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1.
Nat Commun ; 12(1): 4402, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285231

RESUMO

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.


Assuntos
Injúria Renal Aguda/patologia , Ferroptose/fisiologia , Túbulos Renais/patologia , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Feminino , Ferroptose/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HT29 , Transplante de Coração/efeitos adversos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/química , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos , Proteínas Mitocondriais/metabolismo , Células NIH 3T3 , Necrose/tratamento farmacológico , Necrose/etiologia , Necrose/patologia , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Cultura Primária de Células , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia
2.
BMC Nephrol ; 22(1): 251, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34229622

RESUMO

BACKGROUND: The ongoing coronavirus pandemic has major impacts on both patients and healthcare systems worldwide, thus creating new realities. Patients on maintenance dialysis listed for renal transplantation are a vulnerable subgroup with many comorbidities and recurring contacts with the healthcare system. Due to the COVID-19 pandemic transplant numbers have dropped considerably, further increasing waiting times in this high-risk population. On the other hand, knowledge of the severity of SARS-CoV-2 infection in immunocompromised patients, development and persistence of neutralising antibodies in such patients is just emerging. It is unclear how best to address the dilemma of postponing the life-saving transplantation. CASE PRESENTATION: We present a case report of a successful kidney transplantation only 65 days after the recipient was hospitalized for treatment of COVID-19 pneumonia. In a follow up of 9 months, we observed no signs of recurrent disease and transplant function is excellent. Monitoring SARS-CoV-2 antibody response demonstrates stable IgG levels. CONCLUSION: This reassuring case provides guidance to transplant centers how to proceed with kidney transplantation safely during the pandemic. Careful consideration of risks and benefits of the organ offer, full recovery from COVID-19 symptoms and the presence of a positive SARS-CoV-2 IgG antibody test, qualifies for kidney transplantation.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Idoso , COVID-19/complicações , Teste Sorológico para COVID-19 , Feminino , Rejeição de Enxerto/prevenção & controle , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Imunossupressores/uso terapêutico , Falência Renal Crônica/complicações , Diálise Renal , SARS-CoV-2
3.
BMC Nephrol ; 21(1): 503, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33228553

RESUMO

BACKGROUND: Endothelial Progenitor Cells have been shown as effective tool in experimental AKI. Several pharmacological strategies for improving EPC-mediated AKI protection were identified in recent years. Aim of the current study was to analyze consequences of constitutive Atg5 activation in murine EPCs, utilized for AKI therapy. METHODS: Ischemic AKI was induced in male C57/Bl6N mice. Cultured murine EPCs were systemically injected post-ischemia, either natively or after Atg5 transfection (Adenovirus-based approach). Mice were analyzed 48 h and 6 weeks later. RESULTS: Both, native and transfected EPCs (EPCsAtg5) improved persisting kidney dysfunction at week 6, such effects were more pronounced after injecting EPCsAtg5. While matrix deposition and mesenchymal transdifferentiation of endothelial cells remained unaffected by cell therapy, EPCs, particularly EPCsAtg5 completely prevented the post-ischemic loss of peritubular capillaries. The cells finally augmented the augophagocytic flux in endothelial cells. CONCLUSIONS: Constitutive Atg5 activation augments AKI-protective effects of murine EPCs. The exact clinical consequences need to be determined.

4.
Kidney Int ; 98(5): 1120-1134, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866505

RESUMO

Over the past 2 decades, scoring systems for multiple glomerular diseases have emerged, as have consortia of pathologists and nephrologists for the study of glomerular diseases, including correlation of pathologic findings with clinical features and outcomes. However, one important limitation faced by members of these consortia and other renal pathologists and nephrologists in both investigative work and routine practice remains a lack of uniformity and precision in clearly defining the morphologic lesions on which the scoring systems are based. In response to this issue, the Renal Pathology Society organized a working group to identify the most frequently identified glomerular lesions observed by light microscopy and electron microscopy, review the literature to capture the published definitions most often used for each, and determine consensus terms and definitions for each lesion in a series of online and in-person meetings. The defined lesions or abnormal findings are not specific for any individual disease or subset of diseases, but rather can be applied across the full spectrum of glomerular diseases and within the context of the different scoring systems used for evaluating and reporting these diseases. In addition to facilitating glomerular disease research, standardized terms and definitions should help harmonize reporting of medical kidney diseases worldwide and lead to more-precise diagnoses and improved patient care.


Assuntos
Glomerulonefrite , Nefropatias , Biópsia , Consenso , Humanos , Rim , Nefropatias/diagnóstico , Glomérulos Renais , Microscopia Eletrônica
5.
Pediatr Nephrol ; 35(8): 1529-1561, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32382828

RESUMO

Idiopathic nephrotic syndrome newly affects 1-3 per 100,000 children per year. Approximately 85% of cases show complete remission of proteinuria following glucocorticoid treatment. Patients who do not achieve complete remission within 4-6 weeks of glucocorticoid treatment have steroid-resistant nephrotic syndrome (SRNS). In 10-30% of steroid-resistant patients, mutations in podocyte-associated genes can be detected, whereas an undefined circulating factor of immune origin is assumed in the remaining ones. Diagnosis and management of SRNS is a great challenge due to its heterogeneous etiology, frequent lack of remission by further immunosuppressive treatment, and severe complications including the development of end-stage kidney disease and recurrence after renal transplantation. A team of experts including pediatric nephrologists and renal geneticists from the International Pediatric Nephrology Association (IPNA), a renal pathologist, and an adult nephrologist have now developed comprehensive clinical practice recommendations on the diagnosis and management of SRNS in children. The team performed a systematic literature review on 9 clinically relevant PICO (Patient or Population covered, Intervention, Comparator, Outcome) questions, formulated recommendations and formally graded them at a consensus meeting, with input from patient representatives and a dietician acting as external advisors and a voting panel of pediatric nephrologists. Research recommendations are also given.


Assuntos
Glucocorticoides/efeitos adversos , Imunossupressores/uso terapêutico , Síndrome Nefrótica/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Resistência a Medicamentos , Feminino , Glucocorticoides/administração & dosagem , Humanos , Lactente , Recém-Nascido , Masculino , Síndrome Nefrótica/diagnóstico , Proteinúria/urina , Indução de Remissão/métodos
6.
BMC Nephrol ; 20(1): 296, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382904

RESUMO

BACKGROUND: Transplant failure requires the consideration of numerous potential causes including rejection, acute tubular necrosis, infection, and recurrence of the original kidney disease. Kidney biopsy is generally required to approach these differential diagnoses. However, the histopathological findings on their own do not always lead to a definite diagnosis. Consequently, it is crucial to integrate them with clinical findings and patient history when discussing histopathological patterns of injury. The histopathologic finding of a membranoproliferative glomerulonephritis (MPGN) is one of the most challenging constellations since it does not refer to a specific disease entity but rather reflects a pattern of injury that is the result of many different causes. Whilst MPGN is occasionally classified as immune complex mediated, careful evaluation usually reveals an underlying disorder such as chronic infection, plasma cell dyscrasia, complement disorders, and autoimmune disease. CASE PRESENTATION: We describe the case of a 43-year-old woman who was referred to us because of a slowly rising serum creatinine 4 years after kidney transplantation. As in the native kidney, the biopsy revealed an MPGN pattern of injury. The cause of this finding had not been established prior to transplantation leading to a classification as idiopathic MPGN in the past. Further workup at the time of presentation and allograft failure revealed chronic infection of a ventriculoatrial shunt as the most probable cause. CONCLUSION: This case underlines the fact that MPGN is not a disease but a histopathological description. Consequently, the causative disorder needs to be identified to avoid kidney failure and recurrence after transplantation.


