Les connaissances actuelles en pathologie hépatique suggèrent que HCV n'est pas directement oncogénique mais expose les patients au risque de cancer du foie dans un contexte inflammatoire associé à une réponse UPR (Unfolded Protein Response) et une régénération hépatique. La nétrine-1, le ligand canonique de la famille des DRs (Récepteurs à dépendance), est une protéine anti-apoptotique impliquée dans le développement, l'inflammation et la tumorigenèse. Les DRs induisent l'apoptose en absence de leurs ligands. A ce jour, il n'existe aucune donnée reliant le concept de DR et les virus oncogènes. Au cours de ma thèse, j'ai contribué à démontrer que la fonctionnalité des DRs était altérée au cours de l'infection par HCV in vitro et in vivo. Nous avons montré que la surexpression de la nétrine-1 augmente l'infectivité des virions et promeut leur entrée via l'activation et la diminution du recyclage de l'EGFR. De son coté, HCV augmente l'expression de la nétrine-1 suite à l'activation de l'épissage de son ARN pré-messager. Nous avons aussi montré que l'expression du récepteur à la nétrine-1, UNC5A, était diminuée au cours de l'infection suite à des diminutions transcriptionnelle et traductionnelle. Dans ce cadre, la nétrine-1 joue le rôle de facteur proviral en inhibant une potentielle voie de signalisation antivirale induite par le récepteur UNC5A non lié. Nous avons ensuite voulu savoir quelles conséquences cette surexpression de nétrine-1 pourrait avoir en physiopathologie hépatique en contexte non infectieux. Un stress du RE (Réticulum Endoplasmique) est observé au cours de l'infection par HCV. Le stress du RE entraîne l'activation de la réponse UPR qui induit l'apoptose médiée par la DAPK1 en cas de stress prolongé. Le fait que le récepteur UNC5B active aussi l'apoptose via l'activation de la DAPK1 nous a conduit à étudier l'implication de la nétrine-1 dans la survie cellulaire au cours de la réponse UPR en contexte hépatique. Nous avons démontré à la fois in vitro et in vivo que l'expression de la nétrine-1 pourrait protéger les cellules contre l'apoptose induite par la réponse UPR suite à sa liaison aux récepteurs UNC5A et C qui entraîne l'inhibition de la DAPK1. De nombreuses études ont également reporté des rôles de la nétrine-1 dans l'inflammation et la néoangiogenèse. Nous avons montré que la nétrine-1 inhibe la migration transendothéliale hépatique des PBMCs (Peripheral Blood Mononucleated Cells) et accélère la tubulogenèse des cellules endothéliales intrasinusoïdales hépatiques. Dans leur ensemble, mes travaux de thèse suggèrent que la nétrine-1 via ses récepteurs UNC5s joue des rôles délétères en pathophysiologie hépatique favorables à la persistance virale et à la résistance à la mort cellulaire / Current knowledge in hepatic pathology suggests that HCV is not directly oncogenic but puts patients at risk for liver cancer in a context associated with a chronic inflammation, UPR (Unfolded Protein Response) and liver regeneration. Netrin-1, the canonical ligand of the DR (Dependence Receptor) family, is an antiapoptotic secreted factor implicated in development, cancer and cancer-associated inflammatory diseases. DRs induce cell death when unbound. No data linking the DR system to oncogenic viruses are available to date. During the first part of my PhD, I contributed to demonstrate that HCV infection alters DR functionality both in vitro and in vivo. We found that Netrin-1 conditions HCV virion infectivity and promotes virion entry by increasing the activation and decreasing the recycling of the EGFR. In turn, HCV increases Netrin-1 expression through enhanced Netrin-1 pre-mRNA splicing. The Netrin-1 UNC5A receptor expression was decreased upon HCV infection through diminished transcription and translation. In this setting, Netrin-1 acts as a proviral factor by inhibiting a putative antiviral signaling pathway conveyed by the unbound UNC5A receptor. In this context, we wanted to determine what consequences such Netrin-1 up-regulation could induce in non-infectious hepatic pathophysiology. Chronic ER (endoplasmic reticulum) stress is observed during HCV infection. ER stress leads to UPR activation which triggers apoptosis via DAPK1 activation upon prolonged stress. The fact that the UNC5B receptor induces apoptosis through DAPK1 activation led us to investigate Netrin-1 implication in cell survival upon UPR in the liver. During the second part of my PhD, I have demonstrated both in vitro and in vivo in mice that Netrin-1 translation during UPR could protect cells against UPR-related cell death after binding to UNC5A and C, in a DAPK1-mediated fashion. Several studies have also identified Netrin-1 roles in inflammation and neo-angiogenesis. We found that Netrin-1 inhibits hepatic transendothelial migration of PBMCs (Peripheral Blood Mononucleated Cells) and accelerates tubulogenesis of liver sinusoidal endothelial cells. Netrin-1’s role in a hepatic inflammation and neoangiogenesis, both events being tightly associated with viral hepatitis, remains to be thoroughly elucidated. Altogether, our results suggest that Netrin-1 plays UNC5-dependent deleterious roles in hepatic pathophysiology, leading to viral persistence as well as resistance to cell death

