Orientadores: Márcio Lopes Miranda, Joaquim Murray Bustorff Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:35:16Z (GMT). No. of bitstreams: 1 Barros_RogerioFortunatode_D.pdf: 7693178 bytes, checksum: 55f02301a0933dc093626f6cdc21d65b (MD5) Previous issue date: 2012 / Resumo: Objetivo: Elevações da pressão intra-abdominal durante o pneumoperitônio podem ocasionar oligúria ou anúria em mamíferos. Possível lesão renal decorrente ainda não foi bem documentada na literatura médica. O objetivo deste trabalho é avaliar o impacto do pneumoperitônio no parênquima renal em um modelo experimental de ratos jovens, através da expressão da neutrophil gelatinase-associated lipocalin (N-GAL), um biomarcador de lesão renal precoce. Materiais e Métodos: Vinte ratos machos jovens Sprague-Dowley foram utilizados no trabalho. Dezesseis ratos foram previamente anestesiados, traqueostomizados, flebotomizados e ventilados mecanicamente. Para análise, foram distribuídos em 4 grupos: Pneumoperitônio 1hora(h), Controle 1h, Pneumoperitônio 2h, Controle 2h. O quinto grupo, de quatro ratos, foi submetido à lesão renal através da administração de cisplatina para testar o biomarcador. Após 24h, todos os ratos foram submetidos à coleta de urina por 2 horas em gaiola metabólica; nefrectomia esquerda para quantificação por western blotting e nefrectomia direita para qualificação por immunofluorescência utilizando o biomarcador N-GAL Resultados: Os resultados foram analisados em 5 grupos de 4 ratos: Pneumoperitônio 1 e 2h, Controle 1 e 2h e Cisplatina. A expressão do N-GAL estava significantemente aumentada no grupo Cisplatina. Não houve diferenças estatisticamente significantes entre os grupos Pneumoperitônio 1 e 2h e Controle 1 e 2h (P>0,05). . Conclusão: O Pneumoperitônio controlado de 1 e 2 horas em ratos não promoveu lesão renal aguda / Abstract: Objective: Elevations of intra-abdominal pressure during pneumoperitoneum can lead to oliguria or anuria in mammals. Consequent kidney injury has not been well demonstrated in the literature. The aim of this study is to investigate the post-operative kidney status after pneumoperitoneum in a rat model through expression of neutrophil gelatinase-associated lipocalin (N-GAL), an early kidney injury biomarker. Materials and methods: Twenty male Sprague-Dowley rats were used in this experiment. Sixteen rats were previously anesthetized, tracheostomized, phlebotomized and mechanically ventilated were distributed in 4 groups: Pneumoperitoneum 1hour (h), Control 1h, Pneumoperitoneum 2h and Control 2h. The fifth group, composed of four rats, was kidney injuried with cisplatine to test the biomarker. After 24 hours all rats were submitted to a urine 2 hours output measurement, left nefrectomy to western blotting quantification and a right nefrectomy to immunofluorescence qualification of N-GAL. Results: The results were analyzed within 5 groups: Pneumoperitoneum 1 and 2h, Control 1 and 2h and Cisplatine group. The N-Gal expression was increased in the Cisplatine group. There weren't significant statistical difference between Pneumoperitoneum 1 and 2h and Control 1 and 2h groups (P>0,05). Conclusion: The 1 and 2hours controlled pneumoperitoneum isn't related to acute renal injury / Doutorado / Ciências da Cirurgia / Doutor em Ciências

INTRODUÇÃO: Lesão renal aguda (LRA) é uma complicação comum da cirrose frequentemente desencadeada por infecções bacterianas. A mortalidade de pacientes com cirrose e LRA varia de 10 a 100% a depender do estádio da cirrose, etiologia e progressão da LRA e tratamento recebido. Pacientes com LRA que progride possuem mortalidade intra-hospitalar consideravelmente maior do que aqueles que não progridem. Os critérios diagnósticos da LRA baseiam-se na creatinina sérica. Porém, esse biomarcador tem acurácia diagnóstica limitada, já que demora até 48 horas para se alterar e não distingue a etiologia da LRA. Essa última limitação é particularmente importante em pacientes com suspeita de síndrome hepatorrenal (SHR), já que o seu tratamento envolve o uso de medicações de alto custo (albumina e terlipressina) e eficácia limitada. O NGAL (neutrophil gelatinase-associated lipocalin) é um biomarcador de necrose dos túbulos renais e sua dosagem tem sido proposta como marcador diagnóstico mais acurado da LRA na cirrose, pois se altera mais precocemente e seus níveis urinários variam conforme a etiologia e gravidade da LRA. OBJETIVOS: Os objetivos primários do trabalho foram avaliar a acurácia do NGAL urinário para predizer: a) desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; b) resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR. Os objetivos secundários foram: a) acurácia de marcadores de disfunção hemodinâmica em cirrose (atividade plasmática de renina e noradrenalina sérica) na predição do desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; b) acurácia de marcadores de função hepática, parâmetros hemodinâmicos, marcadores inflamatórios e testes laboratoriais de lesão renal tradicionalmente utilizados na prática clínica na predição do desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; c) acurácia de marcadores de disfunção hemodinâmica em cirrose (atividade plasmática de renina e noradrenalina sérica) na predição de resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR; d) acurácia