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.

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.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2015-07-22T20:49:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A angiotensina II (Ang II), um dos mais relevantes peptídeos angiotensinérgicos, tem um importante papel na fisiologia e na fisiopatologia dos tecidos renal e cardíaco. Existem diversas enzimas capazes de gerar Ang II. Uma delas é a tonina, capaz de formar a Ang II a partir da angiotensina I (Ang I) ou diretamente do AGT. Nosso objetivo foi caracterizar o RAS no rim e no coração do camundongo transgênico que expressa tonina de rato (TGM(rTon)). Foram utilizados camundongos machos C57bl/6 (grupo controle, CT) e camundongos transgênicos [TGM(rTon)]. Vinte e quatro horas após o implante de cânulas, animais acordados, com 12 semanas de idade, foram submetidos a procedimentos de avaliação hemodinâmica. Os dados mostraram que não existem diferenças estatísticas entre os grupos quanto aos parâmetros hemodinâmicos. Após determinação da pressão arterial, os camundongos foram sacrificados por decapitação e os órgãos (rim e coração) foram retirados. O coração foi dividido em átrios (AT) [direito + esquerdo], ventrículo direito (VD) e ventrículo esquerdo (VE). Usando o tetradecapeptídeo sintético como substrato, a atividade tonina foi avaliada nos rins e nas estruturas cardíacas. A atividade da enzima conversora de angiotensina (ECA) foi determinada usando os substratos ZPhe-His-Leu (específico para o domínio N da ECA) e Hip-His-Leu (específico para o domínio C). Tanto a atividade da tonina, quanto da ECA nos rins e no AT foram significantemente maiores no grupo TGM(rTon) quando comparado ao dos camundongos CT. Já entre as estruturas cardíacas o AT mostrou atividade significantemente maior em ambos os grupos, quando comparados aos ventrículos. A expressão da isoforma de 65 kDa da ECA foi significantemente maior no grupo TGM(rTon) nos rins e no AT. Apenas no rim foi analisada a expressão de ECA e não foi observada diferença estatística entre os grupos. As concentrações da angiotensina 1-7 [Ang-(1-7)] e da Ang I foram significantemente diminuídas no grupo TGM(rTon) quando comparadas ao CT. Entretanto, não foi observada diferença estatística nos níveis da Ang II entre os grupos. Sugerimos que o ambiente com abundância em tonina pode aumentar a atividade N-domínio da ECA por meio de uma secretase, explicando os baixos níveis da Ang-(1-7) encontrados no grupo transgênicoPela primeira vez foi mostrado um importante papel fisiológico da tonina comomodulador do SRA renal e cardíaco. / Angiotensin II (Ang II), one of the most relevant angiotensinergic peptides, has an important role in the renal and cardiac physiology. There are many enzymes that generate Ang II. One of them is tonin, that is able to liberate AII from angiotensin I (Ang I) or directly from angiotensinogen (AGT). Our goal was to characterize the RAS in the kidney and heart of transgenic mouse that express rat tonin [TGM(rTon)]. Twenty-four hours after implantation of cannulas, 12 weeks old awake animals were subjected to hemodynamic evaluation. Data showed no statistical differences for the hemodynamic parameters analyzed between transgenic and the wild-type (control, CT). After that mice were sacrificed by decapitation and their organs (kidney and heart) removed. Heart was separated into atria [right plus left (AT)], right ventricle (RV), and left ventricle (LV)]. Using the synthetic tetradecapeptide renin substrate, tonin activity was evaluated in the kidney and cardiac structures. The angiotensin converting enzyme (ACE) activity was determined using the substrates Z-Phe- His-Leu (specific for N-domain ACE active site) and Hip-His-Leu (specific for Cdomain active site). Both the activity of tonin and the ACE, in the kidneys and AT were significantly higher in TGM(rTon) when compared with CT mice. Among the cardiac structures AT showed significantly greater activity in both groups when compared to the ventricles.The expression of the 65 kDa ACE isoform was