Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein. / Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity.

Schwankungen des arteriellen Blutdrucks (BP) im niederfrequenten Bereich (LF, 0.02-0.2 Hz) sind möglicherweise Ausdruck von endokrinen Regulationssystemen bei der Aufrechterhaltung der kardiovaskulären Homöostase, wie beispielsweise des Renin-Angiotensin-Systems (RAS). Bei Untersuchungen an Ratten mit sogenannter "one-clip, two-kidney" (1C-2K) Goldblatt Hypertonie wurde ein Anstieg der LF Komponente des BP-Powerspektrums unter aktiviertem RAS gefunden. Trotz der bisherigen Untersuchungen blieb jedoch die Frage ungeklärt, ob dieser Anstieg LF(BP)-Power auf einer Stimulation des RAS beruht, oder durch die Blutdruckerhöhung selbst bedingt ist. Aus diesem Grund wurden die Auswirkungen eines stimulierten RAS auf BP-Oszillationen im LF-Bereich in dieser Studie untersucht, während ein Blutdruckanstieg pharmakologisch verhindert wurde. Zweiundzwanzig normotensive Wistar- und siebzehn normotensive Brown-Norway-Katholiek-Ratten wurden aus diesem Grunde chronisch mit einem Telemetriesender instrumentiert. Es erfolgten Blutentnahmen zur Reninaktivitsbestimmung, der BP wurde jeden zweiten Tag telemetrisch aufgezeichnet. Nach drei Wochen wurden die Tiere in zwei Behandlungsgruppen aufgeteilt. Entweder wurden die Tiere einer Behandlung mit Placebo zugewiesen (n=14 bei den Wistar-Ratten und n=8 bei den Brown-Norway-Katholiek-Ratten), oder sie erhielten eine Behandlung mit Hydralazin (n=8 bei den Wistar-Ratten und n=9 bei den Brown-Norway-Katholiek-Ratten, 40-120 mg/kg/Tag). Ein Silberclip (innerer Durchmesser 200 Mikrometer) wurde auf die linke Nierenarterie plaziert, eine erneute Blutentnahme erfolgte und der BP wurde für weitere drei Wochen gemessen. Hiernach wurde die Behandlung beendet, der Clip von der Nierenarterie entfernt und eine letzte Blutabnahme erfolgte. Abschließende Blutdruck-Registrierungen wurden über einen Zeitraum von drei Wochen durchgeführt. Die Power im LF-Bereich wurde aus den aufgezeichneten Blutdrucksignalen berechnet. Nach Implantation eines Nierenclips stieg der BP bei den mit Placebo behandelten Tieren signifikant an (+37 +/- 5.7 mmHg bei den Wistar-Ratten und +50 +/- 7.4 mmHg bei den Brown-Norway-Katholiek-Ratten, p / Low frequency (LF, 0.02-0.2 Hz) blood pressure (BP) fluctuations may result from cardiovascular regulation by endocrine systems such as the renin-angiotensin system (RAS). Studies employing one-clip, two-kidney (1C-2K) Goldblatt hypertension in rats demonstrated an increase in the LF component of the BP power spectrum. However it remains controversial, whether this increase in LF(BP)-Power is due to the stimulation of the RAS or to the elevation in BP itself. Therefore, we investigated the effect of RAS stimulation on LF(BP) fluctuation while the increase in BP was prevented pharmacologically. Twenty-two normotensive Wistar and seventeen Brown-Norway Katholiek rats were chronically instrumented with telemetric BP sensors, blood samples for measurement of the renin activity were taken and BP was monitored every other day. Three weeks later, rats were subjected to oral treatment with either placebo (n=14 in the Wistar rats and n=8 in the Brown-Norway Katholiek rats) or hydralazine (n=8 in the Wistar rats and n=9 in the Brown-Norway Katholiek rats, 40-120 mg/kg/day). A stainless steel clip (inner diameter 200 micrometer) was placed on the left renal artery, again blood samples were taken and BP was recorded for another three weeks. Finally, treatment was discontinued, the clip was removed from the renal artery, a last blood sample was taken and BP was monitored for a final period of three weeks. LF spectral power was calculated off-line from the recorded BP signal. After renal artery clipping BP significantly increased in placebo-treated rats (+37 +/- 5.7 mmHg in the Wistar rats, +50 +/- 7.4 mmHg in the Brown-Norway Katholiek rats, p

