Le récepteur minéralocorticoïde (MR), activé par l’aldostérone, exerce de nombreuses fonctions pléïotropes, notamment au niveau rénal où il régule l’homéostasie hydrosodée. Des dysfonctionnements de la signalisation minéralocorticoïde sont impliqués dans des pathologies majeures chez l’Homme. Dans ce travail, nous avons identifié par ChIP sequencing le premier cistrome du MR dans une lignée cellulaire rénale humaine. La caractérisation des cibles génomiques a permis de décrire l’élément de réponse spécifique du MR, et de démontrer l’existence de deux modes d’action pour le MR : par liaison directe à l’ADN, ou indirecte via la liaison à d’autres facteurs de transcription. Le MR est physiologiquement confronté à une dualité face au récepteur glucocorticoïde (GR) avec lequel il partage un ligand, le cortisol, et des cibles génomiques, dont le gène PER1. Sur ce dernier, les deux récepteurs se distinguent par des recrutements dynamiques et cycliques différents, variants selon l’hormone, et contemporains de celui de partenaires transcriptionnels, régulant ainsi des effets à court ou à long-terme. Enfin, par ChIP en série et en tandem, nous avons montré que le MR et le GR agissent sous forme d’homodimères ou d’hétérodimères.L’identification du cistrome du MR, et la caractérisation de ses mécanismes d’action moléculaires, améliore notre compréhension de la physiopathologie de la signalisation minéralocorticoïde, et pourrait aboutir, notamment par le développement d’antagonistes sélectifs du MR comme la Finérénone, à de nouvelles stratégies thérapeutiques. / The mineralocorticoid receptor (MR), activated by aldosterone, exhibits numerous pleiotropic functions, most notably at the renal level where it regulates electrolytic homeostasis. Dysfunctions in the mineralocorticoid signaling pathway are involved in major diseases in Human. During this work, we have identified by ChIP sequencing the first MR cistrome in a human renal cell lineage. The characterization of the identified genomic targets allowed us to define a specific MR responsive element, and to demonstrate the existence of two transactivation processes for MR: through direct binding to DNA or through indirect interaction via binding to other transcription factors. MR is physiologically confronted with a duality with the glucocorticoid receptor (GR), since they share a common ligand, cortisol, and some of their genomic targets, whose PER1 gene. On the latter, MR and GR are distinguished by different dynamic and cyclical recruitment, varying according to hormone, and coordinated with the one of transcriptional partners, translating into the regulation of short-term and long-term effects. Finally, by serial and tandem ChIP experiment, we have demonstrated that MR and GR act as homodimer and as heterodimer.Identification of new MR genomic targets and characterization of its molecular mechanisms of action, improve our understanding of the pathophysiology of the mineralocorticoid signaling pathway. This could ultimately, notably through the development of selective MR antagonists like Finerenone, lead to new therapeutic strategies.

Part I. The aldosterone synthase gene (CYP11B2) polymorphism T-344C in blood pressure and left ventricular hypertrophy. BACKGROUND: Aldosterone is a key cardovascular hormone, it significantly influences volume, pressure and electrolyte balance. Aldosterone plays an important role in development of left ventricular (LV) hypertrophy and myocardial fibrosis. The aldosterone synthase gene (CYP11B2) is an important candidate gene region in essential hypertension. DESIGN AND METHODS: We assessed the influence of the T-344C polymorphism of aldosterone synthase - the rate-limiting enzyme in aldosterone biosynthesis - on the structure of the left ventricle in young normotensive men. The population included 113 normotensive mid-European Caucasian men aged 18-40 years (mean 27 +/- 5 years). We also studied the association of -344T/C polymorphism of the CYP11B2 gene with the presence and severity of hypertension in 369 individuals, of whom 213 were hypertensive patients (139 controlled hypertensive, 74 resistant hypertensive) and 156 were healthy normotensive subjects. The genotype was assessed using polymerase chain reaction with subsequent cleavage with restriction enzyme HAEIII (restriction fragment length polymorphism method) and visualization with ethidium bromide. Plasma renin activity (PRA) and plasma...

La restriction de croissance intrautérine (RCIU) est associée à l’apparition de maladies à l’âge adulte et le phénotype de la condition pathologique peut être différent selon le sexe. Notre laboratoire a développé un modèle de RCIU chez le rat en administrant une diète faible en sodium lors du dernier tiers de la gestation entraînant une réduction de l’expansion volémique maternelle et de la perfusion utéroplacentaire. L'activité rénine et la concentration d'aldostérone plasmatique sont augmentées chez la mère et les foetus RCIU. Antérieurement, notre laboratoire a démontré une augmentation de l’expression génique et protéique rénale de la Na+-K+-ATPase-α1 uniquement chez les foetus femelles RCIU. Ainsi, nous émettons l’hypothèse que la diminution du volume circulant chez la rate gestante entraîne une augmentation et une expression différentielle, selon le sexe, des éléments de la cascade de signalisation du récepteur des minéralocorticoïdes (MR) dans les reins de foetus RCIU. L’expression des gènes est réalisée par qRT-PCR et celle des protéines par immunobuvardage de type Western. Bien que les résultats démontrent que la transcription génique de SGK1, α-ENaC et GILZ soit augmentée dans les reins de foetus RCIU, l’expression protéique de SGK1, pSGK1(Thr 256) et α-ENaC est similaire à celle des témoins. La protéine GILZ est indétectable. Pour CNKSR3, aucune différence de l’ARNm ou de la protéine n’a été observée entre les deux groupes. Par contre, même si l’expression génique du MR n’est pas différente, l’expression protéique est diminuée chez les RCIU. Aucun effet du sexe n’a été observé. En conclusion, l’augmentation d'aldostérone plasmatique chez les foetus ayant subi une RCIU stimule la transcription des gènes associés à la voie de réabsorption sodique, mais la quantité protéique demeure inchangée. Ceci suggère qu’il peut avoir des mécanismes de régulation post-transcriptionnelle ou une dégradation accélérée des protéines. Malgré la pertinence du sexe dans le développement de maladies, le sexe n’influence pas l’expression des composantes de la voie de rétention sodique chez le foetus. Il serait important de suivre cette voie en fonction de l’âge et de corréler les expressions génique et protéique avec l’apparition de maladies. / Intrauterine growth restriction (IUGR) contributes to the development of diseases in adulthood, of which some are influenced by sex. Our laboratory has developed an IUGR model in the rat by administering a low-sodium diet during the last third of gestation which leads to a reduced volume expansion and uteroplacental perfusion. Plasma renin activity (PRA) and aldosterone concentration are increased in IUGR dams and foetuses. Previously, our laboratory has shown that gene and protein expression of Na+-K+-ATPase-α1 was increased solely in female IUGR kidneys. Therefore, we hypothesize that the reduced maternal volume will result in an increase and differential expression, influenced by foetal sex, of elements from the mineralocorticoid receptor (MR) pathway. Gene expression is determined by qRT-PCR and for protein, Western blotting is performed. Although results show that gene expression of SGK1, α-ENaC and GILZ is increased in IUGR foetal kidneys, protein expression for SGK1, pSGK1 (Thr256), and α-ENaC is similar to controls. GILZ protein is not detected. For CNKSR3, no difference in mRNA or protein expression is observed between the groups. Gene expression of MR is unchanged, while its protein expression is decreased in IUGR foetuses. In conclusion, the increase in plasma aldosterone in IUGR foetuses stimulates gene transcription of several components of the sodium reabsorption pathway without affecting protein expression. These results suggest that there may be post-transcriptional regulation or a higher protein turnover. Despite the importance of biological sex in the development of disease, it did not affect the expression of elements from the sodium reabsorption pathway in the foetus. It would be important to verify this same pathway in animals of different ages to correlate gene and protein expression with the appearance of diseases.

Ainda são escassos estudos que avaliem biomarcadores para diferenciação de transtorno depressivo maior (TDM) e transtorno bipolar (TB), principalmente relativo à etiologia desses transtornos e sua relação com os receptores glicocorticoides (GR) e, principalmente, com os receptores mineralocorticoides (MR). Objetivo: Encontrar biomarcadores genéticos e/ou hormonais e observar sua associação entre si e/ou a fatores externos (estresse precoce - EP) para compreender melhor sua fisiopatogenia e auxiliar no diagnóstico diferencial entre TDM e TB. Material e Métodos: Participaram deste estudo N=273 sujeitos, sendo n=113 controles, n=78 unipolares e n=82 bipolares. A triagem diagnóstica de todos os sujeitos foi realizada por meio do MINI PLUS, checagem de história de trauma na infância pela CTQ, avaliação de sintomas depressivos pela GRID-HAM-D21, e demais comorbidades pela BAI, BHS e BSI. Na busca de biomarcador genético, observou-se as frequências genotípicas e alélicas de 3 polimorfismos de receptor de glicocorticoide (GR) (N363S, R22/23K e BclI) e de 2 polimorfismos de MR (MI180V e -2G/C) após realizada a discriminação alélica por reação em cadeia da polimerase quantitativa (qPCR). Foram avaliados de forma intragrupo as variáveis genéticas e endócrinas (e combinadas) e o efeito do EP sobre tais variáveis. Também, as variáveis