京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13060号 / 論医博第2118号 / 新制||医||1018(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 一山 智, 教授 古川 壽亮, 教授 小川 修 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’activation du récepteur minéralocorticoïde par l’aldostérone joue un rôle majeur dans le remodelage cardiovasculaire en participant à l’hypertension, à la fibrose et à l’inflammation. Notre laboratoire a récemment mis en évidence le rôle critique de la Neutrophil Gelatinase-Associated Lipocalin (NGAL), une nouvelle cible du MR, dans les effets délétères de son activation. Afin de mieux comprendre le rôle de NGAL dans ces effets, nous avons réalisé différents modèles profibrotiques chez des souris présentant une invalidation génique totale de NGAL ou une déplétion de NGAL dans leurs cellules immunitaires spécifiquement. Nos résultats démontrent un rôle primordial de la NGAL produite par les cellules immunitaires dans les lésions cardio-rénales induite par l’activation du MR. De plus, nos données suggèrent que le contexte inflammatoire pourrait être un élément déterminant de l’implication physiopathologique de NGAL. / Mineralocorticoid receptor (MR) activation by aldosterone plays a major role in cardiovascular remodeling by participating in hypertension, fibrosis and inflammation. Our group has recently evidenced a critical implication of the Neutrophil Gelatinase-Associated Lipocalin (NGAL), a new target of the MR, in the deleterious effects of its activation. In order to better understand the role of NGAL in these effects, we carried out several models of fibrosis in mice presenting a genetic invalidation for NGAL or in mice lacking NGAL in their immune cells specifically. Our results demonstrate that NGAL produced by immune cells plays a pivotal role in MR mediated cardiac and renal damage. Furthermore, our data suggest that inflammatory context could represent a key factor in the pathophysiological implications of NGAL.