de marcadores de função hepática, parâmetros hemodinâmicos, marcadores inflamatórios e testes laboratoriais de lesão renal tradicionalmente utilizados na prática clínica na predição de resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR; e) comparar o conceito tradicional de LRA em cirrose com a nova classificação ICA-AKI para predizer mortalidade intra-hospitalar, em 30 e 90 dias em pacientes com cirrose e infecção bacteriana; f) acurácia do NGAL para predizer mortalidade intra-hospitalar em pacientes com cirrose e infecção bacteriana. PACIENTES E MÉTODOS: Critérios de inclusão: a) cirrose; b) ascite e/ou hidrotórax hepático; c) idade maior que 18 anos; d) concordância em participar no estudo; e) LRA e/ou infecção bacteriana. Critérios de exclusão: a) comorbidades graves; b) choque; c) nefropatia intrínseca; d) uso de drogas nefrotóxicas; e) diálise prévia; f) transplante hepático. Foram coletadas amostras de urina para dosagem de NGAL e sangue para dosagem de atividade plasmática de renina e noradrenalina sérica no momento da inclusão do paciente no estudo. Os pacientes com SHR receberam o tratamento padrão atual com albumina e terlipressina. RESULTADOS: Foram incluídos 199 pacientes: 179 com infecção bacteriana para avaliação de desenvolvimento ou progressão da LRA e 58 com SHR para avaliação da resposta ao tratamento (38 pacientes foram avaliados nas duas partes do estudo). O NGAL urinário apresentou associação com a progressão da LRA (AUC: 0,67; p=0,002), mas não com o seu desenvolvimento (p=0,973). Outras variáveis associadas à progressão da LRA foram INR (p=0,033), MELD (p=0,012), FEUr (p=0,026) e relação proteinúria/creatinina urinária (p=0,023). Houve associação entre NGAL urinário e a resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR (AUC: 0,70; p=0,007). Outras variáveis associadas à resposta ao tratamento da SHR foram INR (p=0,028), MELD (p=0,004) e relação proteinúria/creatinina urinária (p=0,003). Combinando o MELD com o NGAL urinário foi possível identificar subgrupos de pacientes com taxas de resposta ao tratamento com albumina e terlipressina distintas (9,1% x 48,1% x 80,0%, p < 0,001). Tanto o conceito tradicional de LRA na cirrose quanto a classificação ICA-AKI foram capazes de predizer mortalidade intra-hospitalar, em 30 e 90 dias (p < 0,05). O NGAL urinário também foi capaz de predizer mortalidade intra-hospitalar (AUC: 0,71; p < 0,001). CONCLUSÕES: a) NGAL urinário aumentado foi associado à progressão da LRA em pacientes com cirrose e infecção bacteriana; b) atividade plasmática de renina e noradrenalina sérica não se correlacionaram ao desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; c) NGAL urinário foi preditor de resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR; d) atividade plasmática de renina e noradrenalina sérica não se correlacionaram à resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR; e) tanto o conceito tradicional de LRA em cirrose quanto a classificação ICA-AKI mostraram-se adequados para predizer mortalidade intra-hospitalar, em 30 e 90 dias em pacientes com cirrose e infecção bacteriana; f) NGAL urinário aumentado foi associado à maior mortalidade intra-hospitalar em pacientes com cirrose e infecção bacteriana / INTRODUCTION: Acute kidney injury (AKI) is a frequent complication of cirrhosis, and is often triggered by bacterial infection. The mortality of patients with cirrhosis and AKI varies from 10 to 100%, depending on the stage of cirrhosis, etiology and progression of AKI, and treatment. Patients with AKI that progresses have a higher in-hospital mortality than those with AKI that does not progress. AKI diagnostic criteria are based on serum creatinine. However, this biomarker has limited diagnostic accuracy, taking up to 48 hours to rise, and does not distinguish the etiology of AKI. This latter limitation is particularly important in patients with suspected hepatorenal syndrome (HRS), because treatment involves high cost medication (albumin and terlipressin) with limited efficacy. NGAL (neutrophil gelatinase-associated lipocalin) is a biomarker of renal tubular necrosis which has been proposed as a more accurate AKI biomarker in cirrhosis because it rises earlier than creatinine and its urinary levels vary according to the etiology of AKI. OBJECTIVES: The primary endpoints were the establishment of the accuracy of urinary NGAL to predict: a) development or progression of AKI in patients with cirrhosis and bacterial infection; b) response to treatment with albumin and terlipressin in patients with diagnosed HRS. The secondary endpoints were: a) accuracy of biomarkers of hemodynamic dysfunction in cirrhosis (renin plasma activity and serum noradrenaline) to predict AKI development or progression in patients with cirrhosis and bacterial infection; b) accuracy of biomarkers of hepatic function, hemodynamics, inflammation and kidney injury routinely used in clinical practice to predict AKI development or progression in patients with cirrhosis and bacterial infection; c) accuracy of biomarkers of hemodynamic dysfunction in cirrhosis (renin plasma activity and serum noradrenaline) to predict response to treatment with albumin and terlipressin in patients with diagnosis of HRS; d) accuracy of biomarkers of