Orientador: Heitor Moreno Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T16:28:07Z (GMT). No. of bitstreams: 1 Martins_LuizClaudio_D.pdf: 1037559 bytes, checksum: 4d9767531662913714e7c20456c8e484 (MD5) Previous issue date: 2010 / Resumo: Introdução Hipertensão resistente é definida como a pressão arterial que permanece acima da meta pressórica, apesar do uso de 3 classes de antihipertensivos sendo um deles um diurético. Recentes diretrizes da American Heart Association (AHA-2008) consideraram também hipertensos resistentes os pacientes que usam 4 ou mais classes e possuem suas pressões controladas. No entanto, é desconhecido se as repercussões cardíacas e vasculares nos pacientes hipertensos resistentes não controlados (HARNC) e controlados (HARC) são semelhantes e qual a influência do envelhecimento sobre esses subgrupos de hipertensos resistentes (HAR). Objetivo Comparar as repercussões cardíacas e vasculares nos HARNC e HARC e avaliar a influência do envelhecimento nesses subgrupos. Métodos Após a triagem e adesão rigorosa, noventa pacientes foram separados em HARNC (n=47) e HARC (n=43). Pressão arterial sistólica (PAS), pressão arterial diastólica (PAD), pressão de pulso (PP), velocidade de onda de pulso (VOP), índice de massa do ventrículo esquerdo (IMVE), índice de massa corpórea (IMC), concentração plasmática de aldosterona (CPA) e atividade renina plasmática (ARP) foram avaliados. Realizaram-se testes estatísticos paramétricos e não paramétricos e regressão linear multivariada (RLM) e univariada (RLU). Resultados A PAS, PAD, PP, VOP, IMVE, IMC e CPA foram maiores nos HARNC (PAS=170/150/204 mmHg; PAD=100/88/130 mmHg; PP=69/40/91 mmHg; VOP=10.9/8/15.3 m/s; IMVE=179±49.2 g/m2; IMC=32±2.1 Kg/m2 e CPA=24.4±3.2 ng/dL), comparados com os HARC (PAS=143/130/163 mmHg; PAD=90/70/116 mmHg; PP=54.5/30/75 mmHg; VOP=9.5/6.8/11.3 m/s; IMVE=140.3±30.1 g/m2; IMC=28.3±1.5 Kg/m2 e CPA=19.7±2.6 ng/dL)(p<0.001). A ARP foi maior nos HARC (ARP=4.3±1.2 ng/mL/h) comparado com os HARNC (ARP=1.08±0.32 ng/mL/h)(p<0.001). A RLM mostrou que a VOP foi significativamente dependente da idade em ambos os grupos, porém a influência do envelhecimento foi mais importante nos HARNC. A RLU mostrou que a CPA se correlaciona com a VOP somente nos HAR com mais de 60 anos. Conclusões Duas características estruturais cardiovasculares não foram semelhantes nos dois grupos estudados: a hipertrofia do ventrículo esquerdo e a rigidez arterial foram maiores nos HARNC. A influência do envelhecimento foi mais significativa neste último grupo. Além disso, a CPA e o IMC foram também maiores nos HARNC. Em seu conjunto, estes dados destacam a importância da aldosterona, envelhecimento e obesidade na patogênese da hipertensão arterial resistente controlada e não controlada. / Abstract: Introduction Resistant hypertension (RH) is defined as blood pressure that remains above goal in spite of the concurrent use of three antihypertensive agents of different classes. Ideally, one of the three agents should be a diuretic and all agents should be prescribed at optimal dose amounts. Recent American Heart Association guidelines include patients who are well controlled but are also considered as having resistant hypertension if they require four or more medications. However, we do not know if both "uncontrolled" (UCRH) and "controlled" CRH) resistant hypertension patients have a similar impact on the cardiac and vascular structure and function, and how the aging influence these groups. Methods After confirming compliance to treatment, 90 patients were divided in two subgroups: UCRH (n=47) and CRH (n= 43) patients. Office blood pressure (BP), pulse wave velocity (PWV), left ventricular mass index (LVMI), body mass index (BMI), plasma aldosterone concentration (PAC) and plasma renin activity (PRA) were evaluated. Parametric and nonparametric statistical tests and multivariate and univariated linear regression analysis were performed. Objective The aim of this study was to identify patient characteristics that distinguish individuals with CRH from individuals with UCRH, We especially wanted to compare the effects of aging, RAAS activation, and vascular stiffness on lack of BP control. Results Office systolic blood pressure (SBP), diastolic blood pressure (DBP), Pulse Pressure (PP), PWV, LVMI, PAC and PRA were higher in UCRH (SBP=170/150/204mmHg; DBP=100/88/130mmHg; PP=69/40/91mmHg, PWV=10.9/8/15.3m/s; LVMI=179±49.2g/m2; BMI=32±2.1Kg/m2; PAC=24.4±3.2ng/dL) than in CRH group (SBP=143/130/163mmHg; DBP=90/70/116mmHg; PWV=9.5/6.8/11.3 m/s; LVMI=140.3±30.1g/m2; BMI=28.3±1.5Kg/m2 and PAC=19.7±2.6 ng/dL) (p<0.001). PRA was higher in CRH (PRA=4.3±1.2ng/mL/h) than in UCRH (PRA=1.08±0.32ng/mL/h)(p<0.001). Multivariate linear regression analysis of variables in function of age indicated that the PWV was significantly dependent of age in both UCRH and CRH patients; moreover, the influence of aging was more important in the former group. Univariate linear regression analysis showed that PAC correlates with PWV only in RH subjects over than 60 years old. Conclusions Clearly, at least two cardiovascular structural characteristics were not similar in the two studied groups: LV hypertrophy and arterial stiffness were more prominent in UCRH individuals and the influence of aging was more important in this group. Also, PAC and BMI were higher elevated in the UCRH group; taken together these results reinforce the concept that links obesity and RH through the hyperaldosteronism, and the important role of aldosterone in the pathogenesis of the metabolic syndrome and controlled and uncontrolled resistant hypertension. / Doutorado / Doutor em Farmacologia

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Background: Osteopontin, also known as cytokine Eta-1, plays an important role in postmyocardial infarction remodeling by regulating collagen accumulation. Aldosterone promotes collagen synthesis and structural remodeling of the heart. The role of osteopontin in aldosterone-induced fibrosis and myocardial remodeling is unknown. Osteopontin expression and left ventricular structural and functional remodeling were determined in wild-type and osteopontin knockout mice after aldosterone infusion. Methods and Results: Immunohistochemical analyses showed increased interstitial osteopontin protein in the wild-type left ventricle after 7 days of aldosterone infusion. After 4 weeks of aldosterone infusion, heart rate was unchanged, and there were similar increases in blood pressure (BP) and heart-to-body weight ratio in both wild-type and knockout mice. Left ventricular end-diastolic diameter was significantly higher, whereas percent fractional shortening was significantly lower (P < .05) in knockout versus wild-type mice after 4 weeks of aldosterone infusion. Aldosterone infusion increased fibrosis and apoptosis (TUNEL-positive) in both wild-type and knockout mice. However, the increase in the extent of fibrosis and apoptosis was significantly lower in knockout hearts. Conclusions: Increased osteopontin plays an important role in the regulation of aldosterone-induced remodeling with effects on left ventricular dilation, fibrosis, and apoptosis.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

Na+ sensitive hypertension in Dahl salt sensitive rats (Dahl S) or spontaneously hypertensive rats (SHR) is linked to intrinsic changes in the brain that favour increased Na+ entry into the cerebrospinal fluid (CSF) followed by increases in sympathetic hyperactivity and hypertension (Huang et al 2004). Similar responses are observed in salt resistant and Wistar rats that receive an intracerebroventricular (icv) infusion of Na+ rich artificial cerebrospinal fluid (aCSF) (Huang et al 2001, 2006). Downstream to increased CSF[Na+], a pathway has been described involving mineralocorticoid receptors (MRs), benzamil sensitive Na+ channels, “ouabain”, and angiotensin II type 1 receptors (AT1-R) (Huang et al 1998, Zhao et al 2001, Wang and Leenen 2003, Huang et al 2008). Blood pressure (BP) responses to increased CSF[Na+] may involve activation of AT1-R in the subfornical organ (SFO) as the BP response to injection of NaCl into a lateral ventricle can be blocked by AT1-R blockade in the SFO (Rohmeiss et al 1995a). The role of aldosterone and AT1-R in the SFO was investigated in mediating the BP and heart rate (HR) response to increases in CSF[Na+] and local [Na+]. Results show that infusion of 0.45M and 0.6M Na+ rich aCSF into the SFO increases BP but not HR. The BP is unchanged by infusion of a mannitol solution osmotically equivalent to 0.6M Na+ rich aCSF indicating that the SFO is Na+ sensitive. The BP response to a lower concentration of Na+ (0.45M) is enhanced by prior infusion of aldosterone while BP response to 0.6M is not further enhanced suggesting that the SFO may have maximal responsiveness to acute increases in [Na+] at 0.6M. The BP responses to Na+ rich aCSF in the SFO and the enhancement of those responses by aldosterone can be blocked by infusion of the AT1-R blocker Candesartan in the SFO. This response appears therefore to be mediated in the SFO through AT1-R activation, likely through Ang II release in the SFO. ICV infusion of Na+ rich aCSF increases BP but not HR and this response is partially blocked by infusion of the AT1-R blocker Candesartan in the SFO. This indicates that nearly half the BP responses to icv infusion of Na+ rich aCSF is mediated through AT1-R activation in the SFO. Lastly, contrary to icv, PVN and MnPO studies (Huang and Leenen 1996, Budzikowski and Leenen 2001, Gabor and Leenen 2009) ouabain in the SFO does not increase BP or HR. In conclusion, these results show that the SFO is Na+ sensitive and mediates half the BP responses to changes in CSF[Na+] through a mechanism that involves AT1-R activation. The SFO is further sensitized to Na+ by aldosterone presumably through its genomic effects. Lastly, ouabain in the SFO does not increase BP or HR suggesting that endogenous ouabain in the SFO is not involved in modulating BP or HR responses.

Na+ sensitive hypertension in Dahl salt sensitive rats (Dahl S) or spontaneously hypertensive rats (SHR) is linked to intrinsic changes in the brain that favour increased Na+ entry into the cerebrospinal fluid (CSF) followed by increases in sympathetic hyperactivity and hypertension (Huang et al 2004). Similar responses are observed in salt resistant and Wistar rats that receive an intracerebroventricular (icv) infusion of Na+ rich artificial cerebrospinal fluid (aCSF) (Huang et al 2001, 2006). Downstream to increased CSF[Na+], a pathway has been described involving mineralocorticoid receptors (MRs), benzamil sensitive Na+ channels, “ouabain”, and angiotensin II type 1 receptors (AT1-R) (Huang et al 1998, Zhao et al 2001, Wang and Leenen 2003, Huang et al 2008). Blood pressure (BP) responses to increased CSF[Na+] may involve activation of AT1-R in the subfornical organ (SFO) as the BP response to injection of NaCl into a lateral ventricle can be blocked by AT1-R blockade in the SFO (Rohmeiss et al 1995a). The role of aldosterone and AT1-R in the SFO was investigated in mediating the BP and heart rate (HR) response to increases in CSF[Na+] and local [Na+]. Results show that infusion of 0.45M and 0.6M Na+ rich aCSF into the SFO increases BP but not HR. The BP is unchanged by infusion of a mannitol solution osmotically equivalent to 0.6M Na+ rich aCSF indicating that the SFO is Na+ sensitive. The BP response to a lower concentration of Na+ (0.45M) is enhanced by prior infusion of aldosterone while BP response to 0.6M is not further enhanced suggesting that the SFO may have maximal responsiveness to acute increases in [Na+] at 0.6M. The BP responses to Na+ rich aCSF in the SFO and the enhancement of those responses by aldosterone can be blocked by infusion of the AT1-R blocker Candesartan in the SFO. This response appears therefore to be mediated in the SFO through AT1-R activation, likely through Ang II release in the SFO. ICV infusion of Na+ rich aCSF increases BP but not HR and this response is partially blocked by infusion of the AT1-R blocker Candesartan in the SFO. This indicates that nearly half the BP responses to icv infusion of Na+ rich aCSF is mediated through AT1-R activation in the SFO. Lastly, contrary to icv, PVN and MnPO studies (Huang and Leenen 1996, Budzikowski and Leenen 2001, Gabor and Leenen 2009) ouabain in the SFO does not increase BP or HR. In conclusion, these results show that the SFO is Na+ sensitive and mediates half the BP responses to changes in CSF[Na+] through a mechanism that involves AT1-R activation. The SFO is further sensitized to Na+ by aldosterone presumably through its genomic effects. Lastly, ouabain in the SFO does not increase BP or HR suggesting that endogenous ouabain in the SFO is not involved in modulating BP or HR responses.

The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

Eight well-trained male and female cyclists were studied to determine the effect of sodium and/or water intake on plasma aldosterone during six hours of cycling (55% VO2max) in a warm environment (Tdb = 350C WBGT = 30°C). Each subject randomly completed three trials (water = W; saline = S and no fluid = NF) at one week intervals. Venous blood samples were obtained before dehydration, at 2, 4, 5 and 6 hours during exercise, and also after dehydration. Plasma samples were analyzed for hemoglobin, sodium, potassium, aldosterone and osmolality. Sweat and urine samples were also collected and analyzed for sodium content. Plasma volume based on hemoglobin decreased significantly ( P < 0.01 ) at 15 min in all three trials (Trial W = -7.6%±1.12%; Trial S = -8.6%±1.42% and Trial NF = -6.7%±0.88%) and continued to decrease significantly in Trial NF during exercise ( -10.99% ± 1.3% at the 2th hr; -15.5% ±1.3% at the 4th hr and -16.8%±1.32% at the 5th hr).No significant differences were found betwee trials. Plasma sodium concentration [Na+] decreased over time in Trials W and S and increased in Trial NF due to plasma volume loss. Significant differences in [ Na+ ] were found between Trial NF and Trials W or S. Plasma sodium [Na+ ] adjusted by plasma volume change decreased significantly at 2 hours ( P < 0.01) in the three trials. Average total sodium content of plasma decreased by 125.9 mEq during Trial S, 223.1 mEq during Trial W and 147.1 mEq during Trial NF. Plasma potassium increased significantly (P < 0.01) at 2 hours in all three trials. Plasma osmolality increased significantly (P < 0.01) during prolonged exercise (Trial W = 287.1±2.4 mEq/l; Trial S = 289.4±1.17 mEq/1 and Trial NF = 306±1.6 mEq/1). No significant differences were found between Trials W and S although osmolality was lower in Trial W than in Trial S. A significant difference in osmolality was obtained between Trial NF and Trials W and S (P < 0.01). Plasma aldosterone increased significantly (P < 0.01) during exercise and decreased after exercise. No significant differences existed between Trials W and S although aldosterone levels were lower in Trial S than in Trial W. However, a significant difference was found between Trial NF and Trials W or S. The results of this study suggest that plasma aldosterone has an inverse relationship with plasma volume changes and total sodium concentrations. An increase in plasma potassium and a decrease in plasma sodium during prolonged exercise in a warm environment significantly enhanced plasma aldosterone concentration. The intake of water significantly decreased plasma aldosterone during prolonged exercise in a warm environment, but the intake of sodium had no significant effect in this study. / School of Physical Education

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.