Angiotensinogen Gene Research Articles

Allele-Specific regulation of the human angiotensinogen gene by dexamethasone: implications for human hypertension

Allele-Specific regulation of the human angiotensinogen gene by dexamethasone: implications for human hypertension

Reduced miR‐133a Results in Upregulation of Angiotensinogen in the Paraventricular Nucleus of Rats with Chronic Heart Failure

Activation of renin-angiotensin system (RAS) within the paraventricular nucleus (PVN) of the hypothalamus has been suggested to enhance sympathetic activation in chronic heart failure (CHF) condition. The contribution due to changes in angiotensinogen (AGT) per se remains to be examined. In this study, we observed a 12-fold increase in levels of AGT gene in the PVN of CHF rats compared to sham-operated controls. In order to elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of 3'-UTR of AGT gene and found potential binding site for microRNA (miR)-133a. We found a 1.9-fold decrease in mir-133a expression in the PVN of rats with CHF. We hypothesized that decreased expression of mir-133a may be responsible for the increased expression of AGT in the PVN. To test this hypothesis we used NG108 (neuroblastoma x glioma) cell line, which endogenously expresses RAS, as our in vitro model. Overexpression of miR133a (pEZX-MR03-miR133a) in NG108 cells resulted in 1.4 and 1.5 fold decreases in AGT and AT1R mRNA levels, respectively. To further validate miR-133a binding to 3'-UTR of AGT, luciferase reporter assay was performed in NG108 cells co-transfected with reporter gene construct (hLuc/hRLuc) containing the 3'-UTR of AGT gene and increasing doses of miR-133a (0, 1 and 2ug). Our data shows that miR-133a inhibits AGT luciferase activity in dose dependent manner. There was a 3.4-fold reduction in luciferase activity with the highest concentration of miR133a. These results suggest that AGT is targeted by miR-133a, revealing a novel and unique role for miR-133a in the regulation of Ang II within the PVN, which potentially contributes to sympathoexcitation during CHF.

Dynamic regulation of the angiotensinogen gene by DNA methylation, which is influenced by various stimuli experienced in daily life.

Angiotensinogen (AGT) has a central role in maintaining blood pressure and fluid balance. DNA methylation is an epigenomic modification maintaining a steady pattern in somatic cells. Herein we summarize the link between AGT regulation and DNA methylation. DNA methylation negatively regulates AGT expression and dynamically changes in response to continuous AGT promoter stimulation. High-salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT enhancer-binding protein-binding sites, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue. Salt-dependent hypertension may be partially affected by increased adipose AGT expression. Because angiotensin II is a well-established aldosterone-releasing hormone, stimulation of adipose AGT by aldosterone creates a positive feedback loop. This effect is pathologically associated with obesity-related hypertension, although it would be physiologically favorable for humans to efficiently retain their body fluid. The clear difference in DNA demethylation patterns between aldosterone and cortisol indicates a difference in the respective target DNA-binding sites between mineralocorticoid and glucocorticoid receptors in the AGT promoter. Stimulation-induced interactions between transcription factors and target DNA-binding sites trigger DNA demethylation. Dynamic changes in DNA methylation occur in relaxed chromatin regions both where transcription factors actively interact and where transcription is initiated. In contrast to rapid histone modifications, DNA demethylation and remethylation will progress relatively slowly over days or years. A wide variety of stimuli in daily life will continue to slowly and dynamically change DNA methylation patterns throughout life. Wise choices of beneficial stimuli will improve health.

Dexamethasone Promotes Hypertension by Allele-specific Regulation of the Human Angiotensinogen Gene