hepatic function, hemodynamics, inflammation and kidney injury routinely used in clinical practice to predict response to treatment with albumin and terlipressin in patients with diagnosis of HRS; e) compare the traditional definition of AKI in cirrhosis with the new classification ICA-AKI to predict in-hospital, 30-day and 90-day mortality in patients with cirrhosis and bacterial infection. f) accuracy of urinary NGAL in predicting in-hospital mortality in patients with cirrhosis and bacterial infection. PATIENTS AND METHODS: Inclusion criteria: a) diagnosis of cirrhosis; b) presence of ascites and/or hepatic hydrothorax; c) age over 18 years old; d) consent to participate in the study; e) AKI and/or bacterial infection. Exclusion criteria: a) severe systemic comorbidities; b) shock; c) intrinsic nephropathy; d) nephrotoxic drug use; e) previous dialysis; f) liver transplantation recipient. Urine and blood samples were collected for urinary NGAL and renin plasma activity and serum noradrenaline measurement at the inclusion. Patients with HRS received standard treatment with albumin and terlipressin. RESULTS: 199 patients were included: 179 with bacterial infection for the analysis of AKI development or progression and 58 with HRS for the analysis of response to treatment (38 patients were analyzed in both parts of the study). Urinary NGAL was associated with AKI progression (AUC: 0.67, p=0.002), but not with AKI development (p=0.973). Other variables associated with AKI progression were INR (p=0.003), MELD (p=0.012), FEUr (p=0.026) and proteinuria/urinary creatinine ratio (p=0.023). There was also an association of urinary NGAL with response to HRS treatment with albumin and terlipressin (AUC: 0.70, p=0.007). Other variables associated with response to HRS treatment were INR (p=0.028), MELD (p=0.004) and proteinuria/urinary creatinine ratio (p=0.003). Combining MELD and urinary NGAL we could identify subgroups of patients with distinct response rates to treatment with albumin and terlipressin (9.1% x 48.1% x 80.0%, p < 0.001). Both the traditional definition of AKI in cirrhosis and the classification ICA-AKI predicted in-hospital, 30-day and 90-day mortality (p < 0.05). Urinary NGAL was also associated with in-hospital mortality (AUC: 0.71, p < 0.001). CONCLUSIONS: a) High urinary levels of NGAL were associated with AKI progression in patients with cirrhosis and bacterial infection; b) renin plasma activity and serum noradrenaline were not associated with AKI development or progression in patients with cirrhosis and bacterial infection; c) urinary NGAL was associated with response to combined treatment with albumin and terlipressin in patients with HRS; d) renin plasma activity and serum noradrenaline were not associated with response to combined treatment with albumin and terlipressin in patients with HRS; e) both traditional definition ok AKI in cirrhosis and ICA-AKI classification were able to predict in-hospital, 30-day and 90-day mortality in patients with cirrhosis and bacterial infection; f) high urinary levels of NGAL were associated with in-hospital mortality in patients with cirrhosis and bacterial infection

LOPAP, proteína isolada da toxina de lagartas Lonomia obliqua, possui ação ativadora de protrombina, efeito pró-coagulante e ação citoprotetora em células do endotélio humano, em cultura. Tem cadeia única com 181 resíduos de aminoácidos e 21 kDa. Sua estrutura terciária é formada por oito folhas-b fechadas em uma extremidade, mantidas juntas por pontes de hidrogênio, em formato de barril. Está classificada como pertencente ao grupo das Lipocalinas (proteínas de transporte). Neste trabalho estudou-se o LOPAP, que foi produzido recombinante em cultivo de Pichia pastoris em biorreator e purificado. Avaliou-se sua estabilidade quanto às atividades enzimática e citoprotetora, e sua estrutura secundária. Não foi detectada ativação de protrombina para o r-LOPAP obtido, mas foi observada ação citoprotetora. Considerando estes resultados e a análise de sua estrutura secundária por dicroísmo circular, concluiu-se que a proteína foi expressa com tamanho e sequência corretos, mas sem uma estrutura terciária correta, o que é determinante para a atividade enzimática. / LOPAP, a protein isolated from the toxin of Lonomia obliqua caterpillars, has prothrombin activation action, procoagulant effect and cytoprotection action in human endothelium cells culture. It has only chain with 181 amino acid residues and 21 kDa of size. Its tertiary structure is made by eight b-sheets closed at one end, hold together by hydrogen bonds, barrel-shaped. It is classified as belonging to the Lipocalin group (proteins of transport). This work studied the LOPAP, which was produced recombinant in Pichia pastoris culture in bioreactor, was purified, and it was evaluated its stability related to enzymatic and cytoprotection activities, and its secondary structure. It was not detected prothrombin activation for the r-LOPAP obtained, but it was observed a cytoprotective effect. Regarding these results and the analysis of its secondary structure, by circular dichroism, it was concluded that the protein was expressed with correct size and sequence, but without a correct tertiary structure, which is determinant for the enzymatic activity.