polimorfismos, níveis hormonais e exposição a EP foram comparadas entre grupos para avaliar se havia diferença de prevalência, de perfil endócrino, ou se havia suscetibilidade maior por parte dos unipolares ou bipolares para alteração dos níveis hormonais e/ou intensidade do quadro depressivo frente a EP ou a determinado genótipo. Resultados: Todos os sujeitos unipolares e bipolares mostraram piora de seus sintomas depressivos frente a EP e seus subtipos, sendo eles unipolares ou bipolares. Como biomarcador hormonal, comparando-se controles x unipolares x bipolares, ou apenas unipolares x\' bipolares, foi possível observar que os níveis de cortisol e os níveis de aldosterona apresentaram-se os altos em unipolares e os baixos mais em bipolares, quando estes pacientes estavam com depressão grave ou gravíssima. Também, bipolares expostos a EP global, abuso físico e emocional mostraram níveis mais baixos de aldosterona que bipolares que não foram expostos. Frente a exposição a esses EP global e abuso físico, os bipolares tenderam a se mostrar mais suscetíveis que os unipolares a alteração dos níveis de aldosterona. Para biomarcador genético, frequência de genótipos ou alelos não diferenciaram unipolares de bipolares. Entretanto, houve maior prevalência do genótipo heterozigoto AG de GR N363S em pacientes depressivos uni e bipolares quando comparados com controles. Combinando-se os biomarcadores genéticos e hormonais, unipolares apresentaram níveis mais baixos de cortisol e de aldosterona quando carregavam genótipo variante GG de MR -2G/C, enquanto bipolares mostraram tendência a redução de cortisol quando carregavam o alelo variante G de MR MI180V. Quando comparados os genótipos por si só, intragrupo, novamente o polimorfismo MR -2G/C mostra influência sobre o fenótipo unipolar. Em unipolares, presença do alelo variante G de MR -2G/C piora significativamente o quadro depressivo, mas o alelo variante G de MI180V mostrou-se protetor frente a EP. Tanto os unipolares frente aos outros 4 polimorfismos, quanto os bipolares frente a todos os polimorfismos estudados, apresentaram piora significativa de seu quadro depressivo se expostos a EP. Bipolares mostraram uma tendência a ser mais suscetíveis que unipolares a alterações endócrinas (aldosterona) quando expostos a EP global e abuso físico. Conclusão: Tendo em vista os vários achados significativos a cerca dos polimorfismos de MR, tanto para unipolar quanto para bipolar, sua influência sobre os níveis de aldosterona e cortisol basais, reforça-se a importância do papel dos receptores MR dentro da etiologia dos transtornos depressivos unipolares e bipolares, e a forma diferente de funcionamento do MR para a distinção entre TDM e TB / There are still few studies assessing biomarkers for differentiation of major depressive disorder (MDD) and bipolar disorder (TB), mainly related to the etiology of these disorders and its relationship with glucocorticoid receptors (GR) and, manily, with mineralocorticoid receptors (MR). Aim: Finding genetic and / or hormonal biomarkers and observing their association to each other and / or external factors (early-life stress - ELS) for better comprehend their pathophysiology and, then, assisting in differential diagnosis between MDD and TB. Material and Methods: A total of N = 273 subjects composed the study sample, being n = 113 control, n = 78 unipolar, and n = 82 bipolar subjects. The diagnostic screening of all subjects was performed applying MINI PLUS, for history of ELS, CTQ; assessment of depressive symptoms, GRID-HAM-D21; and assessment of other comorbidities, BAI, BHS, and BSI. Researching for genetic biomarker, genotypic and allelic frequencies of 3 GR polymorphisms (N363S, R22 / 23K and BclI) and 2 MR polymorphisms (MI180V and -2G/C) were evaluated after allelic discrimination by quantitative polymerase chain reaction (qPCR). Genetic and endocrine variables (and their combination), and the effect of ELS over these variables were assessed intragrups. Also, polymorphisms, hormonal levels and history to ELS were compared between groups to assess whether there was difference in prevalence, endocrine profile, or whether there was greater susceptibility on the part of unipolar or bipolar for alteration of hormonal levels and / or severity of depressive symptoms considering history of ELS and/or a specific genotype. Results: All unipolar and bipolar subjects showed worsening of their depressive symptoms in the presence of ELS and its subtypes. As hormonal biomarker, comparing unipolar x bipolar x control subjects, or comparing unipolar x bipolar, cortisol and aldosterone levels were higher in unipolar subjects, and lower in bipolar subjects, when these patients presented severe or very severe depressive symptoms. Also, bipolar subjects\' exposed to global ELS, physical and emotional abuse showed lower basal levels of aldosterone than did bipolar who were not exposed to ELS. Concerning global ELS and physical abuse, bipolar tended to be more susceptible than unipolar for aldosterone levels to change. For genetic biomarker, frequency of genotypes or alleles did not distinguished unipolar from bipolar sample. However, there was a higher prevalence of GR N363S heterozygous genotype (AG) in unipolar and bipolar depressive patients when compared to controls. Combining the genetic and hormonal biomarkers, unipolar had lower levels of cortisol and aldosterone when carrying GG variant genotype of MR-2G / C, while