Le syndrome métabolique est constitué d'une constellation d'anomalies métaboliques telles que l'obésité centrale, une altération de la glycémie à jeun, une hypertriglycéridémie, un faible taux de cholestérol HDL et de l'hypertension artérielle. Les maladies cardiovasculaires caractérisées par une dysfonction endothéliale sont le résultat clinique primaire du syndrome métabolique. De plus, les microparticules (MP), de petites vésicules membranaires libérées de la membrane plasmique des cellules activées et / ou apoptotiques ont été décrites comme étant impliquées dans la pathogenèse du syndrome métabolique car elles induisent une dysfonction endothéliale par la diminution du monoxyde d’azote (NO). D'autre part, des MPs générées à partir de cellules T apoptotiques sont capables induire une dysfonction endothéliale par la diminution de la production de NO. Cependant, les mécanismes par lesquels les MPs humaines induisent cette dysfonction endothéliale ne sont pas complétement élucidés. Ainsi, l'objectif de cette étude est d'étudier les mécanismes par lesquels les MPs humaines induisent une dysfonction endothéliale. / Metabolic syndrome (MetS) consists of a constellation of metabolic abnormalities such as central obesity, impaired fasting glucose, hypertriglyceridemia, low HDL cholesterol and hypertension. Cardiovascular diseases are the primary clinical outcome of MetS whereas endothelial dysfunction represents a primary disturbance in cardiovascular events. Recently, it has been shown that microparticles (MPs), small membrane vesicles released from the plasma membrane of activated and/or apoptotic cells, are involved in the pathogenesis of MetS by inducing endothelial dysfunction through the decrease of nitric oxide (NO) production. Also, MPs from apoptotic T cells induce endothelial dysfunction by decreasing NO production. However, the mechanism through which this endothelial dysfunction takes place is not completely elucidated. Thus, the objective of this study is to study the mechanisms through which human MPs induce endothelial dysfunction.

No description available.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes.

Le syndrome d’apnées obstructives du sommeil (SAOS) est un problème de santé publique majeur qui est considéré comme un facteur indépendant de risque de survenue d’un infarctus du myocarde (IM). Les altérations cardiovasculaires associées au SAOS sont principalement dues à l’hypoxie intermittente (HI) chronique. En particulier, l’HI induit l’activation du facteur de transcription hypoxia-inducible factor-1 (HIF-1), susceptible d’être impliqué dans la vulnérabilité accrue du myocarde à l’ischémie-reperfusion. L’objectif de cette thèse était d’étudier le rôle de HIF-1 dans les mécanismes induits par l’HI et impliqués dans l’augmentation de la taille de l’infarctus suite à une ischémie-reperfusion. Ces travaux ont mis en évidence deux nouveaux effets délétères de l’HI, à savoir l’induction d’un stress du réticulum endoplasmique (RE) et d’altérations mitochondriales. A travers, l’inhibition génétique et/ou pharmacologique de HIF-1, nous avons montré que HIF-1 apparaît comme un acteur primordial dans l’ensemble des mécanismes délétères de l’HI, incluant ceux découverts lors de cette thèse. De plus, HIF-1 joue un rôle majeur dans l’augmentation de la taille de l’IM induite par l’HI chronique. Parallèlement, son activation myocardique est corrélée à l’index d’apnées-hypopnées chez des patients apnéiques atteints d’une maladie coronarienne (comparativement aux non-apnéiques). Par conséquent, l’activation de HIF-1 pourrait être utilisée comme marqueur diagnostic du SAOS chez les patients à risque cardiovasculaire. HIF-1 pourrait également représenter une cible pour le développement de nouvelles thérapies complémentaires ou substitutives aux traitements actuels. / Obstructive sleep apnea syndrome (OSAS) is a major public health problem that is considered an independent risk factor for the occurrence of myocardial infarction (MI). The cardiovascular alterations associated with OSA are mainly due to the chronic intermittent hypoxia (IH). In particular, activation by IH, the hypoxia-inducible factor-1 (HIF-1) transcription factor likely contributes to enhance myocardial vulnerability to ischemia-reperfusion injury. The aim of this thesis was to study the role of HIF-1 in the mechanisms involved in the increase in MI induced by chronic IH. This work has highlighted two new deleterious consequences of IH exposure, namely endoplasmic reticulum (ER) stress and mitochondrial alterations. Through genetic and/or pharmacological inhibition of HIF-1, we have shown that HIF-1 appears to be a primordial actor in all the deleterious mechanisms of IH, including those discovered during this thesis. HIF-1 also appears to play a major role in the IH-induced increase in MI size. In parallel, its myocardial activation is correlated with the apnea-hypopnea index in apnoeic, compared to non-apnoeic, patients with coronary heart disease. Therefore, HIF-1 activation could serve as a diagnostic marker of OSA in patients with cardiovascular risk. HIF 1 could also be a target for new therapeutic approaches, in complement or replacement of standard treatments.

Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens. / Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

O remodelamento vascular é um determinante fundamental do lúmen em doenças vasculares, porém os mecanismos envolvidos não estão completamente elucidados. Nós investigamos o papel da chaperona redox residente do retículo endoplasmático Dissulfeto Isomerase Proteica (PDI) e sua fração localizada na superfície celular (peri/epicelular=pecPDI) no calibre e arquitetura vascular durante reparação à lesão. Em artérias ilíacas de coelho submetidas à lesão in vivo, houve importante aumento do mRNA e expressão proteica (~25x aumento 14 dias pós-lesão vs. controle) da PDI. O silenciamento da PDI por siRNA (cultura de órgãos) acentuou o estresse do retículo e apoptose, diferentemente da inibição da pecPDI com anticorpo neutralizante (PDI Ab). Bloqueio in vivo da pecPDI por aplicação de gel perivascular contendo PDI Ab no 12° dia após lesão, com análise após 48 h, promoveu ca.25% redução no calibre vascular analisado por arteriografia e diminuição similar na área total do vaso detectada por tomografia de coerência óptica. Neste processo, não ocorreu alteração no tamanho da neoíntima, indicando assim, que PDI Ab acentuou remodelamento constrictivo. Neutralização da pecPDI promoveu importantes alterações na arquitetura da matriz de colágeno e citoesqueleto, resultando em fibras com orientação invertida e desorganizadas. Diminuição na produção de espécies reativas de oxigênio e óxidos de nitrogênio também ocorreu. Análise de propriedades viscoelásticas nas artérias indicou redução na ductilidade vascular, evidenciada pela menor distância para ruptura. As alterações subcelulares no citoesqueleto observadas in vivo após PDI Ab foram recapituladas em um modelo de estiramento cíclico em células musculares lisas vasculares, com importante redução na formação das fibras de estresse. Em modelo de migração randômica de células musculares lisas, a exposição a PDI Ab reduziu a resiliência de regulação da polaridade. Embora a neutralização da pecPDI não tenha afetado a atividade global de RhoA, ela promoveu alterações no padrão de marcação em resposta ao estiramento, na redistribuição de RhoA na superfície celular e na associação com regiões contendo caveolina. Além disso, em aterosclerose nativa em humanos, a expressão da PDI correlacionou-se inversamente com remodelamento constrictivo. Dessa forma, PDI é fortemente expressa após a lesão e sua fração peri/epicelular remodela a arquitetura da matriz e citoesqueleto, promovendo um efeito anti-remodelamento constrictivo / Whole-vessel remodeling is a critical lumen caliber determinant in vascular disease, but underlying mechanisms are poorly understood. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI(peri/epicellular=pecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and pecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis, contrarily to pecPDI neutralization with PDI-antibody(PDI Ab). PecPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. PecPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and disorganization. Decreased ROS and nitrogen oxide production also occurred. Viscoelastic artery properties assessment showed decreased ductility, evidenced by decreased distance to rupture. Subcellular cytoskeletal disruption by PDI Ab was recapitulated in vascular smooth muscle cell stretch model, with marked decrease in stress fiber buildup. Also, PDI Ab incubation promoted decreased regulation resilience of vascular smooth muscle migration properties. While pecPDI neutralization did not affect global RhoA activity, there was altered RhoA redistribution to the cell surface and association with caveolin-containing clusters, which mislocalized after stretch. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect

Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens. / Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD