Made available in DSpace on 2016-04-28T20:18:29Z (GMT). No. of bitstreams: 1 2008 - Daniel de Castro Trindade.pdf: 735793 bytes, checksum: 3d6718dbe1ec23e27fbeb49005d34856 (MD5) Previous issue date: 2009-02-28 / The myocardial infarction was induced in rats by the permanent occlusion of left coronary artery in two different parts. In the first, infarcted rats were treated (CAPoral, 2g/L water) or not (INF) with oral captopril immediately after infarct and the whole experiment (21 days). In the second part, the infarcted rats were treated with captopril (CAPicv, 2 ?L 25 mg/mL/ 12-12 hours) or saline (SAL) intracerebroventricular (icv) during five consecutive days. The functional assessments were performed by electro (ECG) and echocardiogram before and after the experiment. The behavior study of water or hypertonic saline ingestion was performed in individual metabolic cages during the whole period of icv injections. The post-mortem assessment was performed in the end of each part. The ECG recorded from INF, CAPoral, SAL and CAPicv showed similar and indicative values of large myocardial infarction: decrease of QRS index amplitude, presence of Q wave in D1 and rightward deviation of the QRS axis. The main differences in the end of the treatment between INF and CAPoral groups were the prevention of P wave increase and attenuation in rightward deviation of the QRS axis in CAPoral. In the second part, there were no significant differences in ECG exam between infarcted groups. The ECO performed in the first part showed attenuation of the left atrial and ventricular dilatation, ejection fraction improvement and normalization of left ventricular filling only in CAPoral group. In the second part, ECO also showed that captopril treatment induced significative attenuation of left ventricular dilatation and improvement of ventricular filling similarly as captopril treatment by oral route. The study of fluids ingestion showed that CAPicv group exhibited less water ingestion if compared to SAL group. The hypertonic saline ingestion was not different between SAL and CAPicv groups. CAPoral group exhibited smaller scar tissue if compared to INF group. On the other hand, CAPicv group showed similar infarcted area to SAL group in histological study. / O infarto do mioc?rdio foi induzido em ratos pela oclus?o permanente da art?ria coron?ria esquerda em duas diferentes etapas. Na primeira, os ratos infartados foram tratados (CAPoral, 2g/L ?gua) ou n?o (INF) com captopril por via oral imediatamente ap?s o infarto e durante todo per?odo do experimento (21 dias). Na segunda etapa, os ratos infartados foram tratados com captopril (CAPicv, 2 ?L 25mg/mL/ 12-12 horas) ou salina (SAL) intracerebroventricular (icv) durante cinco dias consecutivos. As avalia??es funcionais foram realizadas por eletrocardiograma (ECG) e ecocardiografia (ECO) antes e ao final do experimento. O estudo comportamental de ingest?o de ?gua ou salina hipert?nica foi realizado em gaiolas metab?licas individuais durante todo per?odo de inje??es icv. A avalia??o post-mortem foi realizada no final de cada etapa. Os ECGs dos grupos INF, CAPoral, SAL e CAPicv apresentaram valores similares e indicativos de presen?a de infarto extenso do mioc?rdio como: diminui??o da amplitude do ?ndice QRS, presen?a de onda Q em D1 e desvio do vetor QRS para direita. As principais diferen?as ao final do tratamento entre os grupos INF e CAPoral foram a preven??o do aumento da onda P no grupo CAPoral e a atenua??o do desvio do vetor QRS para direita. Em rela??o aos animais da segunda etapa, n?o houve diferen?as significativas entre os grupos. No ECO realizado na primeira etapa, o grupo CAPoral mostrou atenua??o das dilata??es do ?trio e ventr?culo esquerdos, melhora na fra??o de eje??o e normaliza??o do padr?o de enchimento ventricular analisados pela t?cnica de Doppler. Na segunda etapa, o ECO mostrou que o tratamento com captopril promoveu redu??o significativa da dilata??o do ventr?culo esquerdo e melhora do enchimento ventricular. O estudo da ingest?o de fluidos mostrou que o grupo CAPicv apresentou menor ingest?o de ?gua quando comparado ao grupo SAL. O consumo de salina hipert?nica n?o foi significativamente diferente entre os grupos SAL e CAPicv. O grupo CAPoral apresentou menor tamanho de infarto quando comparado ao grupo INF, o que n?o foi observado no grupo CAPicv, que apresentou tamanho de infarto similar ao grupo SAL no estudo histol?gico.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-24T16:23:42Z No. of bitstreams: 1 2015 - Eriane de Lima Caminotto.pdf: 1339748 bytes, checksum: 753de2c181b04ca05748c103c2d6ec8b (MD5) / Made available in DSpace on 2017-01-24T16:23:42Z (GMT). No. of bitstreams: 1 2015 - Eriane de Lima Caminotto.pdf: 1339748 bytes, checksum: 753de2c181b04ca05748c103c2d6ec8b (MD5) Previous issue date: 2015-10-21 / Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do RJ - FAPERJ / Glaucoma is a disease that causes more blindness in dogs and has no cure, only treatment. The difficulty in the drainage of aqueous humor (AH) results in an increase in intraocular pressure (IOP), representing a significant risk factor for the occurrence of this condition, leading to irreversible damage to the progressive loss of visual field and vision as a whole death of retinal ganglion cells (RGC), loss of axons in the optic nerve and excavation of the same. In addition to this, several mechanisms may contribute to the development and progression of this disease, for example, unbalances