O córtex pré-límbico (PL) é uma importante área límbica envolvida em vários processos funcionais correlatos ao estresse, tais como respostas cardiovasculares, hormonais e comportamentais. O modelo de estresse de restrição (ER) foi padronizado na literatura como uma situação aversiva capaz de promover aumento da pressão arterial e frequência cardíaca, queda da temperatura cutânea e estimulação do eixo hipotálamo-pituitária-adrenal (HPA). Trabalhos da literatura evidenciaram que ratos submetidos ao ER apresentavam aumento da atividade neuronial no PL, sugerindo que essa estrutura module respostas ao ER. Assim, a inibição temporária de sinapses no PL potencializou a resposta taquicárdica induzida pelo ER, sem alterar a resposta pressora. Além do controle cardiovascular, outros trabalhos demonstraram que o PL também participa do controle hormonal durante o ER. O ER agudo também está envolvido com a produção de espécies reativas de oxigênio (EROs), fator que pode estar envolvido nas alterações a longo prazo observadas após exposição a uma situação aversiva. O sistema renina angiotensina (SRA) central modula respostas cardiovasculares, inclusive aquelas induzidas por situações aversivas, além de ter um papel reconhecido na produção de EROs. Além disso, foi demonstrado que o PL possui SRA funcional com presença dos peptídeos a ele relacionados. Baseado nos fatos mencionados acima, a hipótese do presente projeto é que a neurotransmissão angiotensinérgica do PL está envolvida na modulação de respostas autonômicas (aumento de pressão arterial e frequência cardíaca, e queda da temperatura cutânea) e hormonal (aumento plasmático de corticosterona) evocadas pelo ER, e que essa via envolveria a formação de EROs. A microinjeção do inibidor da enzima conversora de angiotensina (ECA) lisinopril no PL, nas doses de 0,5 e 1nmol/100nL, reduziu a resposta pressora, sendo a dose de 1nmol/100nL de lisinopril também capaz de reduzir a resposta taquicárdica induzida pelo ER; porém nenhuma dose utilizada ocasionou mudanças na queda da temperatura cutânea evocada pelo ER. O pré-tratamento do PL com o antagonista de receptores do subtipo AT1 candesartan reduziu o efeito pressor induzido pelo ER, porém não alterou a resposta taquicárdica e queda da temperatura cutânea associadas ao ER. Por sua vez, o pré-tratamento com o antagonista de receptores do subtipo AT2, PD123177, reduziu a resposta taquicárdica sem alterar a resposta pressora e a queda da temperatura cutânea evocadas pelo ER. Em adição, o estresse de restrição agudo e os pré-tratamentos realizados não foram capazes de alterar a atividade da enzima NADPH oxidase no PL. Em conclusão, os presentes resultados sugerem a participação do SRA na modulação da resposta cardiovascular ao ER, através da ativação de receptores AT1, e AT2 do PL, afetando respectivamente, o componente vascular e o cardíaco da resposta autonômica causada pelo ER. Além disso, os resultados da atividade da enzima NADPH oxidase no PL sugerem que o ER agudo, os receptores AT1, AT2 e a ECA não modulam o status oxidativo local. / The prelimbic cortex is an important limbic structure involved in several stressrelated functional processes, such as cardiovascular, hormonal and behavior responses. Restraint stress (RS) was standardized in literature as an aversive situation able to promote blood pressure and heart rate increases, reduction in tail temperature and stimulation of the hypothalamic-pituitary-adrenal axis (HPA). Previous studies demonstrated that rats submitted to RS exhibited increased neuronal activity in the PL, suggesting that this structure modulates RS-evoked responses. Temporary, synaptic temporary inhibition in the PL markedly increased the RS-evoked tachycardiac response, without affecting the pressor one. Beyond cardiovascular control, other studies demonstrated that PL also participates in hormonal control during RS. Acute RS is also involved in the production of reactive oxygen species (ROS), which could be involved in long- term changes observed after exposure to an aversive situation. The central renin-angiotensin system (RAS) modulates cardiovascular responses, including those induced by aversive situations. In addition, this system has a well-known role in ROS production. Furthermore, the presence of angiotensinergic peptides in PL has also been demonstrated, suggesting the existence of a functional RAS in this structure. Based on the facts mentioned above, the hypothesis of the present study was that the angiotensinergic neurotransmission in PL is involved in the modulation of autonomic responses (blood pressure and heart rate increase, and reduction in tail temperature) evoked by RS, and this pathway would involve ROS formation. Microinjection of lisinopril (0.5 and 1nmol/100nL), an inhibitor of angiotensinconverting enzyme (ACE), into PL reduced the pressor response, and the dose 1nmol/nL was also able to reduce the tachycardiac response induced by RS; however, none of doses changed the reduction in tail temperature evoked by RS. PL treatment with candesartan, an AT1 receptors antagonist, reduced the RS-evoked pressor response, but did not affect the RS-evoked tachycardiac response and reduction in tail temperature. In addition, pretreatment with PD123177, an AT2 receptors antagonist, reduced the RS-evoked tachycardiac response, without affecting the pressor response or the RS-evoked reduction in tail temperature. In addition, neither acute RS or local treatments affected NADPH oxidase activity in the PL. In conclusion, the present results suggests the involvement of the central RAS in the modulation of the cardiovascular responses caused by RS, through the activation of both AT1 and AT2 receptors in the PL. The PL AT1 receptors modulating the vascular, and the AT2 modulating the cardiac component of RS-evoked autonomic response. Furthermore, our study suggests that neither acute RS or local AT1, AT2 and ACE affect oxidative status in the PL.

O alto consumo de sódio é o principal fator de risco para o desenvolvimento de doenças cardíacas. A sobrecarga de sal na dieta aumenta o conteúdo de angiotensina II no coração. Muitos estudos tem avaliado o papel da angiotensina II no desenvolvimento da hipertrofia e fibrose cardíaca. A angiotensina II age através de dois receptores: o receptor de angiotensina II tipo 1 (AT1) e o receptor de angiotensina tipo 2 (AT2). Embora muitos estudos têm elucidado o papel do receptor AT1 e sobrecarga de sal na dieta no desenvolvimento da hipertrofia, os estudos envolvendo o receptor AT2 ainda são controversos. Com o objetivo de entender melhor o papel do AT2 em modelos de sobrecarga de sal na dieta no desenvolvimento da hipertrofia cardíaca, ratos Wistars machos foram alimentados com uma dieta normal ou hipersódica desde o desmame até a décima oitava semana de idade. Ambos os grupos foram subdivididos em dois subgrupos. A partir da sétima semana de idade cada um dos subgrupos recebeu o tratamento com composto 21 (3mg/kg por dia, n=16), um agonista do AT2. Peso corporal, pressão arterial caudal, consumo de ração, ingestão hídrica, volume urinário, hematócrito, massas ventriculares, diâmetro transverso do cardiomiócito, porcentagem de fibrose intersticial, expressão gênica e proteica dos componentes do sistema renina-angiotensina foram avaliados. O C21 preveniu o desenvolvimento da hipertrofia e fibrose cardíaca em ratos alimentados com dieta hipersódica. O C21 preveniu o incremento da pressão arterial dos ratos alimentados com dieta hipersódica / High salt intake is one of the main risk factors for the development of cardiovascular diseases. Dietary salt overload was found to increase cardiac angiotensin II content. Many studies have evaluated the role of angiotensin II on the development of cardiac hypertrophy and fibrosis. Angiotensin II acts through two main receptors: angiotensin II type 1 (AT1) and type 2 (AT2) receptors. Though there are many studies pointing to the effects of the AT1 and high salt diet, the role of AT2 and its effects in dietary salt overload model is still not elucidated. Aiming to better understand the role of AT2 receptor in models of salt overload on cardiac hypertrophy and fibrosis, male Wistar rats were fed normal or high salt diet from weaning up to 18 weeks of age. Both groups were divided into two subgroups. Starting at 7 weeks of age they were treated or not with compound 21 (3mg/kg per day, n=16), an AT2 receptor agonist. Body weight, blood pressure, food intake, water intake, urine volume, plasma and urinary sodium and potassium, cardiomyocyte transverse diameter, percentage of cardiac fibrosis, gene and protein expression of renin, angiotensinogen, angiotensin converting enzyme, AT1 and AT2 were measured. Compound 21 prevented the development of cardiac hypertrophy and fibrosis in rats that received high salt diet. Compound 21 also reduced the increased blood pressure, prevented the lower weight gain in animals fed with high salt diet

Treinamento aeróbio (T) reduz o elevado RNAm de angiotensinogênio (AGT), no NTS de SHR. Este trabalho investiga a sequência temporal dos efeitos do T sobre a pressão arterial, freqüência cardíaca, RNAm e proteína de AGT e AT1 no PVN, NTS e RVL. WKY e SHR, submetidos a T de baixa intensidade por 0, 1, 2, 4, 8 ou 12 semanas, tiveram a PA e FC basais registradas e após o sacrifício determinou-se o RNAm (RT-PCR) e imunofluorescência para AGT ou AT1 e NeuN. SHR (vs WKY) apresentaram PA (174±2 vs 123±2 mmHg) e FC elevadas e maior RNAm de AT1 no NTS(+67%). T reduziu a PA em S de T4 à T12 e FC em S em T4 e WKY em T8. Nos SHR, T reduziu o RNAm de AGT no PVN (-34% em T2) e NTS (-30% em T4), e de AT1 apenas em T12 (-56% e -39%, PVN e NTS). Nos WKY, T reduziu o RNAm de AGT(-49%) e AT1(-58%) no RVL a partir de T8. A imunofluorescência localizou estas proteínas, confirmando as alterações pelo T. Estes dados indicam que a queda de PA e FC são progressivas e mais precoces em SHR que em WKY e acompanhadas por redução de AGT no PVN e NTS dos S e redução de AGT e AT1 no RVL dos WKY. / The aerobic training (T) for 12 weeks was effective in reducing the mRNA of angiotensinogen(AGT) in NTS of SHR. We evaluate in SHR and WKY time-course changes of T on blood pressure, heart rate, AGT and AT1 mRNA and protein (PVN, NTS and RVL). WKY and SHR were T for 0,1,2,4,8 or 12 weeks, the baseline BP and HR were recorded and mRNA(RT-PCR) and Immunofluorescence for AGT, AT1 and NeuN were made. SHR(vs. WKY) had high BP(174±2 vs 123±2 mmHg), HR and mRNA of AT1 in the NTS(+67%). T reduced the BP only in SHR(-6.5% from T4 to T12) and HR in SHR in T4 and WKY in T8. In the SHR, T reduced the AGT mRNA within the PVN (-34% in T2) and NTS (-30% in T4), and AT1 mRNA only in T12 in PVN(-56%) and NTS(-39%). In WKY, T decreased of both AGT(-49%) and AT1(-58%) mRNA in the RVL at T8. Immunofluorescence localized these proteins, confirming the mRNA content changes induced by T. Our data indicate that the fall of BP and HR are progressive and appear earlier in SHR than in WKY and are accompanied by reduced AGT in the PVN and NTS of the SHR and reduced AGT and AT1 in RVL of WKY.