bipolar showed tendency to reduce cortisol when carrying the variant G allele of MR MI180V. When comparing the genotypes (intragroup), again, MR-2G/C polymorphism shows influence on the unipolar phenotype. In unipolar, the presence of the variant G allele of MR-2G / C significantly worsens the depressive condition, unlike variant G allele of MI180V has shown to be protective against ELS. Both the unipolar compared to the other 4 polymorphisms, and the bipolar ones against all polymorphisms studied, presented a significant worsening of their depressive condition if exposed to ELS. Bipolar tend to be more susceptible than unipolar to endocrine changes (aldosterone) when exposed to global ELS and physical abuse. Conclusion: Considering the several significant findings regarding MR polymorphisms, for both unipolar and bipolar subjects, and their influence on basal aldosterone and cortisol levels, we highlight importance of the role of MR receptors within the etiology of depressive unipolar and bipolar disorders, and different way of MR functioning in each disorder for assisting the distinction between MDD and TB

Chez les patients présentant un syndrome métabolique, le développement des comorbidités cardiaques et rénales associées au diabète de type 2 sont liées à des altérations au niveau vasculaire. Afin d’évaluer l’effet protecteur rénal et cardiaque de nouvelles molécules, le modèle de rat Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) semblait approprié. Cependant, son développement lent et les impacts rénaux et cardiaques modestes en limitaient son utilisation. Notre but fut d’exacerber ces altérations par deux méthodes. Nous avons d’abord effectué une néphrectomie unilatérale chez le rat ZSF1 et évalué l’évolution des fonctions cardiaque et rénale en fonction de l’âge. Seule une exacerbation de la dysfonction rénale a été mise en évidence. Néanmoins nous avons pu démontrer l’effet protecteur rénal de l’inhibiteur de l’enzyme de conversion de l’angiotensine lisinopril ainsi que d’un composé antioxydant, le bardoxolone. Notre seconde stratégie a consisté à infuser de l’angiotensine II (AngII) à des rats ZSF1. L’hypertension déjà existante dans ce modèle a été fortement accrue, et le niveau d’aldostérone circulante a été significativement augmenté. Dans ce contexte, les fonctions cardiaque et rénale ont été dégradées de manière importante. Enfin nous avons montré que dans ce modèle, un inhibiteur de l’aldostérone synthase induisait une meilleure protection rénale que l’antagoniste des récepteurs à l’aldostérone éplérénone. Nous avons donc mis en évidence que le rat ZSF1-AngII est un modèle de dysfonction cardio-rénale permettant d’évaluer l’effet protecteur de composés sur les fonctions rénale et cardiaque, dans un contexte de syndrome métabolique / In the context of metabolic syndrome, development of Type 2 Diabetes is associated with (and influenced by) cardiac and renal comorbidities linked to micro- and macro-vasculature alterations. To assess the efficacy of new compounds on targeted organs in the context of metabolic syndrome, the Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model could be suitable assuming cardiorenal alterations would develop in a short timeframe. Actually, the ZSF1 rat model recapitulates features of human metabolic syndrome, but develops relatively late (1year-time) and moderate cardiac and renal dysfunctions. The aim of our work was to exacerbate cardiorenal impairments in terms of onset and extent. Two options were explored. On one hand, unilateral nephrectomy was performed in ZSF1 rats, and cardiac and renal functions were longitudinally assessed. This surgical insult only significantly deteriorated renal function, which was prevented by standard of care, lisinopril and new renal protective agent, bardoxolone. On the other hand, subcutaneous infusion of angiotensin II (AngII) was used in the aim to induce hemodynamic and hormonal stress and thus to enhance cardiorenal impairments. AngII-infused ZSF1 rats displayed significant hypertension and increased levels of circulating aldosterone. Moreover, renal and cardiac functions dropped, concomitantly. We showed in this model that an aldosterone synthase inhibitor induced overall better renal protection than the mineralocorticoid receptor antagonist eplerenone. Our data showed that ZSF1-AngII rat is a suitable model to evaluate the cardio and renoprotective effects of drugs in the context of metabolic syndrome

A nicotina é o principal componente do tabaco e dos cigarros eletrônicos. A exposição crônica a nicotina, em quantidades semelhantes às atingidas pelo tabagismo humano, é responsável por piora da lesão renal aguda e da doença renal crônica. O gene klotho, predominantemente expresso no rim, foi descoberto após uma mutação insercional, com o surgimento de um fenótipo semelhante ao envelhecimento humano nos camundongos homozigotos para esse transgene. A proteína Klotho transmembrana tem ação de co-receptor do fator de crescimento fibroblástico 23 (FGF-23) e sua forma secretada atua em diversas vias intracelulares e em órgãos a distância. A deficiência de Klotho ocorre no envelhecimento, em situações que levam a lesão renal aguda e na doença renal crônica. A expressão reduzida de Klotho também agrava lesão renal aguda e participa da progressão da doença renal crônica, enquanto o seu aumento, ou a sua reposição, protegem dos processos inflamatórios e do estresse oxidativo. Neste estudo, objetivamos