Background. The kidney is one of the major organs involved in whole-body homeostasis and it is well understood that chronic renal impairment is further complicated with deranged carbohydrate metabolism, dyslipidemia, altered abdominal fat distribution and the activation of renin-angiotensin system (RAS). Recently, RAS blockades of angiotensinconverting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB) have been noticed for their potential effects on improve glucose and lipid metabolisms and lowering the risk of new-onset diabetes. However, underlying cellular and molecular mechanisms are not fully established. / Conclusions. (1) UNX induces progressive renal impairment and dysregulation of pancreatic and renal RAS in rats. (2) Pancreatic RAS activation leads to intra-islet fibrosis, insulin-secreting beta-cell deficit and insulin secretory deficiency. (3) Renal cortex RAS dysregulation induces ectopic adipocyte differentiation and lipid infiltration, in combination with lipodystrophy and lipid peroxidation, results to insulin resistance. (4) Pancreatic insulin-secretion deficit and insulin resistance contribute to the development of glucose intolerance and hyperglycemia. (5) Kidney impacting on glucose and lipid metabolism by affecting pancreatic islet and adipocyte, suggesting an essential role of the kidney in maintaining the whole-body homeostasis. (6) RAS blockade with ACEI or ARB may prevent the development of chronic renal impairment and glucose and lipid dysmetabolisms in UNX rats. (7) Common pathways modulating blood pressure, glucose and lipid metabolism warrant future studies for the better management of the global epidemic of metabolic syndrome. / Materials and methods. Chronic renal impairment and RAS disturbance were induced by unilateral nephrectomy (UNX) in adult Sprague-Dawley rats undergoing as long as 10 months of observation. Three-month old male rats were randomized into 4 groups: (1) sham operated control rats (n=10), (2) untreated UNX model rats (n=10), (3) ACEI---lisinopril treated UNX rats (n=10), and (4) ARB-olmesartan treated UNX rats (n=10). Blood glucose levels during fasting and oral glucose tolerance test (OGTT) conditions, lipids, insulin and renal function were measured at 3, 6, 8 and 10 months after operation. Histological changes of kidney, pancreas, liver, and adipose tissue were examined at 10 months post-operation. / Objectives. (1) To set up a rat model with persistent chronic renal impairment and RAS activation. (2) To examine changes of fasting blood glucose, glucose tolerance, blood lipids and insulin sensitivity. (3) To examine changes of pancreatic islets and the factors contributing to pancreatic islet damage such as RAS, transforming growth factor (TGF)-beta and alpha-smooth muscle actin (SMA). (4) To examine changes of systemic and renal adipose tissue and the factors contributing to adipopathy such as RAS, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and hydroxy-3-methylglutary coenzyme A reductase (HMGCR). (5) To investigate preventive effect of RAS blockades by the ACEI-lisinopril (4 mg/kg body weight) and ARB-olmesartan (4 mg/kg body weight) on the rat model of progressive renal deficiency. / Results. (1) UNX rats developed time-dependent progressive renal functional impairment and marked glomerulosclerosis and tubulointerstitial lesions. (2) UNX rats showed fasting hyperglycemia, progressive glucose intolerance, hyperlipidemia and insulin resistance. (3) UNX rats demonstrated insulin secretory deficiency in parallel to pancreatic islet fibrosis, beta-cell deficit, and overexpression of RAS components, TGF-beta, and alpha-SMA. (4) UNX rats displayed adipopathy evidenced by shifts the subcutaneous and visceral fats to the ectopic fat with lipid accumulation, lipofuscin pigmentation and adipocytes transformation. The adipopathy associated with down-regulation of AT1R and over-expression of angiotensin, AT2R, PPAR-gamma and HMGCR in the remnant kidney. (5) Treatment with lisinopril and olmesartan significantly attenuated the development of chronic renal impairment, RAS dysregulation and aberrant proteins expression, islet damage, adipose redistribution, and glucose and lipid dysmetabolism. / Sui, Yi. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3422. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 195-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In the course of this study, several endogenous compounds and model substances were used to mimic the conditions in patients suffering from hypertension. As endogenous compounds, angiotensin II and aldosterone were chosen. As model substances, 4-nitroquinoline-1-oxide (NQO), hydrogen peroxide and phorbol 12-myristate 13-acetate (PMA) were selected. Benfotiamine as well as α-tocopherol proved in the course of the experiments to be able to prevent angiotensin II-induced formation of oxidative DNA strand breaks and micronuclei. This could be due to a prior inhibition of the release of reactive oxygen species and is in contrast to results which were achieved using thiamine. Furthermore, experiments in which cells were pre-incubated with benfotiamine followed by incubation with NQO showed that benfotiamine was not able to prevent the induction of oxidative stress. The hypothesis that benfotiamine has, like α-tocopherol, direct antioxidative capacity was fortified by measurements in cell free systems. In brief, a new working mechanism for benfotiamine in addition to the ones already known could be provided. In the second part of the study, angiotensin II was shown to be dose-dependently genotoxic. This effect is mediated via the angiotensin II type 1 receptor (AT1R) which. Further experiments were extended from in vitro settings to the isolated perfused kidney. Here it could be shown that angiotensin II caused vasoconstriction and DNA strand breaks. Co-perfusion of kidneys with angiotensin II and candesartan prevented vasoconstriction and formation of strand breaks. DNA strand break formation due to mechanical stress or hypoxia could be ruled out after additional experiments with the thromboxane mimetic U 46619. Detailed investigation of the DNA damage in vitro revealed that angiotensin II induces single strand breaks, double strand breaks and 8-hydroxydeoxyguanosine (8-oxodG)-adducts as well as abasic sites. Investigations of the effects of aldosterone-treatment in kidney cells showed an increase of oxidative stress, DNA strand breaks and micronuclei which could be prevented by the steroidal mineralocorticoid receptor antagonist eplerenone. Additional experiments with the non-steroidal mineralocorticoid receptor antagonist (S)-BR-4628 revealed that this substance was also able to prevent oxidative stress and genomic damage and proved to be more potent than eplerenone. In vivo, hyperaldosteronism was imitated in rats by aid of the deoxycorticosteroneacetate (DOCA) salt model. After this treatment, levels of DNA strand breaks and chromosomal aberrations in the kidney could be observed. Furthermore, an increase in the release of ROS could be measured. Treatment of these animals with spironolactone , BR-4628 and enalaprile revealed that all antagonists were effective BR-4628 was the most potent drug. Finally, rosuvastatin was investigated. In HL-60 cells phorbol 12-myristate 13-acetate caused oxidative stress. Rosuvastatin was able to prevent the release of ROS and subsequent oxidative DNA damage when co-incubated with PMA. Furthermore, not only an inhibition of PMA-induced oxidative stress but also inhibition of the unspecific release of ROS induced by hydrogen peroxide was observable. Addition of farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), and mevalonate, intermediates of the cholesterol pathway, caused only a marginal increase of oxidative stress in cells treated simultaneously with PMA and rosuvastatin, thus indicating the effect of rosuvastatin to be HMG-CoA-reductase-independent. Investigation of the gene expression of subunits of NAD(P)H oxidase revealed a down-regulation of p67phox following rosuvastatin-treatment. Furthermore, it could be shown that rosuvastatin treatment alone or in combination with PMA increased total glutathione levels probably due to an induction of the gene expression and enzyme activity of