Assuntos
Derivações do Líquido Cefalorraquidiano/efeitos adversos , Glomerulonefrite Membranoproliferativa/etiologia , Transplante de Rim , Complicações Pós-Operatórias/etiologia , Infecções Estafilocócicas/complicações , Adulto , Biópsia , Creatinina/sangue , Feminino , Glomerulonefrite Membranoproliferativa/sangue , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Hidrocefalia/sangue , Hidrocefalia/cirurgia , Rim/patologia , Recidiva , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis , Staphylococcus hominis , Derivação Ventriculoperitoneal
7.
Cell Death Dis ; 10(3): 235, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850581

RESUMO

Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation.


Assuntos
Anticorpos Monoclonais/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Lisossomos/efeitos dos fármacos , Necrose/imunologia , Citoesqueleto de Actina/metabolismo , Apoptose/efeitos dos fármacos , Células Endoteliais/metabolismo , Rejeição de Enxerto , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon gama/farmacologia , Lisossomos/enzimologia , Lisossomos/imunologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo
8.
J Pathol ; 247(5): 697-707, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30714148

RESUMO

Necrosis of a cell is defined by the loss of its plasma membrane integrity. Morphologically, necrosis occurs in several forms such as coagulative necrosis, colliquative necrosis, caseating necrosis, fibrinoid necrosis, and others. Biochemically, necrosis was demonstrated to represent a number of genetically determined signalling pathways. These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis. Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features. Necroptosis can be specifically detected by immunohistochemistry and immunofluorescence employing antibodies to phosphorylated MLKL. Likewise, it is possible to generate cleavage-specific antibodies against epitopes in gasdermin protein family members. In ferroptosis, however, specific detection requires quantification of oxidative lipids by mass spectrometry (oxylipidomics). Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions. These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials. Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Necrose/patologia , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Membrana Celular/patologia , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Ferro/metabolismo , Camundongos
10.
Cell Death Dis ; 9(3): 359, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29500402

RESUMO

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.


Assuntos
Anticonvulsivantes/farmacologia , Necrose/prevenção & controle , Fenitoína/farmacologia , Injúria Renal Aguda/patologia , Animais , Biópsia , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia
11.
Proc Natl Acad Sci U S A ; 114(45): E9618-E9625, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078325

RESUMO

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by (i) pharmacologic inhibition of RIPK1 or (ii) enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Apoptose/imunologia , Armadilhas Extracelulares/imunologia , Necrose/imunologia , Neutrófilos/imunologia , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Linhagem Celular , Ativação do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Células Endoteliais/imunologia , Feminino , Humanos , Rim/imunologia , Rim/metabolismo , Masculino , Camundongos , Necrose/metabolismo , Ativação de Neutrófilo/imunologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
12.
Cell ; 169(2): 286-300.e16, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388412

RESUMO

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here, we show that, during necroptosis, MLKL-dependent calcium (Ca2+) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM "bubbles" with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules and promote antigenic cross-priming of CD8+ T cells.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Necrose/metabolismo , Animais , Cálcio/metabolismo , Sobrevivência Celular , Células HT29 , Humanos , Células Jurkat , Camundongos , Células NIH 3T3 , Fosfatidilserinas , Proteínas Quinases/metabolismo , Transdução de Sinais
13.
Transplantation ; 100(5): 1004-14, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26895216

RESUMO

BACKGROUND: Antibody-mediated rejection is a leading cause for renal transplant loss. Rodent models are useful to dissect pathomechanisms and to develop treatment strategies. Although used for decades as a model, glomerular histopathological findings of Fischer-344 kidneys transplanted into Lewis rats have never been comprehensively described. METHODS: Kidneys from Fischer-344 rats were transplanted into Lewis rats as life-sustaining allografts without immunosuppression. Lewis isografts and normal Fischer-344 kidneys served as controls. Grafts were harvested at 9 days, 6 and 26 weeks. Histopathological examination included light microscopy, immunohistochemistry, and morphometry. Findings were compared with 51 human biopsies with transplant glomerulopathy. RESULTS: Most glomerular findings in rat allografts resembled human acute and chronic antibody-mediated rejection with glomerulitis, microthrombosis, microaneurysms, glomerular hypertrophy, podocyte loss, glomerular basement membrane splitting, and secondary focal and segmental glomerulosclerosis. In line with previous reports on nonendothelial antigens, glomerular immunoglobulin and C4d deposition was mostly nonendothelial. Only in 26-week allografts, we found mesangial and subendothelial immune complex-type electron-dense deposits. Similar deposits were found in 8 of 51 human biopsies with transplant glomerulopathy after rigorous exclusion of immune complexes of other cause, particularly recurrent glomerulonephritis and hepatitis C. CONCLUSIONS: Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.