of the renin-angiotensin-system (RAS), and cardiovascular and renal diseases. The measurement of IOP and the examination of the fundus are the methods used to diagnose this condition. The treatment of choice is based on eye drops that stimulate drainage of HA and / or decrease their production resulting in IOP control; however in many cases the treatment does not attain the desired effect, culminating in blindness. In an attempt to change this therapeutic perspective, this study compared, in healthy and normotensive Beagle dogs, systemic and ocular action of three drugs: timolol maleate 0.5% (non-selective beta-blocker used in cases of glaucoma) , captopril 0.1% and 0.5% (ECA1 inhibitor never before studied in greater concentration in dogs) and aceturato of diminazene - DIZE 0.1% and 0.5% (endogenous activator of ACE2, never before researched in eyes canines). The latter two drugs were formulated for ophthalmic use for the reduction of IOP, since it is known about the existence of SARS eye level, and that they have been successfully administered systemically at reducing systemic arterial pressure (SAP). Thus, before and after treatment, 18 dogs underwent the Schirmer Tear Test (TLS1) and blood collection for measuring the concentration of angiotensin-converting enzyme (ECA1) serum. For three consecutive days before treatment and at three times (6h, 12h and 18h) all animals have gone through four exams, always following the same order: evaluation of pupil size, IOP measurement, measurement of HR and SAP. After obtaining the normal range, the animals had the left eye subjected to predetermined protocols initially with 1 drop every 12 hours, 7 days a lower concentration and, in the other seven days, in the highest concentration. The adelfos eyes were control and every day in the three different times dogs went through the same initial exams. All drugs were good penetration and no adverse eye level. As for tear production, captopril is the most suitable for glaucoma patients and patients with keratoconjunctivitis sicca, while timolol is contraindicated for them. All decreased IOP values, and in the second week of treatment, with higher concentrations, reductions were more significant. The bradycardia was observed in captopril-treated animals 0.5%, with a reduction of almost 9% in HR compared to captopril in lower concentrations / O glaucoma ? uma das doen?as que mais causa cegueira em c?es e n?o possui cura, apenas tratamento. A dificuldade na drenagem do humor aquoso (HA) resulta no aumento da press?o intra-ocular (PIO) que representa um fator de risco significativo para a ocorr?ncia dessa afec??o, conduzindo ? danos irrevers?veis como a perda progressiva do campo visual e da vis?o como um todo, morte das c?lulas ganglionares da retina (CGR), perda de ax?nios do nervo ?ptico e escava??o do mesmo. Al?m deste, diversos mecanismos podem contribuir para o desenvolvimento e progress?o desta doen?a como, por exemplo, os desequil?brios do sistema-renina-angiotensina (SRA) e as doen?as cardiovasculares e renais. A mensura??o da PIO e o exame do fundo de olho s?o os m?todos mais utilizados para diagnosticar essa afec??o. O tratamento de escolha se baseia em col?rios que estimulam a drenagem do HA e/ou diminuam sua produ??o resultando no controle da PIO; por?m em muitos casos a terapia n?o atinge o efeito desejado e culmina em cegueira. Na tentativa de mudar essa perspectiva terap?utica, o presente trabalho comparou, em c?es sadios e normotensos da ra?a Beagle, a a??o sist?mica e ocular de tr?s f?rmacos: maleato de timolol 0,5% (beta-bloqueador n?o seletivo usado nos casos de glaucoma), captopril 0,1% e 0,5% (inibidor de ECA1 nunca antes estudado nesta maior concentra??o em c?es) e aceturato de diminazeno ? DIZE 0,1% e 0,5% (ativador end?geno de ECA2, nunca antes pesquisado em olhos caninos). Os dois ?ltimos f?rmacos foram formulados para uso oftalmol?gico visando a redu??o da PIO, uma vez que ? sabido sobre a exist?ncia do SRA a n?vel ocular e que os mesmos j? foram administrados sistemicamente com sucesso na redu??o press?o arterial sist?mica (PAS). Dessa forma, antes e ap?s os tratamentos, 18 c?es foram submetidos ao teste lacrimal de Schirmer (TLS1) e ? coleta sangu?nea para dosagem da concentra??o da enzima conversora de angiotensina (ECA 1) do soro.Durante tr?s dias consecutivos antes dos tratamentos e em tr?s hor?rios distintos (6h, 12h, e 18h) todos os animaispassaram por quatro exames, seguindo sempre a mesma ordem: avalia??o do di?metro pupilar, aferi??o da PIO, aferi??o da FC e da PAS.Ap?s a obten??o dos valores normais, os animais tiveram o olho esquerdo submetido aos protocolos pr?-determinados inicialmente com 1 gota, a cada 12 horas, por 7 dias na menor concentra??o e, nos demais 7 dias, na maior concentra??o. Os olhos adelfos foram o controle e, todos os dias nos tr?s hor?rios distintos os c?es passaram pelos mesmos exames iniciais. Todos os f?rmacos tiveram boa penetrabilidade e aus?ncia de efeitos adversos a n?vel ocular. Quanto ? produ??o lacrimal, o captopril ? o mais indicado para os pacientes glaucomatosos e portadores de ceratoconjuntivite seca, enquanto que o timolol ? contra-indicado para os mesmos. Todos diminu?ram os valores da PIO, sendo que na segunda semana de tratamento, com concentra??es maiores, as redu??es foram mais significativas. A bradicardia foi observada nos animais tratados com captopril 0,5%, com uma redu??o de quase 9% na FC, quando comparado com o captopril em menor concentra??o.