avaliar os efeitos renais, hemodinâmicos e sobre a expressão de Klotho da exposição crônica a nicotina e quais os efeitos dessa exposição nos animais haploinsuficientes para o transgene klotho (Kl+/-). Utilizamos para estas avaliações camundongos Kl+/- e seus controles wild type (Kl+/+), que foram expostos a nicotina (200 mcg/mL) ou veículo (sacarina 2%) diluídos em água por 28 dias. Ao final do estudo foram avaliados diurese, eletrólitos plasmáticos e urinários, ureia, aldosterona, ADH, FGF-23 e PTH intactos plasmáticos, expressão protéica renal de Klotho, alfa7-nAchR, NHE3, ENaC, NKCC2, AQP2, e-NOS, VEGF, MnSOD e renina, expressão genica renal de klotho, interleucinas, TBARS e GSH em tecido renal, taxa de filtração glomerular por FITC-inulina, pressão arterial e frequência cardíaca invasivas, sensibilidade baroreflexa e modulação autonômica cardíaca e periférica por análise espectral. Após a exposição a nicotina, os animais Kl+/+ apresentaram redução da expressão renal da proteína e do RNAm de Klotho e uma tendência a aumento dos níveis plasmáticos de FGF-23, associados a uma queda da diurese e da taxa de filtração glomerular, sem alteração dos níveis de ADH. Esses animais Kl+/+ também apresentaram aumento da sensibilidade barorreflexa em resposta ao nitroprussiato e um predomínio da modulação simpática cardíaca, com redução da expressão renal dos alfa7-nAchR. Os animais Kl+/- tiveram níveis renais ainda menores de Klotho após a exposição a nicotina, com aumento de TBARS, IL-6, uréia e aldosterona em relação aos Kl+/- não expostos. A diurese, a taxa de filtração glomerular e a expressão dos alfa7-nAchR não se reduziram e não houve aumento da sensibilidade barorreflexa após exposição a nicotina, com um predomínio da modulação parassimpática cardíaca, nesses animais Kl+/-. A ingesta hídrica, a pressão arterial e a frequência cardíaca foram semelhantes entre os 4 grupos. A proteinúria foi maior nos animais Kl+/- do que nos animais Kl+/+ após a exposição a nicotina. Podemos concluir que a exposição crônica à nicotina reduz a expressão renal de Klotho, estimula vias de inflamação, fibrose e estresse oxidativo renais e tem efeitos renais e sistêmicos diferentes de acordo com os níveis basais de Klotho / Nicotine is a major compound of tobacco and electronic cigarettes. Chronic exposure to nicotine concentrations that are similar to human smoke worsens acute kidney injury and chronic kidney disease. The klotho (Kl) gene is expressed predominantly by the kidney and was discovered after an unintentional insertional mutation that resulted, in transgenic homozygous mice, in a phenotype similar to human aging. Klotho transmembrane protein acts as a co-receptor to fibroblastic growth factor 23 (FGF-23) and the secreted form interacts in multiple intracellular pathways, with effects in distant organs. Klotho deficiency occurs in aging and in multiple acute kidney injury and chronic kidney disease etiologies, whereas klotho upregulation and replacement protect from inflammation and oxidative stress. Here, we investigated renal and hemodynamic effects of chronic nicotine exposure, its effects over renal expression of Kl, and compared wild type (Kl+/+) and Kl haploinsufficient mice (Kl+/-) in terms of the effects of that exposure. Kl+/- and Kl+/+ mice received nicotine (200 ?g/ml) or vehicle (saccharine 2%) in drinking water for 28 days. We evaluated diuresis, ions in serum and urine, urea, plasma and urinary levels of cotinine, aldosterone, plasma antidiuretic and parathyroid hormone, plasma FGF-23, protein expression of (immunoblotting for) Klotho and ?7 nicotinic acetylcholine receptor, NHE3, NKCC2, ENaC, aquaporin-2, e-NOS, VEGF and renin, klotho mRNA, kidney interleukines, TBARS and GSH, glomerular filtration rate by fluorescein isothiocyanate-inulin clearance, mean arterial pressure, heart rate, baroreflex sensitivity and autonomic cardiac and peripheral modulation by spectral analysis. After nicotine exposure, Kl+/+ mice showed decreased Klotho protein and mRNA and a tendency towards an elevation in plasma FGF-23, which were associated with both diuresis and glomerular filtration rate reductions, without modifications in ADH levels. Besides that, Kl+/+ animals increased baroreflex sensitivity after nitroprusside, a predominant sympathetic cardiac modulation and lower alfa7-nAchR kidney expression. Kl+/- mice reduced even more Klotho renal expression, with higher levels of TBARS, IL-6, urea and aldosterone. Diuresis, glomerular filtration rate, alfa7-nAchR expression and baroreflex sensitivity were the same of their controls. Cardiac parasympathetic modulation predominated in Kl+/- mice. Fluid intake, mean arterial pressure and heart rate were similar across the 4 groups. Renal protein excretion was higher in Kl+/- than in their controls after nicotine exposure. We can conclude that chronic nicotine exposure downregulates Klotho kidney expression induces inflammation and oxidative stress and stimulates fibrosis, with different renal and systemic responses according to basal Klotho levels