INTRODUÇÃO: Lesão renal aguda (LRA) é uma complicação comum da cirrose frequentemente desencadeada por infecções bacterianas. A mortalidade de pacientes com cirrose e LRA varia de 10 a 100% a depender do estádio da cirrose, etiologia e progressão da LRA e tratamento recebido. Pacientes com LRA que progride possuem mortalidade intra-hospitalar consideravelmente maior do que aqueles que não progridem. Os critérios diagnósticos da LRA baseiam-se na creatinina sérica. Porém, esse biomarcador tem acurácia diagnóstica limitada, já que demora até 48 horas para se alterar e não distingue a etiologia da LRA. Essa última limitação é particularmente importante em pacientes com suspeita de síndrome hepatorrenal (SHR), já que o seu tratamento envolve o uso de medicações de alto custo (albumina e terlipressina) e eficácia limitada. O NGAL (neutrophil gelatinase-associated lipocalin) é um biomarcador de necrose dos túbulos renais e sua dosagem tem sido proposta como marcador diagnóstico mais acurado da LRA na cirrose, pois se altera mais precocemente e seus níveis urinários variam conforme a etiologia e gravidade da LRA. OBJETIVOS: Os objetivos primários do trabalho foram avaliar a acurácia do NGAL urinário para predizer: a) desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; b) resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR. Os objetivos secundários foram: a) acurácia de marcadores de disfunção hemodinâmica em cirrose (atividade plasmática de renina e noradrenalina sérica) na predição do desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; b) acurácia de marcadores de função hepática, parâmetros hemodinâmicos, marcadores inflamatórios e testes laboratoriais de lesão renal tradicionalmente utilizados na prática clínica na predição do desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; c) acurácia de marcadores de disfunção hemodinâmica em cirrose (atividade plasmática de renina e noradrenalina sérica) na predição de resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR; d) acurácia de marcadores de função hepática, parâmetros hemodinâmicos, marcadores inflamatórios e testes laboratoriais de lesão renal tradicionalmente utilizados na prática clínica na predição de resposta ao tratamento combinado com albumina e terlipressina em pacientes com diagnóstico estabelecido de SHR; e) comparar o conceito tradicional de LRA em cirrose com a nova classificação ICA-AKI para predizer mortalidade intra-hospitalar, em 30 e 90 dias em pacientes com cirrose e infecção bacteriana; f) acurácia do NGAL para predizer mortalidade intra-hospitalar em pacientes com cirrose e infecção bacteriana. PACIENTES E MÉTODOS: Critérios de inclusão: a) cirrose; b) ascite e/ou hidrotórax hepático; c) idade maior que 18 anos; d) concordância em participar no estudo; e) LRA e/ou infecção bacteriana. Critérios de exclusão: a) comorbidades graves; b) choque; c) nefropatia intrínseca; d) uso de drogas nefrotóxicas; e) diálise prévia; f) transplante hepático. Foram coletadas amostras de urina para dosagem de NGAL e sangue para dosagem de atividade plasmática de renina e noradrenalina sérica no momento da inclusão do paciente no estudo. Os pacientes com SHR receberam o tratamento padrão atual com albumina e terlipressina. RESULTADOS: Foram incluídos 199 pacientes: 179 com infecção bacteriana para avaliação de desenvolvimento ou progressão da LRA e 58 com SHR para avaliação da resposta ao tratamento (38 pacientes foram avaliados nas duas partes do estudo). O NGAL urinário apresentou associação com a progressão da LRA (AUC: 0,67; p=0,002), mas não com o seu desenvolvimento (p=0,973). Outras variáveis associadas à progressão da LRA foram INR (p=0,033), MELD (p=0,012), FEUr (p=0,026) e relação proteinúria/creatinina urinária (p=0,023). Houve associação entre NGAL urinário e a resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR (AUC: 0,70; p=0,007). Outras variáveis associadas à resposta ao tratamento da SHR foram INR (p=0,028), MELD (p=0,004) e relação proteinúria/creatinina urinária (p=0,003). Combinando o MELD com o NGAL urinário foi possível identificar subgrupos de pacientes com taxas de resposta ao tratamento com albumina e terlipressina distintas (9,1% x 48,1% x 80,0%, p < 0,001). Tanto o conceito tradicional de LRA na cirrose quanto a classificação ICA-AKI foram capazes de predizer mortalidade intra-hospitalar, em 30 e 90 dias (p < 0,05). O NGAL urinário também foi capaz de predizer mortalidade intra-hospitalar (AUC: 0,71; p < 0,001). CONCLUSÕES: a) NGAL urinário aumentado foi associado à progressão da LRA em pacientes com cirrose e infecção bacteriana; b) atividade plasmática de renina e noradrenalina sérica não se correlacionaram ao desenvolvimento ou progressão da LRA em pacientes com cirrose e infecção bacteriana; c) NGAL urinário foi preditor de resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR; d) atividade plasmática de renina e noradrenalina sérica não se correlacionaram à resposta ao tratamento combinado com albumina e terlipressina em pacientes com SHR; e) tanto o conceito tradicional de LRA em cirrose quanto a classificação ICA-AKI mostraram-se adequados para predizer mortalidade intra-hospitalar, em 30 e 90 dias em pacientes com cirrose e infecção bacteriana; f) NGAL urinário aumentado foi associado à maior mortalidade intra-hospitalar em pacientes com cirrose e infecção bacteriana / INTRODUCTION: Acute kidney injury (AKI) is a frequent complication of cirrhosis, and is often triggered by bacterial infection. The mortality of patients with cirrhosis and AKI varies from 10 to 100%, depending on the stage of cirrhosis, etiology and progression of AKI, and treatment. Patients with AKI that progresses have a higher in-hospital mortality than those with AKI that does not progress. AKI diagnostic criteria are based on serum creatinine. However, this biomarker has limited diagnostic accuracy, taking up to 48 hours to rise, and does not distinguish the etiology of AKI. This latter limitation is particularly important in patients with suspected hepatorenal syndrome (HRS), because treatment involves high cost medication (albumin and terlipressin) with limited efficacy. NGAL (neutrophil gelatinase-associated lipocalin) is a biomarker of renal tubular necrosis which has been proposed as a more accurate AKI biomarker in cirrhosis because it rises earlier than creatinine and its urinary levels vary according to the etiology of AKI. OBJECTIVES: The primary endpoints were the establishment of the accuracy of urinary NGAL to predict: a) development or progression of AKI in patients with cirrhosis and bacterial infection; b) response to treatment with albumin and terlipressin in patients with diagnosed HRS. The secondary endpoints were: a) accuracy of biomarkers of hemodynamic dysfunction in cirrhosis (renin plasma activity and serum noradrenaline) to predict AKI development or progression in patients with cirrhosis and bacterial infection; b) accuracy of biomarkers of hepatic function, hemodynamics, inflammation and kidney injury routinely used in clinical practice to predict AKI development or progression in patients with cirrhosis and bacterial infection; c) accuracy of biomarkers of hemodynamic dysfunction in cirrhosis (renin plasma activity and serum noradrenaline) to predict response to treatment with albumin and terlipressin in patients with diagnosis of HRS; d) accuracy of biomarkers of hepatic function, hemodynamics, inflammation and kidney injury routinely used in clinical practice to predict response to treatment with albumin and terlipressin in patients with diagnosis of HRS; e) compare the traditional definition of AKI in cirrhosis with the new classification ICA-AKI to predict in-hospital, 30-day and 90-day mortality in patients with cirrhosis and bacterial infection. f) accuracy of urinary NGAL in predicting in-hospital mortality in patients with cirrhosis and bacterial infection. PATIENTS AND METHODS: Inclusion criteria: a) diagnosis of cirrhosis; b) presence of ascites and/or hepatic hydrothorax; c) age over 18 years old; d) consent to participate in the study; e) AKI and/or bacterial infection. Exclusion criteria: a) severe systemic comorbidities; b) shock; c) intrinsic nephropathy; d) nephrotoxic drug use; e) previous dialysis; f) liver transplantation recipient. Urine and blood samples were collected for urinary NGAL and renin plasma activity and serum noradrenaline measurement at the inclusion. Patients with HRS received standard treatment with albumin and terlipressin. RESULTS: 199 patients were included: 179 with bacterial infection for the analysis of AKI development or progression and 58 with HRS for the analysis of response to treatment (38 patients were analyzed in both parts of the study). Urinary NGAL was associated with AKI progression (AUC: 0.67, p=0.002), but not with AKI development (p=0.973). Other variables associated with AKI progression were INR (p=0.003), MELD (p=0.012), FEUr (p=0.026) and proteinuria/urinary creatinine ratio (p=0.023). There was also an association of urinary NGAL with response to HRS treatment with albumin and terlipressin (AUC: 0.70, p=0.007). Other variables associated with response to HRS treatment were INR (p=0.028), MELD (p=0.004) and proteinuria/urinary creatinine ratio (p=0.003). Combining MELD and urinary NGAL we could identify subgroups of patients with distinct response rates to treatment with albumin and terlipressin (9.1% x 48.1% x 80.0%, p < 0.001). Both the traditional definition of AKI in cirrhosis and the classification ICA-AKI predicted in-hospital, 30-day and 90-day mortality (p < 0.05). Urinary NGAL was also associated with in-hospital mortality (AUC: 0.71, p < 0.001). CONCLUSIONS: a) High urinary levels of NGAL were associated with AKI progression in patients with cirrhosis and bacterial infection; b) renin plasma activity and serum noradrenaline were not associated with AKI development or progression in patients with cirrhosis and bacterial infection; c) urinary NGAL was associated with response to combined treatment with albumin and terlipressin in patients with HRS; d) renin plasma activity and serum noradrenaline were not associated with response to combined treatment with albumin and terlipressin in patients with HRS; e) both traditional definition ok AKI in cirrhosis and ICA-AKI classification were able to predict in-hospital, 30-day and 90-day mortality in patients with cirrhosis and bacterial infection; f) high urinary levels of NGAL were associated with in-hospital mortality in patients with cirrhosis and bacterial infection

LOPAP, proteína isolada da toxina de lagartas Lonomia obliqua, possui ação ativadora de protrombina, efeito pró-coagulante e ação citoprotetora em células do endotélio humano, em cultura. Tem cadeia única com 181 resíduos de aminoácidos e 21 kDa. Sua estrutura terciária é formada por oito folhas-b fechadas em uma extremidade, mantidas juntas por pontes de hidrogênio, em formato de barril. Está classificada como pertencente ao grupo das Lipocalinas (proteínas de transporte). Neste trabalho estudou-se o LOPAP, que foi produzido recombinante em cultivo de Pichia pastoris em biorreator e purificado. Avaliou-se sua estabilidade quanto às atividades enzimática e citoprotetora, e sua estrutura secundária. Não foi detectada ativação de protrombina para o r-LOPAP obtido, mas foi observada ação citoprotetora. Considerando estes resultados e a análise de sua estrutura secundária por dicroísmo circular, concluiu-se que a proteína foi expressa com tamanho e sequência corretos, mas sem uma estrutura terciária correta, o que é determinante para a atividade enzimática. / LOPAP, a protein isolated from the toxin of Lonomia obliqua caterpillars, has prothrombin activation action, procoagulant effect and cytoprotection action in human endothelium cells culture. It has only chain with 181 amino acid residues and 21 kDa of size. Its tertiary structure is made by eight b-sheets closed at one end, hold together by hydrogen bonds, barrel-shaped. It is classified as belonging to the Lipocalin group (proteins of transport). This work studied the LOPAP, which was produced recombinant in Pichia pastoris culture in bioreactor, was purified, and it was evaluated its stability related to enzymatic and cytoprotection activities, and its secondary structure. It was not detected prothrombin activation for the r-LOPAP obtained, but it was observed a cytoprotective effect. Regarding these results and the analysis of its secondary structure, by circular dichroism, it was concluded that the protein was expressed with correct size and sequence, but without a correct tertiary structure, which is determinant for the enzymatic activity.

Les maladies rénales chroniques sont devenues un enjeu majeur de santé publique. Qu’elle qu’en soit la cause initiale, la MRC est caractérisée par une réduction néphronique progressive, aboutissant au remplacement des néphrons sains par un tissu fibreux et au déclin de la fonction rénale. Les mécanismes de progression de la MRC sont encore mal compris, mais il a été suggéré que le développement des lésions tubulo-interstitielles joue un rôle essentiel dans le déclin de la fonction rénale. Deux éléments physiopathologiques cruciaux dans le développement de ces lésions sont représentés par (i) l’activation de la voie du récepteur à l’EGF (epidermal growth factor) (EGFR), et (ii) la protéinurie et ses conséquences pour les cellules tubulaires. Les médiateurs communs à ces deux phénomènes ne sont pas connus. Mon travail de thèse a consisté à caractériser une protéine commune à ces deux voies d’activation, ie la lipocaline2 (Lcn2), petite protéine de transport de fer, en étudiant ses voies d'activation et ses conséquences physiopathologiques. Nous montrons que le rôle pathologique de la voie de l’EGFR est gouverné par la surexpression de Lcn2. En effet, dans le contexte de réduction néphronique chirurgicale, (i) les animaux invalidés pour Lcn2 sont protégés du développement des lésions, et (ii) les souris exprimant un dominant négatif de l’EGFR dans le tubule rénal présentent une diminution de l’expression de Lcn2. Nous montrons également que l’invalidation de Lcn2 permet de ralentir la progression de la MRC dans un modèle de polykystose rénale dépendante de l’EGFR, les souris jck (juvenile cystic kidney). Parallèlement, nous montrons que la protéinurie induit également l’expression de Lcn2 dans les cellules tubulaires rénales dans différents modèles expérimentaux. De plus, nous montrons le rôle majeur de Lcn2 dans la progression de la MRC protéinurique, l’invalidation de Lcn2 limitant le développement des lésions rénales et la mortalité des animaux protéinuriques. Si le rôle délétère de Lcn2 est démontré dans différents modèles de néphropathie chronique, nous montrons que les voies moléculaires impliquées dans l’activation de Lcn2 et le rôle de cette protéine dépendent du contexte cellulaire. Nous prouvons que Lcn2 est un médiateur de l'effet mitogénique de l'EGFR, phénomène essentiel de la progression de la MRC, et nous montrons que l’activation de Lcn2 via l’EGFR est dépendante du facteur HIF1α. Cependant, nous démontrons également que l'expression de Lcn2 dans le contexte de protéinurie est dépendante du facteur ATF4 activé par le stress du reticulum endoplasmique (ER), et que Lcn2 est un médiateur de l'apoptose dépendante du stress de l'ER. Enfin, nous prouvons que l’inhibition pharmacologique du stress de l'ER permet une réduction de l’expression de Lcn2 dans les cellules tubulaires, et surtout, un ralentissement du déclin de la fonction rénale des animaux protéinuriques. Nous démontrons également l’importance de ces résultats chez les patients atteints de MRC. Nous identifions NGAL, l'analogue humain de Lcn2, comme un biomarqueur de progression dans la polykystose rénale dominante, et nous montrons qu’elle est fortement surexprimée dans le tissu rénal de patients protéinuriques. L’ensemble de ce travail permet de montrer que Lcn2 est un nouveau médiateur essentiel de multiples néphropathies chroniques. Lcn2 est impliquée dans l’effet mitogénique de l’EGFR ou la réponse apoptotique associée à la protéinurie durant la MRC. Nous ouvrons également de nouvelles perspectives thérapeutiques avec l'utilisation d'inhibiteurs du stress de l'ER dans les néphropathies protéinuriques humaines / Chronic kidney disease (CKD) is now a major public health concern. Whatever the initial kidney injury, CKD is characterized by progressive nephron reduction and kidney function decline. Tubulointerstitial lesions are an essential component of CKD progression, and are mediated by two crucial pathophysiologic elements: epidermal growth factor receptor (EGFR) activation, and proteinuria responsible of tubular cell damage. The aim of this study was to describe a common mediator of both these pathways, ie lipocalin2, an iron carrier protein, by identifying its activation pathways and its pathophysiologic consequences. We show the deleterious effects of the EGFR pathway during nephron reduction is mediated by the activation of Lcn2, which controls the mitogenic effect of EGFR. In fact, after nephron reduction, animals invalidated for Lcn2 are protected from lesions developpement. Moreover, a similar protective effect is seen in jck (juvenile cytic kidney) mice invalidated for Lcn2, a model of polycystic kidney disease EGFR-dependant. Otherwise, we show proteinuria induces Lcn2 expression in tubular cells of different experimental models, and Lcn2 invalidation slows lesion developpement and reduces mortality of proteinuric mice. We demonstrate that the Lcn2 role and activation pathways are dependant of these different models. We show Lcn2 is a mediator of the mitogenic effect of the EGFR, and Lcn2 activation is dependant of HIF1α stabilisation. However, we also show ATF4 is an activator of Lcn2 during endoplasmic reticulum (ER) stress induced by proteinuria in tubular cells. In this context, Lcn2 controls ER stress-induced apoptosis. Pharmacologic inhibition of ER stress in proteinuric animals decreases Lcn2 overexpression, and slows renal function decline. In patients suffering from CKD, we demonstrated NGAL (neutrophil gelatinase-associated lipocalin), the human analog of Lcn2, appears as a critical biomarker of autosomal dominant polycystic kidney disease progression. NGAL is also highly overexpressed in tubular cells in kidney biopsies of proteinuric patients. This work demonstrates Lcn2 is an essential mediator of multiple pathophysiologic components of CKD progression. Moreover, we open new therapeutic perspectives with the use ER stress modulators in proteinuric CKD