γ-glutamylcysteine synthetase (γ-GCS). / Im Zuge dieser Studie wurden sowohl endogene Substanzen als auch Modellsubstanzen eingesetzt, um die pathologischen Verhältnisse in Patienten, die an Bluthochdruck leiden, zu imitieren. Als endogene Substanzen wurden Angiotensin II und Aldosteron ausgewählt. Als Modellsubstanzen wurden 4-Nitrochinolin-1-oxid (NQO), Wasserstoffperoxid und Phorbol-12-myristat-13-gewählt. Der erste Teil dieser Arbeit beschäftigt sich mit zwei Vitaminen, nämlich Benfotiamin und α-Tocopherol. Sowohl Benfotiamin als auch α-Tocopherol zeigten im Laufe der Experimente, dass sie in der Lage sind, durch Angiotensin II verursachte DNA-Strangbrüche und chromosomale Aberrationen zu verhindern. Dies ist möglicherweise auf eine ebenfalls beobachtbare vorausgegangene Inhibition der Freisetzung reaktiver Sauerstoffspezies zurückzuführen. Zusammenfassend konnte ein neuer Wirkmechanismus für Benfotiamin vorgestellt werden. Im zweiten Teil dieser Studie konnte nachgewiesen werden, dass Angiotensin II eine dosisabhängige Gentoxizität verursacht. Dieser Effekt wird durch den Angiotensin II-Rezeptor Typ 1 vermittelt. Im weiteren Verlauf der Studie wurden die in vitro Experimente auf das Modell der isolierten perfundierten Mäuseniere ausgeweitet. Hier konnte gezeigt werden, dass Angiotensin II Vasokonstriktion und DNA-Strangbrüche verursacht. Co-Perfusion der Nieren mit Angiotensin II und Candesartan verhinderte hingegen die Vasokonstriktion und die Bildung von DNA-Strangbrüchen. Die Verursachung von Strangbrüchen durch mechanischen Stress oder Hypoxie konnte ausgeschlossen werden. Die Untersuchung der ex vivo beobachteten DNA-Schäden in vitro ließ erkennen, dass Angiotensin II Einzelstrangbrüche, Doppelstrangbrüche, die Bildung des DNA-Addukts 8-OxodG und abasische Stellen induziert. Ein Reparatur-Comet Assay, parallel durchgeführt mit der Messung des phosphorylierten Histons 2AX (γ-H2AX) über 24 h, zeigte eine vollständige Reparatur der Einzelstrangbrüche, wohingegen die Zahl der Doppelstrangbrüche in diesem Zeitraum sogar zunahm. Untersuchungen der Effekte, die eine Aldosteron-Behandlung auf Nierenzellen hat, zeigten einen Anstieg des oxidativen Stress, der DNA Strangbrüche und der Mikrokerne. Diese Effekte konnten durch Eplerenon verhindert werden. Weitere Experimente mit dem nicht-steroidalen Mineralocorticoid Rezeptor-Antagonisten (S)-BR-4628 zeigten, dass auch diese Substanz oxidativen Stress und DNA Schäden verhindern konnte, im Gegensatz hierzu hatte das (R)-Isomer, das keine Aktivität am Mineralocorticoid Rezeptor zeigt, keine präventiven Effekte. In vivo wurde der Hyperaldosteronismus mit Hilfe des Deoxycorticosteronacetat- (DOCA) Salzmodells nachgeahmt. Unter dieser Behandlung konnten Level an DNA-Strangbrüchen und chromosomalen Aberrationen beobachtet werden. Des Weiteren konnten in den DOCA-Tieren erhöhte Level an oxidativem Stress gemessen werden. Wurden die Versuchstiere zusätzlich zur DOCA-Behandlung mit Spironolacton, BR-4628 und dem Enalapril behandelt, konnte gezeigt werden, dass BR-4628 potenter war als Spironolacton Enalapril. Zuletzt wurde mit Rosuvastatin eine Substanz untersucht, die die antioxidative Abwehr der Zellen aktivieren kann. In der humanen Leukämie-Zelllinie HL-60 verursachte Phorbol-12-myristat-13-acetat (PMA) oxidativen Stress. Rosuvastatin war in der Lage, die Freisetzung von ROS und daraus resultierende DNA-Strangbrüche bei Co-Inkubation mit PMA zu verhindern. Außerdem konnte gezeigt werden, dass Rosuvastatin nicht nur PMA-induzierten oxidativen Stress, sondern auch die unspezifische Wasserstoffperoxid-induzierte Freisetzung von ROS verhinderte. Die Untersuchung der Genexpression von Untereinheiten der NAD(P)H Oxidase ergab, dass p67phox nach Rosuvastatin-Behandlung herabreguliert wurde. Behandlung mit Rosuvastatin allein oder zusammen mit PMA konnte außerdem die Glutathion-Spiegel erhöhen. Dies ist vermutlich auf die Induktion der Genexpression und der Enzymaktivität der γ-Glutamylcystein-Synthetase (γ-GCS), des Schrittmacherenzyms des Glutathionsystems, zurückzuführen.

