Plasma Angiotensinogen Concentration Research Articles

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.

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.

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 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 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.

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 …

Overexpression of mouse angiotensinogen in renal proximal tubule causes salt-sensitive hypertension in mice

The role of proximal tubule (PT) angiotensinogen (AGT) in modulating blood pressure has previously been examined using mice expressing PT human AGT and human renin, or rat AGT. These animals are hypertensive; however, the question remains whether alterations in mouse PT AGT alone affects arterial pressure. Mouse AGT cDNA was knocked-in to the endogenous kidney androgen protein (KAP) gene using an internal ribosomal entry site (IRES)-based strategy. The KAP-mAGT animals showed kidney-specific KAP-AGT mRNA expression; renal in situ hybridization detected KAP-AGT mRNA only in PT. Urinary AGT was markedly increased in KAP-mAGT mice. On a high Na diet, radiotelemetric arterial pressure showed a systolic pressure elevation; no significant difference in arterial pressure was observed on a normal diet. Plasma renin concentration (PRC) was reduced in KAP-mAGT animals given a high Na diet, but was not different between mouse lines during normal Na intake. Plasma AGT concentration was not altered by overexpression of PT mouse AGT. In summary, PT overexpression of mouse AGT leads to salt-sensitive hypertension without recruitment of the systemic renin-angiotensin system.

Hypercontrols in Genotype-Phenotype Analysis Reveal Ancestral Haplotypes Associated With Essential Hypertension

The angiotensinogen gene locus has been associated with essential hypertension in most populations analyzed to date. Increased plasma angiotensinogen levels have been proposed as an underlying cause of essential hypertension in whites; however, differences in the genetic regulation of plasma angiotensinogen levels have also been reported for other populations. The aim of this study was to analyze the relationship between angiotensinogen gene polymorphisms and haplotypes with plasma angiotensinogen levels and the risk of essential hypertension in the Mexican population. We genotyped 9 angiotensinogen gene polymorphisms in 706 individuals. Four polymorphisms, A-6, C4072, C6309, and G12775, were associated with increased risk, and the strongest association was found for the C6309 allele (χ(2)=23.9; P=0.0000009), which resulted in an odds ratio of 3.0 (95% CI: 1.8-4.9; P=0.000006) in the recessive model. Two polymorphisms, A-20C (P=0.003) and C3389T (P=0.0001), were associated with increased plasma angiotensinogen levels but did not show association with essential hypertension. The haplotypes H1 (χ(2)=8.1; P=0.004) and H5 (χ(2)=5.1; P=0.02) were associated with essential hypertension. Using phylogenetic analysis, we found that haplotypes 1 and 5 are the human ancestral haplotypes. Our results suggest that the positive association between angiotensinogen gene polymorphisms and haplotypes with essential hypertension is not simply explained by an increase in plasma angiotensinogen concentration. Complex interactions between risk alleles suggest that these haplotypes act as "superalleles."

Abstract 5457: Angiotensinogen Hypomorphic Mice Have Markedly Reduced Hypercholesterolemia-induced Atherosclerosis

Background and Objective: The renin angiotensin system exerts profound effects on experimental atherosclerosis. Although multiple enzymes are proposed to generate the increasingly complex array of angiotensin peptides, angiotensinogen is the unique precursor of all angiotensin peptides. Angiotensinogen deficiency in mice leads to poor neonatal survival rate and multiple organ dysfunctions that present barriers to atherosclerosis studies. Therefore, we generated a hypomorphic angiotensinogen (hypoAGT) mouse model to determine the effects of reduced angiotensinogen on atherosclerosis in LDL receptor −/− mice. Methods and Results: hypoAGT mice were developed by insertion of a neomycin resistance cassette in the second intron of the angiotensinogen gene. Although hypoAGT mice have almost undetectable plasma angiotensinogen concentrations and markedly higher renin concentrations compared to the wild type control, these mice have normal neonatal survival rate and grossly normal growth development. To determine the contribution of angiotensinogen to atherosclerosis, male wild type and hypoAGT mice in an LDL receptor −/− background were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for 12 weeks. Body weight was monitored weekly. hypoAGT mice had modestly lower body weight at the initiation of the study. When fed the saturated fat-enriched diet for 12 weeks, wild type mice increased body weight by 49% compared to a 6% increase in hypoAGT mice. Systolic blood pressure (SBP) was measured using a non-invasive tail-cuff system on conscious mice. hypoAGT mice had lower SBP at baseline and when fed the saturated fat-enriched diet. Both wild type and hypoAGT mice were hypercholesterolemic, although hypoAGT mice had lower plasma cholesterol concentrations (wild type versus hypoAGT: 2789±115 versus 1647±106 mg/dl). Atherosclerotic lesion areas were measured on the intima of the aortic arch by the en face technique. hypoAGT mice had a virtual ablation of the development of atherosclerotic lesions (wild type versus hypoAGT: 20.5±2.4 versus 1.1±0.8%). Conclusion: Profound reduction of angiotensinogen strikingly attenuates development of hypercholesterolemia-induced atherosclerosis in LDL receptor −/−mice.

Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.

Genetic Variation and Activity of the Renin-Angiotensin System and Severe Hypoglycemia in Type 1 Diabetes

Background The deletion-allele of the angiotensin-converting enzyme (ACE) gene and elevated ACE activity are associated with increased risk of severe hypoglycemia in type 1 diabetes. We explored whether genetic and phenotypic variations in other components of the renin-angiotensin system are similarly associated. Methods Episodes of severe hypoglycemia were recorded in 171 consecutive type 1 diabetic outpatients during a 1-year follow-up. Participants were characterized at baseline by gene polymorphisms in angiotensinogen, ACE, angiotensin-II receptor types 1 (AT1R) and 2 (AT2R), and by plasma angiotensinogen concentration and serum ACE activity. Results Three risk factors for severe hypoglycemia were identified: plasma angiotensinogen concentration in the upper quartile (relative rate [RR] vs. lower quartile 3.1, 95% confidence interval [CI,] 1.4-6.8), serum ACE activity in the upper quartile (RR vs. lower quartile 2.9, 95% CI, 1.3-6.2), and homo- or hemizygosity for the A-allele of the X chromosome-located AT2R 1675G/A polymorphism (RR vs. noncarriers 2.5, 95% CI, 1.4-5.0). The three risk factors contributed independently to prediction of severe hypoglycemia. A backward multiple regression analysis identified a high number of renin-angiotensin system-related risk factors and reduced ability to perceive hypoglycemic warning symptoms (impaired hypoglycemia awareness) as predictors of severe hypoglycemia. Conclusions High renin-angiotensin system activity and the A-allele of the AT2R 1675G/A polymorphism associate with high risk of severe hypoglycemia in type 1 diabetes. A potential preventive effect of renin-angiotensin system blocking drugs in patients with recurrent severe hypoglycemia merits further investigation.

Novel sandwich ELISA for human angiotensinogen

We recently reported that urinary excretion rates of angiotensinogen (U(AGT)) provide a specific index of intrarenal renin-angiotensin (ANG) system (RAS) status in ANG II-dependent hypertensive rats. When this is shown to be applicable to human subjects, a diagnostic test to identify those hypertensive patients most likely to respond to an RAS blockade could provide useful information to allow a mechanistic rationale for selection of an optimized approach to treatment of hypertensive patients. However, simple and accurate methods to measure human angiotensinogen (hAGT) are unavailable. For future studies of human subjects, we developed antibodies and a sensitive and specific quantification system for hAGT using a sandwich ELISA. We raised two antibodies against hAGT: a mouse monoclonal antibody and a rabbit polyclonal antibody. The standard curve of this ELISA exhibited a high linearity (0.31-20 ng/ml). The correlation coefficient was >0.99. Plasma angiotensinogen concentrations of healthy volunteers ranged from 28 to 71 microg/ml (n = 10). The ratio of U(AGT) to urinary creatinine concentration ranged from 5.0 to 30 microg/g (n = 7). Intra- and interassay coefficients of variation ranged from 4.4 to 5.5% and from 4.3 to 7.0%, respectively. This ELISA system had no cross-reactivity with major proteins in proteinuric urine samples, such as human albumin, immunoglobulin, or transferrin. Moreover, the cross-reactivity of the system with angiotensin peptides was also negligible. This hAGT ELISA will be a useful tool to investigate the relationship of U(AGT) and reactivity to antihypertensive drugs in hypertensive patients.

Laughter therapy modulates the parameters of renin-angiotensin system in patients with type 2 diabetes