O diabetes mellitus tipo 2 (DM2), uma doença que afeta milhões de pessoas em todo o mundo, é marcado pela presença de complicações micro e macrovasculares, as quais estão associadas à disfunção endotelial, inflamação e fibrose. A aldosterona, cujos níveis plasmáticos estão elevados em pacientes e modelos experimentais de DM2, aumenta a geração de espécies reativas de oxigênio (ERO) e a expressão de marcadores inflamatórios. As citocinas IL-1? e IL-18 são liberadas principalmente após ativação de plataformas moleculares denominadas inflamassomas, as quais incluem os receptores NLRP3. Recentemente, demonstramos que o receptor NLRP3 contribui para a disfunção vascular induzida pela aldosterona. Considerando a existência de evidências que a aldosterona, via receptor mineralocorticoide (MR) e NLRP3, induz a produção de mediadores inflamatórios e, consequentemente, ativação do inflamassoma, podendo assim contribuir para o processo inflamatório no diabetes, nós hipotetizamos que o bloqueio de receptores MR e NLRP3 previne a ativação do inflamassoma e reduz o desenvolvimento das alterações vasculares funcionais associadas ao DM2. Observamos que artérias mesentéricas de animais diabéticos apresentam aumento da expressão/ativação de caspase-1 e IL-11?, aumento dos níveis plasmáticos de IL-1?, aumento da atividade da caspase- 1 em macrófagos do lavado peritoneal e prejuízo no relaxamento dependente de endotélio, comparativamente a artérias do grupo controle. Os tratamentos com espironolactona (antagonista MR) e MCC950 (inibidor de receptor NLRP3) atenuam a disfunção vascular, reduzem a expressão e a atividade da caspase-1 e diminuem os níveis plasmáticos de IL-1?. Células de músculo liso vascular e macrófagos derivados da medula estimulados com aldosterona também apresentam aumento da expressão dos componentes do inflamassoma, o que poderia contribuir para as alterações observadas em animais diabéticos. Pacientes com DM2 apresentam correlação positiva entre os níveis de aldosterona e de IL-1? e/ou glicemia. Em conclusão, nosso estudo demonstra que a aldosterona induz disfunção vascular e processo inflamatório no diabetes tipo 2 através da ativação de MR e do inflamassoma NLRP3. / Type 2 diabetes mellitus (T2DM), a disease that affects millions of people around the world, is marked by the presence of micro and macrovascular complications, which are associated with endothelial dysfunction, inflammation and fibrosis. Aldosterone excess aggravates endothelial dysfunction in diabetes by promoting insulin resistance, fibrosis, oxidative stress and inflammation. Aldosterone activates the molecular platform inflammasome in cells of the immune system, an event that contributes to vascular dysfunction induced by the mineralocorticoid hormone. However, it is unclear whether activation of the inflammasome contributes to the effects of aldosterone in diabetes-associated vascular abnormalities. In the present study we tested the hypothesis that aldosterone induces vascular dysfunction in T2DM via activation of mineralocorticoid receptors (MR) and assembly of the NLRP3 inflammasome. To determine whether aldosterone activates the NLRP3 inflammasome and NLRP3 activation contributes to diabetes-associated vascular dysfunction, mesenteric arteries from control mice (db/m) and mice with type 2 diabetes (db/db), treated with vehicle, spironolactone (MR antagonist) or the NLRP3 antagonist MCC950, were used. Db/db mice exhibited increased vascular expression/activation of caspase-1 and IL-1?, increased plasma IL-1? levels, increased number of caspase-1-positive macrophages in the peritoneal lavage as well as reduced acetylcholine (ACh) vasodilation, compared to control db/m mice. Treatment of db/db mice with spironolactone and MCC950 reduced vascular caspase-1, decreased plasma IL-1? levels and partly restored ACh responses. Spironolactone treatment also reduced the number of caspase-1-positive-macrophages in db/db mice. Vascular smooth muscle cells and bone marrow-derived macrophages stimulated with aldosterone also exhibited increased expression of inflammatory components, which may contribute to diabetes-associated vascular changes. Patients with T2DM exhibited a correlation between aldosterone and IL-1? levels and/or glycemia. In conclusion, our study demonstrates that aldosterone induces vascular dysfunction and inflammatory process in type 2 diabetes through MR receptor activation and NLRP3 inflammation.

Ces dernières décennies ont mis à jour une implication pathologique nouvelle del’aldostérone, via le récepteur aux minéralocorticoïdes (RM) dans le coeur. L’ensemble desdonnées issues des études expérimentales et des essais cliniques suggère une association délétèreentre l’aldostérone et la survenue d’arythmies. L’utilisation d’antagonistes du RM prévient cesarythmies. Cependant, les voies de signalisations, comme les mécanismes moléculaires soustendantces effets bénéfiques du blocage des RM demeurent incertains. Nous avons accumulésdes preuves d’une modulation de la signalisation calcique dans le cardiomyocyte, et en particulierde l’influx calcique (Ca2+) au travers du canal Ca2+ de type L (LTCC). Celui-Ci pourrait être unecible primaire de l’aldostérone et du RM dans les cardiomyocytes ventriculaires. Toutefois, lesmécanismes par lesquels l’aldostérone et le RM régulent l’expression du LTCC restent à définir.Au cours de ces travaux menés sur cardiomyocytes de rats nouveau-Nés, nous avonsétudiés les évènements moléculaires par lesquels l’aldostérone exerce ses effets sur le CaV1.2,qui correspond à la sous-Unité principale du LTCC formant le pore du canal ; cette protéine estcodée par le CACNA1C. Par microscopie confocale, nous avons suivi en temps réel le traffickingnucléo-Cytoplasmique du RM couplé à la GFP en réponse à l’aldostérone, démontrant ainsi queles RM cardiaques sont fonctionnels. Le traitement