O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

A lesão por balão cateterismo é um procedimento comumente utilizado para o estudo dos mecanismos de restenose. O estresse mecânico produzido pela passagem do balão promove alterações tanto na artéria lesada quanto na artéria contra-lateral. Observou-se um aumento da densidade de neurônios que contêm neuropeptídeos como a Substância P (SP) e o Peptídeo Relacionado com o Gene da Calcitonina (CGRP) na artéria contra-lateral à lesão, o que evidencia a ocorrência de um processo neurocompensatório. Observou-se ainda um aumento da reatividade desta artéria à fenilefrina (Phe) e à angiotensina II (Ang II) após 4 e 15 dias da lesão, respectivamente. O objetivo do presente trabalho foi estudar as conseqüências dos tratamentos com celecoxibe (inibidor seletivo da enzima COX-2), -tocoferol (Vitamina E, antioxidante natural) e losartan (antagonista dos receptores AT1), sobre a reatividade à Phe, Cloreto de Potássio (KCl), Acetilcolina (ACh), Ang II de artérias ipsilaterais e contra-laterais em relação a artérias controles. Em animais tratados com celecoxibe (10 mg/Kg - 2 vezes ao dia) ou -tocoferol (400 mg/Kg/dia) por 7 dias. Os resultados mostram que os dois tratamentos normalizam os valores de efeito máximo (Emax) da Phe nas artérias contra-laterais com endotélio aos níveis de artérias controle. Em animais tratados com celecoxibe, a artéria ipsilateral não respondeu à Phe, enquanto no tratamento com -tocoferol mostrou valores de Emax reduzidos em relação a animais controle. Os valores de Emax da ACh em artérias de animais controle e tratados com celecoxibe ou -tocoferol, são idênticos. Entretanto, ambos os tratamentos promoveram redução na potência da ACh em artérias controle quando comparadas com as de animais não tratados. A potência da ACh na artéria contra-lateral foi semelhante ao controle em todos os tratamentos. O Emax da Ang II estava aumentado na artéria contra-lateral à lesão. O tratamento com losartan (15 mg/Kg/dia) por 18 dias promoveu redução neste parâmetro na artéria contra-lateral aos níveis do controle. A potência da Ang II em artérias contra-laterais de animais tratados com losartan é igual ao da artéria de animais controle. Nos animais controles tratados com losartan a potência desse peptídeo foi menor do que controle e contra-lateral sem tratamento e contra-lateral tratada. Na artéria contra-lateral o Emax do KCL estava diminuído em relação ao controle e o tratamento com losartan não modificou este parâmetro. O Emax da ACh em artérias controle e contra-laterais não foi alterado pelo tratamento com losartan, entretanto houve aumento da potência deste agonista em artérias controles e contra-laterais em relação a artérias de animais controles. Os resultados obtidos no presente estudo indicam que a produção de espécies reativas de oxigênio, prostanóides vasocontritores e Ang II levam a alterações na reatividade aos agonistas estudados na artéria contra-lateral à lesão por cateter balão. / The injury by balloon catheter is a procedure commonly used to study the mechanisms of restenosis. The mechanical stress produced by the passage of the balloon promotes changes both in the injured artery in the contralateral artery. There was an increased density of neurons containing neuropeptides such as substance P (SP) and the Gene Related Peptide of Calcitonin (CGRP) in the contralateral artery to the lesion, which shows the occurrence of a process neurocompensatory. There was also an increase in the artery reactivity to phenylephrine (Phe) and angiotensin II (Ang II) after 4 and 15 days of injury, respectively. There was also an increase in the artery reactivity to Phe and Ang II after 4 and 15 days of injury, respectively. The objective of this work was to study the consequences of treatment with celecoxib (selective inhibitor of COX-2), -tocopherol (vitamin E, natural antioxidant) and losartan (AT1 receptor antagonist) on the reactivity to Phe, chloride Potassium (KCl), acetylcholine (ACh), Ang II in arteries ipsilateral and contralateral side for control arteries. In animals treated with celecoxib (10 mg / kg - 2 times daily) or -tocopherol (400 mg / kg / day) for 7 days. The results show that both treatments normalize the values of maximum effect (Emax) of Phe in the contralateral arteries with endothelium to the levels of control arteries. In animals treated with celecoxib, the ipsilateral artery did not respond to Phe, whereas the treatment with -tocopherol showed reduced values of Emax for the control animals. The values of Emax of ACh in arteries from control animals and treated with celecoxib or -tocopherol, are identical. However, both treatments promoted reduction in the potency of ACh in control arteries when compared with untreated animals. The potency of ACh in the contralateral artery was similar to control in all treatments. The Emax of Ang II was increased in the contra-lateral artery to the lesion. Treatment with losartan (15 mg / kg / day) for 18 days promoted reduction in this parameter in the contra-lateral artery levels of control. The potency of Ang II in contralateral arteries of animals treated with losartan is equal to the artery of control animals. In control animals treated with losartan the potency of this peptide was lower than control and contra-lateral untreated and control-treated side. Contralateral artery in the Emax of KCL was decreased in the control and treatment with losartan did not modify this parameter. The Emax of ACh in control arteries and contralateral side was not changed by treatment with losartan, however increased the power of this agonist in control arteries and contralateral side on arteries of control animals. The results of this study indicate that the production of reactive oxygen species and vasoconstrictors prostanoids Ang II lead to changes in reactivity to agonists studied in the contra-lateral to the artery by balloon catheter injury.