The human angiotensinogen (hAGT) gene has polymorphisms in its 2.5-kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, and -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension, whereas Hap -6G/-217G reduces cardiovascular risk. Hap -6A/-217A has increased promoter activity with enhanced transcription factor binding, including to the glucocorticoid receptor (GR). Glucocorticoid therapy frequently causes hypertension, the mechanisms for which are incompletely understood. We have engineered double transgenic (TG) mice containing the human renin gene with either Hap of the hAGT gene and examined the physiological significance of glucocorticoid-mediated allele-specific regulation of the hAGT gene. We have also studied the consequential effects on the renin angiotensin system and blood pressure. TG mice with Hap -6A and -6G were treated with and without a low dose of a GR agonist, dexamethasone (2.5 μg/ml), for 72 h. We found greater chromatin-GR binding with increased GR agonist-induced hAGT expression in liver and renal tissues of Hap -6A mice. Additionally, dexamethasone treatment increased circulating hAGT and angiotensin II levels in Hap -6A mice, as compared with -6G mice. Importantly, GR agonist significantly increased blood pressure and redox markers in TG mice with Hap-6A of the hAGT gene. Taken together, our results show, for the first time, that glucocorticoids affect hAGT expression in a haplotype-dependent fashion with SNPs in Hap -6A favoring agonist-induced GR binding. This leads to increased expression of the hAGT, up-regulation of the renin angiotensin system, and increased blood pressure and oxidative stress in Hap -6A mice.

Vitamin D depletion aggravates hypertension and target-organ damage.

BackgroundWe tested the controversial hypothesis that vitamin D depletion aggravates hypertension and target‐organ damage by influencing renin.Methods and ResultsFour‐week‐old double‐transgenic rats (dTGR) with excess angiotensin (Ang) II production due to overexpression of the human renin (hREN) and angiotensinogen (hAGT) genes received vitamin D‐depleted (n=18) or standard chow (n=15) for 3 weeks. The depleted group had very low serum 25‐hydroxyvitamin D levels (mean±SEM; 3.8±0.29 versus 40.6±1.19 nmol/L) and had higher mean systolic BP at week 5 (158±3.5 versus 134.6±3.7 mm Hg, P<0.001), week 6 (176.6±3.3 versus 162.3±3.8 mm Hg, P<0.01), and week 7 (171.6±5.1 versus 155.9±4.3 mm Hg, P<0.05). Vitamin D depletion led to increased relative heart weights and increased serum creatinine concentrations. Furthermore, the mRNAs of natriuretic peptides, neutrophil gelatinase‐associated lipocalin, hREN, and rRen were increased by vitamin D depletion. Regulatory T cells in the spleen and in the circulation were not affected. Ang metabolites, including Ang II and the counter‐regulatory breakdown product Ang 1 to 7, were significantly up‐regulated in the vitamin D‐depleted groups, while ACE‐1 and ACE‐2 activities were not affected.ConclusionsShort‐term severe vitamin D depletion aggravated hypertension and target‐organ damage in dTGR. Our data suggest that even short‐term severe vitamin D deficiency may directly promote hypertension and impacts on renin‐angiotensin system components that could contribute to target‐organ damage. The findings add to the evidence that vitamin D deficiency could also affect human hypertension.

Genotypes of the renin-angiotensin system and glucocorticoid complications.

Angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) are recognized as important regulators of body mass index (BMI) and systemic blood pressure (BP). An association between these single nucleotide polymorphisms (SNP) of AGT and ACE genes and obesity or hypertension has been established. This study examined relationships between the molecular variants of the AGT and ACE genes and bodyweight or BP in children treated with glucocorticoids for nephrotic syndrome. Twenty Japanese children (male, n = 14; female, n = 6; age, 2-13 years) were genotyped for AGT polymorphisms (M235T and A-6G) and the ACE polymorphisms (insertion/deletion: I/D and rs4341). All of the children studied were treated with daily prednisolone 2 mg/kg for 4 weeks and thereafter alternate-day prednisolone for 8 weeks. BMI, BMI z-scores, blood lipids, renal function and BP in each group were evaluated during the study period. BMI and BMI z-scores during the glucocorticoid therapy were significantly higher in the TT genotype of the AGT M235T polymorphisms and the AA genotype of the AGT A-6G polymorphisms compared to other genotypes (P < 0.05). In contrast, the molecular variant of ACE I/D and rs4341 genotypes did not change bodyweight during the glucocorticoid exposure. It was evident, however, that the BP and blood lipids and renal function were not significantly influenced by the AGT and ACE polymorphisms. The TT genotype of the AGT M235T and the AA genotype of the A-6G polymorphisms may predispose children to bodyweight gain when initially treated with glucocorticoids for nephrotic syndrome.

