Made available in DSpace on 2016-01-26T12:51:13Z (GMT). No. of bitstreams: 1 pires_tese.pdf: 363791 bytes, checksum: af03ea5388a25575042d8de8b77224dd (MD5) Previous issue date: 2003-02-23 / Acute Renal Failure can be defined as an abrupt and sustained reduction in the glomerular filtration rate with a consequent retention of nitrogenous waste products. Despite the development in treatment, mortality remains high, varying beetween 50 and 70%. In hospitalised patients the incidence is about 5% but in respect to intensive care units it varies from 10 to 30%. In the last three decades, the characteristics of patients who suffer from acute renal failure changed dramatically. Before the advent of dialytic treatment, the main causes of mortality were uraemia, hyperkalaemia and the cardiac complications arising from volume overload. Nowadays, the causes are sepsis, cardiopulmonary failure, nephrotoxic drugs, and post renal transplantation complications. Multiple organ dysfunction, disseminated intravascular coagulation and diabetes mellitus are morbid conditions that can aggravate the prognosis of acute renal failure in intensive care units. Due to the high prevalence of diabetes mellitus in the population, this study intends to evaluate its influence in the morbidity and mortality of patients suffering from acute renal failure in the intensive care unit of Hospital de Base of São José do Rio Preto, Brazil was made in the period from January 1997 to December 2000. A total of 255 (25%) of the patients were diabetic and 765 (75%) were not. Demographic data, the presence of underlying diseases, aetiology, types, the clinical features and complications of acute renal failure were evaluated. Besides these, the presence of multiple organ failure syndrome was observed. In the study population 64% were male, 46% were more than 60 years old and 85% had one or more concomitant diseases. The ischaemic aetiology predominated in 53% of cases and a clinical cause was the most common type seen at 57%. The means and standard deviations of the Apache II score and creatinine levels (mg/dL) were 20.5 + 6.7 and 3.7 + 2.0 respectively. The prevalence of disseminated intravascular coagulation, shock, liver failure and respiratory failure were 32%, 69%, 15% and 79% respectively. Among the observed complications hyperkalaemia was seen in 35%, acidosis in 70%, sepsis in 61%, systemic arterial hypertension in 14%, bleeding in 22%, central nervous system disfunction in 44% and mortality in 71% of the cases. The demographic data, clinical features, morbidity and mortality due to acute renal failure of diabetic and non-diabetic individuals were compared. The hyperkalaemia, acidosis, respiratory failure, shock, central nervous system dysfunction, hypervolaemia and the bleeding were similar in both groups. A logistic regression analysis did not demonstrate a significant association between diabetes mellitus and mortality. An ischaemic aetiology, the failure of three or more organs, hyponatraemia and acidosis exhibited significant association between mortality and acute renal failure. In conclusion, the diabetic patients were older involving fewer men, with less oliguria, disseminated intravascular coagulation, hyponatraemia and liver failure than the non-diabetic individuals. Diabetes mellitus had no influence on the mortality due to acute renal failure in the intensive care unit. / A insuficiência renal aguda pode ser definida como uma redução abrupta e sustentada da taxa de filtração glomerular com conseqüente retenção de produtos nitrogenados. Apesar da evolução terapêutica, a sua mortalidade ainda continua elevada, variando entre 50 e 70%. Em pacientes hospitalizados, a sua incidência está próxima de 5% e, especificamente, em unidades de terapia intensiva, varia entre 10 e 30%. Nas últimas três décadas, as características dos pacientes acometidos de insuficiência renal aguda alteraram-se profundamente. Antes do advento do tratamento dialítico, as principais causas de mortalidade eram a uremia, a hipercalemia e as complicações cardiológicas decorrentes da sobrecarga de volume. Atualmente, são a sepse, a insuficiência cardiopulmonar, drogas nefrotóxicas e complicações pós-transplante renal. Quanto ao prognóstico de insuficiência renal aguda em unidades de terapia intensiva, a disfunção de múltiplos órgãos, a