Plasma Angiotensinogen Research Articles

Urinary angiotensinogen as a potential biomarker of intrarenal renin–angiotensin system activity in Chinese chronic kidney disease patients

Urinary angiotensinogen (AGT) mainly derives from the AGT produced in proximal tubular cells. Evidence exists that supports the correlation between urinary AGT and circulating AGT. To investigate the role of urinary AGT as a potential biomarker of intrarenal renin-angiotensin system activity in Chinese chronic kidney disease (CKD) patients. ELISA-based method used to quantify urinary AGT. Analyzed the relationship between urinary AGT and intrarenal angiotensin II (Ang II) activity in 128 CKD patients. ELISA was applied to measure the urinary and plasma renin activity, AGT, Ang II and aldosterone. Furthermore expression levels of intrarenal renin, AGT, Ang II and Ang II receptor were examined by immunohistochemistry staining (IHCS) in 72 CKD patients undergoing renal biopsy. The logarithmic transformation Log(urinary AGT/UCre) levels showed a normal distribution. Therefore, Log(urinary AGT/UCre) levels were used for the analyses. Average urinary AGT was 2.02±0.55ng/(mg Cr). Hypertension, urinary protein, urinary Ang II and urinary type IV collagen (Col IV) positively correlated with urinary AGT. Estimated glomerular filtration rate (eGFR), urinary sodium and serum AGT negatively correlated with urinary AGT. Multiple regression analysis indicated that low serum AGT, high urinary protein, urinary Ang II and urinary Col IV correlated significantly with high urinary AGT. We observed positive correlation between urinary AGT and positive IHCS area of AGT, Ang II and Ang II type 1 receptor in renal tissue. These data suggest that urinary AGT might be a potential biomarker of intrarenal Ang II activity in CKD patients.

Increased plasma Angiotensinogen levels and its association with the M235T gene polymorphism and hypertension in Calabar and Uyo cities, Nigeria

Hypertension results from an interaction of many risk genes and environmental factors. The M235T allele of the angiotensinogen gene is thought to increase plasma levels of the angiotensinogen which is associated with hypertension. The angiotensinogen is an important substrate for renin in the RAAS that is finally converted into angiotensin II that plays a key role in the control of blood pressure. This study was designed to measure plasma angiotensinogen levels in an adult population in Calabar and Uyo (South-South), Nigeria in relation to the M235T allele and hypertension. Out of a large population of 1224 participants who had been genotyped for the M235T polymorphism, plasma was collected from a sample of 300 consisting of 150 patients and 150 controls. Protein A sandwich enzyme linked immunosorbent assay was carried out with the plasma samples to measure the angiotensinogen levels. Age, BMI, M235T allele, blood pressure and O.D values were compared between controls and patients using the independent t test. The absorbance values of plasma angiotensinogen were significantly higher in the patients (0.71) with M235T allele than in the controls (0.53). Further research still needs to be carried out to determine the actual concentration of the protein in the participants.

Up-Regulation of Intrarenal Renin-Agiotensin System Contributes to Renal Damage in High-Salt Induced Hypertension Rats

Background/Aims: To investigate the change of intrarenal renin-agiotensin system (RAS) and its role in high-salt induced hypertension. Methods: Wistar rats were divided into normal-salt (NS), high-salt diet (HS) and high-salt diet with Losartan group (HS+L), for 6 weeks. Systolic blood pressure (SBP) was monitored. Blood and urine samples were collected every 2 weeks. Angiotensinogen (AGT) was measured by ELISA. AGT mRNA and protein were measured by real-time PCR and immunohistochemistry. Renin activity and angiotensin II (Ang II) were measured by radioimmunoassay. Results: HS versus NS group, SBP increased from 2^nd week (P<0.05), urinary protein increased at 6^th week (P<0.05). Although plasma renin, AGT and Ang II had no significant changes (P>0.05), renal cortex renin, AGT, and Ang II increased significantly in HS (P<0.05). In HS+L, Losartan failed to reduce SBP (P>0.05) but abolished the increase of proteinuria (P<0.01), renal cortex renin, AGT, Ang II and urinary AGT reduced (P<0.05) while plasma renin, AGT and Ang II enhanced (P<0.05) when compared with HS. Urinary AGT was positively correlated with renal AGT (r=0.592, P <0.01) and Ang II (r=0.726, P <0.01). Conclusion: Inappropriate response of the renal RAS to a high salt diet may contribute to hypertension and renal damage, and urinary AGT could reflect intrarenal RAS activity.

