The mas proto-oncogene encodes a seven transmembrane protein (MAS) which is suggested to function as a receptor for angiotensin. It (MAS) was initially identified in NIH-3T3 cells that were transformed with DNA isolated from a human epidermoid carcinoma. These cells formed foci in culture and tumors when injected into nude mice. On the other hand, untransformed cells did not. Further analysis of these cells showed that transformed cells bind increased levels of angiotensin when compared to untransformed cells. These studies also demonstrated that the Mas protein was structurally similar to the angiotensin receptor transmembrane proteins, AT1 and AT2 . This investigation was undertaken to examine the ability of the Mas protein to function as an receptor for angiotensin and promote cell proliferation. To this end, quantitation of mas genes by Polymerase Chain Reaction (PCR) and serial dilutions, and Southern blot analysis support an increased in mas genes in transformed cells. Northern blot analysis demonstrated an increased expression of the mas gene in transformed cells. No changes in the level of the AT2 angiotensin receptor gene expression was observed in the transformed and untransformed cell lines. Expression of the AT1 angiotensin receptor gene was not observed in these cell lines. Anti-peptide antibodies were generated against the 1st and 2nd extracellular regions of the Mas protein. Flow cytometric analysis using these antibodies indicated an increased presence of the Mas protein on the surf ace of transformed cells recognized by anti-peptide antibodies. Western blot analysis showed two cross-reacting proteins of approximately ll0kd and 66kd in transformed cells; whereas, only a 66kd protein was found in untransformed cells. Transformed cells exposed to mas antisense oligos greatly reduced the synthesis of Mas, decreased cell proliferation and the binding of angiotensin. Binding studies using [3H]-DUP- 753 (a non-peptidyl ligand which recognizes Ang subtype AT1 receptors) showed little binding to transformed cells. Similar studies using PD-123319 (a non-peptidyl ligand that recognizes AT2 subtype receptors) indicated that approximately 60% of [125I]-Ang II was displaced using PD-123319. Further binding analysis of transformed cells suggests that [Sarl]-Ang II (an Ang II antagonist) could not completely displace [ 125I]-Ang II. Taken together, these data suggest that Mas protein is an Ang receptor which functions in the regulation of cell proliferation. Mas appears to be a member of a subtype different from AT1 or AT2.

A new approach to study angiotensin receptor distribution in the brain has been taken by developing antibodies to partial sequence of the angiotensin II (AII) type-1 receptor subtype (AT1) and demonstrating the presence of receptors with immunohistochemical staining. The antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor revealed distinctive punctate immunoreactive staining on cell bodies. The cell bodies were distributed in the forebrain in paraventricular and supraoptic nuclei, the organum vasculosum lamina terminalis, median preoptic area and subfornical organ. In the brainstem, the entire locus coeruleus was stained, together with the adjacent mesencephalic and motor nuclei of the trigeminal nerve. The auditory system including the cochlear nucleus and superior olivary nuclei were stained. In the medulla, all the structures involved in blood pressure control were stained including the nucleus of the solitary tract, the 12th nerve nuclei, the rostroventral lateral area and the nucleus ambiguous. Sites where AT2 receptors are located were not stained or staining was limited to specific area such as the medial accessory nucleus of the inferior olive. Immunocytochemical staining of AT1 receptors provides a new and more precise approach to the cellular localization of AII receptors.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AIIα subtype) or PD123177 (designated AIIβ subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AIIα and AIIβ subtypes.

Hypertension, or high blood pressure (BP), is an ever-growing epidemic in the developing world. Understanding the genetics behind essential hypertension (EH), or hypertension with no known cause, is especially important. In this study, three single nucleotide polymorphisms (SNPs) known to be linked to an increase in susceptibility to EH were quantified from a cohort of Kenyans living in the Kasigau region. The SNPs are located in three genes that are part of the renin angiotensin system, the primary regulatory pathway in humans controlling BP. They include: AGT (rs699), AGTR1 (rs5186), and HSD11β2 (rs5479). Overall, by using a fluorescent-based RT-PCR technique, the genotype distribution of AGT (rs699) was 0.63 C/C, 0.34 C/T, and 0.03 T/T. When evaluated as normotensive, prehypertensive, Stage I, or Stage II categories the allele frequencies for f(C)= 0.77,0.85,0.81, 0.77, respectively, and demonstrated Hardy Weinberg Equilibrium (HWE) as assessed by Χ2, p < 0.05. The genotype distribution of AGTR1 (rs5186) was 0.96 A/A, 0.03 A/C, and 0.00 C/C and the genotype distribution of HSD11β2 (rs5479) was 0.46 A/A, 0.46 A/C, and 0.08 C/C. The majority of genotype frequencies for each SNP were in HWE, with the exception of the AGT (rs699) SNP found in the sublocation of Bughuta suggesting other evolutionary selective pressures may be at work in this subpopulation. The high prevalence of the susceptible C allele for AGT (rs699) likely implies it is a critical factor in the high prevalence of EH observed in this population.

O receptor para Angiotensina II tipo 1 (AT1R) é expresso tanto em membrana apical quanto basolateral dos túbulos proximais renais. Embora haja evidências de diferenças funcionais entre receptores apicais e basolaterais, como, por exemplo, a dependência do processo de internalização de receptores apicais, mas não de basolaterais, para a efetivação dos efeitos fisiológicos da Angiotensina II, os mecanismos envolvidos na determinação dessas diferenças não são conhecidos. Alguns trabalhos já evidenciaram a importância da porção c-terminal do receptor AT1 na sua internalização. Desta forma, com o intuito de identificar proteínas de membrana que possam interagir com tal região, foi feita a clonagem do fragmento de DNA correspondente a esta no vetor pGEX-6P-2. O produto da transcrição e tradução do gene foi uma proteína de fusão (GST-AT1aR) que possui em torno de 35kDa, a qual foi imobilizada em resina de glutationa sefarose e incubada com proteínas de membranas totais de córtex renal de ratos (GST pull-down assay). As amostras foram submetidas à Eletroforese Bidimensional, onde identificamos seis spots correspondentes a proteínas que interagem especificamente com a proteína de fusão, mas não com GST. Estes spots foram recortados e analisados por espectrometria de massa. Cinco diferentes proteínas foram identificadas como provavelmente associadas ao receptor AT1aR: ATP sintase subunidade beta, ATP sintase subunidade alfa mitocondrial, GRP78 (heat shock protein de 78kDa regulada por glicose), HSC70 (heat shock protein de 71kDa) e dipeptidil peptidase 4 (DPPIV). Experimentos subsequentes de GST pull-down e western blotting para as proteínas encontradas, confirmaram interação da cauda C-terminal do receptor com as proteínas ATP sintase subunidade beta, HSC70 (heat shock protein de 71kDa) e GRP78 (heat shock protein de 78kDa regulada por glicose). No entanto, nos estudos de co-imunoprecipitação foi possível confirmar apenas a interação com HSC70, um membro da família HPS70, uma heat shock protein. HSP são também chamadas de chaperonas por estarem envolvidas no dobramento correto de proteínas recém sintetizadas, no redobramento de proteína desnaturadas ou dobradas incorretamente e na degradação de proteínas com danos irreparáveis. No entanto, trabalhos recentes descrevem novos papéis para esta proteína, como a participação em processos de tráfego protéico entre compartimentos intracelulares, reciclagem de proteínas para a membrana plasmática e endocitose mediada por clatrina. Novos estudos serão necessários para se determinar a função fisiológica da interação de HSC70 com a cauda citoplasmática do receptor AT1 e ainda, se essa associação estaria envolvida nas diferenças funcionais observadas quando esse receptor