Association of A-20C polymorphism in the angiotensinogen gene with essential hypertension: a meta-analysis.

The A-20C polymorphism in the angiotensinogen (AGT) gene has been associated with increased risk of essential hypertension in several studies; however, these studies gave inconsistent results. In this study, we performed a meta-analysis to assess the association between AGT A-20C polymorphism and essential hypertension. Published literature was retrieved from PubMed. Pooled odd's ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effect models. A total of 10 case-control studies containing 3653 cases and 3457 controls were enrolled to this meta-analysis. In a combined analysis, the results showed a significant association between the AGT A-20C polymorphism and risk of essential hypertension (AA vs CC: OR = 0.62, 95%CI = 0.46-0.84; recessive model: OR = 0.66, 95%CI = 0.49-0.88). In the subgroup analysis stratified by race, significant associations were found between the AGT A-20C polymorphism and essential hypertension risk in Asians (AA vs CC: OR = 0.59, 95%CI = 0.43-0.80; recessive model: OR = 0.63, 95%CI = 0.46-0.85). In conclusion, the results of this meta-analysis suggested that the AGT A-20C polymorphism was associated with risk of essential hypertension in Asians.

T174M polymorphism in the angiotensinogen gene and risk of myocardial infarction: a meta-analysis

Numerous studies have evaluated the association between the T174M polymorphism in the angiotensinogen (AGT) gene and myocardial infarction (MI) risk. However, the specific association remains controversial because of small sample sizes and varied study designs among different studies. We performed a meta-analysis to assess this correlation. A comprehensive search was conducted to identify all published articles regarding the association between the AGT gene T174M polymorphism and MI risk from different databases. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated, and heterogeneity and publication bias were assessed. A total of 1032 patients with lung cancer and 1286 controls from 6 comparative studies were included in this meta-analysis. The results revealed a significant association between the AGT gene T174M polymorphism and MI risk (MM vs TT: OR = 2.87, 95%CI = 1.71-4.83; dominant model: OR = 1.57, 95%CI = 1.10-2.25; recessive model: OR = 0.41, 95%CI = 0.25-0.66). In subgroup analysis by nationality, we observed a significant association between the AGT gene T174M polymorphism and susceptibility to MI in both Caucasian and Asian populations. In conclusion, the T174M polymorphism in the AGT gene may be related to an increased risk of MI. Further larger studies are needed to confirm these conclusions.

Angiotensinogen Gene M235T and T174M Polymorphisms in Patients with Morbid Obesity and Type 2 Diabetes Mellitus

Background: Angiotensinogen and its cleaved form - angiotensin II are important regulators of adipose tissue metabolism and they may affect adipogenesis. Aim: We studied the correlation between M235T and T174M polymorphisms of angiotensinogen (AGT) gene and morbid obesity and their association with Type 2 Diabetes Mellitus (T2DM). We also investigated the role of haplotypes formed by these polymorphisms in the risk for extreme obesity and obesity- associated T2DM. Material and methods: The study included 335 morbidly obese patients (study groups) and 230 subjects without obesity (control group). The molecular analysis was performed using Polymerase Chain Reaction (PCR) and PCR-restriction fragment length polymorphism technique. Results: Distribution of the genotype for AGT M235T polymorphism differed significantly between the controls and all extremely obese patients (p 0.1). AGT gene polymorphic variants did not display any difference in clinical or metabolic parameters according to each genotype for either the control or the morbidly obese group. In univariate logistic regression analysis, the carriers for T235T and T174M (Hap2Hap3) had lower risk for extreme obesity (p<0.05). Homozygosity for both T235T and T174T (Hap2Hap2) was associated with decreased risk for extreme obesity and concomitant T2DM (p<0.05). Conclusion: M235T but not T174M polymorphism of angiotensinogen (AGT) gene, may be linked with extreme obesity and T2DM in the investigated group of patients. On the basis of our results, we suggest that the risk of extreme obesity, irrespective of T2DM, was lower in Hap2Hap3 haplotype pair carriers. The Hap2Hap2 haplotype pair may be a significant protective factor for morbid obesity with T2DM and T2DM development in extreme obesity status (p<0.05).

Role of ACE and AGT gene polymorphisms in genetic susceptibility to diabetes mellitus type 2 in a Brazilian sample

The aim of the current study was to investigate the association between the InDel polymorphism in the angiotensin I-converting enzyme gene (ACE) and the rs699 polymorphism in the angiotensinogen gene (AGT) and diabetes mellitus type 2 (DM2) in a sample population from Southern Brazil. A case-control study was conducted with 228 patients with DM2 and 183 controls without DM2. The ACE InDel polymorphism was genotyped by polymerase chain reaction (PCR) with specific primers, followed by electrophoresis on 1.5% agarose gel. The AGT rs699 polymorphism was genotyped using a real-time PCR assay. No significant association between the ACE InDel polymorphism and DM2 was detected (P = 0.97). However, regarding the AGT rs699 polymorphism, DM2 patients had a significantly higher frequency of the AG genotype and lower frequency of the GG genotype when compared to the controls (P = 0.03). Our results suggest that there is an association between the AGT rs699 polymorphism and DM2 in a Brazilian sample.

Association between G-217A polymorphism in the AGT gene and essential hypertension: a meta-analysis.

Numerous studies have evaluated the association between the angiotensinogen (AGT) G-217A gene polymorphism and essential hypertension risk. However, the results have been inconsistent. We examined whether the AGT G-217A gene polymorphism confers essential hypertension risk by conducting a meta-analysis. We conducted a literature search of the Google Scholar, PubMed, and China National Knowledge Infrastructure databases for relevant studies that examined the G-217A polymorphism and risk of essential hypertension. Statistical analyses were carried out using Stata 12.0 to combine all relevant studies. Crude odds ratios (ORs) with 95% confidence intervals (95%CIs) were calculated to estimate the strength of this association. A total of 2017 patients with psoriasis and 1708 controls from 7 comparative studies were included in this meta-analysis. We found a significant association between the AGT G-217A gene polymorphism and the risk of essential hypertension (AA vs GG: OR = 2.52, 95%CI = 1.68-3.78; AA vs GA: OR = 2.26, 95%CI = 1.48-3.45; dominant model: OR = 0.38, 95%CI = 0.26-0.57; recessive model: OR = 1.20, 95%CI = 1.03-1.39). Further stratified analyses were conducted by ethnicity and sample size and produced similar results. No evidence of publication bias was found. This meta-analysis confirms that the AGT G-217A gene polymorphism is associated with essential hypertension susceptibility.

16-OR

Objectives To investigate the role of COX-derived prostanoids and the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) on uterine vascular control during early pregnancy in a rodent model of preeclampsia. Methods The transgenic female rat containing the human angiotensinogen (hAGN) gene mated with the male transgenic containing human renin (hREN) is a model of preeclampsia (TgA). Uterine arteries (UA) were isolated from Sprague–Dawley (SD, n = 7) and TgA ( n = 9) rats at 7 days of gestation and mounted in a wire myograph (DMT-USA, 620 M). Concentration response curves were performed in control conditions and after preincubation with indomethacin (Indo, 10 −5 M), or inhibitors of endocannabinoid hydrolysis: URB597 (FAAH) or JZL184 (MAGL) at 10 −6 M. Data were fitted to a dose response curve, maximal responses were expressed as percent of maximal response to 75 mM KCl (% K MAX ) and sensitivity as pD 2 (pD 2 = −Log[EC 50 ]). Results Responses to ACh reached similar maximal relaxations (64 ± 8 vs. 75 ± 6%, p > 0.05), an increased contraction in TgA UA at ACh >10 μM was eliminated by Indo. Contraction to Phe was similar in both groups with an inhibitory effect of Indo in TgA UA ( p MAX 92 ± 7 vs. 109 ± 4; pD 2 8.54 ± 0.03 vs. 8.9 ± 0.08, p MAX 76 ± 9, pD 2 8.58 ± 0.06, p p Conclusions Blockade of COX enzymes inhibited ACh, Phe and Ang II contractions whereas endogenous AEA and 2-AG reduced TgA UA Ang II contraction. A pro-contractile role of COX-derived mediators and a counteracting role of AEA and 2-AG in the enhanced Ang II contraction in TgA UA are evident before the hypertensive phenotype is established. These interactions may control uterine vascular function in early stages of preeclampsia. Disclosures V.M. Pulgar: None. L.M. Yamaleyeva: None. J. Varagic: None. C.M. McGee: None. M. Bader: None. R. Dechend: None. A.C. Howlett: None. K. Brosnihan: None.

Association between angiotensinogen T174M polymorphism and ischemic stroke: A meta-analysis.

Background:Numerous studies have evaluated the association between the angiotensinogen (AGT) T174M polymorphism and ischemic stroke(IS) risk. However, the specific association is still controversial.Materials and Methods:In order to explore this association more deeply, we performed a meta-analysis. All of the relevant studies were identified from PubMed, Embase, and Chinese National Knowledge Infrastructure database up to October 2014. Statistical analyses were conducted with STATA 12.0 software. Odds ratio (OR) with 95% confidence interval (CI) values were applied to evaluate the strength of the association.Results:Six studies with 1290 cases and 1125 controls were included. No significant variation in IS risk was detected in any of the genetic models in the overall (MM vs. TT: OR = 1.64, 95% CI = 0.51-5.28; MT vs. TT: OR = 0.93, 95% CI = 0.66-1.31; dominant model: OR = 1.08, 95% CI = 0.69-1.72; recessive model: OR = 0.61,95% CI = 0.20-1.91). Taking into account the effect of ethnicity, further stratified analyses were performed. The results showed that AGT gene T174M polymorphism might be associated with IS risk in Asians (MM vs. TT: OR = 3.28, 95% CI = 1.79-6.02; recessive model: OR = 0.31, 95% CI = 0.17-0.57).Conclusion:In conclusion, the AGT T174M polymorphism may be a susceptible predictor of the risk of IS in Asians. Further, large and well-designed studies are needed to confirm this conclusion.

Constitutive activation of the renin-angiotensin system reduces visceral fat and improves glucose tolerance in mice.

The renin-angiotensin system (RAS), and particularly angiotensin II, is involved in the control of energy balance, glucose homeostasis and kidney functions. The integrated impact of the RAS on glucose homeostasis is still a matter of debate. We used a model of constitutive RAS activation in double transgenic mice (dTGM) carrying both human angiotensinogen and human renin genes. We evaluated energy balance, measured renal functions, performed glucose and insulin tolerance tests, and used ramipril to inhibit the angiotensin-converting enzyme. dTGM had a lower physical activity and an increased food intake without change in body weight. Renal impairment was characterized by low-grade albuminuria. High urinary output secondary to polydipsia was associated with proximal tubule dysfunction. Compared to controls, dTGM had a lower hyperglycemia induced by an intraperitoneal glucose administration. This decrease was not due to changes in insulin sensitivity and/or secretion. dTGM had an increased creatinine production and a lower epididymal fat mass. Acute inhibition of angiotensin-converting enzyme with ramipril did not suppress this improved glucose tolerance profile. Chronic RAS activation is not sufficient to cause insulin resistance in mice. Moreover, adaptation to constitutive RAS activation in mice results in a better glucose tolerance.

Novel mechanism of blood pressure regulation by forkhead box class O1-mediated transcriptional control of hepatic angiotensinogen.

The renin-angiotensin system is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving 3 components: angiotensinogen, renin, and angiotensin-converting enzyme, which generate angiotensin II as a biologically active product. Angiotensinogen is largely produced in the liver, acting as a major determinant of the circulating renin-angiotensin system, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of angiotensinogen is regulated is not completely understood. Here, we hypothesize that angiotensinogen is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice, which exhibited a reduction in plasma angiotensinogen and angiotensin II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased angiotensinogen expression, whereas hepatocytes lacking Foxo1 demonstrated a reduction of angiotensinogen gene expression and partially impaired insulin inhibition on angiotensinogen gene expression. Furthermore, mouse angiotensinogen prompter analysis demonstrated that the angiotensinogen promoter region contains a functional Foxo1-binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic angiotensinogen gene expression and controls plasma angiotensinogen and angiotensin II levels, modulating blood pressure control in mice.