A doença periodontal corresponde a um grupo de doenças inflamatórias que resultam na destruição das estruturas de suporte dental. São doenças infecciosas e possuem etiologia relacionada a microrganismos gram-negativos podendo manifestar-se de inúmeras maneiras. Estes possuem uma variedade de fatores que permitem o aumento de sua virulência e capacidade de se multiplicarem e persistirem no periodonto. Experimentos recentes de nosso laboratório mostraram que no tecido gengival de rato existe a expressão de RNAm para todos os componentes do Sistema Renina-Angiotensina (SRA), presença da renina e atividade da Enzima Conversora de Angiotensina I (ECA) em tecido gengival de ratos, sugerindo, assim, possível correlação entre o SRA e a doença periodontal (DP). Portanto, o objetivo do presente trabalho foi investigar se o captopril, um inibidor da ECA, altera a progressão da DP induzida experimentalmente em ratos. Para tanto, foi utilizado o modelo de indução da DP por colocação de ligadura ao redor do primeiro molar inferior de ratos divididos em grupos com 10 animais cada, que foram tratados com captopril (via gavagem, 30 mg/kg/dia) ou água (veículo). Foi realizado pré-tratamento com esta droga por 7 ou 14 dias previamente à indução da DP e após este período, o captopril foi administrado por 14 e 21 dias. Além disso, foi realizada cirurgia fictícia para indução da DP (grupo SHAM). As técnicas utilizadas neste trabalho foram: indução da DP em ratos, extração de RNA total, transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RT-qPCR) e análise de perda óssea alveolar. Os dados foram analisados por meio de gráficos. Todos os resultados foram submetidos à análise unidirecional de variância (ANOVA) e representaram médias e respectivos desvios-padrão. Diferenças entre os grupos foram consideradas estatisticamente significativas quando p < 0,05. Com base nos resultados obtidos neste trabalho, foi possível concluir que o captopril não foi capaz de diminuir a perda óssea na doença periodontal induzida experimentalmente em ratos, apesar desta droga ter alterado a expressão de RNAm para um alvo do RAS (AT1a) e alguns mediadores do processo inflamatório no tecido periodontal, tais como COX-2, ECA-2, IL-6, RANKL, VEGF-R1 e VEGF-R2. / Periodontal disease (PD) consists of a group of inflammatory diseases which result in the destruction of tooth supporting structures. They are of infectious nature, with etiological factors related to gram-negative microorganisms, and may have manifestations in several ways. These comprise a variety of factors that allow the increase in its virulence and ability to multiply and persist in the periodontal tissue. Recent findings form our laboratory showed that mRNA expression exists in rat gingival tissue for all components of the Renin-Angiotensin System (RAS), the presence of renin as well as Angiotensin Converting Enzyme I (ACE) activity in rat gingival tissue, thus suggesting a possible correlation between the RAS and periodontal disease. Therefore, the aim of this study was to investigate whether captopril, an ACE inhibitor, alters the progression of experimentally-induced PD in rats. Thus, the model of PD induction by ligature placement around rat lower first molar was used. Animals were divided groups of 10 animals each, which were treated with captopril (via gavage, 30 mg/kg/day) or water (vehicle). Pre-treatment with this drug during 7 or 14 days was performed previously to PD induction, and after this period captopril was administered during 14 or 21 days. In addition, fictitious operation (SHAM group) was performed to induce PD. The techniques used in this study were: PD induction in rats, total RNA extraction, reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) and alveolar bone loss. Data were analyzed by means of graphs. All the results were subjected to one-way analysis of variance (ANOVA) and represented means and respective standard errors. Differences between groups were considered statistically significant when p <0.05. Based on the results obtained in this study, it was concluded that captopril was not able to decrease bone loss in experimentally-induced PD in rats, although this drug altered the expression of mRNA for one RAS target (AT1a) and some mediators of inflammation in periodontal tissue such as, COX-2, ACE-2, IL-6, RANKL, VEGF-R1 and VEGF-R2.