significantly higher in TGM(rTon) in the kidney and AT when compared with CT. ACE2 expression was determined only in the kidney and there was not statistic differences between groups. The levels of angiotensin 1- 7 [Ang-(1-7)] and Ang I was significantly decreased in TGM(rTon) when compared with CT. However, the levels of Ang II were not statistically different between groups. We suggest that the environment of tonin abundance may increase N-domain ACE activity by a secretase activity, which could explain the low levels of Ang-(1-7) in the transgenic animal. Our data show, for the first time, the physiologic role of tonin as an important modulator of renal and cardiac RAS. / FAPESP: 2009/03261-04 / TEDE / BV UNIFESP: Teses e dissertações

A prevalência da obesidade tem aumentado em todo mundo, afetando também mulheres em idade reprodutiva. Estudos têm demonstrado que o acúmulo excessivo de tecido adiposo resulta em prejuízos à reprodução feminina. O mecanismo pelo qual a obesidade diminui a fertilidade não está totalmente estabelecido. Atualmente, são propostas a condição inflamatória e a indução de estresse oxidativo como potenciais mecanismos de ação para as patologias relacionadas à obesidade. Adicionalmente, a angiotensina II (Ang II) é mais um fator que tem sido relacionado com alterações associadas à obesidade, e os efeitos deste peptídeo incluem ações pró-inflamatórias e pró-oxidativas. O presente estudo analizou o efeito da obesidade na ovulação e no metabolismo oxidativo ovariano, e a participação da Ang II como um possível modulador da formação de espécies reativas de oxigênio em ovários de ratas obesas e seus efeitos na ovulação. Foram utilizadas ratas submetidas a uma dieta hipercalórica composta por alimentos palatáveis, conhecida como dieta de cafeteria, a partir do desmame até a idade adulta, por um período de 17 semanas. Os animais foram divididos em grupos: CTL (controle), CTL LOS (controle + losartan), CAF (cafeteria) e CAF LOS (cafeteria + losartan). Avaliamos o consumo de alimentos e líquidos, o peso corporal, o peso da gordura abdominal e retroperitonial, a concentração de insulina plasmática, o número de oócitos, as atividades das enzimas antioxidantes (superóxido dismutase e catalase), concentração de peróxido de hidrogênio (H2O2) e parâmetros de dano oxidativo (lipoperoxidação e oxidação de proteínas). As fêmeas obesas do grupo CAF apresentaram maior consumo de energia e reduzido consumo de água e ração padrão, hiperinsulinemia, aumento das gorduras abdominal e retroperitonial, e aumento de peso corporal. A obesidade não reduziu significativamente a ovulação, mas aumentou a atividade das enzimas antioxidantes e a concentração de H2O2 no ovário. A administração do losartan, em ratas alimentadas com a dieta de cafeteria, reduziu a ingestão energética total, a concentração plasmática de insulina e o ganho de gordura abdominal, mas não evitou o desenvolvimento da obesidade. O losartan inibiu a atividade da enzima antioxidante superóxido dismutase no ovário das ratas obesas do grupo CAF, porém não alterou a atividade da enzima antioxidante catalase. Os resultados deste estudo demonstram que a obesidade resulta em alterações no metabolismo e sugerem a participação da Ang II na formação de espécies reativas de oxigênio no ovário. / The prevalence of obesity has increased worldwide, also affecting women of reproductive age. Studies have shown that excessive accumulation of adipose tissue results in impaired female reproduction. The mechanism by which obesity reduces fertility is not fully established. Currently, proposals are the inflammatory condition and the induction of oxidative stress as a potential mechanism of action for diseases related to obesity. Additionally, angiotensin II (Ang II) is another factor that has been related to changes associated with obesity, and the effects of this peptide include pro-inflammatory and pro-oxidative actions. The present study considers the effect of obesity on ovulation and ovarian oxidative metabolism, and the participation of Ang II as a possible modulator of the formation