A hiperatividade do Sistema Renina Angiotensina (SRA) está intimamente relacionada a hipertensão. Avaliamos os efeitos sequênciais do treinamento aeróbio e sedentarismo sobre a pressão arterial média (PAM), frequência cardíaca (FC) e o SRA vascular em ratos SHR e WKY. Ao início dos protocolos SHR vs WKY apresentaram elevada PAM e FC. Ang II era superior em SHR vs WKY nas artérias renais, femorais e carótidas. O conteúdo de Ang (1-7) em SHR vs WKY era inferior nas renais, superior nas femorais e nas carótidas. O treinamento promoveu já na primeira semana redução de Ang II nas renais e aorta de SHR e WKY, femorais e carótidas de SHR; redução de Ang (1-7) nas renais, carótidas e aorta de SHR e WKY, e femoral de SHR. Redução da PAM foi observada apenas nos SHR a partir da 8ª semana de treinamento, por outro lado SHR e WKY apresentaram bradicardia de repouso na 2ª e 8ª semanas respectivamente. Em linhas gerais o treinamento promoveu imediata, progressiva e mantida redução da Ang II e Ang (1-7) vascular, favorecendo a redução da PAM nos SHR e da FC em ambos os grupos. / Hyperactivity of the Renin Angiotensin System (RAS) is closely related to hypertension. We evaluated the effects of aerobic training and sedentarism on blood pressure (MBP), heart rate (HR) and vascular RAS in SHR and WKY. At the beginning of protocols SHR vs. WKY showed high MAP and HR. Ang II was higher in SHR vs. WKY renal, femoral and carotid arteries. The contents of Ang (1-7) in SHR vs. WKY was lower in renal than in the femoral and carotid arteries. The training caused a reduction in the first week of Ang II in renal artery and aorta of SHR and WKY, femoral and carotid arteries SHR. And reduction of Ang (1-7) in renal artery, carotid and aorta of SHR and WKY, with reduction only in SHR femoral artery. Reduction of MAP was observed only in SHR from 8 weeks of training, on the other hand SHR and WKY had resting bradycardia at 2 and 8 weeks respectively. In general the training caused immediate, sustained and progressive reduction of Ang II and Ang (1-7) vascular, favoring the reduction in MAP in SHR.

O presente trabalho buscou identificar uma possível relação causa-efeito entre exercício, SRA, remodelamento cardíaco e função autonômica na redução da pressão arterial (PA) induzida pelo treinamento aeróbio (T) iniciado na fase pré-hipertensiva. SHR, com 4 semanas de idade, foram submetidos ao T (55% da capacidade física, 1h/dia, 5x/semana) ou mantidos sedentários (S) por 8 semanas. WKY serviram como controle temporal. Os parâmetros foram avaliados nas semanas 0, 1, 2, 4 e 8. Nossos dados indicam que o treinamento iniciado ainda na fase pré-hipertensiva, protege o coração reduzindo a expressão relativa de receptores AT1 e a hipertrofia cardíaca, reduz a FC basal e aumenta sua variabilidade por facilitar o componente vagal alterando o balanço simpato-vagal ao coração. Estas adaptações contribuem para retardar a instalação, reduzindo substancialmente os níveis pressóricos atingidos na fase crônica da hipertensão arterial. A oposição precoce aos efeitos deletérios da hipertensão pelo treinamento é fundamental para minimizar seus prejuízos estruturais e funcionais. / In the present dissertation, we identified a possible cause-effect relation between exercise training, SRA, cardiac remodeling and autonomic function during the establishment of essential hypertension. 4 weeks-old SHR were submitted to aerobic training (5x/week; 1hour/day; 55% of maximal exercise capacity). Factors were assessed on weeks 0,1,2,4 and 8. Exercise training was able to attenuate increase AP, vascular sympathetic activity, cardiac remodeling and cardiac AT1 protein expression; to intensify decrease HR; to increase HR variance and cardiac vagal activity. Our data indicate that aerobic training, during pre-hypertensive phases, attenuates cardiac remodeling associated with decrease of AP, cardiac AT1 protein expression and autonomic dysfunction in SHR. Premature modulation of maladaptative effects of hypertension induced by aerobic tranining seem to be crucial to minimize long-term deleterious effects in cardiac structure and function.

Descrevemos recentemente um novo modelo de DRC, baseado nos efeitos adversos da administração de L na lactação (LLact). Os objetivos do presente estudo foram; caracterizar os mecanismos patológicos envolvidos com a nefropatia do LLact e investigar se o extraordinário efeito renoprotetor obtido com a associação L+H no modelo NX seria reproduzido no modelo LLact. Utilizamos 20 ratas Munich-Wistar lactantes, com 6 filhotes cada. As matrizes receberam L, 250mg/Kg/d durante a amamentação e a droga atingiu a prole via leite materno. Os filhotes machos foram acompanhados até os 7 meses de vida, quando se verificou; pressão caudal, albuminúria, creatinina sérica, glomerulosclerose, expansão intersticial, proliferação celular, presença de miofibroblastos intersticiais e rarefação capilar. Os animais LLact restantes foram divididos em 3 novos grupos: LLact+V, mantido sem tratamento, LLact+L, LLact+H, e LLact+LH. Os parâmetros foram reavaliados após 3 meses nesses grupos e também em animais controle (C). Os ratos, LLact apresentaram hipertensão, albuminuria, glomerulosclerose (GS) e lesão intersticial com inflamação e fibrose aos 10 meses de vida. O tratamento com L+H na vida adulta limitou a hipertensão, albuminúria, GS, proliferação intersticial e infiltração de miofibroblastos. Porém, a renoproteção obtida pela associação foi moderada em relação aos resultados previamente obtidos com o modelo NX, especialmente no tocante ao comprometimento tubulointersticial / We recently standardized a severe CKD model based on impaired nephrogenesis by suppression of angiotensin II (Ang II) activity during lactation (LLact). In the present study we sought to gain further insight into the mechanisms associated with the LLact model and to verify if the renoprotection obtained with the association of the Ang II receptor blocker, Losartan (L), and Hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, could be also obtained in the LLact model. Twenty Munich-Wistar dams, each nursing 6 pups, received L, 250 mg/kg/d, until weaning. The male LLact offspring remained untreated until 7 months of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (LLactPre), followed no further. The remaining rats were divided in groups: LLact+V, untreated, LLact+L, given L, 50 mg/kg/day, now as a therapy, LLact+H, given H, 6mg/kg/day and LLact+LH, given L and H. All the parameters were reassessed 3 months later in these groups and in agematched controls (C). At this time, LLact rats exhibited hypertension, albuminuria, glomerulosclerosis (GS), interstitial expansion and inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. LH therapy normalized blood pressure, albuminuria, GS, and limited interstitial cell proliferation and -smooth muscle actin (-SMA) accumulation. However, LH renoprotection achieved with the LLact model was only mild if compared previous studies with the 5/6 renal ablation model