Neste estudo, avaliamos e caracterizamos o efeito de Ang II em leito venular mesentérico (LVM) e anéis de veia porta (AVP) de wistar e SHR. A reatividade vascular para Ang II foi estudada na presença e ausência de diferentes antagonistas para elucidar os mecanismos envolvidos na venoconstrição induzida por Ang II. Nossos resultados sugerem que a Ang II induz venoconstrição através da ativação de receptores AT1 e AT2 em Wistar e receptor AT1 em SHR. Essa venoconstrição parece ser contrabalanceada pelo receptor B2 em ambas as espécies. NO contribue para este efeito em wistar e metabólitos da COX em SHR. Há redução da venoconstrição induzida por Ang II em AVP de SHR que pode estar relacionado a redução da expressão protéica de receptores AT2. Esses resultados indicam diferentes mecanismos de regulação do tônus venoso de ratos wistar e SHR em resposta a Ang II que podem ter relevância no controle do retorno venoso e débito cardíaco. / In this study we have evaluated and characterized the effect of Ang II in the isolated perfused mesenteric venular bed (MVB) and portal vein rings (PVR) of SHR and wistar rats. Vascular reactivity to Ang II was studied in the absence and presence of different antagonists to elucidate the mechanisms involved in Ang II-induced venoconstriction. Our results suggest that Ang II induces venoconstriction by activation of AT1 and AT2 receptors in wistar and AT1 receptor in SHR. These venoconstriction seems to be counterbalanced by B2 receptor in both species. NO might contribute to this effect in wistar and COX metabolites in SHR. There is a decrease in Ang II induced venoconstriction in PVR of SHR that might be related to the decrease of protein expression of AT2 receptor. These results indicate different mechanisms of regulation of venous tonus of the SHR and wistar rats induced by Ang II that can be relevant in the control of the venous return and cardiac output.

A hipertrofia cardíaca é caracterizada como um aumento da massa cardíaca e é considerada um mau prognóstico e está associada as diversas formas de insuficiência cardíaca. No entanto, outra forma de resposta hipertrófica pode ser gerada por meio do treinamento físico, o qual produz aumento proporcional da espessura da parede, levando ao fenótipo conhecido como \"coração de atleta\", onde há uma preservação ou melhora na função cardíaca. Ambos fenótipos de remodelamento cardíaco estão estritamente associados às vias canônicas e não canônicas do sistema renina angiotensina (SRA). Estudo prévio do nosso grupo demonstrou, que o treinamento físico aeróbio alterou a expressão de microRNAs envolvidos na regulação do SRA, em especial na regulação da expressão das enzimas chave deste sistema, a enzima conversora de angiotensina 1 (ECA) e enzima conversora de angiotensina 2 (ECA2). Porém, ainda pouco se sabe sobre o papel dos microRNAs na modulação do SRA local cardíaco em resposta ao treinamento físico. O presente estudo teve como objetivo traçar o perfil de expressão de microRNAs entre camundongos treinados e com superexpressão de ECA2 cardíaca. Nós utilizamos para isso, camundongos da linhagem C57BL/6 divididos em três grupos experimentais: 1) Sedentário 2) Grupo Treinado e com 3) superexpressão de ECA2 no coração. A superexpressão de ECA2 cardíaca resultou em aumento da massa cardíaca e na alteração de 14 microRNAs em relação ao grupo controle e o treinamento aeróbio embora não tenha apresentado alteração na massa cardíaca, alterou 4 microRNAs em relação ao controle. Destes microRNAs, 3 microRNAs (-133a-5p, -208a-3p e -215) foram confirmados por RT-qPCR. A busca por alvos preditos destes microRNAs gerou uma lista de 418 genes que resultou em 24 vias de sinalização via KEGG Pathway. Destas, atenção foi dada para as vias pró-hipertróficas de PI3K e MAPK que são conhecidas por participarem do remodelamento cardíaco fisiológico e patológico / Cardiac hypertrophy is characterized as an increase in heart mass and is considered a poor prognostic sign, being associated with the various forms of heart failure. However, another form of hypertrophic response can be generated through physical training, which produces a proportional increase in wall thickness, leading to the phenotype known as the \"athlete\'s heart,\" where there is preservation or improvement in cardiac function. Both phenotypes of cardiac remodeling are strictly associated with the canonical and noncanonical pathways of the renin angiotensin system (RAS). A previous