O tratamento combinado com IECA e BRA foi proposto como alternativa para o tratamento da ND. Nosso objetivo foi avaliar se o tratamento IECA+BRA era superior ao tratamento com IECA em termos de proteinúria e excreção urinária de marcadores inflamatórios. Cinqüenta e seis pacientes com ND iniciaram o uso de enalapril. Após 4 meses, os pacientes passaram a receber losartan (Grupo E+L) ou placebo (Grupo E). As incidências de hipercalemia (HK) e deterioração aguda da função renal (DAFR) foram avaliadas. A análise de ANOVA de medidas repetidas não revelou diferença entre os grupos, mas, após ajustes, a progressão da proteinúria foi pior no Grupo E+L. A proteinúria final mostrou-se significativamente maior no Grupo E+L (proteinúria final estimada de 1,2 vs 2,6 g/d/1.73m2, p= 0.03). Os resultados foram confirmados nos modelos de regressão logística. Ocorreram 7 eventos de HK (12,6%) e 9 de DAFR (16,1%). Nossos dados sugerem que, em ND avançada, o tratamento combinado IECA+BRA não seja superior ao tratamento com IECA isoladamente em relação à proteinúria e marcadores inflamatórios. / Combined treatment with an ACE inhibitor (ACEI) and an angiotensin II receptor blocker (ARB) has been proposed for diabetic nephropathy (DN) treatment. In this study we compared the effect of association therapy versus ACEI on proteinuria progression and on urinary inflammatory biomarkers in DN. Fifty-six patients with DN were started on enalapril. After 4 months, losartan (Group E+L) or placebo (Group E) treatment was started. Incidences of hyperkalemia (HK) and acute kidney function deterioration (AKFD) were monitored. Unadjusted repeated measures ANOVA revealed no difference between groups. After adjustment, proteinuria progression was significantly higher in the E+L Group. In addition, final proteinuria was significantly higher in the E+L Group (predicted adjusted final proteinuria 1,2 vs 2,6 g/d/1,73m2, p=0,03). Finally, logistic regression models showed the same results. We observed 7 HK events (12,6%) and 9 AKFD events (16,1%). These results suggest that, at least in advanced DN, association therapy is not superior to ACEI monotherapy in terms of proteinuria and inflammatory biomarkers.

Obesity is a major health problem in the United States and worldwide. It increases the risk for type-2 diabetes and cardiovascular diseases. A chronic low-grade inflammation occurring in white adipose tissue (WAT) is causally linked to the development of insulin resistance (IR), metabolic syndrome and obesity-associated chronic diseases. The aim of this dissertation research was to elucidate the WAT function in metabolic syndrome using genetic (overexpression of an adipose pro-inflammatory hormone, angiotensinogen) and nutritional manipulations/approaches (caloric restriction and omega-3 fatty acids), with specific emphasis on the role of inflammation. Previous research indicates that WAT renin-angiotensin system (RAS) is overactivated in obesity. However, its role in the pathogenesis of IR is hitherto unknown. Using mice overexpressing angiotensinogen (Agt), the only precursor for the hypertensive hormone angiotensin (Ang) II, in WAT, we showed that adipose-specific RAS overactivation leads to systemic IR. This is at least in part due to Ang II, NADPH oxidase and NF-kB-dependent increases in WAT inflammation. Caloric restriction is the main dietary intervention to treat obesity-associated metabolic disorders. While most health agencies recommend a low-fat diet, energy-restricted high-fat diets (HFR) are also claimed to be effective in this regard. Here, we show that weight loss due to HFR is accompanied by improvements of IR but only partial resolution of WAT inflammation. Further, this diet negatively impacted the adipokine profile supporting the current recommendations for low-fat diets. Dietary interventions targeted at reducing WAT inflammation have not been explored in detail. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid of marine origin with anti-inflammatory properties. We show that EPA is able to both prevent and reverse high-fat diet-induced IR and hepatic steatosis via modulation of WAT inflammation. In conclusion, primary changes occurring in WAT, such as overexpression of Agt, can lead to WAT inflammation and systemic IR. Moreover, nutritional interventions targeting at reducing adiposity (caloric restriction) and inflammation (EPA) can both lead to improvements in systemic IR. Our findings support the current recommendation of low-fat diets for improvement in metabolic profile and show that dietary modulation of WAT function can be used to improve metabolic derangements in obesity.