of reactive oxygen species in ovaries of obese rats and their effects on ovulation. Female rats fed a diet consisting of high calorie foods palatable, known as cafeteria diet from weaning to adulthood, for a period of 17 weeks. The animals were divided into groups: Control (CTL) CTL LOS (control + losartan), CAF (cafeteria) and CAF LOS (losartan + cafeteria). We evaluate the consumption of food and fluids, body weight, abdominal and retroperitoneal fat weight, the plasma insulin concentration, the number of oocytes, the activities of antioxidant enzymes (superoxide dismutase and catalase), concentration of hydrogen peroxide (H2O2 ) and parameters of oxidative damage (lipid peroxidation and protein oxidation). The females of the CAF group had higher energy consumption and reduced consumption of water and standard chow, hyperinsulinemia, increased abdominal and retroperitoneal fat, and weight gain. Obesity not significantly reduced ovulation, but increased the activity of antioxidant enzymes and the concentration of H2O2 in the ovary. The administration of losartan in rats fed the cafeteria diet, reduced total energy intake, plasma insulin concentration and abdominal fat gain, but did not prevent the development of obesity. Losartan inhibited the activity of the antioxidant enzyme superoxide dismutase in the ovary of rats CAF group, but did not alter the activity of the catalase antioxidant enzyme. The results of this study demonstrate that the cafeteria diet results in changes in metabolism and suggests the involvement of Ang II in the formation of ROS in the ovary.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este estudo teve como objetivo avaliar o papel da administração crônica de cafeína durante a gestação de camundongos C57BL/6 sobre o remodelamento cardíaco e a expressão de componentes do sistema renina-angiotensina (SRA) na prole macho adulta. Fêmeas C57BL/6 grávidas foram divididas em dois grupos (n = 10): grupo controle (C), progenitoras foram injetadas apenas com o veículo (solução salina NaCl 0,9%), e grupo cafeína (CF), progenitoras receberam diariamente uma injecção subcutânea contendo 20 mg/kg de cafeína (1 mg/ml de solução salina). Após o desmame, os filhotes tiveram livre acesso à ração padrão até 90 dias de idade quando foram sacrificados. Rim e ventrículo esquerdo (VE) foram coletados para análise estrutural e western blotting. O grupo cafeína mostrou uma redução significativa no ganho de massa corporal (MC) (-18%; P <0,0001). O grupo cafeína apresentou ainda um aumento da pressão arterial sistólica (+ 48%; P <0,0001) e freqüência cardíaca (+10%; P <0,01) em relação ao grupo controle. A massa do VE corrigida pela MC no grupo da cafeína foi maior que no grupo C (+10%; P <0,01). O grupo cafeína apresentou um aumento na área de cardiomiócitos (+40%; P <0,05), e reduzida densidade capilar (-25%; P <0,05). No rim, as expressões de renina (128%; P <0,05) e dos receptores 1 da angiotensina II (AT1R) (88%; P <0,05) foram significativamente maiores nos animais do grupo cafeína. No VE, o grupo cafeína demonstrou aumento da expressão de ECA (+30%; P <0,05), angiotensina II (+60%; P <0,01), e AT1R (+77%; P <0,01) e diminuição da expressão do receptor 2 de angiotensina II (-46%; P <0,05). Em conclusão, a administração crônica de cafeína durante a gestação, possivelmente programa a expressão de componentes de sistema renina-angiotensina, levando à ativação persistente do SRA renal e cardíaco local, que por sua vez promove o aumento da pressão sanguínea, remodelação e efeitos cardíacos adversos. / This study aimed to evaluate the role of caffeine chronic administration during gestation of C57BL/6 mice on cardiac remodeling and the expression of components of the renin-angiotensin system (RAS) in male offspring as adults. Pregnant C57BL/6 female mice were divided into two groups (n=10): Control group (C), dams were injected with the vehicle only (saline 0.9% NaCl), and Caffeine group (CF), dams received daily a subcutaneous injection containing 20 mg/kg of caffeine/day (1 mg/ml saline). After weaning, pups had free access