Para avaliar os efeitos de angiotensina II (Ang II) ou endotelina-3 (ET-3), ratos Wistar foram organizados nos grupos: controle, tratados com Ang II ou ET-3 por 42 dias, tratados com losartan, atrasentan ou BQ788 ou co-tratados com Ang II e losartan ou ET-3 e atrasentan ou BQ 788. Foram avaliados: pressão arterial, concentrações plasmáticas e intrarrenais das Angs e ETs, morfologia e função renal. A Ang II induziu hipertensão arterial, aumentou as concentrações plasmáticas das ETs e das Angs e a expressão de RNAm para renina; induziu injuria glomerular e remodelamento das arteríolas renais. O tratamento com losartan reverteu a maioria dos efeitos da Ang II. A ET-3 induziu hipertensão arterial, injúria glomerular, alteração da função renal e aumento de RNAm intrarrenal para os componentes do SRA. O bloqueio do receptor ETA atenuou os efeitos de ET-3 na hipertensão, enquanto o bloqueio de ETB foi melhor nos parâmetros renais. Nossos resultados sugerem uma interação entre os sistemas SRA e endotelinas, induzindo mudanças na estrutura e função renal. / To evaluate the chronic effects of angiotensin II (Ang II) and /or losartan or endothelin-3 (ET-3) and /or atrasentan or BQ788, Wistar rats were organized as: control, treated with Ang II, or ET-3 for 42 days, losartan-treated, co-treated with Ang II and losartan. Treated with ET-3, treated with BQ788 or atrasentan, co-treated with ET-3 and antagonists. Were evaluated: blood pressure, plasma and intrarenal concentrations of Angs and ETs, protein expression, renal morphology and function. Ang II induced hypertension, increased plasma levels of ETs and Angs; increased mRNA for renin; induced glomerular injury and renal arterioles remodeling. Treatment with losartan restored most of the changes induced by Ang II. ET-3 induced hypertension, glomerular injury, renal dysfunction and increased intrarenal mRNA for SRA components. The ETA receptor blockage reverted the ET-3 effects on hypertension, while ETB blockage reverted renal parameters. Our results suggest a crosstalk of renin-angiotensin and endothelin systems, inducing renal structural and functional changes.

A hipertensão arterial é acompanhada de hiperatividade do sistema renina angiotensina (SRA). Demonstramos em ratos SHR que a hiperativação do SRA plasmático antecede o cardíaco. A hiperatividade do SRA foi acompanhada de aumento do estresse oxidativo, perfil inflamatório, hipertrofia cardíaca e deposição de colágeno no ventrículo esquerdo (VE) de SHR. O treinamento aeróbio de baixa a moderada promoveu pronta redução do SRA cardíaco e plasmático seguido por normalização do estresse oxidativo e redução da inflamação no VE. Embora não houve alteração na hipertrofia cardíaca, observamos redução da deposição de colágeno no VE de SHR treinados. Sugerimos que a redução do SRA foi determinante na modulação dos parâmetros analisados contribuindo para a manutenção das estruturas cardíacas e evitando remodelamento cardíaco deletério nos SHR treinados. / The hypertension is followed by renin angiotensin system hyperactivity (RAS). We have showed in SHR rats that plasma RAS hyperactivity precedes the cardiac RAS hyperactivity. Furthermore the RAS hyperactivity was followed by oxidative stress and inflammation increase as well as left ventricle (LV) collagen deposition in SHR. The aerobic training promoted prompt cardiac and plasma RAS activity reduction. It was followed by oxidative stress normalization and inflammatory reduction. Although we did not observe cardiac hypertrophy change, the collagen deposition was reduced in SHR trained group. We suggest the RAS activity reduction as a determinant condition to the beneficial adaptations occurred in the parameters analyzed. Thus contributing to maintenance of cardiac structures and avoids the deleterious cardiac remodeling in SHR.