Targeting of hepatic angiotensinogen using chemically modified siRNAs results in significant and sustained blood pressure lowering in a rat model of hypertension

Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.

Activation of adenosine receptors improves renal antioxidant status in diabetic Wistar but not SHR rats

Background. Diabetes and hypertension independently contribute to renal injury, and the major mechanisms involved are increased reactive oxygen species (ROS) bioavailability and renin-angiotensin system (RAS) activation. We investigated the role of adenosine in controlling ROS production and RAS activation associated with renal dysfunction in hypertension and diabetes.Methods. Fourteen days after induction of diabetes with streptozotocin in 12-week-old male Wistar and spontaneously hypertensive (SHR) rats, animals were treated during 7 days with 2-chloroadenosine (CADO group, 5 mg/kg/d), a stable analogue of adenosine, or underwent a sham operation procedure. At the end of the study (day 21), intra-arterial systolic blood pressure (SBP) was measured, and 24-h urine and plasma samples and renal tissue were collected.Results. CADO treatment decreased the plasma glucose concentration and glucose and protein excretion by more than 30% in both strains. CADO treatment decreased SBP in diabetic SHR rats (143 ± 8 versus 114 ± 4 mmHg, p < 0.05), but not in diabetic Wistar rats. The hypotensive effect of CADO was associated to a ∼70% increase in plasma angiotensinogen (AGT) concentration and a ∼50% decrease in urinary AGT excretion. CADO also caused a decrease in medullary and cortical hydrogen peroxide production of about 40%, which was associated with a proportional increase in glutathione peroxidase (GPx) activity in diabetic Wistar but not in diabetic SHR animals.Conclusions. These results suggest that activation of adenosine receptors improves renal antioxidant capacity in diabetic Wistar but not SHR rats, although it improves glucose metabolism in both strains. Furthermore, activation of adenosine receptors does not seem to be directly influencing AGT production.

ELISA examining urinary angiotensinogen as a potential indicator of intrarenal renin–angiotensin system (RAS) activity: a clinical study of 128 chronic kidney disease patients

In the current study, we measured urinary angiotensinogen (AGT) through enzyme-linked immunoadsordent assay (ELISA) and analyzed its correlation with intrarenal renin-angiotensin system (RAS) activity in 128 chronic kidney disease (CKD) patients. Urinary and plasma renin activity, AGT, angiotensin II (Ang II) and aldosterone levels were also measured by radioimmunoassay (RIA) or ELISA in these participants. Further, the expression level of intrarenal renin, AGT, Ang II and Ang II receptors were examined by immunohistochemistry staining (IHCS) in 72 CKD patients. Their correlations with urinary AGT were also analyzed. We found that the urinary AGT level was positively correlated with hypertension (ρ = 0.28, P < 0.01), urinary protein (r = 0.38, P < 0.01), urinary Ang II (r = 0.29, P < 0.05), urinary type IV collagen (Col IV) (r = 0.56, P < 0.01), and was negatively correlated with estimated glomerular filtration rate (eGFR) (r = -0.28, P < 0.01), urinary sodium (r = -0.22, P < 0.05) and serum AGT (r = -0.27, P < 0.01). Multiple regression analysis indicated low serum AGT (P < 0.01), high urinary protein (P < 0.01), high urinary Ang II (P < 0.05) and high urinary Col IV (P < 0.01) were correlated significantly with high urinary AGT. Urinary AGT level was positively correlated with intrarenal expression level of AGT (ρ = 0.46, P < 0.01), Ang II (ρ = 0.56, P < 0.01) and Ang II type 1 receptor (ρ = 0.32, P < 0.01), as detected by IHCS. Together, these data suggest that urinary AGT might be a potential biomarker of intrarenal RAS and Ang II activities in CKD patients.

Abstract 195: Circadian Rhythm of Plasma and Urinary Angiotensinogen in Patients with Chronic Kidney Disease

