Muitos estudos epidemiológicos existentes na literatura revelaram que insultos que ocorrem durante a vida intra-uterina estão associados com diversas anormalidades, tanto funcionais quanto estruturais na vida adulta. Estes estudos revelaram uma associação entre baixo peso fetal e subseqüente diabetes tipo 2, hipertensão e obesidade[1]. Mães que tiveram uma dieta restrita em nutrientes durante a gestação geraram proles com baixo peso ao nascimento e obesidade na vida adulta. Há ainda um aumento na expressão de genes que estão relacionados ao metabolismo lipídico, alem disso há menor expressão gênica da aminopetidase leucil específica, uma enzima que inativa a AII. AII é capaz de regular e estimular diversos fatores que podem modificar o metabolismo do tecido adiposo marrom e do tecido adiposo branco, como as prostaglandinas, enzimas lipogênicas (GPDH e a FAS), 3\' - 5\' monofosfato de adenosina cíclico, catecolaminas, proteína desacopladora mitocondrial (UCP1), prolactina. É conhecido que na vigência de restrição de sal há ativação do sistema renina-angiotensina (SRA) circulante. Desta forma, dieta hipossódica durante a gestação pode alterar o desenvolvimento fetal através de um efeito da angiotensina II. O objetivo do presente estudo foi avaliar a função do sistema renina-angiotensina circulante e no tecido adiposo, renal e cardíaco em prole adulta cujas mães receberam diferentes conteúdos de sal na dieta. Para tanto, ratas Wistar foram alimentadas a partir do segundo mês de vida com dieta hipo, normo ou hipersódica. Subgrupos de ratas em cada uma das dietas foram tratados com bloqueadores do SRA ou com angiotensina II. A prole teve seu peso acompanhado desde o nascimento até a 12a semana de idade, quando foi sacrificada por decapitação para coleta de sangue e retirada dos tecidos adiposos retroperitoneal, inguinal, marrom, rins e coração que foram armazenados para determinação das atividades de renina plasmática, ECA sérica, ECA renal, ECA cardíaca e Western blot dos componentes do SRA. Restrição de sal no período perinatal induz baixo peso ao nascimento. Maior índice de adiposidade, maior expressão protéica da enzima conversora da angiotensina I na gordura inguinal e menor expressão protéica do receptor AT2 na gordura marrom foram verificados na prole de fêmeas adultas de mães submetidas à restrição de sal durante a gestação e amamentação. A atividade de renina plasmática foi maior na prole de machos adultos cujas mães foram alimentadas com dieta hipossódica durante o período perinatal. Sobrecarga de sal na dieta durante o período perinatal também induziu baixo peso ao nascimento, somente na prole de fêmeas que na idade adulta tem maior massa de tecido adiposo inguinal. Concluindo, sobrecarga e restrição de sal durante o período perinatal induzem alterações no tecido adiposo e no sistema renina - angiotensina na prole adulta de fêmeas, mas não de machos. / Epidemiologic studies reported that insults during the intrauterine life have been associated with many abnormalites such low birth weigth, type 2 diabetes, hypertension and obesity in adulthood[1]. Low birth weight and obesity in adulthood were observed in offspring of undernourished dams. In addition, a high expression of genes related with lipid metabolism, and a low expression of the leucyl-specific aminopetidase gene, an enzyme that inactivates angiotensin II (AII) was also observed in offspring of undernourished dams. AII is capable to regulate and stimulate many factors that can change the brown (BAT) and white adipose tissue (WAT) metabolism, like a prostaglandin, lipogenic enzymes (GPDH and FAS), cAMP, catecholamins, mitochondrial uncoupling protein one (UCP1) and prolactin (PRL). It is well estabilish that low sodium diet stimulates the RAS. Therefore, low sodium diet during pregnancy may alter fetus development due to an effect of AII. The objective of this study was to evaluate the function of the circulating and adipose tissue, kidney and heart RAS in the adult offspring of dams that received differents contents of salt during the pregnancy and lactation. Wistar rats were fed a low (LSD), normal (NSD) or high (HSD:) salt diet since 8 weeks of age. Subgroups that received RAS blockers or AII were also studied. BW was measured since birth until adulthood. At 12 weeks of age, the mesenteric (MES), gonadal (GON), and retroperitoneal (RET) white adipose tissue (WAT), brown adipose tissue, heart and kidney were excised and stored. Low birth weight was observed in offspring of dams on salt restriction during pregnancy and lactation. Higher adiposity index, higher protein expression of the angiotensin I converting enzyme in inguinal fat tissue, and lower protein expression of the AT2 receptor in brown adipose tissue were observed in adult female offspring of salt restricted dams during the perinatal period. Plasma renin activity was higher in adult male offspring of salt restricted dams. Dietary salt overload during the perinatal period also induced lower birth weight but only in female offspring in which higher inguinal adipose tissue mass was observed in adulthood. In conclusion, changes in adipose tissue and renin-angiotensin system occur in female but not in male adult offspring in response to salt overload and restriction during pregnancy and lactation.