Hydronephrosis, due to ureteropelvic junction obstruction, is a common condition in infants with an incidence of approximately 0.5-1%. During the last decade, the surgical management of non-symptomatic hydronephrosis has become more conservative, and the long-term physiological consequences of this new policy are unclear. The overall aim of this thesis was to determine whether there is a link between hydronephrosis and the development of hypertension. Hydronephrosis was induced by partial ureteral obstruction in 3-week old rats or mice. In the adult animals, blood pressure was measured telemetrically during different sodium conditions and the renal function was evaluated. Both species developed salt-sensitive hypertension and histopathological changes (i.e. fibrosis, inflammation, glomerular and tubular changes) that correlated with the degree of hydronephrosis. An abnormal renal excretion pattern with increased diuresis and impaired urine concentrating ability was observed in hydronephrosis. The mechanisms were primarily located to the diseased kidney, as relief of the obstruction attenuated blood pressure and salt-sensitivity. Increased renin angiotensin system activity, due to ureteral obstruction, might be involved in the development but not necessary the maintenance of hypertension. Hydronephrotic animals displayed reduced nitric oxide availability, which might be due to increased oxidative stress in the diseased kidney. Renal nitric oxide deficiency and subsequent resetting of the tubuloglomerular feedback mechanism, appeared to have an important role in the development of hypertension. In conclusion, experimental hydronephrosis, induced by partial ureteral obstruction, provides a new model for studies of salt-sensitive hypertension. Furthermore, the new findings imply that the current conservative treatment strategy in hydronephrosis should be reconsidered in favour of treatment that is more active, in order to prevent the development of renal injury and hypertension in later life.

Το μετατρεπτικό ένζυμο της αγγειοτενσίνης (ACE) είναι μία διπεπτιδυλκαρβοξυπεπτιδάση ψευδαργύρου που ανήκει στην οικογένεια των gluzincin πεπτιδασών της οποίας η θερμολυσίνη θεωρείται ως πρωτότυπο μέλος. Το ένζυμο πήρε το όνομά του από τη δυνατότητά του να μετατρέπει το βιολο- γικώς ανενεργό δεκαπεπτίδιο αγγειοτενσίνη-Ι στο οκταπεπτίδιο αγγειοτεν- σίνη-ΙΙ, το οποίο εμφανίζει ισχυρή αγγειοσυσπαστική δράση. Μία άλλη βασική δυνατότητα του ACE είναι η αδρανοποιήση του εννεαπεπτιδίου βραδυκινίνη που έχει αγγειοδιασταλτική δράση. Αυτές οι δύο σημαντικές ιδιότητες του ACE το καθιστούν ένα από τα σημαντικότερα συστατικά του συστήματος ρενίνης-αγγειοτενσίνης-αλδοστερόνης. Υπάρχουν δύο ισομορφές του ACE που μεταγράφονται από το ίδιο γονίδιο κατά τρόπο ιστοειδικό. Η σωματική ισομορφή του ACE, η οποία εμφανίζεται στην επιφάνεια των ενδοθηλιακών κυττάρων, είναι μία γλυκοπρωτεΐνη η οποία αποτελείται από μία ενιαία, πολυπεπτιδική αλυσίδα 1306 αμινοξέων. Η σπερματική ισομορφή που εμφανίζεται στους όρχεις και στα κύτταρα σπέρματος είναι μία χαμηλότερης-μοριακής μάζας γλυκοπρωτεΐνη 732 αμινοξέων. Η σωματική ισομορφή αποτελείται από δύο ομόλογες περιοχές (περιοχή Ν και C). Κάθε περιοχή περιέχει ένα ενεργό κέντρο με ένα συντηρημένο δεσμευτικό μοτίβο ψευδαργύρου HEXXH, όπου οι δύο ιστιδίνες είναι οι δύο πρώτοι υποκαταστάτες του ιόντος ψευδαργύρου. Μετά από 24 αμινοξέα στην αλληλουχία του μορίου, βρίσκεται ένα γλουταμινικό οξύ που είναι ο τρίτος υποκαταστάτης του ιόντος ψευδαργύρου. Η ύπαρξη αυτών των Ν- και C- περιοχών είναι πιθανότατα το αποτέλεσμα ενός αρχέγονου γεγονότος διπλασιασμού γονιδίων το οποίο έλαβε χώρα κατά την διάρκεια της εξέλιξης των σπονδυλωτών. Οι δύο περιοχές εμφανίζουν εκλεκτικότητα έναντι διαφόρων υποστρωμάτων, αναστολέων και διαφορές στην απαιτούμενη