coagulação intravascular disseminada e o diabete melito são condições mórbidas que podem piorar a sua evolução. Devido à alta prevalência de diabete melito na população, o presente trabalho propôs-se avaliar a sua influência na morbidade e mortalidade da insuficiência renal aguda em unidade de terapia intensiva. Para tal, foram estudados, retrospectivamente, 1020 pacientes com insuficiência renal aguda internados na unidade de terapia intensiva do Hospital de Base de São José do Rio Preto, Brasil, no período de janeiro de 1997 a dezembro de 2000, dos quais, 255 (25%) eram diabéticos e 765 (75%) não diabéticos. Foram avaliados dados demográficos, presença de doenças de base, etiologia, tipos, quadro clínico e complicações de insuficiência renal aguda e ainda a presença de síndrome de disfunção de múltiplos órgãos. Entre a população estudada, 64% eram do sexo masculino, 46% tinham mais de 60 anos de idade, e 85% tinham uma ou mais doenças concomitantes. Nota de Resumo A etiologia isquêmica predominou com 53%, e a causa clínica foi o tipo mais freqüente com 57%. As médias e os desvios padrão de apache II e creatinina (mg/dL) foram 20,56,7 e 3,7+2 O respectivamente. A prevalência de coagulação intravascular disseminada, de choque, de insuficiência hepática e respiratória foi 32%, 69%, 15% e 79%, respectivamente. Entre as complicações, observamos a hiperpotassemia em 35%, a acidose em 70%, a sepse em 61%, a hipertensão arterial sistêmica em 14%, os sangramentos em 22%, a disfunção do sistema nervoso central em 44% e a mortalidade em 71%. Foram comparados, entre os diabéticos e os não diabéticos, os dados demográficos, quadro clínico, morbidade e mortalidade de insuficiência renal aguda. A hipercalemia, a acidose, a insuficiência respiratória, a sepse, o choque, a disfunção do sistema nervoso central, a hipervolemia e os sangramentos foram similares em ambos os grupos. A análise de regressão logística não mostrou associação significante entre diabete melito e a mortalidade. A etiologia isquêmica, a presença de três ou mais insuficiências de órgãos, a hiponatremia e a acidose foram de forma significante associadas com a mortalidade de insuficiência renal aguda. Em conclusão, os diabéticos foram mais idosos, menor prevalência de masculinos, menos oligúria, coagulação intravascular disseminada, hiponatremia e falência hepática do que os não diabéticos. O diabete melito não teve influência na mortalidade da insuficiência renal aguda em unidade de terapia intensiva.

Le contrôle de la transcription constitue le principal niveau de la régulation de l’expression des gènes dans les cellules eucaryotes. Dans le génome de ces derniers, les éléments régulateurs peuvent être localisés à de très grandes distances du gène qu’ils régulent. Le laboratoire a identifié 6 familles indépendantes avec un diabète insipide néphrogénique (DIN) lié à l’X portant de grandes délétions en amont du gène de l’AVPR2. Dans chacune de ces familles, les gènes AVPR2 et AQP2 ont été retrouvés intacts et les hommes sont atteints de DIN lié à l’X dans sa forme rénale « classique ». Le séquençage et l’analyse de 30 et 31 kilobases en amont et en aval de l’AVPR2 ont permis l’identification de 6 zones délétées chez 6 familles indépendantes, dont 5 zones de taille supérieure à 7 kilo bases, et une zone, de 102 paires de bases, commune à l’ensemble des délétions. Chez le patient porteur de cette délétion, l’osmolalité urinaire ne répond pas au dDAVP. Contrairement à ce qui est observé chez les patients atteints de DIN avec mutations de l’AVPR2, celui-ci présente des réponses hémodynamiques et de coagulation, normales. Ceci indique que les récepteurs V2 ne sont pas exprimés dans le tubule collecteur mais le sont au niveau des cellules endothéliales. Le but de notre travail est donc de tenter de comprendre les mécanismes régulateurs du locus de l’AVPR2, et plus précisément d’étudier l’expression « tissu spécifique » de ce gène. Les études réalisées in vivo, dans le système Hprt, confirment le rôle activateur de la séquence de 102 pb : coloration intense des tubules collecteurs avec la construction comportant la zone délétée et absence avec la construction ne la contenant pas. Cependant, les expériences menées in vitro semblent indiquer que cet effet dépende du contexte extracellulaire, isotonique ou hypertonique, de