durant 24 heures des cardiomyocytes avec del’aldostérone montre une augmentation dose-Dépendante des protéines et de l’ARN messager duCaV1.2. L’utilisation de la technique du gène rapporteur de la luciférase permet l’analyse del’activité du promoteur du CaCNA1C. Celui-Ci montre une activité transcriptionnelle dose ettemps dépendante en réponse à l’aldostérone. De plus, ces effets sont dépendant des RM carinhibés en présence de RU28318, un antagoniste sélectif du RM, ou par l’utilisation de siRNAdirigés contre le RM. L’analyse in silico de la séquence du promoteur du CaCNA1C nous a permisd’identifier cinq séquences putatives correspondant à des éléments de réponse auxglucocorticoïdes (GRE). La mutation du site le plus lointain du site d’initiation de la transcriptionne révèle aucun changement dans les réponses transcriptionnelles induites par un RM humainconstitutivement actif (hMRΔ5,6) ou dans les réponses doses-Dépendantes de l’aldostérone ou dela déxaméthasone, un glucocorticoïde de synthèse. La mutation des trois sites GRE putatifssuivants provoque une diminution des réponses au hMRΔ5,6 comme à l’aldostérone, alors que lesréponses à la déxaméthasone sont soit inchangées, soit augmentées. En contraste, la mutation dusite le plus proximal du promoteur augmente de façon importante l’activité transcriptionnelle dupromoteur en réponse au hMRΔ5,6, à l’aldostérone comme à la déxaméthasone.Ces résultats démontrent que le LTCC cardiaque constitue une cible directe del’aldostérone et du RM, et apportent de nouvelles perspectives quant aux conséquencesmoléculaires et fonctionnelles engendrées par l’activation délétère du système minéralocorticoïdedans la défaillance cardiaque. / During the past decades, major novel pathogenic roles of the steroid hormone,aldosterone, via the Mineralocorticoid Receptor (MR) have been identified in heart. Collectively,experimental studies and clinical trials, suggest a detrimental association between aldosteroneand life threatening arrhythmias that may be prevented by MR blockade. However, the signalingpathways and underlying mechanisms still remain elusive. We have accumulated evidence thatmodulation of Ca2+ signaling, especially Ca2+ influx via L-Type Ca2+ channel (LTCC), might bethe primary aldosterone/MR target in ventricular cardiomyocytes. Yet, the molecularmechanisms by which MR regulates expression of LTCC remain to be defined. Here, weinvestigated, in primary cultures of neonatal rat ventricular myocytes, the molecular eventscritical for aldosterone-Mediated cardiac effects on CaV1.2, the pore-Forming main subunit ofLTCC, which is encoded by the CaCNA1C gene.We showed that cardiac MR are functional as demonstrated by aldosterone-Induced MRnucleocytoplasmic trafficking observed by time-Lapse imaging of transfected GFP-Labeled MRusing confocal microscopy. Aldosterone exposure for 24 hours, induced a dose-Dependentincrease in CaV1.2 expression at both mRNA and protein levels. Analysis of the CaCNA1Cpromoter activity using luciferase reporter assays, revealed a dose- and time-Dependent activationby aldosterone. These effects were inhibited in the presence of either RU28318, a selective MRantagonist, or MR siRNA. In silico analyze enabled us to identify five putative GlucocorticoidResponse Elements (GRE) within the CaCNA1C promoter sequence. The mutation of the mostdistal GRE from Transcription Start Site (TSS) did not altered responses either elicited by theconstitutively active human MR (hMRΔ5,6) or dose-Dependent effects of aldosterone anddexamethasone (a synthetic glucocorticoïd with minimal MR effect). Mutations of the three nextones decreased responses to hMRΔ5,6 and aldosterone, whereas dexamethasone responses wereeither unchanged or increased. In sharp contrast, the mutation of the most proximal GRE fromTSS, increased responses to hMRΔ5,6, aldosterone and dexamethasone.These results provide new insights into the molecular mechanisms associated with cardiacMR activation, and suggest that LTCC is a primary MR target, with subsequent molecular andfunctional consequences that could lead to MR-Related cardiac dysfunction.

A angiotensina (Ang) II e aldosterona induzem hipertensão arterial por mecanismos em parte mediados pela imunidade adaptativa, envolvendo linfócitos T auxiliares respondedores (Tresp). Os linfócitos T reguladores (Treg) são capazes de suprimir os efeitos próinflamatórios do sistema imune. O presente estudo avaliou se a transferência adotiva de Treg é capaz de prevenir a hipertensão e a lesão vascular induzidas pela Ang II ou pela aldosterona, em dois protocolos distintos. No protocolo com Ang II, camundongos machos C57BL/6 sofreram a injeção endovenosa de Treg ou Tresp, sendo depois infundidos com Ang II (1&#956;g/kg/min), ou salina (grupo controle) por 14 dias. No protocolo com aldosterona, um outro conjunto de animais sofreu injeções de Treg ou Tresp, sendo depois infundido com aldosterona (600&#956;g/kg/d) ou salina (grupo controle), pelo mesmo intervalo de tempo. O grupo tratado com aldosterona recebeu salina 1% na água. Tanto o grupo Ang II como aldosterona apresentaram elevação da pressão arterial sistólica (43% e 31% respectivamente), da atividade da NADPH oxidase na aorta (1,5 e 1,9 vezes, respectivamente) e no coração (1,8 e 2,4 vezes, respectivamente) e uma redução da resposta vasodilatadora à acetilcolina (de 70% e 56%, respectivamente), quando comparados com os respectivos controles (P<0,05). Adicionalmente, a