συγκέντρωση ιόντων χλωρίου προκειμένου να έχουν καταλυτική δραστικότητα. Υπάρχουν δύο υποστρώματα τα οποία εμφανίζουν εκλεκτικότητα έναντι του Ν-ενεργού κέντρου: το Ν-ακετυλ-σερυλασπαραγυλο-λυσυλ-προλυλ πεπτίδιο, το οποίο ρυθμίζει τη διαφοροποίηση και τον πολλαπλασιασμό των πολυδύναμων αιμοποιητικών κυττάρων και το πεπτίδιο αγγειοτενσίνη-(1-7) που είναι το αποτέλεσμα της δράσης της βραδυκινίνης. Αφ' ετέρου, τα ενεργά κέντρα και των δύο περιοχών καταλύουν την υδρόλυση της αγγειοτενσίνης-Ι και τη βραδυκινίνης με παρόμοια αποτελασματικότητα. Εντούτοις, η αναστολή του Ν-ενεργού κέντρου με το φωσφινικό πεπτίδιο RXP407 δεν έχει καμία επίδραση στην ρύθμιση της αρτηριακής πίεσης. Διαγονιδιακά ποντίκια τα οποία εκφράζουν μόνο το Ν-ενεργό κέντρο εμφανίζουν φαινότυπο παρόμοιο με αυτόν που εμφανίζεται σε ποντίκια στα οποία το γονιδίο του ACE έχει απαλειφθεί πλήρως. Κατά συνέπεια, το C-ενεργό κέντρο φαίνεται να είναι απαραίτητο και σημαντικό για τον έλεγχο της αρτηριακής πίεσης και της καρδιαγγειακής λειτουργίας. Η σπερματική ισομορφή του ACE είναι πανομοιότυπη με την C-περιοχή της σωματικής εκτός από μία μοναδική ακολουθία 36 αμινοξέων που βρίσκεται στο Ν-τελικό άκρο του. Επίσης έχει αποδειχθεί ότι η σπερματική ισομορφή του ACE διαδραματίζει σημαντικό ρόλο στην ωρίμανση του σπέρματος και στη δέσμευση αυτού στο επιθήλιο του ωαγωγού των ωοθηκών. Ο στόχος αυτής της διατριβής ήταν α) η υπερέκφραση, σε βακτηριακά κύτταρα, ο καθαρισμός και η λήψη σε διαλυτή μορφή δύο πεπτιδίων του ACE μεγέθους 108 αμινοξέων(Ala361-Gly468 (ACE_N), Ala959-Ser1066 (ACE_C)). Αυτή η πειραματική προσέγγιση επελέγη λόγω της ευκολίας χειρισμού και καλλιέργειας που εμφανίζουν τα βακτηριακά κύτταρα και λόγω της δυνατότητας της χρήση επισημασμένων με 15Ν ή/και 13C θρεπτικών μέσων. β) Η κατοχή ενός τόσο μεγάλου πεπτιδίου σε διάλυμα, επισημασμένο ή μη, δίνει τη δυνατότητα μελέτης του ως προς τα δομικά χαρακτηριστικά του χρησιμοποιώντας τη φασματοσκοπία κυκλικού διχροϊσμού ή/και πυρηνικού μαγνητικού συντονισμου (NMR). Τα προαναφερθέντα πρωτεϊνικά τμήματα υπερεκφράστηκαν σε βακτηριακά κύτταρα και ελήφθησαν σε καθαρή μορφή. Η καθαρότητά τους ήταν μεγαλύτερη από 99%. Η απόδοση για το πρωτεϊνικό τμήμα ACE_N ήταν 9mg και για το πρωτεϊνικό τμήμα ACE_C ήταν 6mg από 1L καλλιέργειας βακτηριακών κυττάρων. Τα τμήματα αυτά μελετήθηκαν ως προς την δευτεροταγή τους διαμόρφωση με φασματοσκοπία κυκλικού διχρωϊσμού. Τα αποτελέσματα της μελέτης αυτής έδειξαν ότι παρουσία 1,1,1-τριφθοροαιθανόλης, σε συγκέντρωση μεγαλύτερη από 60% και τα δύο τμήματα λαμβάνουν διαμόρφωση η οποία βρίσκεται σε συμφωνία με τη θεωρητικώς υπολογιζόμενη και με αυτή που έχει βρεθεί από κρυσταλλογραφικές μελέτες του ενζύμου. Το αποτέλεσμα αυτό μερικώς επιβεβαιώθηκε για το πρωτεϊνικό τμήμα ACE_N με τη μελέτη αυτού με φασματοσκοπία Πυρηνικού Μαγνητικού Συντονισμόυ. Η πλήρης επιβεβαίωση δεν κατέστει δυνατή λόγω της αδυναμίας λήψης καλής ποιότητας φάσματος 2D-NOESY. Συμπερασματικά, η περιγραφόμενη σε αυτή τη Διατριβή μεθοδολογία εμφανίζει πλεονεκτήματα όσον αφορά την ταχύτητα παραγωγής, τη δυνατότητα καθαρισμού των παραγομένων πεπτιδίων, καθώς και καλή επαναληψιμότητα. Οι in vitro επαναδιατεταγμένες ανασυνδυασμένες πρωτεΐνες εμφάνισαν χαρακτηριστικά δευτεροταγούς δομής, όμοια σχεδόν με αυτά που έχουν αποκαλυφθεί από την κρυσταλλογραφική μελέτη του ACE, έχοντας υψηλό ποσοστό σε α-έλικα. Κατά συνέπεια, αυτή η μελέτη περιγράφει ένα αποτελεσματικό σύστημα για την παραγωγή μεγάλων ποσοτήτων καθαρών πεπτιδίων του ACE που μπορούν να χρησιμοποιηθούν για διάφορες μελέτες. / Angiotensin converting enzyme (ACE) is a gluzincin zinc dipeptidyl carboxypeptidase I, of which thermolysin is considered the prototypical member. This enzyme took its name from its ability to convert the decapeptide Angiotensin-I to octapeptide Angiotensin-II, which is a highly