la nature des cellules, du tubule proximal ou collecteur, ainsi que du promoteur de l’AVPR2. L’identification des protéines liant potentiellement l’une des extrémités de la délétion a révélé la présence, soit de protéines régulatrices, soit de séquences inconnues, toutes exprimées dans le rein. À terme, ces études, ainsi que celles en découlant, permettront de positioner l’AVPR2 comme une cible de choix dans le traitement des diabètes insipides, centraux et néphrogéniques, par thérapie génique. / Transcriptional control is the primary means of regulating genes expression in eukaryotes cells. In the genome of the latter, regulatory elements can be localised with very long distance from the gene which they control. The laboratory identified six independent families with X-linked nephrogenic diabete insipidus (NDI) bearing large deletions upstream of the AVPR2 gene leaving intact AVPR2 and AQP2 coding sequences. Males bearing these deletions have classical renal X-linked NDI. The sequencing and analysis of 30 and 31 kilo bases upstream and downstream, respectively, encompassing the AVPR2 gene had led to identify 6 deletions in 6 ancestrally independent families including, 5 larger than 7 kilo bases and one of 102 base paires shared by the other deletions. In male patient bearing the 102 bp upstream deletion, urinary osmolality was unresponsive to dDAVP but, unlike patients with mutations in the coding sequence, their coagulation and hemodynamic responses to dDAVP were normal. This suggests that V2 receptors are not expressed in renal collecting duct cells but normally expressed in endothelial cells. Our goal is thus to understand the regulatory mechanism controlling the AVPR2 locus and more precisely the tissu specific expression of this gene.. The studies carried out in vivo, in the Hprt system, confirm the enhancer role of the sequence of 102 bp: intense coloration of the collecting tubules with construction comprising the deleted zone and abscence with construction not containing it. However, in vitro undertaken experiments seem to indicate that this effect depends on the extracellular context, isotonic or hypertonic, of the nature of the cells, of the tubule proximal or collecting duct, as well as promoter of the AVPR2. The identification of proteins potentially binding one of the ends of the deletion revealed the presence, either of regulating proteins, or of unknown sequences, all expressed in the kidney. In the long term, these studies, like those while rising, will make it possible to position the AVPR2 gene like a target of choice in the treatment of the diabetes insipidus, central and nephrogenic, by genic therapy. / Thèse réalisée en cotutelle avec l'Université Pierre et Marie Curie, Paris VI, France

INTRODUÇÃO: acredita-se que o controle glicêmico inadequado, a duração do diabetes melito (DM) e a presença de hipertensão arterial e dislipidemia sejam os fatores de risco mais importantes para o desenvolvimento das complicações microvasculares no DM, contudo, existem inúmeras evidências sugerindo que uma predisposição genética participe da suscetibilidade para o desenvolvimento dessas complicações. Vários genes relacionados aos mecanismos dos danos induzidos pela hiperglicemia têm sido investigados. O papel do estresse oxidativo na patogênese das complicações crônicas do DM vem sendo demonstrado e os genes que codificam enzimas que participam dos mecanismos antioxidantes são candidatos a conferirem suscetibilidade ou proteção contra as complicações crônicas. A glutationa é um dos mais importantes antioxidantes endógenos; no entanto, a associação entre polimorfismos em genes que codificam enzimas que participam desse sistema e complicações crônicas do DM foi pouco explorada na literatura. OBJETIVOS: avaliar a associação de polimorfismos em três genes que codificam enzimas relacionadas ao metabolismo da glutationa com o desenvolvimento de nefropatia e retinopatia em pacientes diabéticos tipo 1. Foram estudados: o polimorfismo -129C/T do gene GCLC, o número de repetições do trinucleotídeo GCG no exon 1 do gene GPX1 e o polimorfismo -65T/C do gene GPX3. CASUÍSTICA E MÉTODOS: 299 pacientes (139 do gênero masculino e 160 do gênero feminino) com DM tipo 1 com mais de 15 anos de diagnóstico e mau controle glicêmico foram divididos conforme presença ou ausência das seguintes