologia , Medula Renal/patologia , Chaperonas Moleculares , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Adhesion molecules on lymphocytes ( l -selectin and alpha4beta7) and endothelium (MAdCAM-1 and ICAM-1) direct lymphocytes into the gut-associated lymphoid tissue (GALT) of mice. Parenteral nutrition and MAdCAM-1 blockade reduce GALT cell mass. This study examined the effects on GALT cell mass of blockade of l -selectin, alpha4beta7, and ICAM-1 with saturating doses of monoclonal antibodies. METHODS: In experiment 1, l -selectin and alpha4beta7 expression were measured by flow cytometry in chow-fed mice. In experiment 2, 49 mice randomly received chow, parenteral nutrition, chow + intravenous (IV) anti-CD62L, chow + IV anti-LPAM-1, or chow + IV isotype control antibody. After 4 days, lymphocyte yields in GALT and respiratory and intestinal IgA levels were measured. In experiment 3, 27 mice randomly received chow, parenteral nutrition, chow + IV anti-ICAM-1 monoclonal antibody, or chow + IV isotype control antibody for 5 days. Lymphocyte counts and IgA levels were determined as in experiment 2. RESULTS: Some 80% of all circulating lymphocytes were positive for l -selectin and alpha4beta7. Lymphocyte counts in the Peyer's patches, lamina propria, and intraepithelial space were lower in the l -selectin and alpha4beta7 blockade groups (3.1, 1.8, and 0.9 x 10(6) and 2.1, 1.9, and 0.7 x 10(6) , respectively) than in the chow group (5.9, 3.0, and 1.7 x 10(6) ; P < .02 vs the l -selectin group and P <.001 vs the alpha4beta7 group) and similar to the levels in the parenteral group. Respiratory and intestinal IgA levels are maintained in all groups except the parenteral group ( P <.04 vs the chow group). ICAM-1 blockade did not influence cell counts or IgA levels. CONCLUSION: Most circulating lymphocytes have GALT homing potential. Their distribution into GALT is hindered by blockade of l -selectin or alpha4beta7, but not by ICAM-1.
Assuntos
Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Selectina L/metabolismo , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Movimento Celular , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/metabolismoRESUMO
BACKGROUND: Parenteral nutrition (PN) decreases gut-associated lymphoid tissue (GALT), the intestinal IgA stimulating cytokines IL-4 and IL-10 in gut homogenates, intestinal IgA levels and the expression of Peyer patch (PP) mucosal cellular adhesion molecule-1 (MAdCAM-1), an adhesion molecule found on the high endothelial venules of PP and other tissues. IL-4 in PP stimulates MAdCAM expression in vitro. MAdCAM-1 blockade with MECA-367 reduces GALT cell populations to PN levels but maintains intestinal IgA levels if the animals are chow fed. This study compares IL-4 levels in PP of chow and PN fed mice and measures the effects of MAdCAM blockade on IL-4 and IL-10 levels in gut homogenates of chow fed mice. We hypothesized that in vivo IL-4 levels drop in PP of PN fed mice and IL-4 and IL-10 levels are maintained after MAdCAM-1 blockade in chow fed mice. METHODS: Exp 1: 18 mice received chow or PN for 5 days to determine PP IL-4 levels. Exp 2: 44 mice were randomized to chow + control monoclonal antibody (mAb), chow + MECA-367 (anti-MAdCAM-1 mAb) or PN for 4 days before measurement of IL-4 and IL-10 levels in gut homogenates. RESULTS: Exp 1: IL-4 levels in vivo were lower in PP of PN-fed mice than chow fed mice (92.0 +/- 15.1 pg/mL vs 251.1 +/- 14.8, p = .0003). Exp 2: IL-4 levels were significantly higher in chow + control mAb (187.1 +/- 44.1 pg/mL) and chow + MECA-367 (110.9 +/- 19.1 pg/mL) groups than PN mice (21.8 +/- 30.6 pg/mL, p < .02 vs chow + control or chow + MECA-367). IL-10 levels were significantly lower with PN (23.1 +/- 40.9 pg/mL) with chow+control (174.0 +/- 22.2 pg/mL p < .01), or chow + MECA-367 (181.7 +/- 23.1 pg/mL, p < .02 vs PN). CONCLUSIONS: PN-feeding reduces in vivo IL-4 levels in PP (consistent with lowered MAdCAM-1 expression) and IL-4 and IL-10 levels in gut homogenates compared with chow. Despite MAdCAM-1 blockade, enteral feeding preserved gut IL-4 levels and increased IL-10 levels consistent with preserved IgA levels.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Nutrição Enteral , Imunidade nas Mucosas/imunologia , Imunoglobulinas/imunologia , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Mucoproteínas/imunologia , Nódulos Linfáticos Agregados/metabolismo , Animais , Moléculas de Adesão Celular , Imunoglobulina A/metabolismo , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mucoproteínas/antagonistas & inibidores , Nutrição Parenteral/efeitos adversos , Distribuição AleatóriaRESUMO