potent vasoconstrictor. Another basic ability is to inactivate bradykinin, a vasodilatory peptide. These two major activities render ACE through the renin– angiotensin–aldosterone system. There are two isoforms of ACE that are transcribed from the same gene in a tissue-specific manner. Somatic ACE, which is present in brush-border epithelial cells and endothelial cells, exists as a glycoprotein composed of a single, large polypeptide chain of 1,306 amino acids, whereas in sperm cells it is a lower-molecular-mass glycoform of 732 amino acids. The somatic form consists of two homologous domains (N and C domain). Each domain contains an active site with a conserved HEXXH zinc binding motif, where the two histidines are zinc ligands, with a glutamate 24 residues downstream forming the third ligand. These N- and C-domains most likely are the result of an ancient gene duplication event that occurred during vertebrate evolution. The two domains differ in their substrate specificities, inhibitor, chloride activation profiles, and physiological functions. There are two N-domainspecific substrates: the peptide N-acetyl-serylaspartyl-lysyl-proline, which regulates haematopoietic stem cell differentiation and proliferation; and the bradykinin-potentiating peptide angiotensin-(1-7). On the other hand, the active sites of both domains catalyse the hydrolysis of angiotensin I and the vasodilator bradykinin with similar efficiency. However, inhibition of the N domain with a phosphinic peptide RXP407 has no effect on blood pressure regulation and expression in transgenic mice of the N domain alone produces a phenotype similar to that seen in complete ACE knockout mice. Thus, the C domain seems to be necessary and sufficient for controlling blood pressure and cardiovascular function, suggesting that the C domain is the dominant angiotensin-converting site. Testis ACE is identical to the Cterminal half of somatic ACE, except for a unique 36-residue sequence constituting its amino terminus. It has also been shown that testis ACE is thought to play a role in sperm maturation and the binding of sperm to the oviduct epithelium. Objective of this thesis was the overexpression, in bacterial cells, purification and solubilazation of two ACE peptides of 108 aa (Ala361-Gly468 (ACE_N), Ala959-Ser1066 (ACE_C)). This experimental approach was chosen because of the ease of culturing bacterial cells and the advantage of using label mediums with 15N and/or 13C. Such large peptides labelled or nonlabelled, can be studied for their structural features using circular dichroism and/or NMR spectroscopy. The above mentioned protein fragments overexpressed in bacteria and purified. Their purity was greater than 99%. The yield was 9mg for ACE_N and 6mg for ACE_C protein fragment from 1L bacterial culture. Their secondary structure was studied using circular dichroism spectroscopy. Deconvolution of ACE_N and ACE_C CD spectra had shown that the presence of trifluoroethanol, at concentrations of 60% or higher, is necessary for the correct folding of the protein. This result was partially confirmed for ACE_N protein fragment by Nuclear Magnetic Resonance spectroscopy. Complete conformation was not succeded due to the inability of recording the 2DNOESY spectrum of ACE_N. Conclusively, the described procedure in this research proved to be advantageous in speed and facility of purification. It demonstrated good reproductively for ACE peptides during purification. The in vitro refolded recombinant proteins had almost identical secondary features compared with these found using crystallographic data, with a high content in a-helix secondary structure motif. Thus, this study offers an effective system for producing large amounts of pure ACE peptides which can be used for several studies.