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.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-20T13:14:40Z No. of bitstreams: 1 hussenmachado.pdf: 992962 bytes, checksum: 6f5d0b2997eefc4508c4d64ee629e6bc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T11:21:23Z (GMT) No. of bitstreams: 1 hussenmachado.pdf: 992962 bytes, checksum: 6f5d0b2997eefc4508c4d64ee629e6bc (MD5) / Made available in DSpace on 2016-07-02T11:21:23Z (GMT). No. of bitstreams: 1 hussenmachado.pdf: 992962 bytes, checksum: 6f5d0b2997eefc4508c4d64ee629e6bc (MD5) Previous issue date: 2012-12-14 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Introdução: O tratamento da hipertensão arterial (HA) em indivíduos com síndrome metabólica (SM) é um desafio, uma vez que terapias não medicamentosas são de difícil implementação e o tratamento farmacológico ideal não está totalmente estabelecido.Objetivo: O presente trabalho objetivou avaliar o bloqueio do sistema renina angiotensina aldosterona (SRAA) na pressão arterial (PA), na função e na morfologia renais em modelo experimental de SM, induzida por dieta hiperlipídica.Material e métodos: Ratos Wistar receberam ração hiperlipídica a partir da quarta semana de vida, por 20 semanas. Os grupos tratados receberam Losartana (10mg/kg/dia), Espironolactona (40mg/Kg/dia) ou quercetina (10mg/kg/dia), por gavagem, a partir da oitava semana de vida. Avaliou-se semanalmente o peso corporal e a PA de cauda por pletismografia. Ao final do experimento, foram realizados testes orais de tolerância à glicose, perfil lipídico, clearance de creatinina, medida direta da PA e análise morfométrica de área e volume glomerular.Resultados: A administração de dieta hiperlipídica se associou ao desenvolvimento de SM, caracterizada por acúmulo central de gordura, hipertensão arterial, hiperglicemia e hipertrigliceridemia. Nesse modelo, não foram observadas alterações da histomorfometria glomerular. O bloqueio do receptor AT1 da angiotensina II preveniu o desenvolvimento da HA. O bloqueio mineralocorticóide não apresentou eficácia anti-hipertensiva, porém se associou à redução da gordura abdominal. A quercetina, por sua vez, não alterou de forma significante o perfil nutricional, metabólico e pressórico dos animais. Conclusão: Ratos Wistar alimentados com dieta hiperlipídica desenvolveram Síndrome Metabólica, porém não foram observadas alterações morfológicas e funcionais renais. / Introduction: The treatment of arterial hypertension (AH) in patients with metabolic syndrome (MS) is a challenge, since non drug therapies are difficult to implement and optimal pharmacological treatment is not fully established. Objectives: The present study had as objective to evaluate the blockade of the rennin angiotensin aldosterone system (RAAS) in blood pressure (BP) in renal function and morphology in an experimental model of MS induced by high fat diet. Material e methods: Wistar rats were fed on high fat diet from the fourth week of life, for 20 weeks. The groups received Losartan (10mg/Kg/day), Spironolactone (40mg/Kg/day) or Quercetin (10mg/Kg/day) from the eighth week of life. We weekly evaluated the body weight and BP by tail plethysmography. At the end of the experiment oral glucose tolerance, lipid profile, creatinine clearance tests, and the direct measurement of BP were performed. A morphometric kidney analysis was performed. Results: The administration of high-fat diet was associated with the development of MS, characterized by central fat accumulation, hypertension, hyperglycemia and hypertriglyceridemia. In this model there were no changes in renal histomorphometry. The blockade of angiotensin II receptor AT1 prevented the development of hypertension. The mineralocorticoid blockage did not have antihypertensive efficacy but was associated with reduction of abdominal fat. Quercetin, in turn, does not significantly alter animals nutritional, pressure and metabolic profile. Conclusion: Wistar rats fed high fat diet developed metabolic syndrome but no changes were observed morphological and functional kidney.

Orientadores: Nicola Amanda Conran Zorzetto, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T23:15:59Z (GMT). No. of bitstreams: 1 Santos_AlissonFernandesdos_D.pdf: 3501020 bytes, checksum: 082754f565197f1a75d8da113de08ea3 (MD5) Previous issue date: 2014 / Resumo: A anemia falciforme (AF) é uma doença genética causada pela substituição de um ácido glutâmico por uma valina na posição 6 da cadeia globina 'beta'. A mutação de ponto origina a hemoglobina S (HbS), que sob condições de deoxigenação se polimeriza tornando os eritrócitos mais propensos à falcização. A fisiopatologia da AF resulta em processos recorrentes de vaso-oclusão e hemólise, causando numerosas complicações clínicas, incluindo a danificação dos rins e problemas cardiovasculares. A angiotensina II (Ang II), um peptídeo vasoconstritor derivado do sistema renina-angiotensina (SRA), controla a pressão arterial e o equilíbrio dos fluidos. A Ang II também participa na geração de espécies reativas de oxigênio, diminuindo a biodisponibilidade do óxido nítrico (NO), um gás vasodilatador, podendo contribuir para alterações no endotélio. A hidroxiureia (HU), agente quimioterápico importante no tratamento dos indivíduos portadores de AF, exerce seu efeito benéfico por meio do aumento de hemoglobina fetal (HbF), reduzindo a falcização dos eritrócitos, diminuindo o número de leucócitos, além de possuir a capacidade de gerar NO. O objetivo deste estudo foi verificar se a produção e expressão das proteínas do SRA estão alteradas na AF e os efeitos da terapia com HU nestes parâmetros. Para analisar a atividade do SRA na AF e um possível papel para a Ang II no processo inflamatório, foram quantificadas as concentrações plasmáticas de Ang II, enzima conversora de angiotensina (ACE), molécula de adesão vascular-1, molécula de adesão intercelular-1, endotelina-1 (ET-1), metabólitos de NO, guanosina monofosfato cíclico (GMPc), interleucina-6, interleucina-8, fator de necrose tumoral-'alfa' e inibidor do ativador do plasminogênio-1 nas amostras de sangue dos indivíduos portadores de AF e indivíduos sadios controles. A produção de Ang II e expressão de algumas proteínas do SRA também foram estudadas em um modelo animal de AF. Adicionalmente, camundongos com AF foram tratados com a HU (50 e 75 mg/kg/dia) por 4 semanas. Não foram encontradas diferenças significativas nos níveis plasmáticos de Ang II nos indivíduos portadores de AF e indivíduos controles, porém a Ang II mostrou correlação positiva com níveis plasmáticos de HbF e ET-1. Uma correlação negativa entre níveis da Ang II e GMPc também foi encontrada no plasma. As concentrações plasmáticas de ACE foram encontradas significantemente menores em indivíduos portadores de AF em comparação aos indivíduos controle. Em camundongos com AF, as concentrações plasmáticas de Ang II estão significativamente diminuídas quando comparadas aos camundongos controles. O tratamento de camundongos AF com HU (75 mg/kg/dia) aumentou significativamente os níveis de Ang II. Diferenças significativas nas expressões dos genes AT1R e ACE1 (que codificam o receptor de angiotensina II tipo 1 e a enzima ACE, respectivamente) foram detectadas em camundongos com AF sem tratamento de HU quando comparados aos camundongos controles, sendo que as expressões dos genes foram menores nos rins e maiores no fígado. Os dados obtidos no estudo sugerem que pode haver alterações no SRA em indivíduos portadores de AF, apesar de não encontrar associações entre alterações em Ang II com parâmetros inflamatórios. Futuros estudos poderiam indicar se alterações na expressão das proteínas do SRA contribuem para algumas manifestações da AF ou refletem danos teciduais nestes indivíduos / Abstract: Sickle cell disease (SCD) is caused by a point mutation that results in the substitution of glutamic acid for valine at the sixth position of the 'beta'-globin chain, leading to the production of hemoglobin S (HbS). HbS polymerizes under conditions of low oxygen concentration, causing the erythrocyte to adopt a sickled shape. The pathophysiology of SCD results in recurrent vaso-oclusion and hemolysis, causing clinicals complications, including kidney damage and cardiovascular problems. Angiotensin II (Ang II), a peptide and vasoconstrictor derived from the action of the renin-angiotensin system (RAS), controls blood pressure and fluid balance. Ang II also participates in the generation of reactive oxygen species, decreasing the bioavailability of the vasodilatory gas nitric oxide (NO), and potentially causing endothelial alterations. Hydroxyurea (HU), an important chemotherapeutic drug employed in the treatment of SCD, has numerous benefits that include augmentation of fetal hemoglobin, reduction of erythrocyte sickling and decreased leukocyte numbers; furthermore, HU also generates NO in vivo. The aim of this study was to investigate whether the production and expression of proteins of the RAS are altered in SCD and the effects of HU therapy on these parameters. To analyse alterations in Ang II and possible associations with inflamatory processes in sickle cell anemia (SCA), the following were quantified in the plasma of SCA patients on and off HU and in healthy control individuals; Ang II, angiotensin