BACKGROUND: There is a need for new immunosuppression strategies to minimize calcineurin inhibitor (CNI) toxicity while effectively preventing antibody-mediated rejection (AMR). METHODS: We tested the efficacy of an investigational proteasome inhibitor, ixazomib, alone and in a CNI minimization strategy in a rat kidney transplant model of transfusion-elicited acute AMR. Nonsensitized (naïve) and sensitized allograft recipients were randomized into 4 treatment groups (8 groups total, n = 3 to 6 in each group) and treated for 1 week. Groups included: no treatment, full dose cyclosporine (CsA, 10 mg/kg per day), ixazomib (0.25 mg/kg on days -5, -2 and +2) alone, and half dose CsA (5 mg/kg per day) + ixazomib. RESULTS: Compared to untreated animals, ixazomib alone or in combination with ½ dose CsA reduced donor-specific antibody, intragraft transcripts for chemokines CCL-21 and CXCL-13, and CD19 expression in both sensitized and naïve transplants. Compared to full dose CsA, the CNI minimization strategy with ixazomib inhibited AMR and allograft injury as evidenced by reduced C4d staining in peritubular capillaries, microcirculation inflammation, splenic plasma cells, circulating B cell activating factor, and intragraft transcripts for major histocompatibility complex class II, Toll-like receptors (TLR-1, TLR-10, and TLR-12) and CCL-21 and CXCL-13 in sensitized animals, indicating downregulation of B cell activation, antigen presentation and T-cell and B-cell signaling. CONCLUSIONS: These studies suggest that CNI minimization strategies including ixazomib are effective to prevent AMR including in sensitized kidney allograft recipients. Clinical studies are needed to determine the role of novel proteasome inhibitors for the prevention and treatment of AMR.
Assuntos
Compostos de Boro/farmacologia , Inibidores de Calcineurina/farmacologia , Ciclosporina/farmacologia , Glicina/análogos & derivados , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunossupressores/farmacologia , Isoanticorpos/sangue , Transplante de Rim , Rim/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Doença Aguda , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Compostos de Boro/toxicidade , Inibidores de Calcineurina/administração & dosagem , Inibidores de Calcineurina/toxicidade , Ciclosporina/administração & dosagem , Ciclosporina/toxicidade , Modelos Animais de Doenças , Quimioterapia Combinada , Regulação da Expressão Gênica , Glicina/farmacologia , Glicina/toxicidade , Rejeição de Enxerto/sangue , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Imunossupressores/administração & dosagem , Imunossupressores/toxicidade , Rim/enzimologia , Rim/imunologia , Rim/patologia , Transplante de Rim/efeitos adversos , Masculino , Inibidores de Proteassoma/toxicidade , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
BACKGROUND: Bombesin, the amphibian analog of mammalian gastrin-releasing peptide, reverses total parenteral nutrition (TPN)-induced atrophy of gut-associated lymphoid tissue and increases intestinal and respiratory immunoglobulin A (IgA) levels. Structure-activity studies suggested that the biologically active portion of bombesin is a C-terminal heptapeptide (7AA). This study investigates the effect of 7AA on lymphocytes counts of the Peyer's patches (PP), the lamina propria (LP) and the intraepithelial layer (IE). METHODS: Forty-eight male mice were randomized to receive chow (n = 13), TPN only (n = 9), TPN + 15 microg 7AA 3 times per day (n = 13) or TPN + 150 microg 7AA 3 times per day (n = 13). After 5 days of feeding, PP, LP, and IE lymphocytes were determined. Intestinal IgA levels were measured with ELISA. Groups were compared with ANOVA. RESULTS: All TPN-fed mice lost more weight than mice fed chow (p < .04). Lymphocyte counts in PP, LP, and IE were significantly lower in the TPN group than in the 3 other groups but did not differ between the groups fed chow, TPN + 15 microg 7AA 3 times per day, or TPN + 150 microg 7AA 3 times per day. Intestinal IgA levels were higher in chow-fed mice (148.4 +/- 16.9) than in mice fed TPN (98.4 +/- 14.0, p = .008), TPN + 15 microg 7AA 3 times per day (96.9 +/- 7.7, p = .003) or TPN + 150 microg 7AA 3 times per day (87.3 +/- 6.7, p = .001). CONCLUSIONS: The C-terminal heptapeptide of bombesin prevented the TPN-induced decrease in intestinal lymphocyte populations but not the reduction in intestinal IgA levels.