study of our group demonstrated that aerobic physical training altered the expression of microRNAs involved in the regulation of RAS, especially in the regulation of the expression of the key enzymes of this system, angiotensin converting enzyme 1 (ACE) and angiotensin converting enzyme 2 (ACE2). However, little is known about the role of microRNAs in modulating cardiac local RAS in response to physical training. The present study aimed to identify the expression profile of microRNAs between trained mice and mice with overexpression of cardiac ACE2. We used C57BL/6 lineage mice divided into three experimental groups: 1) Sedentary 2) Trained Group and 3) overexpression of cardiac ACE2. The overexpression of cardiac ACE2 resulted in an increase in cardiac mass and in the alteration of 14 microRNAs in relation to the control group and the aerobic training, although it did not present alterations in the cardiac mass, altered 4 microRNAs in relation to the control. Of these microRNAs, 3 microRNAs (-133a-5p, -208a-3p and -215-5p) were confirmed by RT-qPCR. The search for predicted targets of these microRNAs generated a list of 418 genes that resulted in 24 signaling pathways via KEGG Pathway. Of these, attention was given to the pro-hypertrophic pathways of PI3K and MAPK that are known to participate in physiological and pathological cardiac remodeling

SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype. The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1. Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage. Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis. To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress. / SPRED-Proteine sind Inhibitoren des hochkonservierten und in allen Geweben verbreiteten Ras/ERK/MAPK-Signalwegs, welcher Proliferation, Differenzierung und das Wachstum von Zellen reguliert. Um physiologische Konsequenzen der SPRED2-Defizienz im lebenden Modellorganismus aufzuklären, haben wir SPRED2-KO-Mäuse mithilfe der „gene trap“-Methode generiert. Eine erste Studie zur phänotypischen Charakterisierung mit KO-Mäusen bis zu einem Alter von fünf Monaten identifizierte SPRED2 als Regulator der Chondrozytendifferenzierung und des Knochenwachstums. So bewirkt der Verlust der SPRED2-Proteinfunktion eine erhöhte FGFR-vermittelte ERK-Aktivität, was wiederum einen Hypochondroplasie-ähnlichen Minderwuchs verursacht. Allerdings offenbarten Langzeitbeobachtungen älterer KO-Mäuse einen im Allgemeinen sehr schlechten Gesundheitszustand und weitere facettenreiche Symptome, darunter exzessives Putzverhalten mit schweren, selbst zugefügten Wunden, einen abnorm hohen täglichen Wasserkonsum, klare morphologische Anzeichen einer Nierenschädigung und eine reduzierte Überlebenswahrscheinlichkeit durch plötzlichen Tod. Ziel dieser Studie war es, basierend auf unseren Beobachtungen, einen Auslöser für diesen komplexen und vielseitigen Phänotyp zu finden. Die beobachtete Nierendegeneration in unseren SPRED2-KO-Mäusen war auf eine Hydronephrose zurückzuführen, welche durch schwere Atrophie des Nierengewebes und Apoptose von Nierentubuluszellen gekennzeichnet war. Aufgrund des Nierenschadens haben wir Trinkverhalten und gängige Serumparameter analysiert. Trotz der Polydipsie, die sich durch eine nahezu verdoppelte tägliche Wasseraufnahme manifestierte, konnten signifikant erhöhte Na+- und Cl--Werte und die daraus resultierende Hyperosmolalität im Serum der SPRED2-KOs nicht kompensiert werden. Weil Salz- und Wasserhaushalt zum größten Teil unter der hormonellen Kontrolle von Aldosteron und ADH stehen, haben wir beide Hormonspiegel untersucht. Während die ADH-Werte im Serum von WT- und KO-Mäusen vergleichbar waren, insbesondere nach experimentellem Wasserentzug und einem extremen Verlust von Körperflüssigkeit, waren die Serumspiegel von Aldosteron in den SPRED2-KO-Mäusen verdoppelt. Die systematische Untersuchung übergeordneter regulatorischer Hormonachsen ergab, dass sich der Hyperaldosteronismus unabhängig von einer erhöhten Aktivität des Renin-Angiotensin-Systems entwickelte, da die Serum-Ang II-Spiegel in den SPRED2-KOs etwa um die Hälfte reduziert waren. Die Expression der Aldosteronsynthase in der Nebenniere war jedoch wesentlich erhöht. Für Kortikosteron, das wie Aldosteron von der Nebennierenrinde freigesetzt wird, konnten wir ebenfalls mehr als doppelt so hohe Werte im Serum der KO-Tiere detektieren. Die Aldosteron-Produktion steht, ähnlich wie bei Kortikosteron, zumindest teilweise