The effects of angiotensinogen gene polymorphisms on cardiovascular disease outcomes during antihypertensive treatment in the GenHAT study.

Previous studies have reported that risk of cardiovascular morbidity and mortality substantially increases in hypertensive patients, especially among those with inadequate blood pressure control. Two common antihypertensive drug classes including thiazide diuretics and angiotensin-converting enzyme (ACE) inhibitors affect different enzymes in the renin-angiotensin-aldosterone system (RAAS). In the RAAS, angiotensinogen is converted into angiotensin II which increases blood pressure through vasoconstriction. Using a case-only design with 3448 high-risk hypertensive individuals from the Genetics of Hypertension Associated Treatment (GenHAT) study, we examined whether seven single nucleotide polymorphisms (SNPs) in the angiotensinogen gene (AGT) interact with three classes of antihypertensive drugs including chlorthalidone (a thiazide diuretic), lisinopril (an ACE inhibitor), and amlodipine (a calcium channel blocker) to modify the risk of incident coronary heart disease (CHD) and heart failure (HF) among Caucasian and African American participants, separately. We found no gene by treatment interactions to be statistically significant after correction for multiple testing. However, some suggestive results were found. African American participants with the minor allele of rs11122576 had over two-fold higher risk of CHD when using chlorthalidone compared to using amlodipine, or lisinopril compared to amlodipine (p = 0.006 and p = 0.01, respectively). Other marginal associations are also reported among both race groups. The findings reported here suggest that rs11122576 could contribute to future personalization of antihypertensive treatment among African Americans though more studies are needed.

Catalase overexpression prevents nuclear factor erythroid 2-related factor 2 stimulation of renal angiotensinogen gene expression, hypertension, and kidney injury in diabetic mice.

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.

Abstract 529: Treatment With Telmisartan Inhibits Kidney Injury in High Salt-loading Tsukuba Hypertensive Mice With Attenuation of Sympathetic Nervous System

Objective: It is well known that excessive salt intake and/or activation of renin-angiotensin system (RAS) increases blood pressure in part via an increase in sympathetic nerve activity. To further examine the interaction of these factors, we employed transgenic mice carrying both the human renin and angiotensinogen genes (Tsukuba hypertensive mice) and studied the effects of high salt loading and telmisartan on mortality, blood pressure and kidney functions. Design and Method: Ten-week-old male Tsukuba hypertensive mice (TH) were administrated control chow or the chow containing 8% NaCl with or without telmisartan (1 mg/kg/day) and PPAR-γ antagonist, GW9662, in drinking water for 8 weeks. Blood pressure was assessed by radio telemetry method. Urine samples were obtained before and after salt-loading. Concentrations of urinary adrenalin and noradrenalin were measured by ELISA. Expressions of ENaC-α, P47 and p67-phox mRNA in the kidney were measured by RT-PCR. Results: Survival rate 8 weeks after treatment was decreased in salt-loading TH (sTH) mice compared with control TH (cTH). This decrease was improved by the treatment with telmisartan. Blood pressure in sTH mice was significantly higher compared with cTH mice and the treatment with telmisartan mice decreased blood pressure. Urinary sodium concentration was higher in sTH mice compared with cTH mice and treatment with telmisartan further increased urinary sodium concentration in sTH mice. Expression of ENaC-α in did not between sTH mice and cTH mice. Treatment with telmisartan decreased expression of ENaC-α. Urinary adrenalin and noradrenalin concentrations were higher in sTH mice compared with cTH mice. These increases were attenuated by treatment with telmisartan. Expressions of p47 and p67-phox in sTH mice were increased more markedly compared with cTH mice. These increase were also attenuated by treatment with telmisartan. However, these possible protective effects of telmisartan were not influenced by the co-treatment with GW9662. Conclusion: These results suggested that salt-loading enhanced an increase in blood pressure, superoxide anion production, sympathetic activity and mortality in concert with activated RAS.