Adipositas und ihre Folgeerkrankungen stellen eine wachsende medizinische Herausforde- rung globalen Ausmaßes dar. Im Rahmen der Adipositasforschung wurde das Fettgewebe als endokrines Organ identifiziert. Von ihm sezernierte Proteine, die sogenannten Adipo- kine, beeinflussen maßgeblich Insulinresistenz und Gefäßverletzbarkeit. Adipositas geht im Fettgewebe mit einer subklinischen chronischen Entzündung einher, die zu einer erhöhten Sekretion von proinflammatorischen Adipokinen führt. Die verstärkte Anwesenheit dieser Proteine ist mit den Komplikationen der Adipositas assoziiert. Die vorliegende Arbeit be- fasst sich mit dem Einfluss von Interleukin (IL)-1β, einem wichtigen Entzündungsmediator des Organismus, auf die Sekretion der proinflammatorischen Adipokine tissue inhibitor of metalloproteinase (TIMP)-1, serum amyloid A (SAA)-3, Lipocalin-2 und Chemerin. Die zugrundeliegenden Untersuchungen wurden mit 3T3-L1- und braunen Adipozyten durch- geführt. Es erfolgte der Nachweis auf mRNA- sowie auf Proteinebene. Der Einsatz von spe- zifischen Inhibitoren erlaubte den Rückschluss auf grundlegende Signalwege. Für alle vier untersuchten Adipokine konnte eine signifikante dosis- und zeitabhängige Steige- rung der mRNA- und Proteinexpression durch IL-1β nachgewiesen werden. Die Transduktion des IL-1β-Signals erfolgte im Falle von Lipocalin-2 und SAA-3 über nuclear factor (NF)-κB und janus kinase (Jak)-2, bei TIMP-1 lediglich über Jak-2 und in Bezug auf Chemerin über NFκB, Jak-2, p44/42 mitogen-activated protein kinase und Phosphatidylinositol-3-Kinase. Die in dieser Arbeit nachgewiesenen Expressions- und Sekretionssteigerungen von TIMP-1, SAA-3, Lipocalin-2 und Chemerin in braunen und weißen Adipozyten festigen IL-1β als einen entscheidenden Mediator proinflammatorischer Prozesse im Fettgewebe. Eine umfassende Bewertung der Funktion von IL-1β im Fettgewebe, insbesondere im Zustand der Adipositas, muss jedoch in weitergehenden Studien erfolgen.

Introduction: L’approche endovasculaire pour la réparation d’anévrysmes aortiques s’associe à une utilisation importante de produit de contraste, qui peut causer une néphropathie induite par le produit de contraste (NIC) en postopératoire. L’hydratation intraveineuse peut réduire l’incidence de NIC, mais quel produit utiliser reste incertain. Nous avons évalué le bicarbonate de sodium, comparé au NaCl 0,9%, pour réduire l’incidence de NIC. Méthode: Nous avons mené une étude prospective, randomisée et contrôlée à double insu chez 34 patients subissant une chirurgie endovasculaire pour anévrysme aortique. Les patients des deux groupes (17 patients par groupe) ont reçu du bicarbonate de sodium ou du NaCl 0,9% à raison de 3 mL/kg/h pour une heure avant l’intervention puis 1 mL/kg/h jusqu’à 6 h après la fin de la chirurgie. Tous les patients ont reçu du N-acétylcystéine. L’objectif principal était l’incidence de NIC, définie comme une élévation de plus de 25% de la créatinine sérique 48 h suivant l’exposition au produit de contraste. Des biomarqueurs précoces de lésion rénale ont été mesurés. Résultats: Une NIC s’est développée chez 1 patient (5,88%) appartenant au groupe bicarbonate, comparé à aucun patient (0%) dans le groupe NaCl 0,9% (P = 0,31). Les biomarqueurs de lésion rénale étaient significativement augmentés dans les deux groupes après l’exposition au produit de contraste. Conclusions: Nous avons démontré un faible taux d’insuffisance rénale suivant une chirurgie endovasculaire aortique, que l’hydratation soit effectuée avec du bicarbonate ou du NaCl 0,9%, malgré une élévation des biomarqueurs de lésion rénale. / Background: The endovascular approach for the repair of aortic aneurysm involves the administration of large quantities of contrast media, which can cause contrast-induced nephropathy (CIN) in the post operative period. The only proven strategy to prevent CIN is intravenous hydration, but what type of infusion to use is not clear. We evaluated the efficacy of sodium bicarbonate, compared with NaCl 0.9%, to reduce the incidence of postoperative renal failure. Methods: We conducted a prospective, controlled, double-blind, randomized study in patients presenting for endovascular aortic aneurysm surgery. Patients in group A (n = 17) received sodium bicarbonate 3 mL/kg/h for 1 h before the procedure and then 1 mL/kg/h until 6 h after surgery, whereas patients in group B (n= 17) received the same amount of NaCl 0.9%. All patients received N-acetylcysteine. The primary end point was CIN, defined by serum creatinine greater than 25 % above baseline 48 h post operatively. Biomarkers of renal injury were measured. Results: CIN developed in one patient in the bicarbonate group (5,88%), compared with no patient in the NaCl 0,9% group (0%) (difference 5.88%;95% CI -5.3% to 17.06%, P = 0.31). Interleukin-18, N-acetyl-β-D-glucosaminidase and Kidney Injury Molecule-1 increased significantly in both groups after exposure to contrast media. Conclusions: We demonstrated a low rate of renal failure following endovascular aortic surgery using contrast media, regardless of whether bicarbonate or NaCl 0.9% was used for hydration, despite significant elevation in biomarkers of renal injury.