unter der Kontrolle des hypophysären Hormons ACTH, dessen Sekretion wiederum übergeordnet durch die Freisetzung von CRH aus dem Hypothalamus geregelt wird. Tatsächlich war die Stresshormon-Sekretion entlang dieser gesamten Hypothalamus-Hypophysen-Nebennierenrinden-Achse erhöht, da Serum-ACTH, das mittlere, hypophysäre Hormon, und hypothalamisches CRH, der übergeordnete hormonelle Induktor der HPA-Achse, in den SPRED2-KOs auch um 30% erhöht waren. Zusätzlich waren die ERK-Aktivität ebenso wie die CRH-mRNA-Spiegel im paraventrikulären Nukleus des Hypothalamus in unseren SPRED2-KO-Mäusen deutlich höher. In vitro Studien mit der Hypothalamus-Zelllinie mHypoE-44 zeigten weiterhin, dass sowohl SPRED1 als auch SPRED2 die Aktivität des CRH-Promotors, die CRH-Sekretion und die Ets-Faktor-abhängige CRH-Transkription reduzieren können. Passend dazu enthält die CRH-Promotorregion zahlreiche verschiedene Bindungsstellen für Transkriptionsfaktoren der Ets-Familie, speziell für Ets1. Somit zeigt diese Studie zum ersten Mal, dass die durch SPRED2-vermittelte Hemmung der Ras/ERK/MAPK-Signalkaskade mittels Unterdrückung der ERK-Aktivität zu einer Herunterregulation der Ets1-Faktor-abhängigen Transkription führt, was eine Hemmung der CRH-Promotoraktivität, der CRH-Transkription und der CRH-Freisetzung aus dem Hypothalamus zur Folge hat. Die daraus resultierende Hyperaktivität der gesamten HPA-Achse in unseren SPRED2-KO-Mäusen spiegelt eine erhöhte endogene Stress-Reaktion wider und äußert sich durch übermäßiges Putzverhalten und durch selbst zugefügte Hautläsionen auf der einen Seite; auf der anderen Seite erklärt dies, in Kombination mit der erhöhten Aldosteronsynthase-Expression, den Hyperaldosteronismus und das damit verbundene Ungleichgewicht in Salz- und Wasserhaushalt. Weiterhin tragen sowohl Hyperaldosteronismus als auch Polydipsie sehr wahrscheinlich zu den beobachteten Nierenschädigungen bei. Zusammengefasst ist diese Studie ein erster Hinweis, dass SPRED2 wesentlich an der adäquaten Regulation der HPA-Achsen-Aktivität beteiligt ist und essentiell ist für die Aufrechterhaltung der Homöostase im Körper. Um die Folgen von SPRED2-Defizienz in Mäusen und vor allem im Menschen weiter aufzuklären und zu vergleichen, erforschen wir in zwei Folgeprojekten die Funktion von SPRED2 speziell im Gehirn und im Herzen und führen parallel ein genetisches Screening zur Identifikation von funktionellen SPRED2-Mutationen im Menschen durch.

The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo. In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise. The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats. The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 % of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair. / Der Nachweis von oxidativen Stressmarkern hat bei der Untersuchung von Krankheiten wie Diabetes, Krebs und Hypertonie an großer Bedeutung gewonnen. Vor allem 8-Oxo-2’-desoxyguanosin (8-oxodG) wird gezielt mit verschiedenen Methoden gemessen und als Marker für oxidativen Stress herangezogen. Daneben haben 8 Oxoguanin (8-oxoGua), als Produkt aus der Basenexzisionsreparatur der DNA, sowie 8-Oxoguanosin (8-oxoGuo), als Biomarker für oxidativ geschädigte RNA, bisher weniger Aufmerksamkeit bekommen. Das Renin-Angiotensin Aldosteron System (RAAS) spielt eine wichtige Rolle in der Regulierung des Blutdrucks. Im Falle einer Hypertonie werden Angiotensin II (Ang II) und Aldosteron (Aldo) über einen langen Zeitraum in erhöhter Konzentration ausgeschüttet. Dieser Umstand bewirkt eine nicht physiologische Wirkung der Hormone des RAAS, welche zu einer Induktion von oxidativem Stress führt. Die Zielsetzung dieser Arbeit ist es, die oxidative Schädigung, ausgelöst durch Ang II und Aldo, in der DNA und der RNA in vitro und in vivo nachzuweisen und dabei speziell den Biomarker 8-oxodG zu untersuchen. In-vitro-Experimente wurden mit LLC PK1-Zellen, einer Schweinenierenzelllinie, durchgeführt. Ang II und Aldo lösten einen dosisabhängigen Anstieg der DNA Schäden in LLC PK1 Zellen aus. Eine Zeitabhängigkeit wurde für die ersten 30 Minuten gezeigt. Für die restliche Zeit (4 h) blieb der nachgewiesene DNA Schaden konstant. Der FPG Comet-Assay und die immunzytochemische Färbung zeigten jeweils eine signifikante Zunahme von 8-oxodG in LLC-PK1-Zellen an, während die HPLC MS/MS Messung nur geringe Veränderungen nachwies. Das FPG Enzym erkennt neben 8-oxodG auch andere oxidierte Purine und sorgte so für eine Überbestimmung