é expresso em membrana apical ou basolateral / The angiotensin II receptor type 1 (AT1R) is expressed in both apical and basolateral membranes in the renal proximal tubules. Although there are evidences that they have functional differences, such as the dependence on internalization for apical, but not basolateral, receptors to trigger physiological effects of angiotensin II, the mechanisms of this peculiar behavior are not clear. The carboxy-terminal tail of the AT1 receptor was shown to be involved in its internalization. Thus, in order to identify possible AT1R c-terminal interacting proteins, we have inserted the cDNA coding the last 53 amino acids of the C-terminus into pGEX-6P-2 vector. The gene translation product was a fusion protein (GST-AT1aR) weighting approximately 35 kDa which was immobilized on Glutathione Sepharose resin and incubated with rat renal cortex total membrane proteins (GST pull-down assay). The samples were then subjected to two dimensional gel electrophoresis. We identified six protein spots that specifically interacted with GST-AT1aR. These spots were cut and analyzed by mass spectrometry. Five different proteins were identified as probably associated with AT1aR, ATP synthase beta subunit, ATP synthase alpha subunit, GRP78 (glucose regulated protein of 78kDa), HSC70 (Heat shock cognate 71kDa protein) and dipeptidyl peptidase 4 (DPPIV). The interaction with ATP synthase beta subunit, HSC70 and GRP78 was confirmed by GST pull-down and western blotting. However, immunoprecipitation of total protein of renal cortex followed by immunobloting only confirmed the interaction with HSC70. This protein is a member of the Heat Shock Proteins family HSP70 also called chaperones, because their involvement in correct folding of newly synthesized proteins, refolding of partially denatured or misfolded proteins, and in protein degradation of irreparably damaged proteins. Recent studies have described new roles for HSC70, such as the participation in protein trafficking between intracellular compartments, recycling of proteins to the plasma membrane and endocytosis mediated by clathrin. Further studies are necessary to determine the physiological role of this interaction and whether this association is involved in the functional differences observed regarding the activation of the receptor in apical or basolateral membranes

Chronic graft dysfunction (CGD) is an important post-transplant complication. CGD can be considered as an impaired repair process, which ultimately leads to the loss of graft function.To study non-immunological factors contributing to the development of CGD in kidney grafts we used in vitro and in vivo models, and clinical studies. We studied the actions of hyperlipidemia in vitro. LDL induced increased expression of TGF-β1 and TGF-β receptors type I and type II. Smad2 phosphorylation could be induced by conditioned medium from mesangial cells incubated with LDL. The effects of Fluvastatin and AT1 receptor blocker Candesartan cilexetil on aortic graft arteriosclerosis in the rat were evaluated. Fluvastatin neither alone nor in combination with Cyclosporine A affected allograft remodelling, but reduced neointima formation in isografts. Candesartan cilexetil treatment reduced graft arteriosclerosis. The effect is explained by the reduction of TGF-β1 expression. We investigated the effects of Carvedilol in patients with CGD. Carvedilol failed to alter the CGD progression despite the efficient control of blood pressure, and a beneficial effect on lipid pattern and oxidation. Close control of CyA blood levels is recommended due to interaction between CyA and Carvedilol. Measurement of Ab-oxLDL in kidney graft recipients demonstrated that these patients had lower Ab-oxLDL levels as compared with the control group. Decreased Ab-oxLDL levels were associated with graft loss due to acute rejection and with ischemic heart disease. In this thesis we have addressed several important complex issues, which are interconnected: (1) development of chronic graft dysfunction (2) lipoproteins and their role in inducing pathological conditions like atherosclerosis and graft damage, (3) oxidation, (4) TGF-β and its' role in different pathological conditions, including renal and vascular damage.

Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-29T12:59:34Z No. of bitstreams: 1 cp118956.pdf: 11152798 bytes, checksum: 413c607598471e1abd0db7c49883c8ac (MD5) / Approved for entry into archive by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-29T13:00:23Z (GMT) No. of bitstreams: 1 cp118956.pdf: 11152798 bytes, checksum: 413c607598471e1abd0db7c49883c8ac (MD5) / Made available in DSpace on 2016-06-29T13:00:23Z (GMT). No. of bitstreams: 1 cp118956.pdf: 11152798 bytes, checksum: 413c607598471e1abd0db7c49883c8ac (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Com o crescente aumento da prevalência mundial de diabetes mellitus, tem-se buscado modelos experimentais para melhor compreensão de sua fisiopatologia e tratamento que atendam de maneira mais adequada à preservação de células beta, proteção de órgãos-alvo e atenuação da aterosclerose. Objetivos: Desenvolver modelo experimental de diabetes mellitus tipo 2 induzido por meio de dieta, e utilizá-lo para examinar os efeitos de um inibidor da enzima conversora de angiotensina (IECA) e de um bloqueador do receptor de angiotensina (BRA) na proteção de órgãos-alvo. Métodos: Coelhos machos Nova Zelândia (n=49) receberam dieta acrescida de banha (10%), sacarose (40%) durante todo o protocolo do estudo além de colesterol (0,5% nos três primeiros meses e 0,1% nos meses subseqüentes). Os animais receberam aleatoriamente: apenas a dieta sem fármacos (G1), olmesartana 5 mg (G2), quinapril 30 mg (G3), ou a combinação de ambos (G4), acrescidos à mesma dieta por seis meses. Foram analisados lípides, frutosamina, glicose e insulina em jejum com cálculo dos índices para resistência à insulina e função de células beta pancreáticas. Foram ainda examinadas as áreas sob as curvas de insulina e glicose, após infusão de glicose intraperitoneal. Angiofluoresceinografias e análises histopatológicas avaliaram lesões em órgãos-alvo. Resultados: Os coelhos ganharam peso, e houve aumento dos níveis de glicose, colesterol total, LDL-C e triglicérides e redução do HDL-C (p <0,05 vs. basal). A frutosamina e o HOMA-IR se elevaram, enquanto houve redução do HOMA-β (p <0,05 vs. basal). Sinais precoces de retinopatia diabética foram observados a partir do terceiro mês, progredindo até o final do experimento (p<0,0005). Lesões ateroscleróticas em aorta, esteatofibrose hepática e infiltrado glomerular de macrófagos constituíram os principais achados histomorfológicos. O bloqueio do sistema renina-angiotensina modificou favoravelmente a glicemia e o HOMA-β (p<0,05) e houve atenuação do número e grau dos microaneurismas pelo tratamento com BRA isoladamente ou combinado com IECA (p<0,05 vs. G1). Conclusões: Nosso modelo reproduziu várias características glucometabólicas do diabetes mellitus tipo 2 humanóide, incluindo déficit de secreção e resistência à insulina. O bloqueio do sistema renina-angiotensina atenuou algumas alterações bioquímicas e as lesões microvasculares em retina. / With the increasing prevalence of diabetes mellitus worldwide, new experimental models are required to better understand the pathophysiology of this disease and to offer therapeutic options that can preserve pancreatic beta-cells, protect target organs and attenuate atherosclerosis. Objective: The aims of this study were to develop an experimental model of type 2 diabetes mellitus induced by diet and assess on this model the effects of an angiotensin-converting enzyme inhibitor (ACEI) and an antagonist of the angiotensin II type1 receptor (AT1R) on target organ protection. Methods: New Zealand male white rabbits (n=49) were fed high-fat/high-sucrose (10/40%) during the study protocol and cholesterol-enriched diet (0.5% in the first three months followed by 0.1% until the end of the study). These animals were randomized to receive: diet alone (G1), olmesartan 5 mg (G2), quinapril 30mg (G3), or combination of both drugs (G4), added to the same diet for six months. Fasting lipids, fructosamine, glucose and insulin, with calculation of insulin resistance and beta-cell function indexes were evaluated. The areas under the curves for glucose and insulin were obtained after intraperitoneal glucose bolus injection. Fluorescein angiography and histopathological analyses were performed to assess target-organs lesions. Results: The animals gained weight, and there were increases in blood glucose, total cholesterol, LDL-C