Recent basic and clinical data demonstrated that the intrarenal renin-angiotensin system (RAS) plays an important role in the progression of chronic kidney disease (CKD). The urinary angiotensinogen (AGT) excretion rate could be a novel biomarker for the activity of the RAS in the kidney. We previously reported that the healthy volunteers do not have a circadian rhythm of AGT level in urine (Hypertension. 2011;57:e78) or in plasma (Hypertension. 2012;60:A399). However, the circadian rhythm of AGT level in urine and in plasma in patients with CKD has not been reported yet. Therefore, this study was performed to investigate the circadian rhythm of AGT level in urine and in plasma in patients with CKD. We recruited 6 CKD patients with continuous proteinuria admitted to the Kagawa University Hospital from 06/2011 to 10/2011 for the purpose of diagnostic renal biopsy. Plasma samples were collected at 06:00, 12:00, and 18:00. Urine samples were collected at 06:00, 09:00, 12:00, and 18:00. Plasma renin activities (PRAs), plasma and urinary AGT concentrations, and urinary albumin (Alb) concentration were measured using commercially available kits. The urinary concentrations of AGT and Alb were normalized by the urinary concentration of creatinine (Cr) (UAGT/Cr and UAlb/Cr, respectively). PRA (2.07 +/- 0.77 ng of angiotensin I/mL/hr at 06:00, 2.45 +/- 0.77 at 12:00, and 2.58 +/- 0.76 at 18:00, P = 0.8853) or plasma AGT (19.9 +/- 2.4 μg/mL at 06:00, 24.1 +/- 3.1 at 12:00, and 23.0 +/- 3.9 at 18:00, P = 0.6300) did not show a circadian rhythm. Moreover, UAlb/Cr (511 +/- 319 mg/g Cr at 06:00, 827 +/- 459 at 09:00, 1479 +/- 862 at 12:00, and 1370 +/- 818 at 18:00, P = 0.6964) or UAGT/Cr (54 +/- 22 μg/g Cr at 06:00, 119 +/- 41 at 09:00, 350 +/- 194 at 12:00, and 198 +/- 104 at 18:00, P = 0.3035) did not show a circadian rhythm. In conclusion, in addition to healthy volunteers, patients with CKD do not have a circadian rhythm of AGT level in urine or in plasma.

Abstract 27: Deficiency of Angiotensinogen in Liver Markedly Reduces Blood Pressure in Lean and Obese Male C57bl6 Mice

Objectives: We demonstrated that adipose tissue is a major source of elevated plasma angiotensin II (AngII) in obese hypertensive mice. The liver is the primary source of systemic angiotensinogen (AGT), the AngII precursor. The purpose of this study was to define the contribution of liver-derived AGT on obesity-hypertension in male C57BL/6 mice. Methods and results: Male Agt fl/fl mice expressing albumin-driven Cre recombinase were bred to female mice to generate littermate control ( Agt fl/fl ) or liver-AGT deficient mice ( Agt Alb ). Mice (n=10-13/group) were fed a low fat (LF, 10% kcal as fat) or HF diet (HF, 60 % kcal as fat) for 16 weeks. At baseline, there were no differences in body weight or systolic blood pressure (SBP) between genotypes. Liver AGT deficiency had no effect on the development of obesity ( Agt fl/fl , 48.0±0.4; Agt Alb , 47.5±0.4 g; P&gt;0.05). HF-fed control mice had increased plasma AGT concentrations compared to LF-fed controls (LF: Agt fl/fl , 2.0 ± 0.3; HF, Agt fl/fl , 3.2 ± 0.3 μg/ml; P&lt; 0.05). Plasma AGT concentrations were markedly reduced in liver AGT-deficient mice fed either diet (LF: Agt Alb , 0.5 ± 0.1; HF: Agt Alb , 0.5 ± 0.1μg/ml; P&gt; 0.05). At 9 weeks, there was no effect of HF feeding on SBP in control mice; however, SBP of Agt Alb mice was significantly decreased compared to controls fed either diet (LF: Agt fl/fl , 105 ± 2; Agt Alb , 90 ± 2; HF: Agt fl/fl , 106 ± 2; Agt Alb , 87 ± 6 mmHg, P genotype &lt; 0.05, P diet &gt;0.05) . At 15 weeks, we implanted carotid artery catheters and radiotelemetry implants into anesthetized mice. HF-fed Agt fl/fl mice exhibited a significant increase in SBP compared to LF-fed controls (LF: Agt fl/fl , 119 ± 4; HF: Agt fl/fl , 139 ± 3 mmHg, P&lt; 0.05). Moreover, SBP was significantly decreased in HF-fed Agt Alb mice compared to HF-fed Agt fl/fl controls (HF: Agt fl/fl , 139 ± 3; HF: Agt Alb , 85 ± 4 mmHg, P&lt; 0.05) Surprisingly, LF-fed Agt Alb mice did not survive surgery for radiotelemetry, potentially resulting from marked reductions in blood pressure. Conclusions: Liver AGT deficiency markedly decreases systemic AGT concentrations and blood pressure in male mice fed a LF or HF diet.

Urinary renin and angiotensinogen in type 2 diabetes

Urinary levels of renin-angiotensin-aldosterone system (RAAS) components may reflect renal RAAS activity and/or the renal efficacy of RAAS inhibition. Our aim was to determine whether urinary angiotensinogen and renin are circulating RAAS-independent markers during RAAS blockade. Urinary and plasma levels of angiotensinogen, renin, and albumin were measured in 22 patients with type 2 diabetes, hypertension, and albuminuria, during 2-month treatment periods with placebo, aliskiren, irbesartan, or their combination in random order in a crossover study. Aliskiren and irbesartan both increased plasma renin 3-4-fold, and above 10-fold when combined. Irbesartan decreased plasma angiotensinogen by approximately 25%, and no changes in plasma angiotensinogen were observed during the combination. Urine contained aliskiren at micromolar levels, blocking urinary renin by above 90%. Both blockers reduced urinary angiotensinogen, significant for irbesartan only. Combination blockade reduced urinary angiotensinogen even further. Reductions in urinary angiotensinogen paralleled albuminuria changes, and the urine/plasma concentration ratio of angiotensinogen was identical to that of albumin under all conditions. In contrast, urinary renin did not follow albumin, and remained unaltered after all treatments. Yet, the urine/plasma concentration ratio of renin was more than 100-fold higher than that of angiotensinogen and albumin, and approximately 4-fold reduced by single RAAS blockade, and more than 10-fold by dual RAAS blockade. Aliskiren filters into urine and influences urinary renin measurements. The urine/plasma renin ratio, but not urinary renin alone, may reflect the renal efficacy of RAAS blockade. Urinary angiotensinogen is a marker of filtration barrier damage rather than intrarenal RAAS activity.

The link between the fibroblast growth factor-23—klotho—vitamin D3 axis and the renin—angiotensin—aldosterone axis in the development and progression of obesity-related kidney disease

Abstract Introduction and aim of the work Obesity is established as an important contributor of increased diabetes mellitus, hypertension and cardiovascular disease, all of which can promote chronic kidney disease (CKD). Recently, there is a growing appreciation that even in the absence of these risks, obesity itself significantly increases CKD and accelerates its progression. The aim of this work is to evaluate the link between Renin-Angiotensin-Aldosterone System (RAAS) and FGF23-Klotho-1,25D3 axis and their impact in obese and non-obese CKD patients. Patients and methods In a cross sectional randomized multi centers study, two hundred twenty six CKD patients stage III and IV (eGFR20–60 ml/min/m2) have enrolled in this study as follows: group I; 87 non diabetic CKD patients aged 20–40 years with body mass index (BMI) between 20–25 kgm/m2; group II; 130 non diabetic CKD patients aged 20–40 years with (BMI) &gt;30 kgm/m2 and group III; 89 CKD patients aged &gt;60 years. All patient have been tested for plasma leptinlevels, 1,25-dihydrocholicalciferole (1,25D3), plasmaparathormone (PTH) Serum calcium (Ca), serum phosphorus (PO4), and plasma FGF-23 , plasma renin activity (PRA), plasma angiotensinogen receptor 1 &amp; 2 (AT1 &amp; AT2) and plasma aldosterone (ALD) and pulse wave velocity (PWV). Results The eGFR was significantly reduced in the obese group II (eGFR = 37.7 ± 13.6) when compared with eGFR of the lean group I (eGFR = 49.3 ± 7.51) were P &lt; 0.001, but not significant when compared with the old age group III (eGFR = 41.0 ± 13.47). The obese group II shows significant increase in the ALD/PRA ratio when compared with the lean group I and old age group III (43.23 ± 14.9) for group II vs 11.29 ± 4.1 for group I, P &lt; 0.001 and 24.91 ± 12.1 for group III, P &lt; 0.05 ). Regarding the FGF23-Klotho-vitD3 axis, its components of the obese group II (FGF23 259.55 ± 138.6 Ru/ml; PTH 77.63 ± X32.4 pg/ml; S.PO 4.74 ± 1.61 mg/dl) were significantly elevated when compared to the lean group I (FGF23 132.81 ± 126.1 Ru/ ml; PTH 59.18 ± 24.7 pg/ml; S.PO4 3.85 ± 0.92 mg/dl); the P values were &lt;0.001, &lt;0.01 and &lt;0.05 respectively , while when compared with the old age group III (FGF23 179.33 ± 237.4 Ru/ml; PTH 70.94 ± 15.26 pg/ml; S.PO4 4.09 ± 0.42 mg/dl), values were of less significance. Plasma insulin levels were significantly high in the obese group II (insulin = 13.73 ± 2.38fg/l) than the lean group I (insulin = 5.59 ± 2.31 fg/l) and P &lt; 0.001 and in group III p. insulin level was 10.7 ± 1.68 (P &lt; 0.05). Conclusion Obesity per se is an independent risk factor in the development and progression of chronic kidney disease specially in young age patients.