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No. of bitstreams: 1 rocha_ep_me_bot.pdf: 1338051 bytes, checksum: 9bf40b5727bebead9b29d331b3ca8fbe (MD5) Previous issue date: 2016-12-16 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: O idoso se apresenta como principal grupo etário acometido pela lesão renal aguda (LRA) por possuir diversos fatores de risco para o seu desenvolvimento como a nefroesclerose senil, a presença de comorbidades, o uso de medicamentos nefrotóxicos e a susceptibilidade à sepse. Além disso, conforme se envelhece, perde-se massa muscular e por isso a creatinina plasmática basal do idoso pode ser mais baixa que o normal de modo a mascarar o aumento patológico decorrente de lesão renal e assim levar ao subdiagnóstico ou diagnóstico tardio, o que justifica a busca por biomarcadores de lesão mais precoce, como o NGAL. Não há estudos que avaliaram a LRA em pacientes sépticos e idosos admitidos em unidade de terapia intensiva (UTI). Objetivo: Este estudo teve como objetivo principal avaliar a eficácia do NGAL urinário (u) como predito diagnóstico e prognóstico da LRA associada à sepse em pacientes idosos em UTI. Pacientes e métodos: Foram estudados de modo prospectivos pacientes idosos admitidos com sepse ou que evoluíram com sepse em três UTIs do HC-FMB (UNESP) durante o período de 01 de outubro de 2014 a 20 de novembro de 2015. A avaliação de função renal foi realizada diariamente por meio da dosagem da creatinina sérica e verificação de débito urinário. A dosagem do NGALu foi realizada nas primeiras 48h do diagnóstico de sepse (amostra 1) e entre 48 e 96h (amostra 2). Os resultados foram apresentados utilizando estatística descritiva e área sob a curva ROC para determinar a capacidade do NGALu discriminar o diagnóstico e prognóstico da LRA. Todos os resultados dos testes de hipótese foram discutidos no nível de 5% de significância (p<0.05). Resultados: Foram incluídos 75 pacientes idosos sépticos, sendo que 47 (62,7%) desenvolveram LRA. Na regressão logística foram identificados como fatores associados à LRA a presença de DRC (OR= 0,051, IC=0,017-0,601, p=0,045), menor PAM à admissão (OR=0,816, IC=0,130– 0,472; p=0,047) e maior NGALur1 (OR= 2,002, IC=1,205-3,325, p=0,007). O NGAL u apresentou-se como excelente preditor diagnóstico de LRA, com área sob a curva >0,95, e sensibilidade e especificidade superiores a 0,89, antecipando o diagnóstico de LRA em 2,1±0,3 dias. Os fatores associados ao óbito na regressão logística foram a presença de LRA (OR= 2,14, IC= 1,421–3,982, p=0,04), a doença pulmonar obstrutiva crônica (OR=9,376, IC=1,790 – 49,1, p= 0,008) e o uso de droga vasoativa (OR= 2,066, IC=0,987 – 1,021, p=0,04). A acurácia do NGALu1 e 2 como preditor de óbito foi intermediária, com área sob a curva de 0,61 e 0,62; sensibilidade entre 0,65 e 0,77 e especificidade inferior a 0,6. Conclusão: O NGAL u mostrou-se excelente preditor de LRA em pacientes idosos sépticos em UTI, podendo antecipar o diagnóstico de LRA em 2,1 dias. / Background: The elderly is the main age group affected by acute kidney injury (AKI). There are several risk factors for its development as renal structural and functional degeneration, comorbidities, use of nephrotoxic drugs and susceptibility to sepsis. Besides that, in the process of ageing there is loss of muscular mass and therefore, the basal plasmatic creatinine levels of the elderly can be lower than the normal values, masking an increase in kidney injury pathologies, leading to delayed or late diagnosis, justifying the search for biomarkers of early injury biomarkers, such as NGAL. There are no studies that assessed AKI in elderly septic patients admitted to Intensive Care Units (ICUs). Objective: This study aimed to evaluate the efficacy of urinary NGAL (u) as predictor of diagnosis and prognosis in elderly septic patients admitted to ICUs. Patients and Methods: We prospectively studied elderly patients with sepsis admitted to ICUs from October 2014 to November 2015. Assessment of renal function was performed daily by serum creatinine and urine output. The dosage of NGALu was performed within the first 48 hours of the diagnosis of sepsis (NGAL 1) and between 48 and 96 hours (NGAL 2). The results were presented using descriptive statistics and area under the receiver operating characteristic curve (AUC-ROC) by comparing AKI patients with all non-AKI patients and by comparing survivor patients with those nonsurvivor patients. In all tests, differences were considered significant at 5%. Results: Seventy-five septic elderly patients were included, 47 (62.7%) developed AKI. In logistic regression were identified as factors associated with AKI the presence of chronic kidney injury (OR = 0.051, CI = 0.017 to 0.601, p = 0.045), lower mean blood pressure at admission (OR = 0.816, CI = 0,130- 0.472; p = 0.047) and higher NGALur1 (OR = 2.002, CI = 1.205 to 3.325, p = 0.007). The NGAL u was excellent predictor of AKI diagnosis (AUC-ROC > 0.95, and sensitivity and specificity greater than 0.89), anticipating the AKI diagnosis in 2.1 ± 0.3 days. Factors associated with mortality in the logistic regression were the presence of AKI (OR = 2.14, CI = 1.421 to 3.982, p = 0.04), chronic obstructive pulmonary disease (OR = 9.376, CI = 1.790 to 49.1, p = 0.008) and vasoactive drugs (OR = 2.066, CI = 0.987 to 1.021, p = 0.04). The accuracy of NGALu1 and 2 as predictors of death was intermediate, with AUC-ROC of 0.61 and 0.62; sensitivity between 0.65 and 0.77 and a specificity lower than 0.6. Conclusion: The uNGAL was excellent predictor of AKI in septic elderly patients in ICUs and can anticipate the diagnosis of AKI in 2.1 days. / FAPESP: 2012/16713-3