and triglycerides, and decrease in HDL-C (p<0.05 vs. baseline). Fructosamine levels and the homeostasis model assessment of insulin resistance (HOMA-IR) were increased, while there was a reduction in the HOMA-β (p<0.05 vs. baseline). Early clinical features of diabetic retinopathy were seen since the third month, progressing up to the end of the experiment (p<0.0005). Aortic atherosclerosis, hepatic steatofibrosis and glomerular macrophage infiltration were the main histomorphologic findings of this study. The renin-angiotensin system (RAS) blockade favorably modified blood glucose and the HOMA- β (p<0.05) and promoted attenuation of the number and grade of microaneurysms in retina in the group of animals receiving AT1R antagonist or combined therapy with the ACEI (p<0.05 vs. G1). Conclusion: Our model reproduced several glucometabolic characteristics of humanoid type 2 diabetes, including decreased insulin secretion and insulin resistance. The RAS blockade attenuated some biochemical abnormalities and the diabetic retinopathy. / FAPESP: 07/51058-8

O receptor para Angiotensina II tipo 1 (AT1R) é expresso tanto em membrana apical quanto basolateral dos túbulos proximais renais. Embora haja evidências de diferenças funcionais entre receptores apicais e basolaterais, como, por exemplo, a dependência do processo de internalização de receptores apicais, mas não de basolaterais, para a efetivação dos efeitos fisiológicos da Angiotensina II, os mecanismos envolvidos na determinação dessas diferenças não são conhecidos. Alguns trabalhos já evidenciaram a importância da porção c-terminal do receptor AT1 na sua internalização. Desta forma, com o intuito de identificar proteínas de membrana que possam interagir com tal região, foi feita a clonagem do fragmento de DNA correspondente a esta no vetor pGEX-6P-2. O produto da transcrição e tradução do gene foi uma proteína de fusão (GST-AT1aR) que possui em torno de 35kDa, a qual foi imobilizada em resina de glutationa sefarose e incubada com proteínas de membranas totais de córtex renal de ratos (GST pull-down assay). As amostras foram submetidas à Eletroforese Bidimensional, onde identificamos seis spots correspondentes a proteínas que interagem especificamente com a proteína de fusão, mas não com GST. Estes spots foram recortados e analisados por espectrometria de massa. Cinco diferentes proteínas foram identificadas como provavelmente associadas ao receptor AT1aR: ATP sintase subunidade beta, ATP sintase subunidade alfa mitocondrial, GRP78 (heat shock protein de 78kDa regulada por glicose), HSC70 (heat shock protein de 71kDa) e dipeptidil peptidase 4 (DPPIV). Experimentos subsequentes de GST pull-down e western blotting para as proteínas encontradas, confirmaram interação da cauda C-terminal do receptor com as proteínas ATP sintase subunidade beta, HSC70 (heat shock protein de 71kDa) e GRP78 (heat shock protein de 78kDa regulada por glicose). No entanto, nos estudos de co-imunoprecipitação foi possível confirmar apenas a interação com HSC70, um membro da família HPS70, uma heat shock protein. HSP são também chamadas de chaperonas por estarem envolvidas no dobramento correto de proteínas recém sintetizadas, no redobramento de proteína desnaturadas ou dobradas incorretamente e na degradação de proteínas com danos irreparáveis. No entanto, trabalhos recentes descrevem novos papéis para esta proteína, como a participação em processos de tráfego protéico entre compartimentos intracelulares, reciclagem de proteínas para a membrana plasmática e endocitose mediada por clatrina. Novos estudos serão necessários para se determinar a função fisiológica da interação de HSC70 com a cauda citoplasmática do receptor AT1 e ainda, se essa associação estaria envolvida nas diferenças funcionais observadas quando esse receptor é expresso em membrana apical ou basolateral / The angiotensin II receptor type 1 (AT1R) is expressed in both apical and basolateral membranes in the renal proximal tubules. Although there are evidences that they have functional differences, such as the dependence on internalization for apical, but not basolateral, receptors to trigger physiological effects of angiotensin II, the mechanisms of this peculiar behavior are not clear. The carboxy-terminal tail of the AT1 receptor was shown to be involved in its internalization. Thus, in order to identify possible AT1R