des DNA-Schadens. Bei der immunzytochemischen Färbung entsteht die Überbestimmung durch Kreuzreaktionen des 8 oxodG Antikörpers mit oxidierten Strukturen in der DNA. Der Vorteil beider Analysemethoden ist die direkte Messung von Schädigungen in der Zelle, während die HPLC-MS/MS eine Isolierung der Nukleinsäuren voraussetzt. Bei diesem Schritt kann es zur Oxidation der Marker für oxidativen Stress kommen, welche einen genauen Nachweis erschwert. In vivo-Versuche hatten zum Ziel, die oxidativen Stressmarker 8-oxoGua, 8-oxodG und 8-oxoGuo im Urin nachzuweisen. Die Behandlung der C57BL/6-Mäuse und Sprague Dawley-Ratten (SD-Ratten) mit den Hormonen des RAAS zeigten einen Anstieg des Blutdrucks, erhöhte DNA Schäden durch oxidativen Stress sowie erhöhte Exkretionsraten der oxidativen Stressmarker. Durch eine Inhibierung des Angiotensin II-Typ1- oder Mineralkortikoidrezeptors sowie die Mutation des Gens AT1a konnte gezeigt werden, dass die Schädigungen unabhängig vom Blutdruck sind. Zudem konnte gezeigt werden, dass neben NOX4 auch andere NADPH Oxidasen für den oxidativen Stress verantwortlich sein müssen. Eine Aktivierung des Nrf2 Signalweges in den SD-Ratten hat Einfluss auf die Wirkung von Aldo. Die Exkretionsrate der oxidativen Biomarker im 20-h-Urin der behandelten Tiere zeigen, wie sich das Gleichgewicht zwischen DNA-Reparatur und oxidativem Stress verändert. Da 80 % der DNA in RNA umgeschrieben werden, ist der Nachweis von 8 oxoGuo in den Fokus gerückt. In der praktischen Anwendung kann mit der Messung von 8 oxodG und 8-oxoGuo ein Krankheits- oder Heilungsprozess auf nicht invasive Weise verfolgt werden. Der Nachweis von 8-oxodG und 8-oxoGuo in den Nukleinsäuren stellt einen Einstieg für die Grundlagenforschung dar, da sie nur eine Momentaufnahme der Nukleinsäureschädigung in der Zelle zeigen. Meist findet eine Überbestimmung, ausgelöst durch die Messmethode, statt. In Gewebeproben kann eine Unterbestimmung vorliegen, falls nicht alle Zelltypen vom oxidativen Stress betroffen sind. Daher sollte es ein vorrangiges Ziel sein, ein stabileres Oxidationsprodukt des Guanins nachzuweisen, um das Gleichgewicht der DNA-Oxidation und Reparatur besser zu verstehen.

SPRED2 ist ein Inhibitor des Ras/ERK-MAPK-Signalwegs. Um die Folgen einer SPRED2-Defizienz zu erforschen, wurden im Rahmen vorheriger von Ullrich et al. durchgeführter Untersuchungen mittels Gene-Trap-Methode bereits mannigfaltige Auffälligkeiten im Phänotyp der SPRED2-Mäuse festgestellt. So zeigten die Tiere einen Hypochondroplasie-ähnlichen Zwergenwuchs, Verhaltensauffälligkeiten, einen krankhaft gesteigerten Wasserkonsum und nicht zuletzt eine deutlich reduzierte Lebenserwartung im Vergleich mit den WT-Tieren. Des Weiteren fielen erhöhte Aldosteronspiegel auf, die bei näheren Untersuchungen nicht einer erhöhten Aktivität des RAAS geschuldet zu sein schienen. Vielmehr zeigte sich eine deutlich erhöhte Aldosteron-Synthase-Expression in der Nebennierenrinde. Erste Hinweise darauf, dass die SPRED2-Defizienz auch Auswirkungen auf den kardiologischen Phänotyp haben könnte, ergaben sich bereits bei initialen Untersuchungen von Ullrich et al. So konnte bei den SPRED2-KO-Tieren neben hämodynamischer Auffälligkeiten eine gesteigerte Herz-Körpergewicht-Ratio festgestellt werden. Die im Rahmen dieses Folgeprojekts durchgeführten Untersuchungen sollten die Frage klären, ob die Defizienz des SPRED2-Gens Auswirkungen auf die Herzleistung hat und hierüber die verkürzte Lebenserwartung der KO-Tiere verschulden könnte. Hierfür wurden zunächst Untersuchungen der elektrischen kardialen Aktivität mittels EKG und Elektrophysiologischer Untersuchung durchgeführt. Die Ermittlung von Herzrhythmusstörung und die Quantifizierung derselben spielte hierbei eine besondere Rolle. Des Weiteren sollte mit der Durchführung von PSR-Färbungen zur Bestimmung des kardialen Kollagengehaltes histologischen Fragestellungen Rechnung getragen werden. Aufgrund des bereits aus den vorherigen Studien bekannten Hyperaldosteronismus der KO-Tiere stellte sich darüber hinaus die Frage, ob die im Rahmen der Studie feststellbaren kardiologischen Auffälligkeiten als Konsequenz der gesteigerten Aldosteronwerte, oder aber als direkte Folge des Genotyps gewertet werden müssen. Aus diesem Grund wurden alle oben genannten Untersuchungen mit Tieren, welche einer Behandlung mit dem Aldosteronantagonisten Eplerenon zugeführt worden waren, wiederholt. Bei der Auswertung der basalen Ruhe- und Stress-EKGs