converting enzyme (ACE), vascular adhesion molecule-1, intercellular adhesion molecule-1 and endothelin-1 (ET-1), NO metabolites, cyclic guanylate monophosphate (cGMP), interleucin-6, interleucin-8, tumor necrosis factor-'alfa' and plasminogen activator inhibitor-1. The production of Ang II and the expressions of some RAS proteins were also studied in an animal model of SCD. In addition, SCD mice were treated, or not, with hydroxyurea (50 and 75 mg/kg/day) for 4 weeks. Plasma levels of Ang II of SCA patients did not differ from those of controls; however, Ang II demonstrated positive correlations with fetal hemoglobin and ET-1; and a negative correlation with plasma cGMP. Plasma levels of ACE were significantly lower in SCA individuals compared with control individuals. In SCD mice, plasma levels of Ang II were significantly decreased when compared to control mice. Treatment with HU (75 mg/kg/day) in the SCD mice increased levels of Ang II significantly. Significant differences in the gene expressions of AT1R and ACE1 (encoding the angiotensin II receptor type 1 and ACE) were detected in SCD mice not treated with HU, when compared to control mice, where these gene expressions were lower in the kidneys and higher in the liver. These results suggest that some alterations in the RAS may occur in SCD individuals. Although we found no association between plasma Ang II levels with inflammatory parameters in patients, further studies should indicate whether alterations in the RAS may contribute to some of the manifestations of SCD or reflect tissue damage in these individuals / Doutorado / Clinica Medica / Doutor em Clínica Médica

Orientador: José Antonio Rocha Gontijo / Tese (doutorado) - Universidade Estaual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T13:09:43Z (GMT). No. of bitstreams: 1 Lima_MarceloCardosode_D.pdf: 7673135 bytes, checksum: e538dba1f2c8583b18739df7c2e1a919 (MD5) Previous issue date: 2012 / Resumo: O presente estudo avaliou a repercussão da restrição protéica gestacional sobre os receptores da Angiotensina II (AT1R e AT2R) no sistema nervoso central e seu impacto na programação da hipertensão arterial na vida adulta. Os resultados deste estudo demonstraram que o ambiente intrauterino inadequado, desencadeou nos animais do grupo hipoprotéico (LP), um baixo peso ao nascimento, uma expressão significativamente menor de receptores AT1R no extrato de tecido hipotalâmico aos 12 dias de idade e não significativa na 16a semana de vida quando comparados com os animais do grupo normoprotéico (NP). Inversamente, foi observado uma maior e significativa expressão de receptores AT2R no estrato total de hipotálamo de animais do grupo LP com 12 dias de vida, permanecendo nos animais com 16 semanas de vida, quando comparados ao grupo NP. Adicionalmente, observou-se a redução na resposta dipsogênica frente à administração Intracerebroventricular (i.c.v.) de Angiotensina II (AngII) nos animais LP em comparação com os animais do grupo NP. Estes resultados podem estar relacionados à expressão diminuída de vasopressina (AVP) hipotalâmico, demostrada aqui pela imunohistoquímica, na prole submetida à restrição protéica gestacional. A presente investigação também demonstrou uma menor expressão de receptores mineralocorticóide (MR) e glicocorticóide (GR) associada a uma elevação de propiomelanocortina (POMC) e hormônio adrenocorticotrófico (ACTH), assim sugerindo uma participação de mineralocorticoides e glicocorticóides como resposta compensatória ao longo da vida para atenuar as alterações na fisiologia renal comprovadas pela queda acentuada na excreção fracional de sódio urinário (FENa+) nos animais do grupo LP sem ocorrer alteração na filtração glomerular estimada pelo clearance de creatinina (CCr). O Clearance de lítio demonstrou que esta excreção urinária aumentada foi devida à perda da capacidade reabsortiva do néfron proximal a despeito do aumento na reabsorção pós-proximal. Estes efeitos foram associados com um significativo aumento na pressão arterial no grupo LP, mas, o mecanismo exato destes fenómenos permanecem desconhecidos / Abstract: Recent studies have shown that the central nervous system, during development, can be influenced by alterations in the intrauterine environment. This organizational phenomenon is termed 'early-life programming'. Here, in maternal protein-deprived offspring model, we focus on adult hypertension development, hypothalamic changes and renal function disorders as an outcome and confirm the hypothalamus as a structure in which there are early and permanent changes that underlie the developing hypertension. The present study shows that LP male pup body weight was significantly reduced when compared to that of NP pups. However, the body masses at 12-days-old and 16-wk-old were similar to observed in NP age-matched group. Furthermore, the immunoblotting analysis in the current study demonstrated a significantly decreased expression of type 1 AngII receptors in the entire hypothalamic tissue extract of LP rats at 12 days of age and unaltered expression in16-wk-old rats, compared to that observed in NP offspring. The unchanged AT1R expression by blotting in the whole hypothalamic extract of 16- wk LP rats may results