c-terminal interacting proteins, we have inserted the cDNA coding the last 53 amino acids of the C-terminus into pGEX-6P-2 vector. The gene translation product was a fusion protein (GST-AT1aR) weighting approximately 35 kDa which was immobilized on Glutathione Sepharose resin and incubated with rat renal cortex total membrane proteins (GST pull-down assay). The samples were then subjected to two dimensional gel electrophoresis. We identified six protein spots that specifically interacted with GST-AT1aR. These spots were cut and analyzed by mass spectrometry. Five different proteins were identified as probably associated with AT1aR, ATP synthase beta subunit, ATP synthase alpha subunit, GRP78 (glucose regulated protein of 78kDa), HSC70 (Heat shock cognate 71kDa protein) and dipeptidyl peptidase 4 (DPPIV). The interaction with ATP synthase beta subunit, HSC70 and GRP78 was confirmed by GST pull-down and western blotting. However, immunoprecipitation of total protein of renal cortex followed by immunobloting only confirmed the interaction with HSC70. This protein is a member of the Heat Shock Proteins family HSP70 also called chaperones, because their involvement in correct folding of newly synthesized proteins, refolding of partially denatured or misfolded proteins, and in protein degradation of irreparably damaged proteins. Recent studies have described new roles for HSC70, such as the participation in protein trafficking between intracellular compartments, recycling of proteins to the plasma membrane and endocytosis mediated by clathrin. Further studies are necessary to determine the physiological role of this interaction and whether this association is involved in the functional differences observed regarding the activation of the receptor in apical or basolateral membranes

G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that perform transmembrane signal transduction. Due to their pivotal role in a wide range of essential physiological functions GPCRs represent a high proportion of all drug targets. High resolution X-ray structures of GPCRs are however underrepresented in the Protein Data Bank. This is due to their instability in detergent, low expression levels and the presence of misfolded receptors in many heterologous expression systems. The objective of this project was to engineer the angiotensin II type 1 receptor (AT1R), a human GPCR, to make it suitable for structural studies. It was determined that detergentsolubilised AT1R was thermostable with antagonist bound with an apparent Tm of ~45°C, which was sufficiently stable for purification without further thermostabilisation by rational mutagenesis. Two expression systems were then evaluated for large-scale production of AT1R, namely baculovirus-mediated expression in insect cells and mammalian expression in HEK293 cells. Radioligand binding assays showed that only the mammalian system produced sufficient quantities of active AT1R for structural studies. Expression in the mammalian system was further optimised to approximately 6 mg/L. An AT1R-GFP fusion was created to examine membrane localisation using confocal laser scanning microscopy, to assay expression levels, to select highly expressing monoclonal cell lines using fluorescence activated flow cytometry and to develop a fluorescence size-exclusion chromatographybased assay to examine the suitability of 12 different ligands for co-crystallization. AT1R was also engineered to facilitate crystallisation, including C-terminal truncations to remove predicted disordered regions and bacteriophage T4-lysozyme being added to the third intracellular loop to provide additional points of contact for crystallisation, which increased the apparent Tm by approximately 10°C. All modified versions of AT1R were assessed for expression, stability and monodispersity. Additionally a rapid western blotting based assay was developed for the detection of unfolded membrane proteins, which will have wide applicability in the field.

Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1, isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1, isoleucine8 angiotensin II binding was strongly inhibited by 30 mM β-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AIIα) 125I-sarcosine1, isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AIIβ) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AIIα) receptor subtype.