to the standard chow until 90 days of age when they were killed. Kidney and left ventricle (LV) were collected for structural analysis and Western blot. Caffeine group showed a significant reduction in body mass (BM) gain (-18%;P<0.0001). Caffeine group had increased systolic blood pressure (+ 48%;P<0.0001) and higher heart rate (+10%;P<0.01) than control group. LV mass adjusted by BM in caffeine group was greater than in C group (+10%;P<0.01). Caffeine group had increase in the area of cardiomyocytes (+40%;P<0.05), and reduced capillary density (-25%;P<0.05). In the kidney, the expressions of renin (+128%; P<0.05) and angiotensin II receptor 1(AT1R) (+88%;P<0.05) were significantly greater in caffeine mice. In the LV, caffeine group showed increased expression of ACE (+30%; P <0.05), angiotensin II (+60%;P<0.01), and AT1R (+77%;P<0.01), and decreased expression of angiotensin II receptor 2 (-46%;P<0.05). In conclusion, chronic administration of caffeine during gestation possibly programs the expression of renin-angiotensin system components, leading to persistent activation of local renal and cardiac RAS, which in turn promotes increased BP and adverse cardiac remodeling.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A hipertensão essencial humana, bem como a hipertensão desenvolvida em Ratos Espontaneamente Hipertensos (SHR), são caracterizadas pelo desenvolvimento de Pressão Arterial (PA) elevada na medida em que a idade avança, sem identificação da causa primária. Está bem estabelecido que este modelo animal apresenta estresse oxidativo (EOx) concomitante a hipertensão. O mecanismo pelo qual o antioxidante reduz a pressão não está claro, por essa razão, é necessário avaliar o comportamento destas enzimas envolvidas na homeostase da PA. Ratos Wistar-Kyoto (WKY) e SHR machos receberam ácido ascórbico, 200 mg / kg / dia por sonda orogástrica durante cinco semanas. A PA, a Hipertrofia Ventricular Esquerda (HVE), o Sistema Renina-Angiotensina (SRA), o Peptídeo Natriurético Atrial (ANP) e o EOx foram comparados entre os grupos por pletismografia, estereologia, microscopia confocal de varrimento a laser, microscopia eletrônica de transmissão, western blotting e análise do RT-qPCR. Os SHR tratados com ácido ascórbico reduziram a PA e a HVE. Além disso, as enzimas envolvidas na homeostase da PA, a renina e a Enzima Conversora de Angiotensina (ECA) normalizaram-se, bem como os Receptores tipo 1 de Angiotensina II (AT1). A grande quantidade de grânulos de ANP no grupo SHR foi reduzida pelo tratamento com ácido ascórbico. O balanço oxidativo foi restabelecido nos SHR tratados com este antioxidante. O EOx nos SHR eleva os níveis de renina e de PA. Estas espécies reativas de oxigênio podem ser envolvidas no mecanismo de sinalização para aumentar a expressão de ANP nos miócitos atriais. Estes dados também mostram que o tratamento com o antioxidante (vitamin C) reduz o EOx e normaliza a PA ao menos parcialmente pela redução de taxas de renina. / The essential hypertension, as well as the Spontaneously Hypertensive Rat (SHR), is characterized by the development of high BP (BP) with advancing age, with no identified primary cause. It is well established that this animal model presents OxS concomitant hypertension. The mechanism, by which the antioxidant reduces the pressure, is not clear, for this reason, it is necessary to evaluate the behavior of enzymes involved in the homeostasis of BP. Male Wistar-Kyoto (WKY) rats and SHR received ascorbic acid, 200 mg / kg / day by orogastric gavage with lasted five weeks. The BP, Left Ventricular Hypertrophy (LVH), renin-angiotensin system (RAS), Atrial Natriuretic Peptide (ANP) and OxS results have been extensively compared among groups by plethysmography, stereology, confocal laser scanning microscopy, transmission electron microscopy, western blotting and RT-qPCR analysis. The SHR treated with ascorbic acid reduced BP and LVH. Also, the enzymes involved in the homeostasis of BP, renin and Angiotensin Converting Enzyme (ACE) normalized, as well as Angiotensin II type 1 receptor (AT1R). The large amount of ANP granules in SHR group was reduced by treatment with ascorbic acid. Oxidative balance was reestablished in SHR treated with this antioxidant. OxS in SHR elevates renin levels and BP. These reactive oxygen species may be involved in the signaling mechanism for increased expression in ANP atrial myocytes. These data also show that treatment with antioxidant (vitamin C) reduces OxS and normalizes BP at least partly by reducing rates of renin.