of uneven expression of protein, revealed by immunohistochemistry, of different analyzed hypothalamic structures. Conversely, the expression of the type 2 AngII receptors in 12-days and 16-wk-old LP hypothalamus was significantly increased, when compared with the NP agematched group. The current data shows the influence of central AngII administration on spontaneous water consumption in a concentration-dependent fashion, but also demonstrated that the water intake response to graded AngII concentrations was strikingly attenuated in 16-wk-old LP, compared with agematched NP controls. These results may be related with decreased brain AVP expression showed in maternal protein-restricted offspring. The present investigation also shows an early and pronounced decrease in fractional urinary sodium excretion in maternal protein-restricted offspring. It also shows a decreased central expression of MR and GR associated, reciprocally, with enhanced immunoreactivity to POMC e ACTH. The decreased FENa was accompanied by a fall in FEPNa and occurred despite unchanged CCr and an enhanced FEPPNa. All these effects was associated with a significant enhance in arterial blood pressure in the LP group but, the precise mechanism of these phenomena remains unknown / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Abstract Heart failure (HF) is a complex pathological state, involving simultaneous alterations in several signalling pathways and changes in gene programming. In HF, activation of the neurohumoral factors and renin-angiotensin-aldosterone (RAA) system occurs as a compensatory mechanism to combat the abnormal ventricular function. Developments in cardiac gene delivery methods have exerted a significant impact to treat HF and to discover the novel molecular mechanisms associated with HF and other cardiac diseases. This study demonstrated that adenovirus–mediated gene delivery of B-type natriuretic peptide (BNP) into the anterior wall of the left ventricle decreased myocardial fibrosis and increased capillary density. Post-infarction BNP improved systolic function associated with normalization of cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA) 2 expression and phospholamban phosphorylation at Thr17. On the other hand, (Pro)renin receptor ([P]RR) gene delivery resulted deleterious effects on cardiac function and (P)RR activation induced distinct angiotensin II (Ang II)-independent extracellular matrix remodelling and worsening of cardiac function. (P)RR gene delivery resulted in Ang II-independent activation of extracellular-signal regulated (ERK1/2) phosphorylation and increased myocardial fibrosis. In conclusion, the present study indicates that myocardial BNP gene delivery can achieve pleiotropic, context-dependent, favourable effects on cardiac function and that BNP can act locally as a mechanical load–activated regulator of angiogenesis and fibrosis. These results also implicate that (P)RR blockers may display additional cardiac effects in addition to its ability to evoke effective RAA system blockade. Overall, the findings of this study provide a better understanding of the molecular mechanisms involved in the biological actions of BNP and (P)RR, and identify BNP and (P)RR as potential novel drug targets for the treatment of HF. / Tiivistelmä Neuroendokriinisellä aktivaatiolla, jonka seurauksena aiheutuu muun muassa verisuonten supistumista ja laajenemista sekä nesteen kertymistä elimistöön, on tärkeä merkitys sydämen vajaatoiminnan kehittymisessä. Neuroendokriininen aktivaatio kompensoi sydämen vajaatoiminnan seurauksena tapahtuvaa kammioiden poikkeavaa toimintaa. Yksi keskeisimmistä verisuonia supistavista tekijöistä on reniini-angiotensiini-aldosteroni (RAA) -järjestelmä, ja verisuonia laajentaviin tekijöihin kuuluvat muun muassa natriureettiset peptidit, kuten B-tyypin natriureettinen peptidi (BNP) ja A-tyypin natriureettinen peptidi. Geeninsiirtomenetelmillä on ollut merkittäviä vaikutuksia uusien hoitomenetelmien kehittämisessä, sydämen vajaatoiminnan syiden selvittämisessä ja uusien kohdegeenien tunnistamisessa sydämen vajaatoiminnan hoitoon. Väitöskirjan tutkimustulokset osoittivat, että suora adenovirusvälitteinen geeninsiirto rotan sydämen vasemman kammion etuseinään on toimiva menetelmä uusien kohdegeenien löytämiseksi sydämen vajaatoiminnan hoitoon. BNP:n geeninsiirto vähensi merkitsevästi fibroosin muodostumista ja lisäsi verisuonten uudismuodostumista sydämessä. Sydäninfarktin jälkeen BNP paransi sydämen systolista toimintaa, johon liittyi aktiivisen kalsiumpumpun, SERCA2:n ja fosfolambaani-proteiinin fosforylaation normalisoituminen. (Pro)reniini reseptorin ([P]RR) geeninsiirto aiheutti angiotensiini II:sta riippumatonta solunulkoisen matriksin uudelleenmuotoutumista ja sydämen toiminnan huonontumista sekä lisääntynyttä sydämen fibroosia. Väitöskirjatutkimus antaa uutta tietoa solunsisäisistä molekulaarisista mekanismeista sydänsoluissa. BNP geeninsiirto aiheutti sydämen tautitilasta riippuvia suotuisia tapahtumia, ja se toimi paikallisesti muun muassa fibroosia ehkäisevänä tekijänä. (P)RR geeninsiirtotulosten perusteella voidaan olettaa, että (P)RR:n salpaus saattaa olla uusi tehokas hoitokeino sydämen vajaatoiminnan hoitoon.