Abstract 352: Adeno-Associated Virus-mediated Restoration of Angiotensinogen Function in Mice With Hepatocyte-specific Deficiency

Background and Objective We have reported previously that genetic or pharmacological inhibition of angiotensinogen (AGT) leads to resistance to dietary fat-induced body weight gain. This phenotype has not been observed in mice subject to pharmacological inhibitors of other components of the renin angiotensin system including renin, ACE, and AT1 receptors. To investigate whether restoration of AGT reverses this lean phenotype, we injected an adeno-associated virus (AAV) 2/8 vector carrying wild type AGT to mice with hepatocyte-specific deficiency of AGT (hepAGT -/-). Methods and Results HepAGT -/- mice were generated by disrupting AGT gene in hepatocytes via albumin promoter driven Cre recombinase expressed in mice with floxed AGT alleles. An AAV AGT vector was constructed with the insertion of wild type AGT. AAV null vector was used as control. AAV expression was driven by hepatocyte-specific thyroxine-binding globulin promoter. Five groups of male LDL receptor -/- mice at age of 8 - 10 weeks were studied: wild type (hepAGT +/+) mice received either no AAV (PBS) or null AAV, and hepAGT -/- mice received low dose (1 x 1010 genome copy), high dose (3 x 1010 genome copy), or null AAV. Four weeks after AAV injection (i.p.), all mice were fed a saturated fat-enriched diet (42% calories from milk fat) for 12 weeks. Plasma samples were collected through retro-orbital bleeding at weeks 2, 4, 8, and 16 after AAV injection. Plasma AGT concentrations peaked between weeks 2 and 4, then declined modestly, irrespective of dosage. However, at the high dose, plasma AGT concentrations in hepAGT -/- mice were comparable to those in hepAGT +/+ mice. Plasma total cholesterol concentrations, body weight gains, and systolic blood pressures were not different between hepAGT +/+ and hepAGT -/- mice receiving either dose of AGT AAV. Atherosclerosis, quantified as percent lesion area covering the intima of the aortic arch, was equivalent between hepAGT -/- mice receiving either dose of AGT AAV and hepAGT +/+ mice, whereas it was nearly absent in hepAGT -/- mice receiving null AAV. Conclusions AGT in an AAV 2/8 vector efficiently recovers plasma AGT concentration in hepAGT -/- mice in a dose-dependent manner. This approach permits the elucidation of structural mutations on AGT function.

Abstract TP115: The Increase In Plasma Angiotensinogen Is Associated With The Reduction Of Neurological Severity In Ischemic Stroke

Background and Purpose: Activation of the renin-angiotensin system (RAS) is a crucial factor leading to the development of hypertension and atherosclerosis, thereby causing brain infarction. However, the significance of RAS in the acute phase of brain infarction has not been fully understood. Our goal was to elucidate the significance of RAS in the acute phase of ischemic stroke. Methods: We measured plasma angiotensinogen (AGTN), angiotensin-converting enzyme (ACE) and some associated biomarkers, and examined clinical information in 171 ischemic stroke patients at 5 time points (days 0, 3, 7, 14 and 90) after the onset and 171 age- and gender-matched healthy subjects. Results: plasma AGTN values were increased in all stroke subtypes with different profiles in the acute phase of brain infarction: they were continuously elevated in atherothrombotic brain infarction, while they fluctuated in cardioembolic and lacunar infarction. On the other hand, plasma ACE values remained decreased from the onset to the 90 day in all subtypes, suggesting that the final product Angiotensin II (Ang II) was increased in the plasma and suppressed the production of ACE in a feedback mechanism during the period. AGTN values were not significantly associated with blood pressure. Among the biomarkers we tested, AGTN was highly associated with adrenocorticotropic hormone (ACTH) (r = 0.62, p &lt; 0.0001), but not with other pituitary hormones and inflammatory cytokines (IL-1b, IL-6 and TNFa). Neurological severity assessed by NIH Stroke Scale at day 0 was associated negatively with the AGTN values, while positively with ACE. Conclusions: Ang II production may be increased in the acute phase of brain infarction with decreased plasma ACE in all subtypes and different profiles of AGTN according to stroke subtypes. Increased production of AGTN leading to the increase in Ang II appears to be associated with the reduction of neurological severity in ischemic stroke.