Introduction: L’approche endovasculaire pour la réparation d’anévrysmes aortiques s’associe à une utilisation importante de produit de contraste, qui peut causer une néphropathie induite par le produit de contraste (NIC) en postopératoire. L’hydratation intraveineuse peut réduire l’incidence de NIC, mais quel produit utiliser reste incertain. Nous avons évalué le bicarbonate de sodium, comparé au NaCl 0,9%, pour réduire l’incidence de NIC. Méthode: Nous avons mené une étude prospective, randomisée et contrôlée à double insu chez 34 patients subissant une chirurgie endovasculaire pour anévrysme aortique. Les patients des deux groupes (17 patients par groupe) ont reçu du bicarbonate de sodium ou du NaCl 0,9% à raison de 3 mL/kg/h pour une heure avant l’intervention puis 1 mL/kg/h jusqu’à 6 h après la fin de la chirurgie. Tous les patients ont reçu du N-acétylcystéine. L’objectif principal était l’incidence de NIC, définie comme une élévation de plus de 25% de la créatinine sérique 48 h suivant l’exposition au produit de contraste. Des biomarqueurs précoces de lésion rénale ont été mesurés. Résultats: Une NIC s’est développée chez 1 patient (5,88%) appartenant au groupe bicarbonate, comparé à aucun patient (0%) dans le groupe NaCl 0,9% (P = 0,31). Les biomarqueurs de lésion rénale étaient significativement augmentés dans les deux groupes après l’exposition au produit de contraste. Conclusions: Nous avons démontré un faible taux d’insuffisance rénale suivant une chirurgie endovasculaire aortique, que l’hydratation soit effectuée avec du bicarbonate ou du NaCl 0,9%, malgré une élévation des biomarqueurs de lésion rénale. / Background: The endovascular approach for the repair of aortic aneurysm involves the administration of large quantities of contrast media, which can cause contrast-induced nephropathy (CIN) in the post operative period. The only proven strategy to prevent CIN is intravenous hydration, but what type of infusion to use is not clear. We evaluated the efficacy of sodium bicarbonate, compared with NaCl 0.9%, to reduce the incidence of postoperative renal failure. Methods: We conducted a prospective, controlled, double-blind, randomized study in patients presenting for endovascular aortic aneurysm surgery. Patients in group A (n = 17) received sodium bicarbonate 3 mL/kg/h for 1 h before the procedure and then 1 mL/kg/h until 6 h after surgery, whereas patients in group B (n= 17) received the same amount of NaCl 0.9%. All patients received N-acetylcysteine. The primary end point was CIN, defined by serum creatinine greater than 25 % above baseline 48 h post operatively. Biomarkers of renal injury were measured. Results: CIN developed in one patient in the bicarbonate group (5,88%), compared with no patient in the NaCl 0,9% group (0%) (difference 5.88%;95% CI -5.3% to 17.06%, P = 0.31). Interleukin-18, N-acetyl-β-D-glucosaminidase and Kidney Injury Molecule-1 increased significantly in both groups after exposure to contrast media. Conclusions: We demonstrated a low rate of renal failure following endovascular aortic surgery using contrast media, regardless of whether bicarbonate or NaCl 0.9% was used for hydration, despite significant elevation in biomarkers of renal injury.

Conditions which promote reversible growth arrest, such as hypoxia and high cell density, lead to activation of a diverse network of proteins known as growth arrest specific (GAS) genes. Fatty acid binding protein 4 (FABP4), a lipid chaperone involved in the regulation of metabolic and inflammatory responses, has been shown to be part of the GAS program. While the induction of FABP4 in oxygen-deprived environments is well characterized, its functionality and regulation in such conditions remains unclear. In this study, we describe how mis-expression of FABP4 affects cell viability and survival within low oxygen conditions. Loss of FABP4 using shRNA was shown to be associated with a significant increase in oxidative stress and lipid peroxidation, a reduction in lipid droplet formation and a greater incidence of apoptosis. Hypoxia-mediated expression of FABP4 was also found to be positively correlated with cellular levels of C/EBP-beta, an essential activator of p20K in quiescence. FABP4 and p20K are both lipocalins that have been shown to share similar induction patterns and ability to assist in the maintenance of lipid trafficking in cellular stress circumstances. Unexpectedly, the depletion of FABP4 or p20K results in loss of the other in limited oxygen concentrations. This occurs independently of disruption to the broad GAS gene program, suggesting the two proteins may be co-regulated in a shared hypoxic-signalling pathway. C/EBP-beta appears to be the transcriptional activator shared by FABP4 and p20K in quiescence, and the three may be part of an intricate system to sense and respond to reactive oxygen species and lipid radicals. However, the forced expression of either FABP4 or p20K when the other is repressed only moderately restores cell survival through alleviating oxidative stress, indicating the two are both necessary for optimal response to hypoxia. In all, these studies suggest that analogous to the p20K lipocalin, FABP4 plays a critical role in lipid homeostasis and cell survival in conditions of limited oxygen concentrations, and its stimulation is dependent on C/EBP-beta activity. / Thesis / Master of Science (MSc) / A study investigating the role of FABP4 and p20K in conditions of reversible growth arrest with an emphasis on cell survival, lipid homeostasis and mitigating the effects of oxidative stress, and regulation of the two lipocalins by C/EBP-beta.