De nouveaux composants du système rénine angiotensine (SRA) continuent d'être découverts et leurs fonctions étudiées. Cette thèse présente de nouvelles relations entre enzyme de conversion de l’angiotensine (ECA), thrombose et maladies cardiovasculaires (MCV), d’une part, et d’autre part, un rôle pour le récepteur de la (pro)rénine dans le développement du système nerveux central (SNC). Tout d’abord, nous avons étudié l'association de la double mutation de l’ECA et l’inhibiteur de l’activateur du plasminogène-1 (PAI-1) sur l’incidence d’évènements cardiovasculaires chez les patients Libanais hypertendus et hypercholestérolémiques, par rapport à d'autres gènes cardiovasculaires polymorphes des cytokines pro-inflammatoires dans les cellules mononuclées circulantes. Les résultats ont montré que les patients exprimant une double mutation ECA/PAI-1, respectivement Del/4G, sont à haut risque d’apparition des MCV. Dans une deuxième partie, nous avons étudié la relation entre récepteur de la (pro)rénine dont le gène est appelé Atp6ap2, et la voie de signalisation Wnt/bêta-caténine dans les cellules souches/progénitrices neurales (CSPN). À cette fin, nous avons isolé des CSN à partir d’embryons de souris Atp6ap2-/Y, et étudié leur auto-renouvellement et leur différenciation en neurones, astrocytes et oligodendrocytes in vitro. Nos résultats suggèrent que Atp6ap2 est nécessaire à l’auto-renouvellement des CSN indépendamment de la voie de signalisation Wnt/bêta-caténine chez les mammifères. Enfin, nous avons montré qu’il existait une corrélation entre l’expression ATP6AP2 et le degré de différenciation neuronale des cellules souches mésenchymateuses humaines (CSMh) isolées à partir du tissu adipeux. / New components of the renin angiotensin system (RAS) continue to be discovered and their functions studied. This thesis provides new insights on the role of angiotensin converting enzyme (ACE) in thrombosis and cardiovascular diseases (CVD) and of the (pro)renin receptor in the development of the central nervous system (CNS). First, we studied the role of ACE in CVD by analyzing the impact and the association of a mutation in ACE and in plasminogen activator inhibitor-1 (PAI-1) genes compared to other polymorphic cardiovascular genes of proinflammatory cytokines in peripheral blood mononuclear cells in hypertensive hypercholesterolemic Lebanese patients. We showed that Lebanese patients expressing a double mutation (Del/4G) of ACE and PAI-1, respectively, are at high risk of developing CVD, suggesting that combined ACE/PAI-1 mutations may be considered as a potential marker of CVD onset. In the second part, we studied the relation between the (pro)renin receptor, whose gene is called Atp6ap2, and Wnt/beta-catenin signaling pathway, in neural stem/progenitor cells (NSPC) during development. To this end, we used an in vitro model of neural stem cells (NSC) isolated from Atp6ap2-/Y mice embryos and studied their self-renewal capacity and their differentiation into neurons, astrocytes and oligodendrocytes. Our results suggest that Atp6ap2 is necessary for self-renewal of NSC independently of the Wnt/beta-catenin signaling pathway in mammals. In addition, we showed that the expression of ATP6AP2 in human mesenchymal stem cells (hMSC) isolated from adipose tissue was correlated with their degree of neuronal differentiation.

Evidências sobre a presença do sistema renina-angiotensina (SRA) na medula óssea e a possível participação da enzima conversora de angiotensina-I (ECA) na regulação hematopoética tem despertado o interesse da comunidade científica. Como a ECA também é um componente chave do sistema calicreína-cininas (SCC), é possível que elementos deste sistema, possam estar envolvidos no controle hematopoético. Assim, avaliamos a participação da ECA na regulação hematopoética de animais não modificados (WT) e nocautes dos receptores B1 de cininas (KOB1). Para isso, utilizamos técnicas de cultura de células de medula óssea, a saber: os ensaios clonogênicos em soft-ágar para granulócitos e macrófagos (CFU-GM) e o sistema de cultura líquida de longa duração (CLLD). Os resultados mostraram a presença da ECA em células das CLLD e indicaram a participação da enzima na proliferação de progenitores hematopoéticos possivelmente através do controle dos níveis de AcSDKP, pois o tratamento com o tetrapeptídeo e com captopril, reduziu significativamente o número CFU-GM in vitro e in vivo. Quando adicionado às CLLD, o AcSDKP foi capaz de aumentar significativamente a expressão do mRNA da ECA, sugerindo que seus níveis possam controlar a expressão gênica desta enzima. Em relação aos animais KOB1, os resultados mostraram maior atividade da ECA, acompanhado de aumento não significativo da expressão gênica e protéica da enzima. O tratamento das CLLD de animais WT com agonistas de receptores de cininas, não alterou a expressão gênica e a atividade da ECA. Assim, nossos dados sugerem que a ECA participa da regulação hematopoética neste modelo. No entanto, novos estudos serão necessários para a elucidação dos mecanismos envolvidos na expressão e/ou controle da atividade da ECA pelos receptores de cininas. / Evidences on the presence of the renin angiotensin system in the bone marrow and the possible participation of the angiotensin-I converting enzyme (ACE) in the hematopoietic regulation have aroused interest of the scientific community. As the ACE also is a common element of the kallikrein-kinin system (KKS), it is possible that elements of KKS, can be involved in the hematopoietic control. Thus, we evaluated the participation of the ACE on the hematopoietic regulation of wild-type (WT) and kinin receptor B1 knockout mice (KOB1). For this, we use techniques of bone marrow cell culture, to know the clonogenic assays for granulocyte-macrophage (GM-CFU) and the long term bone marrow cultures (LTBMC). The results shown the presence of the ACE in cells from LTBMC and its possible participation on hematopoietic proliferation through the control of AcSDKP levels, therefore the treatment with AcSDKP and captopril, decreased significantly the GM-CFU number in vitro and in vivo. When added to the LTBMC, the AcSDKP increase significantly the expression of ACE mRNA, suggesting that its levels could control the gene expression of this enzyme. In relation to KOB1 mice, the results shown increase of the ACE activity and not significant increase of the gene and protein expression of the enzyme. The treatment of the LTBMC of WT mice with kinins receptors agonists, did not modify the gene expression and the ACE activity. Thus, our data suggesting that ACE participate of the hematopoietic regulation in this model. However, new studies will be necessary to understand the involved mechanisms in the expression and/or control of ACE activity by kinins receptors.