研究背景和目的 / 高血壓和糖尿病是人群中常見的疾病，兩者常共同存在，其共存的病理生理機制非常複雜，其中腎素血管景張素系統功能紊亂起重要作用。多個研究表明血管緊張素轉化晦抑制劑和血管緊張素II 1 型受體阻滯劑通過調節不同基因的表達，發揮其保護心血管和腎臟功能的效用。然而，目前仍缺乏遠兩類藥物影響全基因表達譜的全面調查。因此，本研究應用全基因表達譜晶片技術，檢測分析了高血壓和糖尿病並發的病人在服用安慰劑、雷米普利(ramipril)和替米沙坦(telmisartan)後的全基因表達譜的變化，從而全面評估了血管緊張素轉化臨抑制劑和血管繁張素II 1 型受體阻滯劑對相關基因的轉錄調控作用。 / 方法 / 11 名患有高血壓和糖尿病的病人(男性5 名)在服用安慰劑最少2 星期后，以隨機吹序接受為期各6 星期的雷米普利和替米沙坦治療，並分別在安慰劑期和2 個藥物治療期結束后提取心A 進行全基因表達譜分析。 / 結果 / 與服用安慰劑時的全基因表達譜相比，雷米普利治療后有267 個基因的表達降低， 99 個基因的表達增強。表達差異幅度為-2.0 至1.3 (P < 0.05) 。表達下降的基因主要與血管平滑肌收縮、炎症反應和氧化壓力相關。表達增強的基因主要與心血管炎症反應負調節相關。基因共表達網絡分析表明， 2 個共表達基因組與雷米普利的降血壓作用相闕， 3 個共表達基因組與肥胖相關。 / 與服用安慰劑時的全基因表達譜相比， 替米拉)、坦治療后有55 個基因表達降低， 158 個基因的表達增強。表達差異幅度為-1. 9 至1.3 (P < 0.05) 。表達增強的基因主要與脂質代謝、糖代謝和抗炎症因子作用相關。基因共表達網絡分析表明， 2 個共表達基因組與替米沙坦對24 小時舒張壓負荷量的作用相關， 2 個共表達基因組則與總膽固醇， 低密度脂蛋白膽固醇和C 反應蛋白相關。 / 結論 / 本論文描述了高血壓和2 型糖尿病病患全基因組表達的總體模式及經藥物治療後表達譜的相應改變， 為今後進一步研究腎素血管緊張素系統抑制劑和高血壓、糖尿病發展進程的相互作用提供了方向。 / Background and aim: Pathophysiological mechanisms underpinning the coexistence of hypertension and type 2 diabetes are complex systemic responses involving dysregulation of the renin-angiotensin system (RAS). We conducted this study to investigate the genome wide gene expression changes in patients with both hypertension and diabetes at three treatment stages, including placebo, ramipril and telmisartan. This study aimed to obtain a panoramic view of interactions between gene transcription and antihypertensive therapy by RAS inhibition. / Methods: 11 diabetic patients (S men) with hypertension were treated with placebo for at least 2 weeks followed by 6 weeks randomised crossover treatment with ramipril Smg daily and telmisartan 40mg daily, respectively. Total RNA were extracted from leukocytes at the end of placebo and each treatment period, and were hybridized to the whole transcript microarray. The limma package for R was used to identify differentially expressed genes between placebo and the 2 active treatments. The weighted gene coexpression network analysis (WGCNA) was applied to identify groups of genes (modules) highly correlated to a common biological function in pathogenesis and progression of hypertension and diabetes. / Results: There were 267 genes down-regulated and 99 genes up-regulated with ramipril. Fold changes of gene expression were ranged from -2.0 to 1.3 (P < 0.05). The down-regulated genes were involved in vascular signalling pathways responsible for vascular smooth muscle contraction, inflammation and oxidative stress. The up-regulated genes were associated with negative regulation of cardiovascular inflammation. The WGCNA identified 17 coexpression gene modules related to ramipril. The midnight blue (57 genes, r < -0.44, P < 0.05) and magenta (190 genes, r < -0.44, P < 0.05) modules were significantly correlated to blood pressure differences between placebo and ramipril. / There were 55 genes down-regulated and 158 genes up-regulated with telmisartan. Fold changes of gene expression were ranged from -1.9 to 1.3 (P < 0.05). The down-regulated genes were mainly associated with cardiovascular inflammation and oxidative stress. The up-regulated genes were associated with lipid and glucose metabolism and anti-inflammatory actions. The WGCNA identified 8 coexpression gene modules related to telmisartan. The black (56 genes, r = 0.46, P = 0.03) and turquoise (1340 genes, r = -0.48, P = 0.02) modules were correlated with diastolic blood pressure