O Sistema Renina-angiotensina (SRA), e um sistema capaz de gerar hormonios peptideos com grande impacto na regulacao cardiovascular e na patogenese das doencas cardiovasculares. Este sistema opera, por meio das acoes da Angiotensina II, tanto em nivel sistemico (endocrino) quanto tecidual (local, paracrino/autocrino) controlando importantes funcoes, varias delas relacionadas a facilitacao da instalacao e progressao do processo inflamatorio. Por este motivo, a producao desta proteina nos tecidos pode estar relacionada a patogenese de muitas doencas, dentre elas a doenca periodontal (DP), tendo em vista seu carater infeccioso-inflamatorio e os achados da literatura que mostram que a inibicao da formacao de Ang II, diminui a perda óssea da DP em animais. Desta forma, o presente trabalho teve como objetivos: Avaliar in vitro, a) A expressao de componentes do SRA (ANGT, RENINA, ECA, ECA-2, AT1, AT2 e Mas) por fibroblastos de gengiva e ligamento periodontal humanos, por RT-qPCR; b) A producao de componentes do SRA (RENINA, ECA, ECA-2) no sobrenadante de culturas de fibroblastos de gengiva e ligamento periodontal humanos, por ELISA; c) A producao dos receptores do SRA (AT1, AT2 e Mas), nestes fibroblastos, por Imunofluorescencia e d) Se a expressao e a producao dos componentes do SRA por fibroblastos de gengiva e ligamento periodontal humanos, se alteram com a estimulacao por LPS de P. gingivalis e E. coli. Apos a coleta, os dados foram analisados com o auxilio do programa GraphPad Prism 5.0. por meio da analise de variancia a 2 criterios (ANOVA-two way) seguida do pos teste de Bonferroni, com nivel de significancia de 5% para a verificacao das possíveis diferencas. Foi detectada a expressao genica para alguns dos componentes do SRA (ANGT, RENINA, ECA, AT1) por fibroblastos tanto de gengiva quanto de ligamento periodontal. Foi detectada ainda uma expressao genica diferenciada entre fibroblastos de gengiva e ligamento periodontal para a ECA, sendo significativamente maior nos fibroblastos da gengiva. Houve imunomarcacao positiva tanto nos fibroblastos de gengiva quanto de ligamento periodontal compativel com a presenca dos receptores AT1 e Mas. Pode-se observar por fim que o contato com LPS de P. gingivalis e E. coli, na concentracao de 10 g/mL/24 h, nao alteram a expressão dos componentes do SRA. Portanto, pode-se concluir que os fibroblastos tanto de gengiva quanto de ligamento periodontal apesar de nao expressarem e produzirem todos oscomponentes do SRA necessarios para a formacao local de Ang II, poderiam contribuir, ainda que parcialmente, com outras celulas do microambiente dos tecidos periodontais para a formacao e acao locais da Ang II, e assim, para a instalacao e progressao da DP. / The Renin-angiotensin system (RAS) can generate hormones that have a high-impact on cardiovascular regulation as well as in the pathogenesis of cardiovascular disease. This system acts through both systemic (endocrine) and local (paracrine/autocrine) effects of Angiotensin II, controlling important functions related to the facilitation of installation and progression of the inflammatory process. For this reason, this proteins production in tissues can be associated to the pathogenesis of many diseases, including periodontal disease (PD). In the PD setting, a infectious-inflammatory characterized disease, the literature findings shows that inhibition of the Ang II formation can decrease the bone loss in animals. In this context, the aims of the present study were: to investigate in vitro: a) the expression of RAS components (ANGT, RENIN, ECA, ECA- 2, AT1, AT2 and Mas) by human gingival and periodontal ligament fibroblasts by RT-qPCR; b) the production of RAS receptors (AT1, AT2 and Mas) by human cultured gingival and periodontal ligament fibroblasts by Immunofluorescence and d) the production of RAS components (RENIN, ECA, ECA-2) if the expression and production of RAS components by gingival and periodontal ligament fibroblasts modify under P. gingivalis and E. coli LPS stimulation. After collected, the data were analysed using GraphPad Prism 5.0, by the two way ANOVA followed by Bonferroni post test with a significance level of 5%. Gene expression was detected for some of the RAS components (ANGT, RENIN, ECA, AT1) by both gingival and periodontal ligament fibroblasts. It was detected a differential gene expression between gingival and periodontal ligament fibroblasts for ECA, being significantly higher in gingival fibroblasts. There was a stain in Immunofluorescence compatible with the production of RAS receptors (AT1 and Mas). It must be noted that the stimulation with P. gingivalis and E. coli LPS, in a concentration of 10 g/mL/24 h, did not altered the expression of RAS components. In conclusion, despite of neither gingival or periodontal ligament fibroblasts express all components of RAS, needed to local formation of Ang II, they might also contribute to the local formation and action of Ang II and in consequence, to the installation and the progression of DP.