zeigten sich einige Parameter bei den KO-Tieren pathologisch verändert. So war das QRS-Intervall, als Korrelat zur intraventrikulären Überleitungszeit, bei den KO-Mäusen verlängert, im Stress-EKG waren darüber hinaus sowohl die Dauer der P-Welle als auch des PQ-Intervalls erhöht. Durch die Behandlung mit Aldosteron waren diese Unterschiede zwischen WT- und KO-Gruppe teilweise nicht mehr feststellbar. Das die atrioventrikuläre Überleitungszeit abbildende PQ-Intervall war sowohl im Vergleich mit dem behandelten WT, als auch mit dem unbehandelten WT nicht mehr signifikant erhöht. Auch die Länge des QRS-Komplexes näherte sich unter Eplerenon-Behandlung dem der unbehandelten WT-Tiere an und sank bei der Stress-EKG-Auswertung sogar unterhalb des Signifkanzniveaus. Bei der EKG-Analyse in Bezug auf Arrhythmien ergab sich bei Gegenüberstellung der basalen WT- und KO-Gruppe eine deutlich gesteigerte Vulnerabilität für Herzrhythmusstörungen bei den KO-Tieren. Durch die Behandlung mit Eplerenon konnte hierbei ein deutlicher Erfolg erzielt werden mit signifikanter Reduktion der Arrhythmieereignisse. Die elektrophysiologische Untersuchung ergab neben unauffälligen Parametern der Funktion des Sinusknotens und der AV-Überleitung ebenfalls Hinweise für eine gesteigerte Empfindlichkeit für Arrhythmien. Die durch EPU induzierten Arrhythmien zeigten sich durch Eplerenon-Behandlung gleichermaßen rückgängig. Mittels Kollagenfärbung konnte der initiale Verdacht, dass die SPRED2-KO-Tiere zu einer vermehrten kardialen Fibrosierung neigen, bestätigt werden. Dabei zeigte sich durch die Behandlung mit Eplerenon eine deutliche Beeinflussung und Reduktion des kardialen Kollagengehaltes. Insgesamt lässt sich schlussfolgern, dass die mannigfaltigen phänotypischen Effekte, die die SPRED2-Defizienz bedingt, nur teilweise dem Hyperaldosteronismus der Tiere geschuldet sind und durch therapeutische Einflussnahme auf diesen auch nur partiell kompensiert werden können. / SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach and heart investigations were systematically performed. An initial phenotypical characterization in studies of Ullrich et al showed a hypochondroplasia-like dwarfism, abnormally high water uptakes, behavioural syndromes with excessive grooming and reduced survival times. Also, investigations revealed hyperaldosteronism in SPRED2 KO mice with doubled serum aldosterone as compared with WT mice. Systematic investigation of contributing upstream hormone axes demonstrated, that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. First indications for cardiac pathologies in SPRED KO mice resulted from initial cardiac tests of Ullrich et al., revealing enlarged hearts with elevated heart weight/body weight ratios, as well as increased stroke volumes in KO mice. To investigate whether the cardiac phenotype and the reduced survival time is a consequence of the genotype or secondary due to hyperaldosteronism, electrocardiograms, electrophysiological studies, both with arrhythmia analysis, as well as PSR-stainings, were performed with untreated mice, as well as animals after eplerenone treatment. Some ECG-parameters showed significant differences, for example QRS-interval was prolonged in KO-mice, as correlation for ventricular conduction time. Under isoproterenol stimulation, p-wave duration as well as PQ-interval revealed to be extended. These differences showed to be reduced due to eplerenone treatment, in case of PQ-time and QRS-interval after isoproterenol stimulation, even in significant dimension. Concerning the arrhythmia analysis in ECG and EPU, results showed a distinctly increased vulnerability for arrhythmias in KO-mice, which could be influenced with eplerenone treatment. EP studies revealed no significant differences regarding function of sinus and atrioventricular node, but, analogous to ECG-studies, significant more and severe arrhythmias could be detected in KO-mice, which could be clearly reduced with eplerone. By use of Picro-sirius red staining as a tool to appraise collagen fibers, a significant higher amount of collagen in heart slices of KO-mice could be proved, while treatment with eplerenone reduced fibrosis distinctly, both in WT and in KO-mice. In summary, manifold phenotypical characterizations of SPRED2 KO mice showed to be only partially result of the hyperaldosteronism and revealed only partial influence due to eplerenone treatment.

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.