load. The blue (1027 genes) module was enriched with genes correlated with total cholesterol (r = - 0.52, P = 0.01), LDL-C (r = - 0.58, P = 0.004), and hsCRP (r = -0.57, P = 0.006). The green module (272 genes) was significantly correlated with LDL-C (r = - 0.44, P = 0.04) and hsCRP (r = - 0.59, P = 0.004). / Conclusion: Genome wide gene expression profiling in this study describes the general pattern and treatment responses in patients with hypertension and type 2 diabetes, which suggests future directions for further investigations on the interaction between actions of the RAS blockers and disease progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Deng, Hanbing. / "December 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 198-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration --- p.i / Publications --- p.ii / Abstract --- p.iv / 論文摘要 --- p.vi / Acknowledgements --- p.viii / Table of Contents --- p.x / List of tables --- p.xiv / List of figures --- p.xv / List of appendices --- p.xvii / List of abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Epidemiology --- p.6 / Chapter 1.2.1 --- Epidemiology of hypertension --- p.9 / Chapter 1.2.2 --- Epidemiology of type 2 diabetes --- p.10 / Chapter 1.3 --- Aetiology --- p.13 / Chapter 1.3.1 --- Ageing --- p.13 / Chapter 1.3.1.1 --- Age-induced artery stiffness --- p.14 / Chapter 1.3.1.2 --- Age-related endothelial dysfunction --- p.14 / Chapter 1.3.2 --- The renin-angiotensin system (RAS) --- p.16 / Chapter 1.3.2.1 --- The local RAS --- p.20 / Chapter 1.3.2.2 --- The RAS and insulin resistance --- p.22 / Chapter 1.3.2.3 --- The RAS and inflammation --- p.26 / Chapter 1.3.2.4 --- The RAS and oxidative stress --- p.28 / Chapter 1.3.3 --- Obesity --- p.31 / Chapter 1.3.3.1 --- Obesity and renin-angiotensin system (RAS) --- p.33 / Chapter 1.3.3.2 --- Obesity and insulin resistance --- p.36 / Chapter 1.3.3.3 --- Obesity and oxidative stress --- p.38 / Chapter 1.3.3.4 --- Obesity and sympathetic nervous system (SNS) --- p.38 / Chapter 1.4 --- Pharmacogenomics of antihypertensive therapy --- p.39 / Chapter 1.4.1 --- Angiotensin-converting enzyme inhibitors (ACEIs) --- p.41 / Chapter 1.4.2 --- Angiotensin II type 1 receptor blockers (ARBs) --- p.43 / Chapter Chapter 2 --- Aim --- p.59 / Chapter Chapter 3 --- Methods --- p.60 / Chapter 3.1 --- Subjects --- p.60 / Chapter 3.1.1 --- Subject recruitment protocol --- p.60 / Chapter 3.1.2 --- Definition of type 2 diabetes --- p.62 / Chapter 3.1.3 --- Definition of obesity --- p.62 / Chapter 3.1.4 --- Definition of dyslipidaemia --- p.63 / Chapter 3.2 --- Study design and procedure --- p.64 / Chapter 3.2.1 --- Blood pressure assessments --- p.65 / Chapter 3.2.2 --- Anthropometric measurements --- p.68 / Chapter 3.2.3 --- Medical history, life style and side effect evaluation --- p.68 / Chapter 3.2.4 --- RNA isolation --- p.68 / Chapter 3.2.5 --- RNA quality assessment --- p.70 / Chapter 3.2.6 --- Oligonucleotide microarrays --- p.71 / Chapter 3.2.7 --- DNA extraction --- p.75 / Chapter 3.2.8 --- Biomedical measurements --- p.76 / Chapter 3.2.8.1 --- Glycosylated haemoglobin Alc (HbA₁c) --- p.77 / Chapter 3.2.8.2 --- Fasting plasma glucose (FP G) --- p.77 / Chapter 3.2.8.3 --- Fasting insulin --- p.77 / Chapter 3.2.8.4 --- Plasma urate --- p.77 / Chapter 3.2.8.5 --- High sensitive C-reactive protein (hsCRP) --- p.78 / Chapter 3.2.8.6 --- Fasting plasma triglycerides (TG) --- p.78 / Chapter 3.2.8.7 --- Fasting plasma cholesterols --- p.78 / Chapter 3.2.8.8 --- Renal and liver functions --- p.78 / Chapter 3.2.8.9 --- Urinary parameters --- p.79 / Chapter 3.3 --- Statistical Analysis --- p.79 / Chapter 3.3.1 --- Statistical analysis of clinical and biomedical data --- p.79 / Chapter 3.3.2 --- Analysis of microarray data --- p.80 / Chapter 3.3.2.1 --- Raw data assessment --- p.80 / Chapter 3.3.2.2 --- Data normalisation --- p.92 / Chapter 3.3.2.3 --- Data filtering --- p.96 / Chapter 3.3.2.4 --- Linear models for assessment of differential expression --- p.96 / Chapter 3.3.2.5 --- Weighted gene coexpression network analysis --- p.101 / Chapter 3.3.2.6 --- Network visualisation and gene ontology analysis --- p.102 / Chapter 3.3.3 --- Sample size calculation --- p.103 / Chapter Chapter 4 --- Results --- p.104 / Chapter 4.1 --- Demographic and biomedical characteristics at baseline --- p.104 / Chapter 4.1.1 --- Hypertension and diabetes status at baseline --- p.108 / Chapter 4.1.2 --- Prevalence of dyslipidaemia --- p.108 / Chapter 4.1.3 --- Prevalence of obesity --- p.109 / Chapter 4.1.4 --- Prevalence of metabolic syndrome --- p.109 / Chapter 4.1.5 --- Inflammation markers --- p.110 / Chapter 4.2 --- Blood pressure response to the RAS blockers --- p.110 / Chapter 4.2.1 --- Clinic blood pressure --- p.110 / Chapter 4.2.2 --- 24-hour ambulatory blood pressure --- p.112 / Chapter 4.3 --- Biomedical characteristics --- p.118 / Chapter 4.4 --- Compliance, side effects and adverse events --- p.120 / Chapter 4.5 --- Gene expression differences between treatments --- p.121 / Chapter 4.5.1 --- Gene expression differences between placebo and ramipril --- p.121 / Chapter 4.5.1.1 --- Expression changes in genes related to regulation of transcription with ramipril --- p.122 / Chapter 4.5.1.2 --- Expression changes with ramipril in genes related to molecular mechanism of cardiovascular changes in hypertension --- p.125 / Chapter 4.5.1.3 --- Expression changes in genes related to blood pressure with ramipril --- p.128 / Chapter 4.5.1.4 --- Expression changes in genes related to fatty acid metabolism with ramipril --- p.130 / Chapter 4.5.1.5 --- Expression changes in genes related to inflammation with ramipril --- p.130 / Chapter 4.5.1.6 --- Expression changes in genes related to oxidative stress with ramipril --- p.133 / Chapter 4.5.1.7 --- Power estimation --- p.133 / Chapter 4.5.2 --- Gene expression differences between placebo and telmisartan --- p.135 / Chapter 4.5.2.1 --- Changes in regulation oftranscription with telmisartan --- p.137 / Chapter 4.5.2.2 --- Expression changes in genes related to glucose metabolism with telmisartan --- p.141 / Chapter 4.5.2.3 --- Expression changes in genes related to lipid metabolism with telmisartan --- p.143 / Chapter 4.5.2.4 --- Expression changes in genes related to inflammation with telmisartan --- p.143 / Chapter 4.5.2.5 --- Power estimation --- p.145 / Chapter 4.5.3 --- WGCNA for comparison between placebo and ramipriI --- p.147 / Chapter 4.5.3.1 --- Midnight blue module and clinical responses to ramipril --- p.152 / Chapter 4.5.3.2 --- Magenta module and blood pressure responses to ramipril --- p.154 / Chapter 4.5.3.3 --- Yellow module and clinical responses to ramipril --- p.158 / Chapter 4.5.3.4 --- Red module and clinical responses to ramipril --- p.161 / Chapter 4.5.3.5 --- Salmon module and clinical responses to ramipril --- p.163 / Chapter 4.5.4 --- WGCNA for comparison between placebo and telmisaItan --- p.168 / Chapter 4.5.4.1 --- Diastolic blood pressure load and gene coexpression modules --- p.168 / Chapter 4.5.4.2 --- Lipids, hsCRP and gene coexpression modules --- p.172 / Chapter Chapter 5 --- Discussion --- p.176 / Chapter 5.1 --- Gene expression changes related to ramipril --- p.177 / Chapter 5.1.1 --- Gene expression changes and blood pressure reduction by ramipri1 --- p.177 / Chapter 5.1.2 --- Gene expression changes and vascular protection by ramipri1 --- p.181 / Chapter 5.1.3 --- Obesity and gene expression changes by ramipril --- p.183 / Chapter 5.2 --- Gene expression changes related to telmisartan --- p.185 / Chapter 5.2.1 --- Blood pressure and coexpressed gene modules with telmisartan --- p.185 / Chapter 5.2.2 --- Lipid metabolism and gene expression changes by telmisartan --- p.187 / Chapter 5.2.3 --- Glucose metabolism and gene expression changes by telmisartan --- p.189 / Chapter 5.2.4 --- hsCRP and gene expression changes by telmisartan --- p.190 / Chapter 5.3 --- Limitations of this study and future directions of research --- p.191 / Chapter Chapter 6 --- Conclusion --- p.194 / References --- p.198 / Appendices --- p.257

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

A proteína dissulfeto isomerase (PDI) tem ganhado destaque em processos de sinalização celular. O objetivo deste trabalho foi investigar o papel da PDI na sinalização redox induzida por TNF-a em células endoteliais e por Angiotensina II (Ang II) em células musculares lisas vasculares (CMLV). Em cultura de células endoteliais isoladas da veia umbilical humana (HUVECs) os dados demonstraram que a PDI e a ERp46 regulam especificamente a fosforilação da ERK 1/2 induzida por TNF-a, possivelmente via alterações redox sobre a GTPase Ras e participa da angiogenese induzida por TNF-a. Em CMLV de artérias de resistência, os dados sugerem a participação da PDI na contração induzida por Ang II, bem como na disfunção vascular associada à hipertensão arterial, através da regulação da expressão e atividade da Nox1. Desta forma, podemos concluir que a PDI apresenta um papel na regulação da sinalização redox induzida por TNF-a e Ang II em células endoteliais e CMLV, respectivamente. Tais resultados apontam para um novo papel da PDI na fisiopatologia do sistema cardiovascular. / Protein disulfide isomerase (PDI), an oxidoreductase of endoplasmic reticulum, has emerged as a key player in cell signaling. The aim of the present study was to investigate the role of PDI in redox signaling induced by TNF-a in endothelial cells and by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs). In human umbilical vein endothelial cells (HUVECs) ERp46 or PDI inhibition reduced specifically TNF-a-induced ERK1/2 phosphorylation, possibly via redox modifications in Ras GTPase and TNF-a-induced angiogenesis. In VSMCs from resistance arteries, our results suggest that PDI positively regulates Nox1 dependent signaling and expression in VSMCs from resistance arteries and could be a new player in the oxidative stress and vascular dysfunction observed in hypertension. Altogether, the results provide evidence for a role for PDI in cardiovascular pathophysiology.

Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.