Orientador: Daniela Ponce / Resumo: Introdução: O idoso se apresenta como principal grupo etário acometido pela lesão renal aguda (LRA) por possuir diversos fatores de risco para o seu desenvolvimento como a nefroesclerose senil, a presença de comorbidades, o uso de medicamentos nefrotóxicos e a susceptibilidade à sepse. Além disso, conforme se envelhece, perde-se massa muscular e por isso a creatinina plasmática basal do idoso pode ser mais baixa que o normal de modo a mascarar o aumento patológico decorrente de lesão renal e assim levar ao subdiagnóstico ou diagnóstico tardio, o que justifica a busca por biomarcadores de lesão mais precoce, como o NGAL. Não há estudos que avaliaram a LRA em pacientes sépticos e idosos admitidos em unidade de terapia intensiva (UTI). Objetivo: Este estudo teve como objetivo principal avaliar a eficácia do NGAL urinário (u) como predito diagnóstico e prognóstico da LRA associada à sepse em pacientes idosos em UTI. Pacientes e métodos: Foram estudados de modo prospectivos pacientes idosos admitidos com sepse ou que evoluíram com sepse em três UTIs do HC-FMB (UNESP) durante o período de 01 de outubro de 2014 a 20 de novembro de 2015. A avaliação de função renal foi realizada diariamente por meio da dosagem da creatinina sérica e verificação de débito urinário. A dosagem do NGALu foi realizada nas primeiras 48h do diagnóstico de sepse (amostra 1) e entre 48 e 96h (amostra 2). Os resultados foram apresentados utilizando estatística descritiva e área sob a curva ROC para determinar ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

1 Abstract The thesis deals primarily with the Tibetan text of The Genealogy of Ya-ngal Family of Dolpo, which is an important document concerning the history of Western Tibet. The text itself has never been fully translated and there are also different manuscript versions of it, which contain partially different reading. The various manuscript versions have been compared and the critical edition of the text has been established. This formed the basis for translation of the entire text into English. This core of the dissertation is preceded by necessary background information on the region of Dolpo, its history and a significance of the role the Ya-ngal family played in it. Second chapter contains the translation of the Genealogy with detailed comments in the footnotes. The entire text has been divided into chapters, whose titles are given in the square brackets indicating thus that they are missing in the original text. The third chapter focuses mainly on the specific abbreviations of written words used across the entire text of the Genealogy, since there is hardly any literature in western languages dealing with this topic. This part is introduced by brief linguistic description of the Dolpo dialect of Tibetan. It is followed by general introduction of the abbreviating styles in Tibetan scriptures. The...

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

Sepsis and septic shock are conditions, with severe outcome or in many cases death. Sepsis is a systemic inflammatory response trigger by bacteraemia but systemic inflammatory response can also be triggered by major trauma, major surgery, pancreatitis, severe burns etc. The systemic inflammatory reaction initiating the evolvement of septic organ dysfunction can be modelled using endotoxin, a Gram-negative bacterial lipopolysaccharide. This thesis used a porcine experimental sepsis model to examine timing of the inflammatory response due to endotoxin infusion (Paper I) and the influence of steroid treatment on the inflammatory response in endotoxemic pigs (Paper II). Timing of steroid treatment and the role of neutrophil granulocyte activation was evaluated with pig specific NGAL assessing neutrophil activation (Paper III). A clinical observational study was performed with the aim to differentiate between sepsis and other inflammatory conditions (e.g. trauma due to major surgery) evaluated by calprotectin as a marker of neutrophil activation (Paper IV). There was a dose-dependency in endotoxin tolerance which was measured with TNF-a. Pre-exposure to endotoxin did not reduce the pulmonary response to endotoxemic challenge. In fact, both PaO2 / FiO2 and static pulmonary compliance were reduced in this group when pre-treated with endotoxin at low dose. Endotoxemic animals treated with hydrocortisone were more stable in circulatory variables than those without such treatment. This was not explained by an ability of steroids to modulate the production of NO (Nitric oxide), which has been suggested to be a mechanism of steroids in this aspect. Pre-treatment with hydrocortisone attenuated the neutrophil granulocyte response and consequently diminished the release of NGAL in plasma. Circulatory derangement was associated with high plasma NGAL levels. Urine NGAL levels did not differ among the four groups. Plasma calprotectin levels on ICU admission is a sensitive marker of systemic inflammation and are markedly increased in patients with sepsis and patients with systemic inflammatory response. Plasma Calprotectin performed better than any of the other inflammatory variables in predicting mortality at 30 days, except from the composite mortality prediction score, SAPS 3.

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20263号 / 医博第4222号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 小川 修, 教授 福原 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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