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La prééclampsie est la première cause de mortalité et de morbidité périnatale et aucun traitement, mis à part l’accouchement, n’est connu à ce jour. Pour mieux comprendre cette maladie, nous avons utilisé trois modèles animaux. Dans un premier temps, nous avons voulu confirmer la présence de prééclampsie chez les souris déficientes en p57kip2, une protéine impliquée dans le cycle cellulaire des trophoblastes. Contrairement au groupe japonais, l’hypertension et la protéinurie au cours de la gestation ne survenaient pas, malgré une perte de structure des trophoblastes dans le labyrinthe ainsi qu’une microcalcification au niveau de leurs placentas. Nous avons alors observé que la diète japonaise induisait à elle seule une diminution de la croissance fœtale, ainsi qu’une dysfonction endothéliale chez ces souris. Nos résultats démontrent que ni les altérations placentaires, ni la génétique ne sont suffisantes pour induire les symptômes de la prééclampsie dans ce modèle, et que la diète peut avoir des effets délétères chez la souris gestante peu importe le génotype. Ensuite, nous avons démontré que les souris hypertendues surexprimant la rénine et l’angiotensinogène humaine développent de la protéinurie et une augmentation de la pression artérielle au cours de la gestation. Leurs placentas sont affectés par de la nécrose et une perte de structure des trophoblastes du labyrinthe en plus de surexprimer le gène du récepteur sFlt-1. Ces souris représentent le premier modèle animal de prééclampsie superposée à de l’hypertension chronique. Finalement, en utilisant des femelles normotendues surexprimant l’angiotensinogène humaine qui développent les symptômes de la prééclampsie lorsqu’elles sont accouplées à des mâles qui surexpriment la rénine humaine, nous avons établi que l’entraînement physique normalisait la hausse de pression ainsi que l’apparition de protéinurie en fin de gestation. Aussi, l'entraînement améliorait la croissance fœtale et placentaire ainsi que la réponse vasculaire indépendante de l’endothélium, et ce, indépendamment du génotype des souris. La présence d’une prolifération exagérée et désorganisée des trophoblastes dans ce modèle était aussi normalisée. L’entraînement physique prévient donc l’apparition des symptômes de la prééclampsie dans ce modèle. Mis ensemble, nos résultats aideront à mieux comprendre les mécanismes à l’origine de la prééclampsie et de sa prévention. / Preeclampsia is the primary cause of maternal and foetal mortality and morbidity and no treatment, apart from delivery are known to date. To better understand this pathology, we investigated three different animal models. First, we needed to confirm preeclampsia-like symptoms in p57kip2 deficient mice, a protein implicated in the trophoblast cell cycle. Conversely to the Japanese group, we observed neither hypertension nor proteinuria in this model. However their placentas showed labyrinthine trophoblast structure loss as well as microcalcification. We therefore studied the impact of Japanese diet, which induced foetal growth restriction and endothelial dysfunction independently from genotype. Our results demonstrate that placental alterations and genetics are not sufficient to induce preeclampsia-like symptoms in this model, and that diet can have deleterious effects on pregnant mice, independently from genotype. We then demonstrated that hypertensive mice overexpressing human angiotensinogen and renin developed de novo proteinuria and had a significant increase of their hypertension during gestation. Their placentas are affected by necrosis and labyrinthine trophoblast structure loss as well as an overexpression of sFlt-1 receptors. These mice represent the first animal model of superimposed preeclampsia on chronic hypertension. Finally, we used normotensive females overexpressing human angiotensinogen, which develop preeclampsia-like symptoms when they are mated with males overexpressing human rennin, to establish that exercise training normalised hypertension and proteinuria at the end of gestation. Moreover, exercise training ameliorates foetal and placental growth as well as endothelium-independent relaxation, independently from the genotype. Exaggerated and disorganised proliferation of trophoblasts in this model is also normalised. Exercise training prevents preeclampsia-like symptoms in this model. Taken together, our results will help a better understanding of this disease and its prevention.