O mecanismo mais importante para a homeostase do K+ é o controle da secreção de K+ no néfron distal. O objetivo deste trabalho foi avaliar em animais submetidos à depleção de K+ por sete dias, a expressão de AT1R da ATRAP e algumas vias de sinalização como as WNK1, KS-WNK1 e WNK4. Estes animais apresentaram menor ganho de peso corporal, hipertrofia renal, isostenúria, e redução da FE de Na+ e K+, com aumento de Ang II, sem alterar a aldosterona. Verificamos aumento da expressão de AT1R mais acentuado em lisado celular e o aumento de ATRAP foram iguais nas frações de lisado total, membranas total e apical. Não detectamos variação nos níveis de RNAm dessas proteínas. A depleção de K+ induziu a fosforilação de c-Src, ERK1/2 e p38, bem como aumento dos RNAm de WNK1 e WNK4, e redução do RNAm de KS-WNK1. Considerando nossos resultados, a depleção aumenta a ação da Ang II, provavelmente devido à hiperexpressão de AT1R, sem diminuir a expressão de ATRAP. A hiperexpressão de WNK1 e WNK4, associada à redução da KS-WNK1. / The most important mechanism for the K+ homeostasis on varying the content of this ion in the diet is the control of K+ secretion in the distal nephron. Since angiotensin II (Ang II) is an important modulator of K+ secretion, the aim of this study was to evaluate, in animals subjected to K+ depletion for seven days, the expression level of angiotensin type 1 receptor (AT1R) and the AT1R-associated protein (ATRAP). Moreover, it was intended to evaluate the possible activation of some signaling pathways triggered by Ang II via AT1R. We also looked for evaluate the expression of ion transporters and \'\'with no lysine kinases\'\' (WNKs) WNK1, KS-WNK1 and WNK4 in these animals, since some of the effects of angiotensin II in the distal tubular segments are mediated by these kinases. The animals subjected to K+ depletion have showed lower body weight gain, renal hypertrophy, marked polyuria, isosthenuria, and significant reduction in FE Na+ and K+, and increased plasmatic Ang II levels, without changing the aldosterone levels. We found that the expression of ATRAP and AT1R is increased in all cell fractions analyzed, with the highest rise in the AT1R in total cell lysate and ATRAP increase was not significant in the apical membrane. We did not detect changes in mRNA levels of these proteins, suggesting no changes in the transcription rate. The mRNA levels of Na+/H+ exchanger isoform 3 (NHE3) and Cl-/Formate (CFEX), abundant in proximal tubuleswere not altered as well. Regarding signaling pathways, K+ depletion induced c-Src, ERK1/2 and p38 phosphorylation, as well as a significant increase in WNK1 and WNK4 mRNA , and reduced KS- WNK1 mRNA. Considering our results, K+ depletion increases Ang II action in renal tissue, probably due to the overexpression of AT1R, and that effect is not associated to the decreased expression of ATRAP. However, the total cell lysate AT1R increasing, was greater than that of ATRAP. The overexpression of WNK1 and WNK4 associated with (to) the reduction of KS - WNK1 appears to be important for K+ secretion inhibition in K+-depleted animals. The inhibitory activity of WNK4 on ROMK channels depends on its dephosphorylation, which depends on the activation of c-Src. The activation of c-Src was evidenced by the increase in K+ -depleted animals phosphorylation.

<p>The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells.</p><p>Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long-term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored.</p><p>In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.</p>

The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells. Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long-term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored. In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.

The kidney regulates the excretion of water and electrolytes, which maintains homeostasis and enables control of arterial blood pressure. Hyaluronan, a large negatively charged interstitial glucosaminoglycan, is heterogeneously distributed within the kidney, primarily found in the medulla. Medullary hyaluronan content changes depending on the state of body hydration and plays a part in fluid regulation through its water binding and viscoelastic properties. The aim of this thesis was to provide new insight into the regulation of intrarenal hyaluronan during different states of body hydration, during completion of kidney development, and during diabetes mellitus. Dehydration reduces medullary interstitial hyaluronan in parallel with reduced hyaluronan synthase 2 gene expression and increased urinary hyaluronidase activity. Acute hydration results in an increase in medullary hyaluronan, an increase that requires nitric oxide and prostaglandins. Urinary hyaluronidase activity decreases during hydration. The elevation of hyaluronan is important for reducing water permeability of the interstitium i.e. favoring diuresis. Changes in hyaluronan concentration constitute a morphoregulatory pathway that plays a key role in nephrogenesis. The reduction in neonatal hyaluronan depended on an angiotensin II mediated process that does not appear dependent on lymph vessel formation. If angiotensin II is blocked with an ACE inhibitor, hyaluronan accumulates, which results in structural and functional abnormalities in the kidney. Renomedullary hyaluronan is elevated during uncontrolled diabetes, which coincides with induction of hyaluronan synthase 2 mRNA, hyperglycemia, glucosuria, proteinuria and overt diuresis. The levels of hyaluronan are probably at a terminus ad quem as no further response was seen during hydration. The higher interstitial expression of hyaluronan during diabetes may be involved in the progression of diabetic nephropathy. This thesis in physiology provides new mechanistic insights into the regulation of renal hyaluronan during various aspects of fluid handling.