A Possible Interaction Between Systemic and Renal Angiotensinogen in the Control of Blood Pressure

Angiotensinogen (AGT) is synthesized in the liver and proximal tubule. AGT overexpression at either site might increase blood pressure (BP). We used transgenic mice with AGT overexpression in proximal tubule (K), liver (L), or both sites (KL) to determine the relative contributions of hepatic- and proximal tubule-derived AGT in modulating BP. Hepatic AGT overexpression was obtained using the albumin enhancer promoter; the kidney androgen protein gene was used for proximal tubule AGT overexpression. BP and renin angiotensin system parameters were examined in male KL, K, L, and wild-type mice on normal and high-sodium diets. Compared with wild-type mice, K and KL mice had higher BP on normal and high-sodium diets. L mice had similar BP to wild-type mice on a normal-sodium diet, but high sodium intake caused hypertension. There were no differences in plasma AGT, plasma renin concentration, urine volume, or urine sodium excretion between the groups. Urine AGT and angiotensin II (Ang II) excretion were higher in KL and K mice than in L or wild-type mice on a normal-sodium diet and increased with high sodium intake. During high sodium intake, urine AGT and Ang II were higher in all transgenic mice vs wild-type mice. Mice with liver AGT overexpression manifest salt-sensitive hypertension, whereas mice with renal AGT overexpression are hypertensive regardless of salt intake. Systemic AGT may stimulate endogenous renal AGT synthesis during high sodium intake, leading to hypertension in L mice. This suggests that systemic and renal AGT may interact to modulate BP.

Cholecalciferol administration blunts the systemic renin–angiotensin system in essential hypertensives with hypovitaminosis D

Vitamin D plasma levels are negatively associated with blood pressure and cardiovascular mortality, and vitamin D supplementation reduces cardiovascular events. Renin-angiotensin system (RAS) suppression may be one of the mechanisms involved. However, there are no interventional prospective studies demonstrating a reduction in circulating RAS components after vitamin D treatment. Fifteen consecutive drug-free patients with essential hypertension and hypovitaminosis D underwent therapy with an oral dose of 25000 I.U. of cholecalciferol once a week for two months, while maintaining a constant-salt diet. In basal conditions and at the end of the study, RAS activity (plasma angiotensinogen, renin, PRA, angiotensin II, aldosterone and urinary angiotensinogen) was investigated, in addition to blood pressure and plasma vitamin D levels (25(OH)D). After cholecalciferol administration, all patients exhibited normalized plasma 25(OH)D values. At the end of the study, a reduction (p < 0.05) in plasma renin and aldosterone, and a decrement, although not significant, of PRA and angiotensin II, was observed. No difference was found in plasma and urinary angiotensinogen or blood pressure values. Our data indicate that in essential hypertensives with hypovitaminosis D, pharmacological correction of vitamin D levels can blunt systemic RAS activity.

Activation of systemic, but not local, renin-angiotensin system is associated with up-regulation of TNF-α during prolonged fasting in northern elephant seal pups

Northern elephant seal pups naturally endure a 2-3 month post-weaning fast that is associated with activation of systemic renin-angiotensin system (RAS), a decrease in plasma adiponectin (Acrp30), and insulin resistance (IR)-like conditions. Angiotensin II (Ang II) and tumor necrosis factor-alpha (TNF-α) are potential causal factors of IR, while Acrp30 may improve insulin signaling. However, the effects of fasting-induced activation of RAS on IR-like conditions in seals are not well described. To assess the effects of prolonged food deprivation on systemic and local RAS, and their potential contribution to TNF-α as they relate to an IR condition, the mRNA expressions of adipose and muscle RAS components and immuno-relevant molecules were measured along with plasma RAS components. Mean plasma renin activity and Ang II concentrations increased by 89 and 1658%, respectively, while plasma angiotensinogen (AGT) decreased by 49% over the fast, indicative of systemic RAS activation. Prolonged fasting was associated with decreases in adipose and muscle AGT mRNA expressions of 69 and 68%, respectively, corresponding with decreases in tissue protein content, suggesting suppression of local AGT production. Muscle TNF-α mRNA and protein increased by 239 and 314%, whereas those of adipose Acrp30 decreased by 32 and 98%, respectively. Collectively, this study suggests that prolonged fasting activates a systemic RAS, which contributes to an increase in muscle TNF-α and suppression of adipose Acrp30. This targeted and tissue-specific regulation of TNF-α and Acrp30 is likely coordinated to synergistically contribute to the development of an IR-like condition, independent of local RAS activity. These data enhance our understanding of the adaptive mechanisms evolved by elephant seals to tolerate potentially detrimental conditions.