Outre les facteurs métaboliques et hémodynamiques, l’inflammation est actuellement considérée comme un facteur pathogénique potentiel de la néphropathie diabétique (ND), pouvant contribuer à l’initiation et à la progression de la maladie. Les mécanismes menant au développement de l’inflammation rénale dans la ND sont encore peu connus, bien qu’une augmentation d’activité des systèmes rénine angiotensine (RAS) et de l’endothéline (ET) semble y contribuer. L’objectif général de cette étude mono-centre, à double aveugle, randomisée et incluant un groupe placebo était de démontrer que l’inhibition simultanée du RAS et du système de l’ET chez des patients avec ND induisait des effets rénoprotecteurs et anti-inflammatoires supérieurs à ceux observés par blocage du RAS seul. L’objectif spécifique de notre étude était d’évaluer la possibilité que l’administration d’un bloqueur des récepteurs de l’ET-1, le bosentan, à des patients atteints de ND et traités par bloqueurs des récepteurs de l’angiotensine II (BRA), réduisait, chez ces derniers, la protéinurie et les marqueurs inflammatoires systémiques et rénaux. Ce travail constitue un rapport d’un cas clinique et illustre les résultats obtenus suite à l’administration pendant 16 semaines du bosentan chez un patient diabétique de type 2 avec néphropathie clinique traité au long cours par BRA. Le protocole de recherche comprenait 6 visites médicales à 4 semaines d’intervalle, la première visite (V1) correspondant au recrutement du patient, la deuxième visite (V2) constituant le temps 0 de l’étude et la dernière visite (V6) représentant la fin de l’étude. Des échantillons de sang et d’urine étaient prélevés à 3 reprises soit à V2, V4 c’est-à-dire 8 semaines après le début du traitement et à V6 soit 16 semaines après le début du traitement pour mesure des taux sériques et urinaires de divers facteurs pro-inflammatoires incluant l’ET-1, le facteur de nécrose tumorale alpha (TNF-α), l’interleukine-6 (IL-6), le facteur chémoattractant des monocytes-1 (MCP-1), la molécule d’adhésion intracellulaire-1 (ICAM-1), la molécule d’adhésion vasculaire-1 (VCAM-1) et la protéine C-réactive (CRP). Un profil lipidique était aussi déterminé au début et à la fin de l’étude. La fonction rénale était mesurée aux visites V1, V2, V4 et V6 par détermination du taux de filtration glomérulaire (TFG) et de l’excrétion urinaire d’albumine (UAE). Des tests biochimiques de routine étaient aussi faits à chaque visite. La corrélation entre les paramètres inflammatoires et rénaux sous étude et la filtration glomérulaire était enfin déterminée. Nos résultats chez ce sujet ont démontré que le bosentan réduisait l’UAE de 32 % et 35% aux semaines 8 et 16, et ce, sans affecter la pression artérielle ou la filtration glomérulaire. L'effet anti-protéinurique du bosentan était associé à une réduction des concentrations urinaires de VCAM-1, ICAM-1, IL-6, TNF-α et d’ET-1 ainsi qu’à une diminution des concentrations sériques de TNF-α. Le changement dans la protéinurie était corrélé de manière positive avec les changements des niveaux urinaires de VCAM-1 (r=0.86), ICAM-1 (r=0.88), ET-1 (r=0.94), et du TNF-α (r=0.96) ainsi qu’avec les changements des niveaux sériques de TNF-α (r=0.98). Ces données suggèrent que l’inhibition du système de l’ET induit dans la ND des effets rénoprotecteurs additifs à ceux observés par blocage du RAS seul. Ils supportent le concept que l’activation du système de l’ET au niveau rénal, par ses effets inflammatoires, puisse jouer un rôle important dans la pathogenèse de la ND. L’effet anti-inflammatoire et anti-protéinurique du bosentan constitue une découverte intéressante susceptible d’engendrer dans le futur une alternative thérapeutique et préventive dans la prise en charge de la ND. / Apart from metabolic and hemodynamic factors, inflammation has recently been introduced as a potential key pathogenic mechanism involved in the development and progression of diabetic nephropathy (DN). The mechanisms by which renal inflammation occurs in DN are still poorly understood, yet increased renal activity of the renin-angiotensin system (RAS) and endothelin (ET) system may play a key role. The main objective of this mono-centre, double blind, randomized, placebo-controlled study was to demonstrate that concomitant blockade of the RAS and ET system in patients with DN produces greater renal protective effects and exerts greater anti-inflammatory changes than those seen with blockade of the RAS system alone. The specific aim of the study was to evaluate whether administration of bosentan to patients with DN on angiotensin II receptor blockers (ARB) reduces systemic and renal inflammation and improves glomerular filtration. The work presented herein illustrates the results obtained in one type 2 diabetic patient with clinical DN and treated with ARB following the administration of bosentan for 16 weeks. The study protocol included 6 medical visits at 4 weeks interval, with the first visit (V1) being the screening visit and the second visit (V2) being the baseline and randomization visit. Blood and urine samples were taken at V2, after 8 weeks of treatment (V4), and at the end of the study (V6) for determination of serum and urinary inflammatory markers including ET-1, tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and C-reactive protein (CRP). Lipid profile was done at the beginning and end of the study. Renal function was assessed at V1, V2, V4 and V6 by determination of glomerular filtration rate and urinary albumin excretion (UAE). Routine biochemical analyses were done at each visit. Correlation between serum and urinary inflammatory markers and UAE was determined. Our results demonstrated that bosentan administration to this patient reduced UAE by 32% and 35% at weeks 8 and 16, respectively, without affecting blood pressure and glomerular filtration. The anti-proteinuric effect of bosentan was associated with a reduction in urinary levels of VCAM-1, ICAM-1, IL-6, TNF- and ET-1 and a reduction in serum TNF- levels. Change in UAE was positively correlated with changes in urinary levels of VCAM-1 (r=0.86), ICAM-1 (r=0.88), ET-1 (r=0.94), and TNF- (r=0.96) and with change in serum TNF- levels (r=0.98). Our data suggest that blockade of the ET system in top of RAS inhibition exerts additive renoprotective effects in DN. They support the notion that activation of the ET system, by promoting renal inflammation, may play a role in the pathogenesis of DN. The anti-inflammatory and anti-proteinuric effect of bosentan represents an interesting finding which may leads in the future to an alternate therapeutic and preventive for the treatment of DN.