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O objetivo deste trabalho foi estudar a ação do fenofibrato, um agonista do receptor ativador da proliferação peroxissomal alfa, no remodelamento cardíaco e na expressão de componentes do sistema renina-angiotensina (SRA) em um modelo de obesidade induzida por dieta. Camundongos machos C57Bl/6 com três meses de idade foram alimentados durante 11 semanas com dieta controle (grupo C, 3,57 kcal/g de dieta) ou dieta hiperlipídica (grupo HL, 5,40 kcal/g de dieta), em seguida foram separados em quatro grupos e estudados durante cinco semanas: C; HL; C-L (C mais fenofibrato) e HL-F (HL mais fenofibrato). Os animais HL foram mais pesados e apresentaram maior pressão arterial (PA) comparados aos animais C, mas HL-F foram mais leves e tiveram PA menor que HL. A resistência insulínica vista nos camundongos HL foi melhorada com fenofibrato nos camundongos HL-F. Fenofibrato reduziu colesterol total, triglicerídeos e aumentou HDL-c. Os animais HL apresentou um ventrículo esquerdo (VE) mais pesado e com espessura da parede maior, como também cardiomiócitos maiores e uma menor razão cardiomiócito/capilares que os animais C. Fenofibrato foi eficiente em melhorar estas alterações. As expressões cardíacas de Angiotensina II (ANG II) e de seu receptor tipo 1 (AT1R) foram maiores, enquanto que a expressão de seu receptor tipo 2 (AT2R) foi menor nos animais HL que nos animais C, e fenofibrato foi eficiente em atenuar estas diferenças. Como conclusão, a dieta HL lidera para a obesidade, elevação da PA, hipertrofia cardíaca, alterações metabólicas e expressão proteica alterada do SRA em camundongos, sugerindo a participação do SRA nestas alterações. Fenofibrato é eficiente em diminuir a PA e controlar a expressão proteica do SRA, assim como no tratamento da resistência insulínica e do remodelamento cardíaco adverso, diminuindo a hipertrofia dos cardiomiócitos e melhorando a vascularização do miocárdio, desta maneira, diminuindo importantes fatores de risco para doenças cardiovasculares / The aim was to study the action of fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, in cardiac remodeling and protein expressions of RAS components in a model of obesity induced by diet. 3-mo-old C57BL/6 male were fed for 11 weeks with standard chow (SC group, 3.57 kcal/g of chow) or high-fat chow (HF group, 5.40 kcal/g of chow), then they were separated into four groups and studied for five weeks: SC; HF; SC-F (SC plus fenofibrate) and HF-F (HF plus fenofibrate). HF was heavier and had higher blood pressure (BP) than SC, but HF-F was lighter and had lower BP than HF. The insulin resistance seen in HF mice was corrected by fenofibrate in HF-F mice. Fenofibrate reduced total cholesterol, triglycerides and raised the HDL-c. HF had thicker and heavier left ventricle (LV) with bigger LV cardiomyocyte and smaller cardiomyocyte-to-capillaries ratio than SC, and fenofibrate was efficient in treating these alterations. Cardiac expressions of angiotensin II (ANG II) and ANG II receptor 1 were higher, and ANG II receptor 2 was lower in HF than in SC, and fenofibrate was efficient in attenuating these differences. In conclusion, HF diet leads to obesity, BP elevation, cardiac hypertrophy, metabolic changes and altered RAS protein expression in mice, suggesting that RAS is involved. Fenofibrate is efficient in decreasing BP and in controlling RAS protein expressions, and treats the insulin resistance and the adverse cardiac remodeling decreasing the cardiomyocyte hypertrophy and improving the myocardial vascularization, therefore, decreasing important cardiovascular risk factors