Abstract 399: The Circadian Rhythm of Plasma Angiotensinogen in Healthy Volunteers

Background: We have previously reported that urinary angiotensinogen (AGT) excretion did not have a circadian rhythm and could be a novel biomarker for the activity of the renin-angiotensin system (RAS) in kidney. However, there have been few reports investigating the circadian rhythm of plasma AGT in human body. Thus, this study was performed to examine the circadian rhythm in plasma AGT in human. METHODS: Evaluating RAS in clinical practice is generally performed in a recumbent position after a 30-minute stabilization period. However, to determine the necessity of recumbent position, we first compared plasma AGT concentrations measured right after waking up and after a 5-minute sitting rest. Next, we examined the circadian rhythm of plasma AGT in 43 healthy volunteers who had shown no abnormalities in the medical examinations in 2011. Plasma AGT was measured at three time points (9 a.m., 1 p.m., and 4 p.m.) in the above volunteers. Blood was collected by a micro hematocrit capillary tube with heparin, frozen for storage after centrifugation, and thawed for the measurement of plasma AGT using an ELISA kit. Results: There was no significant difference between the plasma AGT values of the two measuring methods (P = 0.1202, n = 5). Based on the result, we performed blood sampling after a 5-minute sitting rest in the volunteers consisting of 17 men and 26 women. Average blood pressure was 116.3/75.1 mmHg at 9 a.m., 116.3/71.9 mmHg at 1 p.m., and 115.5/70.1 mmHg at 4 p.m.; average pulse rate was 78.7/min at 9 a.m., 77.1/min at 1 p.m., and 73.3/min at 4 p.m. Blood pressure and pulse rate did not change throughout the day. Average plasma AGT was 20.4 ± 6.0 ng/ml at 9 a.m., 20.7 ± 5.0 ng/ml at 1 p.m., and 19.8 ± 6.4 ng/ml at 4 p.m. Plasma AGT did not show a circadian rhythm (P = 0.3803). Conclusion: We found in this study that plasma AGT did not have a circadian rhythm. We also found that plasma AGT was not affected by daily life actions. Thus, future patients may not be required to rest nor wait for certain time points before measuring plasma AGT. We also have to unveil the normal AGT levels and the influence on the levels by diseases. As we think that plasma AGT and ratio of urinary AGT to plasma AGT can be a new surrogate marker of hypertension and kidney diseases, we further need to go into this research area.

Abstract 233: Evidence for an Interaction Between Systemic and Renal Angiotensinogen in the Control of Arterial Pressure

Both the liver and proximal tubule make angiotensinogen (AGT), and overexpression of AGT in either region increases blood pressure (BP). To determine the relative contributions of renal and systemic AGT in modulating BP, mice were used with AGT overexpression in kidney (K), liver (L) or both sites (KL). Male transgenic and wildtype (WT) controls aged 24 weeks underwent BP recording via telemetry and metabolic cage studies on normal (0.25%) and high (3.2%) sodium diets. Mean BP (MAP) was higher in K and KL vs L and WT mice fed normal salt, while MAP in L mice was higher than controls. High salt intake did not alter MAP in WT, K or KL mice but increased it in L mice. Plasma AGT levels, plasma renin concentration, urine Na excretion and urine volume were similar between groups regardless of sodium intake. Urinary AGT (ng/day) on normal sodium intake was: WT: 5±1, L: 4±1, K: 20±5 and KL: 34±9. High salt increased urine AGT in all groups (WT: 19±4, L: 42±8, K: 290±75, KL: 407± 72). High salt did not alter plasma AGT levels but reduced plasma renin concentration in all groups. Taken together, these data show a correlation between urinary AGT and BP. Mice with renal AGT overexpression may be maximally hypertensive on a normal sodium diet since renal AGT is already high. Mice with liver AGT overexpression have mild hypertension on a normal sodium diet that is exacerbated by high sodium intake; this is associated with a significant increase in urinary AGT as compared to WT animals. Thus, systemic AGT may have the potential to regulate renal AGT and this effect may be a key determinant of BP.