Assuntos
Complexo Antígeno-Anticorpo , Nefropatias/etiologia , Transplante de Rim/efeitos adversos , Animais , Biópsia , Capilares , Complemento C4b/imunologia , Modelos Animais de Doenças , Progressão da Doença , Mesângio Glomerular/imunologia , Rejeição de Enxerto/imunologia , Humanos , Imunoglobulina G/imunologia , Imuno-Histoquímica , Rim/irrigação sanguínea , Rim/patologia , Glomérulos Renais/patologia , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/imunologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Trombose/patologia , Fatores de Tempo , Transplante Homólogo/efeitos adversos
14.
PLoS One ; 10(12): e0145306, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26690352

RESUMO

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Heme Oxigenase-1/metabolismo , Antígenos de Histocompatibilidade Classe I , Células Endoteliais da Veia Umbilical Humana/enzimologia , Quimiocina CCL2/biossíntese , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-8/biossíntese , Molécula 1 de Adesão de Célula Vascular/biossíntese
15.
Xenotransplantation ; 22(4): 284-94, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26216261

RESUMO

BACKGROUND: Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. METHODS: Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. RESULTS: Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. CONCLUSION: We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.


Assuntos
Transtornos da Coagulação Sanguínea/prevenção & controle , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Transplante Heterólogo/efeitos adversos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Células Endoteliais/imunologia , Etanercepte/administração & dosagem , Feminino , Glucose , Sobrevivência de Enxerto , Humanos , Imunossupressores/administração & dosagem , Técnicas In Vitro , Inflamação/prevenção & controle , Rim/imunologia , Rim/patologia , Manitol , Soluções para Preservação de Órgãos , Perfusão/efeitos adversos , Perfusão/métodos , Cloreto de Potássio , Procaína , Suínos , Trombose/etiologia , Trombose/prevenção & controle , Transplante Heterólogo/métodos
16.
Transplantation ; 99(1): 56-62, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25121474

RESUMO

BACKGROUND: Treatment of patients with antibody-mediated rejection (AMR) after kidney transplantation by rituximab and plasmapheresis is ambiguous. Because of its unknown efficiency and serious side effects, biomarkers, which are predictive for responsiveness to this treatment in AMR patients, are required. METHODS: Twenty renal transplant patients were included in this retrospective study. Selection was based on Renal Index Biopsies, classified according to Banff within 3 months before treatment. Patients were categorized into responders (R) and nonresponders (NR) depending on whether they returned to dialysis within 6 months after initiation of rituximab treatment. Clinical, histopathologic (Banff classification) and serologic parameters were compared between both groups by t test, Mann-Whitney U test, or likelihood ratio chi-square test. RESULTS: In comparisons between the groups, the R group showed a 1.5-fold higher level of estimated glomerular filtration rate and a fourfold lower level of proteinuria. By contrast, there were no differences in the histologic scores for chronic transplant lesions between the groups. The t and i scores were higher in NRs, whereas Banff-C4d scores of peritubular capillaries were increased in the Rs. Transplant biopsies in the Rs exhibited more CD138+ cell infiltrates. Serologic determination of human leukocyte antigen antibodies showed higher positivity for human leukocyte antigen class II donor-specific antibodies in the R group. No significant differences in other clinical criteria were found. CONCLUSION: Increased proteinuria, decreased graft function, and a higher grade of tubulitis and inflammation in AMR are negative predictors for responsiveness to rituximab therapy. Rituximab therapy therefore should be initiated in an early phase of AMR.


Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Rejeição de Enxerto/terapia , Antígenos HLA/imunologia , Imunossupressores/uso terapêutico , Isoanticorpos/sangue , Transplante de Rim/efeitos adversos , Plasmaferese , Anticorpos Monoclonais Murinos/efeitos adversos , Biomarcadores/sangue , Biópsia , Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/imunologia , Humanos , Imunossupressores/efeitos adversos , Seleção de Pacientes , Plasmaferese/efeitos adversos , Valor Preditivo dos Testes , Proteinúria/sangue , Proteinúria/imunologia , Estudos Retrospectivos , Fatores de Risco , Rituximab , Fatores de Tempo , Resultado do Tratamento
17.
J Clin Virol ; 61(4): 517-22, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25453330

RESUMO

BACKGROUND: Cytomegalovirus (CMV) infection of the gastrointestinal tract can cause CMV intestinal disease (CMV-ID), a severe complication in immunocompromised patients. Current gold standard for diagnosing CMV-ID requires the analysis of colon biopsies. Testing of fecal samples by CMV PCR might be a non-invasive diagnostic alternative, but data on this method is scarce. OBJECTIVES: To evaluate the use of quantitative CMV real-time PCR in fecal samples for diagnosing CMV-ID. STUDY DESIGN: Fecal samples and lower intestinal tract biopsies from 66 patients were analyzed by quantitative CMV PCR. To evaluate the diagnostic significance of CMV detection by PCR in fecal samples, patients were classified according to the etiology of their intestinal disease (based on results of endoscopy, histopathology and quantitative CMV DNA detection in biopsies) into three groups: "CMV-ID", "non-CMV-ID", and "equivocal". RESULTS: 10/66 fecal samples were tested positive by quantitative CMV PCR, but CMV DNA loads were low (range <1000-11,000copies/ml). CMV detection by PCR in fecal samples was positive in 8/12 patients of the CMV-ID group, resulting in a sensitivity of 67% for diagnosing CMV-ID. With two exceptions, fecal CMV PCR was negative in the non-CMV-ID group (45/47) indicating a good specificity (96%). Moreover, CMV DNA detection in feces was associated with high CMV DNA levels in intestinal biopsies. CONCLUSIONS: Negative CMV PCR results from fecal samples cannot exclude CMV-ID and thus have to be confirmed by analyzing intestinal biopsies. However, positive fecal PCR results are diagnostically useful and might help to circumvent invasive diagnostic procedures as endoscopy.


Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , Fezes/virologia , Enteropatias/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Criança , Pré-Escolar , Estudos de Coortes , Colo/virologia , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Humanos , Lactente , Enteropatias/virologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
18.
Thromb Haemost ; 111(6): 1077-88, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24500083

RESUMO

The antioxidant enzyme heme oxygenase (HO)-1, which catalyses the first and rate-limiting step of heme degradation, has major anti-inflammatory and immunomodulatory effects via its cell-type-specific functions in the endothelium. In the current study, we investigated whether the key endothelial adhesion and signalling receptor PECAM-1 (CD31) might be involved in the regulation of HO-1 gene expression in human endothelial cells (ECs). To this end PECAM-1 expression was down-regulated in human umbilical vein ECs (HUVECs) by an adenoviral vector-based knockdown approach. PECAM-1 knockdown markedly induced HO-1, but not the constitutive HO isoform HO-2. Nuclear translocation of the transcription factor NF-E2-related factor-2 (Nrf2), which is a master regulator of the inducible antioxidant cell response, and intracellular levels of reactive oxygen species (ROS) were increased in PECAM-1-deficient HUVECs, respectively. PECAM-1-dependent HO-1 regulation was also examined in PECAM-1 over-expressing Chinese hamster ovary and murine L-cells. Endogenous HO-1 gene expression and reporter gene activity of transiently transfected luciferase HO-1 promoter constructs with Nrf2 target sequences were decreased in PECAM-1 over-expressing cells. Moreover, a regulatory role of ROS for HO-1 regulation in these cells is demonstrated by studies with the antioxidant N-acetylcysteine and exogenous hydrogenperoxide. Finally, direct interaction of PECAM-1 with a native complex of its binding partner NB1 (CD177) and serine proteinase 3 (PR3) from human neutrophils, markedly induced HO-1 expression in HUVECs. Taken together, we demonstrate a functional link between HO-1 gene expression and PECAM-1 in human ECs, which might play a critical role in the regulation of inflammation.