Estudamos a amostra das ouropretanas com prevalência de hipertensão arterial de (52,7%). O objetivo foi descrever os fenótipos sistólico, diastólico e pressão de pulso e correlacionar as variantes genética da ECA e AGT. Os dados sugerem que a prevalência de hipertensão entre elas estão associadas a idade, classe social, escolaridade, hiperglicemia, circunferência da cintura abdominal, e perfil lipídico. O alelo T foi significante com glicemia {p < 0,02 (OR = 2,2)} e cor preta {p < 0,002 (OR = 2,7)} o alelo D com idade {p < 0,01 (OR = 2,2)} e HDL {p < 0,0007 (OR= 1,5)}. O genótipo DD com a idade {p < 0,03 (OR = 0,7)} e HDL {p < 0,008 (OR = 2,0)}. A influência das variantes genéticas da ECA e do AGT tiveram pouco efeito sobre os fenótipos de pressão arterial, mas influenciaram os níveis de colesterol HDL e a glicemia, respectivamente. / We studied the sample of women from Ouro Preto with a prevalence of arterial hypertension of (52,7%). The objective was to describe systolic, diastolic and pulse pressure phenotypes and correlate the ECA and AGT genetic variants. The data suggests that the prevalence of hypertension among them is related to age, social class, schooling, hyperglycemia, abdominal and lipidic profile. The T allele showed significance with glycemia {p < 0.02 (OR = 2.2) and color black {p < 0.002 (OR = 2.7)}. The D allele with age {p < 0.01 (OR = 2.2)} and HDL {p < 0.0007 (OR=1.5)}. The DD genotype with age {p < 0.03 (OR = 0.7)} and HDL {p<O.008 (OR = 2.0)}. Influence of the ECA and AGT genetic variants had little effect on arterial pressure phenotypes, but influenced the levels of HDL cholesterol and the hyperglycemia respectively.

La néphropathie diabétique (DN) est l’une des premières causes de maladie rénale en phase terminale (ESKD). L’ESKD est un important facteur de risque d'insuffisance cardiaque et d'accidents vasculaires cérébraux. La dysfonction du système rénine-angiotensine intrarénal (iRAS) est considérée comme étant l'une des principales causes du développement de la DN. Tous les composants du iRAS sont identifiés dans les cellules épithéliales des tubules rénaux proximaux (RPTCs), y compris l'angiotensinogène (Agt), le seul précurseur de toutes les angiotensines. Notre laboratoire a rapporté précédemment que la surexpression spécifique de l’Agt dans les RPTCs provoque l’hypertension, la protéinurie, la fibrose rénale, l’apoptose et des lésions rénales. Nrf2 (Nuclear factor erythroid 2-related factor 2) est un facteur de transcription qui est exprimé de façon abondante dans les RPTCs et a été considéré comme étant un régulateur central de l'équilibre redox dans les réponses cytoprotectrices cellulaires. Le rôle de l’activation du Nrf2 dans la DN, toutefois, est controversé. L’objectif général de cette thèse est de comprendre le rôle physiologique du Nrf2 dans la DN et d’étudier le(s) mécanisme(s) moléculaire(s) de l’action de Nrf2. Premièrement, nous avons démontré que la délétion génétique de Nrf2 ou l’inhibition pharmacologique de Nrf2 avec de la trigonelline chez les souris Akita diabétiques de type 1 régule à la hausse la voie Ace2/MasR et supprime l’expression de Agt/ACE dans les RPTCs, ce qui a pour effet d'atténuer l’hypertension systémique et les lésions rénales. Conformément, dans les cellules immoratalisées de tubule proximal de rat (IRPTC) en culture, la transfection de ARNsi ou le traitement à la trigonelline empêche la régulation positive de Agt/ACE induite par le HG, avec une baisse subséquente de l’expression des gènes Ace2/MasR. Ces données identifient un nouveau mécanisme dans lequel l’activation de Nrf2 stimule l’expression et l’activation des gènes du iRAS, menant au développement de l’hypertension et de la néphropathie dans le diabète. Deuxièmement, nous avons généré des souris Nrf2 transgéniques qui surexprime spécifiquement Nrf2 dans les RPTCs (souris Nrf2RPTC Tg), sous le contôle du promoteur KAP (kidney specific androgen-regulated protein). Nous avons ensuite croisé les souris Nrf2RPTC Tg avec les 6 souris Akita Nrf2-/- pour générer des souris Akita Nrf2-/- /Nrf2RPTC Tg. Nous avons trouvé que la surexpression de Nrf2 dans les RPTCs des souris Akita Nrf2-/- augmentait significativement l’expression du gène SGLT2, entraînant une élévation du glucose sanguin, du