Abstract 606: Soluble Guanylate Cyclase (sGC) Stimulator Reduces The Cardiac And Renal Damage In Angiotensin II (Ang II)-induced Diastolic Heart Failure

Diastolic heart failure (DHF) is estimated to account for approximately 40% of heart failure cases. Due to the high prevalence and high mortality rates, diastolic heart failure represents a major challenge in cardiovascular medicine. The novel therapeutic alternative with NO-independent direct stimulators of sGC has recently been approved for the treatment of pulmonary hypertension. We analyzed the role of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced diastolic heart failure. We used 4 week-old male double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). At age 7 weeks, dTGR show striking cardiac hypertrophy with fibrosis, severe diastolic dysfunction but preserved systolic function, albuminuria, and renal failure. We compared vehicle-treated dTGR receiving 10% transcutol, 20% cremophor, 70% water, dTGR receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats (single oral dose per day for 3 weeks). Systolic blood pressure increased progressively in vehicle-treated dTGRs compared with SD rats. BAY 41-8543 significantly reduced the blood pressure (197 ± 11 mmHg vehicle vs 133 ± 4 mmHg BAY 41-8543). Treatment with sGC stimulator ameliorated albuminuria (0.3 ± 0.06 mg/8h at week 7) compared with vehicle-treated dTGRs (12.4 ± 2.6 mg/8h at week 7). In addition BAY 41-8543 prevented fibrosis and inflammation in kidney and heart. Cardiac hypertrophy or myocyte size were not reduced by BAY 41-8543 treatment. Cardiac echocardiography and hemodynamic investigations showed that BAY 41-8543 increased ejection fraction and improved diastolic function including speckle tracking and tissue doppler imaging. Programmed electrical stimulation showed a high non-sustained and sustained ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Finally, BAY 41-8543 treatment resulted in 100% survival at week 7, whereas 76% of vehicle-treated dTGRs died. Our data demonstrate that sGC stimulators ameliorate cardiac and renal end-organ damage including electrical remodeling in a transgenic rat model of hypertension-induced DHF. Treatment of DHF with sGC stimulators offers a novel therapeutic potential.

Abstract 477: Human Ang-(1-12) and Hemodynamic and Cardiac biometrics in Rats Overexpressing Human Angiotensinogen

It has been reported that insertion of the human angiotensinogen (AGT) gene in rats does not cause hypertension as rat renin has no hydrolytic activity on human AGT. Initial studies in transgenic rats expressing the human AGT gene [TGR(hAGT)L1623] demonstrated the presence of human angiotensin-(1-12) [Ang-(1-12)] immunoreactivity in the heart of these rats. The exciting possibility that [TGR(hAGT)L1623] rats have increased precursor availability for Ang II formation from both human and rat origin stimulated further investigation of this model through measurements of arterial pressure and heart rate with implanted telemetry probes (Data Science International, MN), assessment of cardiac structure and function by echocardiography (VevoLAZR; VisualSonics Inc., Canada), and heart weight (n=9). Comparative studies were done in Sprague Dawley rats (SD; n=11). 24 h mean arterial pressures (Figure), but not heart rates, were significantly higher in [TGR(hAGT)L1623] rats compared to SD controls during 4 weeks of continuous measurement (starting at 13 weeks of age). Although there was no difference in systolic or diastolic function between these two strains, [TGR(hAGT)L1623] rats showed increased thickness of left ventricular posterior wall (2.3 ± 0.1 vs. 2.1 ± 0.1 mm; p&lt;0.05) and diameter (8.1 ± 0.2 vs. 7.5 ± 0.1 mm; p&lt;0.05) that was associated with increased heart weight (1.51 ± 0.03 vs 1.16 ± 0.01 g; p&lt;0.001) and heart weight/tibia length ratio (34.6 ± 0.7 vs 28.6 ± 0.4 mg/mm; p&lt;0.001). Detection of hypertension and cardiac Ang-(1-12) in rats with overexpression of the human AGT gene suggests a non-renin dependent enzymatic mechanism able to cleave Ang-(1-12) from the human substrate.