Blood pressure and renin–angiotensin system resetting in transgenic rats with elevated plasma Val5-angiotensinogen

Increases in plasma angiotensinogen (Ang-N) due to genetic polymorphisms or pharmacological stimuli like estrogen have been associated with a blood pressure (BP) rise, increased salt sensitivity and cardiovascular risk. The relationship between Ang-N, the resetting of the renin-angiotensin system, and BP still remains unclear. Angiotensin (Ang) II-induced genetic hypertension should respond to lisinopril treatment. A new transgenic rat line (TGR) with hepatic overexpression of native (rat) Ang-N was established to study high plasma Ang-N. The transgene contained a mutation producing Val(5)-Ang-II, which was measured separately from nontransgenic Ile-Ang-II in plasma and renal tissue. Male homozygous TGR had increased plasma Ang-N (~20-fold), systolic BP (ΔBP+26 mmHg), renin activity (~2-fold), renin activity/concentration (5-fold), total Ang-II (~2-fold, kidney 1.7-fold) but decreased plasma renin concentrations (-46%, kidney -85%) and Ile(5)-Ang-I and II (-93%, -94%) vs. controls. Heterozygous TGR exhibited ~10-fold higher plasma Ang-N and 17 mmHg ΔBP. Lisinopril decreased their SBP (-23 vs. -13 mmHg in controls), kidney Ang-II/I (~3-fold vs. ~2-fold) and Ile(5)-Ang-II (-70 vs. -40%), and increased kidney renin and Ile(5)-Ang-I (>2.5-fold vs. <2.5-fold). Kidney Ang-II remained higher and renin lower in TGR compared with controls. High plasma Ang-N increases plasma and kidney Ang-II levels, and amplifies the plasma and renal Ang-II response to a given change in renal renin secretion. This enzyme-kinetic amplification dominates over the Ang-II mediated feedback reduction of renin secretion. High Ang-N levels thus facilitate hypertension via small increases of Ang II and may influence the effectiveness of renin-angiotensin system inhibitors.

Role of H2O2 in hypertension, renin‐angiotensin system activation and renal medullary disfunction caused by angiotensin II

Activation of the intrarenal renin-angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H(2) O(2) ) contribute to hypertension. We examined whether H(2) O(2) mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II). Ang II (200 ng·kg(-1) ·min(-1) ) or saline were infused in Sprague Dawley rats from day 0 to day 14. Polyethylene glycol (PEG)-catalase (10 000 U·kg(-1) ·day(-1) ) was given to Ang II-treated rats, from day 7 to day 14. Systolic blood pressure was measured throughout the study. H(2) O(2) , angiotensin AT(1) receptor and Nox4 expression and nuclear factor-κB (NF-κB) activation were evaluated in the kidney. Plasma and urinary H(2) O(2) and angiotensinogen were also measured. Ang II increased H(2) O(2) , AT(1) receptor and Nox4 expression and NF-κB activation in the renal medulla, but not in the cortex. Ang II raised plasma and urinary H(2) O(2) levels, increased urinary angiotensinogen but reduced plasma angiotensinogen. PEG-catalase had a short-term antihypertensive effect and transiently suppressed urinary angiotensinogen. PEG-catalase decreased renal medullary expression of AT(1) receptors and Nox4 in Ang II-infused rats. Renal medullary NF-κB activation was correlated with local H(2) O(2) levels and urinary angiotensinogen excretion. Loss of antihypertensive efficacy was associated with an eightfold increase of plasma angiotensinogen. The renal medulla is a major target for Ang II-induced redox dysfunction. H(2) O(2) appears to be the key mediator enhancing intrarenal RAS activation and decreasing systemic RAS activity. The specific control of renal medullary H(2) O(2) levels may provide future grounds for the treatment of hypertension.

Response to Role of Angiotensinogen and Relative Aldosterone Excess in Salt-Sensitive Hypertension

We thank Satoh and colleagues for their interest in our work published recently in Hypertension .1 In response to the Letter to the Editor by this group,2 we acknowledge that, as compared with aldosterone concentrations alone, the aldosterone/renin ratio (ARR) may be more reproducible and, hence, could be used as an index of inappropriate aldosterone activity. With respect to our recent work,1 further analysis did indeed show that plasma angiotensinogen (AGT) concentrations were related to ARR in participants with urinary Na+/K+ above the median ( r =0.21; P <0.0005) but not in those with urinary Na+/K+ below the median ( r =−0.03; P …