Assuntos
Células Endoteliais/metabolismo , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoantígenos/metabolismo , Células L , Camundongos , Mieloblastina/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Regulação para Cima
19.
Virchows Arch ; 464(2): 203-11, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24374461

RESUMO

According to the Banff guidelines for renal transplants, pure endothelialitis without any tubulointerstitial infiltrates (with the Banff components v ≥ 1, i0, t0) has to be called acute cellular rejection (ACR). The pathophysiology of this rare lesion abbreviated as v_only is currently unclear, as well as its clinical, serological, and prognostic implications. Therefore, we conducted this retrospective comparative study. We compared all 23 biopsies with v_only from Hannover Medical School between 2003 and 2010 with 23 matched biopsies with the Banff components v ≥ 1, i ≥ 1, and t ≥ 1 (v_plus) and 23 biopsies with v0, i0, and t0 (v0i0t0). Serological (available in 10, 11, and 14 patients, respectively), histological, and clinical data were compared. Of all biopsies, 0.4 % had findings of v_only. v_only, v_plus, and v0i0t0 only showed minimal differences in the Banff components apart from the cohort-defining components. Endothelialitis in v_only more frequently involved the arcuate arteries than the smaller preglomerular vessels compared to v_plus and vice versa. Combining histopathological data and serological data, v_only more frequently showed criteria for acute humoral rejection than v0i0t0 (albeit not persistent after the Bonferroni-Holm correction in pairwise comparisons), while there was no difference between v_only and v_plus. No difference could be demonstrated regarding clinical presentation at biopsy or outcome. Our results show minimal differences regarding clinical presentation, outcome, and histological features between v_only and v_plus. Patients with v_only should be thoroughly investigated for evidence of acute humoral rejection.


Assuntos
Rejeição de Enxerto/patologia , Transplante de Rim/efeitos adversos , Doença Aguda , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Transplante Homólogo
20.
Am J Physiol Renal Physiol ; 305(3): F314-22, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23678046

RESUMO

Early endothelial outgrowth cells (eEOCs) reproducibly have been shown to act protectively in acute ischemic kidney injury (AKI) and chronic kidney injury. Bone morphogenetic protein-5 (BMP-5) acted antifibrotically in human hypertensive nephropathy. The aim of the current study was to analyze effects of BMP-5 treatment in an eEOC-based therapy of murine AKI and 5/6-nephrectomy. Male C57/Bl6N mice were either subjected to unilateral renal artery clamping postuninephrectomy or to 5/6-nephrectomy. Untreated or BMP-5-pretreated murine eEOCs were injected into recipient animals at the time of reperfusion (AKI) or at 2 and 5 days after 5/6-nephrectomy. Analysis of renal function and morphology was performed at 48 h and at 6 wk (AKI) or at 8 wk (5/6 model). Cellular consequences of eEOC treatment were evaluated using different in vitro assays. AKI was mitigated significantly by injecting BMP-5-pretreated eEOCs. Renal function was improved at 48 h [corrected] after cell therapy. In 5/6-nephrectomy, the cells failed to act renoprotectively, [corrected] but proteinuria was reduced after administering untreated eEOCs." Next, the original version read as "BMP-5 acts as a potent eEOC agonist in murine AKI in the short [corrected] term. Cell effects in 5/6-nephrectomy are heterogenous, but untreated cells act antifibrotically [corrected] without any impact on EnMT.


Assuntos
Injúria Renal Aguda/patologia , Proteína Morfogenética Óssea 5/fisiologia , Células Endoteliais/fisiologia , Insuficiência Renal Crônica/patologia , Animais , Apoptose/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Transição Epitelial-Mesenquimal/fisiologia , Mediadores da Inflamação/fisiologia , Testes de Função Renal , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Necrose , Nefrectomia , Proteinúria/metabolismo
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