taux de filtration glomérulaire, du rapport albumine/créatinine urinaire et de la fibrose tubulo-interstitielle. Dans les cellules tubulaires proximales humaines immortalisées (HK2), le traitement à l’oltipraz ou la transfection de l’ADNc du NRF2 stimule l’expression de l’ARNm du SGLT2 et l’activité de son promoteur. De plus, des tests de retard sur gel et d’immunoprécipitation de chromatine ont montrés que NRF2 se lie au NRF2-RE du promoteur du SGLT2. En outre, une expression plus élevée de NRF2 et SGLT2 est observée dans les RPTCs de reins de patients diabétiques que dans les reins de patients non diabétiques. Ces données ont établi un nouveau mécanisme de la régulation du NRF2 sur l’expression et l’activation du gène SGLT2, menant à une exacerbation du glucose sanguin, de l’hyperfiltration et des lésions rénales dans le diabète. En somme, cette thèse a démontré que le stress oxidatif (hyperglycémie) induisait l’activation du Nrf2 qui stimulait le iRAS et l’expression de SGLT2, contribuant ainsi à la progression de la DN. Ces études suggèrent que le Nrf2 pourrait être une cible thérapeutique potentielle dans le traitement de la DN et pourront fournir de valabless données pré-cliniques pour les essais cliniques en cours avec le bardoxolone méthyle (un activateur de Nrf2). / Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease (ESKD). ESKD is a major risk factor for heart failure and stroke. Dysfunction of intrarenal renin angiotensin system (iRAS) is considered as one of the main reasons that caused the DN. All components of the iRAS are identified in the renal proximal tubule cells (RPTCs), including angiotensinogen (Agt), the sole precursor of all angiotensins. Our lab has previously reported that specific overexpression of Agt in RPTCs induces hypertension, proteinuria, kidney fibrosis, apoptosis and kidney injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that abundantly expresses in RPTCs and has been considered as a master regulator of redox balance in cellular cytoprotective responses. The role of Nrf2 activation in DN, however, is not clear. The overall aim of this study is to understand the physiological role of Nrf2 in DN and investigate the molecular mechanism(s) of Nrf2 action. First, we have demonstrated that genetic deletion of Nrf2 or pharmacological blockade of Nrf2 with trigonelline in type 1 diabetic Akita mice effectively upregulates Ace2/MasR and suppresses Agt/ACE expression in isolated RPTCs, resulting in attenuation of systemic hypertension and kidney injury. Consistently, in cultured IRPTCs, Nrf2 siRNA transfection or trigonelline treatment prevents high glucose-induced upregulation of Agt/ACE with downregulation of Ace2/MasR gene expression. These data identified a novel mechanism in which Nrf2 activation stimulates iRAS gene expression and activation, leading to the development of hypertension and nephropathy in diabetes. Second, we have generated Nrf2 transgenic mice under the kidney specific androgen regulated protein (KAP) promoter which specifically overexpress Nrf2 in RPTCs (Nrf2RPTC Tg mice). We further crossbred the Nrf2RPTC Tg mice with Akita Nrf2-/- mice to generate Akita Nrf2-/- /Nrf2RPTC Tg mice. We have found that overexpression of Nrf2 in RPTCs of Akita Nrf2-/- mice significantly unregulated sodium-glucose transporter-2 (SGLT2) expression, resulting in elevation of blood glucose, glomerular filtration rate, albumin-creatinine ratio and tubulointerstitial fibrosis. In 8 immortalized human proximal tubular cells (HK2), oltipraz treatment or NRF2 cDNA transfection stimulated SGLT2 mRNA expression and its promoter activity. Furthermore, NRF2 bound to NRF2- RE of SGLT2 promoter were identified by gel mobility shift assay and chromatin immunoprecipitation assay. Moreover, human diabetic kidneys exhibited higher expression of NRF2 and SGLT2 in RPTCs than non-diabetic kidneys. These data established a novel mechanism of NRF2’s regulation on SGLT2, leading to exacerbation of blood glucose, hyperfiltration and kidney injury in diabetes. In summary, this study documented that activation of Nrf2 in hyperglycemia contributed to the progression of DN via regulation of iRAS and SGLT2, suggesting that Nrf2 might be a potential therapeutic target in the treatment of DN.