administração de Ang II proporcionou um aumento rigidez vascular (P<0,001), na expressão de VCAM-1 nas artérias mesentéricas (P<0,05), na infiltração aórtica de macrófagos e linfócitos T (P<0,001) e nos níveis plasmáticos das citocinas inflamatórias interferon (INF)-&#947;, interleucina (IL)-6, Tumor necrosis factor (TNF)-&#945; e IL-10 (P<0,05). Ang II causou uma queda de 43% no número de células Foxp3+ no córtex renal, enquanto que a transferência adotiva de Treg aumentou as células Foxp3+ em duas vezes em comparação com o controle. A administração de Treg preveniu o remodelamento vascular induzido pela aldosterona, observado na relação média/lúmen e na área transversal da média das artérias mesentéricas (P<0,05). Todos os parâmetros acima foram prevenidos com a administração de Treg, mas não de Tresp. Estes resultados demonstram que Treg são capazes de impedir a lesão vascular e a hipertensão mediadas por Ang II ou por aldosterona, em parte através de ações antiinflamatórias. Em conclusão, uma abordagem imuno-modulatória pode prevenir o aumento da pressão arterial, o estresse oxidativo vascular, a inflamação e a disfunção endotelial induzidos por Ang II ou aldosterona. / Angiotensin (Ang) II and aldosterone (aldo) induce hypertension through mechanisms in part mediated by adaptive immunity and T responder lymphocytes. T regulatory (Treg) lymphocytes suppress pro-inflammatory mediators of the immune system. We questioned whether Treg adoptive transfer will blunt Ang II or aldo-induced hypertension and vascular injury, by evaluating two distinct protocols. In the Ang II protocol, male C57BL/6 mice were injected i.v. with Treg or T responder cells, and then infused with Ang II (1&#956;g/kg/min) or saline, for 14 days. In the aldosterone protocol, another set of animals was injected with Treg or T responder cells, and then infused with aldosterone (600&#956;g/kg/d) or saline, for the same period. The aldosterone group received saline 1% in drinking water. Both Ang II and aldosterone treated mice presented an increase in systolic blood pressure (43% and 31% respectively), of NADPH oxidase activity in aorta (1.5 and 1.9 fold, respectively) and heart (1.8 and 2.4 fold respectively) and an impaired vasodilatory response do acetylcholine (by 70% and 56% respectively), when compared to their controls (P<0.05). In addition, Ang II administration resulted in increased vascular stiffness (P<0.001), mesenteric artery vascular cell adhesion molecule (VCAM-1) expression (P<0.05), aortic macrophage and T cell infiltration (P<0.001), and the plasma levels of the inflammatory cytokines INF-&#947;, IL-6, TNF-&#945;, and IL-10 (P<0,05). AngII caused a 43% decrease in the number of Foxp3+ cells in the renal cortex, while Treg adoptive transfer increased Foxp3+ cells 2-fold compared to control. Treg administration prevented aldosterone-induced vascular remodelling, as observed by media to lumen ratio and media cross sectional area analysis of mesenteric arteries (P<0,05). All the above were prevented by Treg but not by T responder cell adoptive transfer. These results demonstrate that Treg suppress Ang II or aldo-mediated vascular injury and BP elevation, in part through anti-inflammatory actions. These findings suggest that an immunomodulatory approach can prevent Ang II or aldosterone-induced blood pressure elevation, vascular oxidative stress, inflammation and endothelial dysfunction.

O objetivo do presente trabalho foi analisar o papel do ANP na ação não genômica da Aldosterona sobre o trocador Na+/H+ no segmento S3 do túbulo proximal de rato, isolado, in vitro. Os resultados indicam que o pHi basal do segmento S3 proximal de ratos é 7.20 + ou - 0.009 (n = 47/209). O valor médio da velocidade de extrusão celular de H+ na condição controle é de 0.195 + ou - 0.012 pHi/min (n = 16/96). Os dados confirmam que a aldosterona apresenta um efeito bifásico sobre o NHE1: em baixas doses (10-12 M) o estimula, enquanto que em altas doses (10-6M), o inibe. O ANP (10-6 M) não possui efeito sobre o NHE1; contudo, o ANP previne ambos os efeitos da aldosterona sobre esse trocador. O valor médio da concentração do cálcio no citosol ([Ca2+]i) na condição controle é 100 ± 1 (n = 5) hM Adicionalmente, nossos estudos mostram que o ANP diminui a [Ca2+]i e inibe o efeito estimulatório de ambas as doses de aldosterona sobre esse parâmetro. / The effects of aldosterone and ANP(2 min preincubation) on the intracellular pH recovery rate (pHirr) after the acid load induced by NH4Cl and on the [Ca2+]i were investigated in isolated rat S3 segment. The basal pHi was 7.20 + ou - 0.009(n=47/209) and the basal pHirr via the Na+/H+ exchanger was 0.195 + ou - 0.012 pHi/min(n=16/96). Aldosterone(10-12M) caused an increase in the pHirr, but aldosterone(10-6M) decreased it. ANP(10-6M) alone or plus aldosterone(10-12 or 10-6 M) had no effect on pHirr. The basal [Ca2+]i was 100 + ou - 1(n=5)hM. After 1 min of Aldosterone pi there was a transient and dose-dependent increase of the [Ca2+]i and after 6 min pi there was a new increase of [Ca2+]i. ANP alone decreased the [Ca2+]i and prevented the stimulatory effects of aldosterone(10-12 or 10-6M) on this parameter. The data indicate a nongenomic action of aldosterone and ANP on the Na+/H+ exchanger and on [Ca2+]i and are compatible with stimulation of the this exchanger by increases in [Ca2+]i in the lower range (at10-12M aldosterone) and inhibition by increases at high levels (at10-6M aldosterone).