complicações: nefropatia diabética (ND) avançada, ND, doença renal crônica (DRC) estágios 3 a 5 e retinopatia diabética proliferativa (RDP). Em cada grupo foram avaliadas as freqüências das variantes alélicas dos três genes estudados. RESULTADOS: a distribuição dos genótipos na população estudada foi consistente com o equilíbrio de Hardy-Weinberg para os três genes analisados. A presença de pelo menos um alelo T do polimorfismo -129C/T do gene GCLC conferiu risco independente para a presença de ND avançada (OR = 2,82 ; IC 95% = 1,13 - 7,05; p = 0,026), para ND (OR = 3,64; IC 95% = 1,27 10,36; p = 0,016) e para DRC estágios 3 a 5 (OR = 5,74; IC 95% = 2,17 15,1; p < 0,001) e a presença de pelo menos um alelo C do polimorfismo -65 T/C conferiu risco independente para a presença de ND avançada (OR = 2,62; IC 95% = 1,19 -5,72, p = 0,022) na população estudada. Não houve associação do número de repetições do trinucleotídeo GCG do gene GPX1 com nenhuma das complicações estudadas. O haplótipo CC_TT, composto pelos alelos selvagens dos genes GCLC e GPX3, foi negativamente associado com ND avançada (OR = 0,32, IC 95% = 0,15 0,66; p = 0,002) e DRC (OR = 0,25; IC 95% = 0,11 - 0,55; p = 0,001). CONCLUSÕES: a presença de pelo menos um alelo T do polimorfismo -129 C/T do gene GCLC e de pelo menos um alelo C do polimorfismo -65 T/C do gene GPX3, ambos associados a uma menor atividade transcricional do respectivo gene, conferiram risco para a presença de complicações renais na população de pacientes estudada. / INTRODUCTION: glycemic control, diabetes duration, systemic hypertension and dyslipidemia have been implicated as main risk factors for the development of diabetic microangiopathy, however there is evidence suggesting that genetic predisposition plays a role in the susceptibility to microvascular complications. Based on underlying pathogenesis, polymorphisms of several genes belonging to multiple pathways have been investigated, like the genes related to mechanisms of hyperglycemia-induced damage. The role of oxidative stress in the pathogenesis of diabetic complication has been increasingly demonstrated and genes coding enzymes involved in antioxidant defense are candidates to confer susceptibility or protection against these complications. Glutathione is one the most important endogen antioxidants, however, the association between polymorphisms in genes related to glutathione metabolism and diabetic complications has not been deeply investigated. OBJECTIVES: to study the association between polymorphisms in three genes which code enzymes related to glutathione metabolism and the development of nephropathy and retinopathy in type 1 diabetic patients: the polymorphism -129 C/T of GCLC, the number of trinucleotide GCG repeats at exon 1 of GPX1 and the polymorphism -65 T/C of GPX3. CASUISTIC AND METHODS: 299 type 1 diabetic patients (139 male and 160 female) with at least 15 years from diagnosis and poor glycemic control were studied. The patients were divided in two groups according to the presence or absence of diabetic complications: with and without diabetic nephropathy (DN), advanced DN, chronic kidney disease (CKD) stages 3 to 5 and proliferative diabetic retinopathy (PDR). RESULTS: The allelic distribution of the three studied polymorphisms was consistent with Hardy-Weinberg equilibrium. The presence of at least one T allele of GCLC 129 C/T was an independent risk factor for advanced DN (OR = 2.82 ; CI 95% = 1.13 -7.05; p = 0.026), for DN (OR = 3.64; CI 95% = 1.27 10.36; p = 0.016) and for CKD stages 3 to 5 (OR = 5.74; CI 95% = 2.17 15.1; p < 0.001) and the presence of at least one C allele of GPX3 -65 T/C was an independent risk factor for advanced DN (OR = 2.62; IC 95% = 1.19 -5.72, p = 0.022) in the studied population. There were no associations between GCG trinucleotide repeats of GPX1 and diabetic complications. The haplotype CC_TT, composed by GCLC and GPX3 wild type alleles, was negatively related to advanced DN (OR = 0.32, CI 95% = 0.15 0.66; p = 0.002) and CKD (OR = 0.25; CI 95% = 0.11 0.55; p = 0.001). CONCLUSIONS: the presence of at least one T allele of -129C/T polymorphism of GCLC and one C allele of -65 T/C polymorphism of GPX3, both associated to a lower transcriptional activity of its genes, conferred risk for renal complications in the studied population.

Le DT2 est caractérise par un défaut combiné de la sécrétion et de l’action de l’insuline. Depuis près d’un demi siècle la chirurgie bariatrique et notamment le Roux-en-Y Gastric Bypass (RYGB) ont montré des effets spectaculaires sur le contrôle glycémique remettant en question le paradigme de la prise en charge médicale du DT2. L’exclusion gastro duodénale induite par le RYGB améliore le métabolisme glucidique indépendemment de la perte de poids. Ainsi les modifications du flux biliaire semblent jouer un rôle, cependant les mécanismes sous-jacents ne sont pas clairs. Nous avons réalisés des RYGB chez le miniporc et nous avons montré que l'absorption intestinale du glucose est diminuée dans l’anse alimentaire (AL) dépourvue de bile. L'absorption du glucose dans l’AL était restaurée par l'ajout de la bile, et cet effet était inhibé lorsque le co transport actif sodium glucose 1 (SGLT1) était bloquée par la phlorizine. SGLT1 restait exprimée dans la AL, cependant la teneur dans la lumière de l’intestin en sodium était nettement diminuée. L’ajout de sodium dans l'AL provoquait le même effet que la bile sur l'absorption du glucose et augmentait également l’excursion glycémique post prandiale chez le miniporc au cours d’un repas test vigil. La diminution de l'absorption intestinale du glucose après RYGB a ensuite été confirmée chez l'homme. Nos résultats démontrent que la l’exclusion biliaire affecte le métabolisme post prandiale du glucose par modulation des co transporteurs intestinaux sodium-glucose. / Type 2 diabetes (T2D) is characterized primarily as a combined defect of insulin secretion and insulin action. For nearly a decade, the somewhat mysterious but spectacular benefit of metabolic surgery, and more specifically of Roux-en-Y gastric bypass (RYGB), on glucose control has been caused a questioning the current paradigm of T2D management. Gastro-intestinal exclusion by RYGB improves glucose metabolism, independent of weight loss. Although changes in intestinal bile trafficking have been shown to play a role, the underlying mechanisms are unclear. We performed RYGB in minipigs and showed that the intestinal uptake of ingested glucose is blunted in the bile deprived alimentary limb (AL). Glucose uptake in the AL was restored by the addition of bile, and this effect was abolished when active glucose intestinal transport was blocked with phlorizin. Sodium-glucose cotransporter 1 remained expressed in the AL, while intraluminal sodium content was markedly decreased. Adding sodium to the AL had the same effect as bile on glucose uptake. It also increased postprandial blood glucose response in conscious minipigs following RYGB. The decrease in intestinal uptake of glucose after RYGB was confirmed in humans. Our results demonstrate that bile diversion affects postprandial glucose metabolism by modulating sodium-glucose intestinal cotransport.

réalisé en cotutelle avec l'Université Claude Bernard Lyon 1 / La physiopathologie du diabète de type II se caractérise par de sévères anomalies métaboliques telles que l’hyperglycémie et les dyslipidémies contribuant au développement des maladies cardiovasculaires. Une altération de l’activité de l’AMPK dans les tissus tels que le muscle squelettique et le foie est associée à ces désordres métaboliques alors que son activation pharmacologique permet de les rétablir. Toutefois, le complexe hétérotrimérique αβγ tissu-spécifique de l’AMPK confère une régulation et des rôles distincts qui demeurent inexplorés dans l’intestin, un organe favorisant pourtant l’augmentation de l’absorption des nutriments en situation de diabète de type II. La présente étude démontre une prépondérance du complexe α1β2γ1 de l’AMPK dans les cellules intestinales Caco-2 dont l’un des rôles de la sous-unité α1 est de réguler l’ACC, l’enzyme de synthèse des acides gras. Contrairement à l’AMPK exprimée dans le foie, elle ne régule pas l’HMG-CoA Réductase impliquée dans la synthèse du cholestérol. L’activation de l’AMPK mime l’effet de l’insuline en réduisant l’absorption intestinale du glucose et des lipides alors que son altération en situation d’insulino-résistance (e.g : induite par le 4-HHE dans un modèle cellulaire Caco-2 ou induite par la diète dans le modèle animal Psammomys obesus) favorise l’absorption du glucose et des lipides, ce qui exacerberait l’hyperglycémie et la dyslipidémie postprandiale associées au diabète de type II. L’AMPK au niveau intestinal constitue donc une cible thérapeutique potentielle complémentaire pour la prévention et le traitement du diabète de type II. / Physiopathology of type II Diabetes is characterized by severe metabolic abnormalities such as hyperglycemia and dyslipidemia also implicated in development of cardiovascular diseases. Impaired AMPK activity in tissues such as skeletal muscle and liver is associated with these metabolic disorders whereas its pharmacologic activation is able to restore such abnormalities. Nevertheless, tissue-specific heterotrimeric αβγ AMPK likely confers distinct roles and regulation that remain unexplored in intestine, an organ promoting enhanced nutrients absorption in type II diabetes situation. This study demonstrates α1β2γ1 AMPK complex preponderance in intestinal Caco-2 cells whose α1 subunit role is to regulate ACC enzyme responsible of fatty acid synthesis. Unlike in the liver, AMPK doesn’t regulate HMG-CoA reductase enzyme implicated in cholesterol synthesis. AMPK activation mimics insulin effect by reducing intestinal glucose and lipids absorption whereas its alteration in insulin-resistance situation (e.g.: induced by 4-HHE in Caco-2 cell model or in Psammomys obesus animal model) enhances glucose and lipids absorption which could exacerbate postprandial hyperglycemia and dyslipidemia associated to type II diabetes. Thus, AMPK at the intestinal level could be a potential therapeutic target in prevention and treatment of type II diabetes.

La physiopathologie du diabète de type II se caractérise par de sévères anomalies métaboliques telles que l’hyperglycémie et les dyslipidémies contribuant au développement des maladies cardiovasculaires. Une altération de l’activité de l’AMPK dans les tissus tels que le muscle squelettique et le foie est associée à ces désordres métaboliques alors que son activation pharmacologique permet de les rétablir. Toutefois, le complexe hétérotrimérique αβγ tissu-spécifique de l’AMPK confère une régulation et des rôles distincts qui demeurent inexplorés dans l’intestin, un organe favorisant pourtant l’augmentation de l’absorption des nutriments en situation de diabète de type II. La présente étude démontre une prépondérance du complexe α1β2γ1 de l’AMPK dans les cellules intestinales Caco-2 dont l’un des rôles de la sous-unité α1 est de réguler l’ACC, l’enzyme de synthèse des acides gras. Contrairement à l’AMPK exprimée dans le foie, elle ne régule pas l’HMG-CoA Réductase impliquée dans la synthèse du cholestérol. L’activation de l’AMPK mime l’effet de l’insuline en réduisant l’absorption intestinale du glucose et des lipides alors que son altération en situation d’insulino-résistance (e.g : induite par le 4-HHE dans un modèle cellulaire Caco-2 ou induite par la diète dans le modèle animal Psammomys obesus) favorise l’absorption du glucose et des lipides, ce qui exacerberait l’hyperglycémie et la dyslipidémie postprandiale associées au diabète de type II. L’AMPK au niveau intestinal constitue donc une cible thérapeutique potentielle complémentaire pour la prévention et le traitement du diabète de type II. / Physiopathology of type II Diabetes is characterized by severe metabolic abnormalities such as hyperglycemia and dyslipidemia also implicated in development of cardiovascular diseases. Impaired AMPK activity in tissues such as skeletal muscle and liver is associated with these metabolic disorders whereas its pharmacologic activation is able to restore such abnormalities. Nevertheless, tissue-specific heterotrimeric αβγ AMPK likely confers distinct roles and regulation that remain unexplored in intestine, an organ promoting enhanced nutrients absorption in type II diabetes situation. This study demonstrates α1β2γ1 AMPK complex preponderance in intestinal Caco-2 cells whose α1 subunit role is to regulate ACC enzyme responsible of fatty acid synthesis. Unlike in the liver, AMPK doesn’t regulate HMG-CoA reductase enzyme implicated in cholesterol synthesis. AMPK activation mimics insulin effect by reducing intestinal glucose and lipids absorption whereas its alteration in insulin-resistance situation (e.g.: induced by 4-HHE in Caco-2 cell model or in Psammomys obesus animal model) enhances glucose and lipids absorption which could exacerbate postprandial hyperglycemia and dyslipidemia associated to type II diabetes. Thus, AMPK at the intestinal level could be a potential therapeutic target in prevention and treatment of type II diabetes. / réalisé en cotutelle avec l'Université Claude Bernard Lyon 1