The effect of laughter therapy on the plasma levels of renin, angiotensinogen, and prorenin was investigated in patients with type 2 diabetes. In the diabetic patients, the mean plasma renin concentrations were 24.6+/-12.1 ng/ml/h in the first observation (at the beginning of laughter therapy), 8.2+/-3.4 ng/ml/h in the second observation (three months after the beginning of laughter therapy) and 7.7+/-1.7 ng/ml/h in the third observation (six months after the beginning of laughter therapy). The mean plasma angiotensinogen concentrations in the 1st, 2nd and 3rd observations were 0.19+/-0.08, 0.47+/-0.12, 0.42+/-0.14 microg/ml, respectively. The mean plasma prorenin concentrations in the 1st, 2nd and 3rd observations during the laughter therapy were 195.1+/-66.2, 193.4+/-88.2 and 170.7+/-52.5 pg/ml, respectively. Plasma renin concentrations were significantly decreased (p<0.05) by the therapy. Subnormal concentrations of plasma angiotensinogen were found in the 1st observation and increased significantly (p<0.05) to the normal range after the therapy. Plasma prorenin concentration only slightly changed during the laughter therapy. Other biochemical parameters remained unchanged during the laughter therapy. These results indicated that a long-term laughter therapy changed the plasma components of renin-angiotensin system in patients with diabetes. Thus, laughter therapy can be used as non-pharmacological treatment for the prevention of diabetic microvascular complications.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

Activation of the systemic and adipose renin-angiotensin system in rats with diet-induced obesity and hypertension.

In obesity-related hypertension, activation of the renin-angiotensin system (RAS) has been reported despite marked fluid volume expansion. Adipose tissue expresses components of the RAS and is markedly expanded in obesity. This study evaluated changes in components of the adipose and systemic RAS in diet-induced obese hypertensive rats. RAS was quantified in adipose tissue and compared with primary sources for the circulating RAS. Male Sprague-Dawley rats were fed either a low-fat (LF; 11% kcal as fat) or moderately high-fat (32% kcal as fat) diet for 11 wk. After 8 wk, rats fed the moderately high-fat diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups based on their body weight gain (body weight: OR, 566 +/- 10; OP, 702 +/- 20 g; P < 0.05). Mean arterial blood pressure was increased in OP rats (LF: 97 +/- 2; OR: 97 +/- 2; OP: 105 +/- 1 mmHg; P < 0.05). Quantification of mRNA expression by real-time PCR demonstrated a selective increase (2-fold) in angiotensinogen gene expression in retroperitoneal adipose tissue from OP vs. OR and LF rats. Similarly, plasma angiotensinogen concentration was increased in OP rats (LF: 390 +/- 48; OR: 355 +/- 24; OP: 530 +/- 22 ng/ml; P < 0.05). In contrast, other components of the RAS were not altered in OP rats. Marked increases in the plasma concentrations of angiotensin peptides were observed in OP rats (angiotensin II: LF: 95 +/- 31; OR: 59 +/- 20; OP: 295 +/- 118 pg/ml; P < 0.05). These results demonstrate increased activity of the adipose and systemic RAS in obesity-related hypertension.

Expression of angiotensinogen in proximal tubule as a function of glomerular filtration rate

Proximal tubule (PT) angiotensinogen (AGT) is part of a tubular renin-angiotensin system (RAS) that participates in the regulation of sodium reabsorption along the entire nephron. Physiologic maneuvers affecting AGT expression in PT also affect systemic RAS. Here, we tested the hypothesis that PT AGT is regulated by increased glomerular filtration rate (GFR). Complete unilateral nephrectomy (UNX) in mice was used to induce a sustained increase in GFR in the remaining kidney. AGT expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR). AGT protein in PT was investigated by semiquantitative histology. We also measured AGT concentration in plasma and in 24-hour urine by a specific enzyme-linked immunosorbent assay (ELISA). Seven weeks after nephrectomy, UNX animals exhibited a 2-fold increase in tubular AGT mRNA (P <.001) compared with sham-operated control animals. The proportion of PT sections exhibiting AGT immunostaining was significantly increased at day 3 (P <.05), and remained elevated at seven weeks (UNX = 0.63 +/- 0.09, sham = 0.38 +/- 0.02, P <.01), revealing recruitment of AGT-producing cells along the PT. AGT excretion in final urine corrected for creatinine and kidney weight was also elevated by UNX at seven weeks (UNX = 209 +/- 42 pmol/mg/g, sham = 147 +/- 29 pmol/mg/g, P <.05), with no difference in plasma AGT between UNX and control animals. These observations suggest that AGT expression in PT adapts in the long-term to changes in GFR. In the UNX model, urinary AGT excretion is also elevated as a consequence of increase in net tubular flow.

Angiotensinogen genotype, plasma protein and mRNA concentration in isolated systolic hypertension.

Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(- 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)-PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(- 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.

The effects of pregnancy and maternal nutrition on the maternal renin-angiotensin system in sheep.

Physiological changes occurring in the mother during pregnancy can determine the outcome of pregnancy in terms of birthweight and neonatal viability. Maternal adaptations include plasma volume expansion linked to enhanced activity of the renin-angiotensin system (RAS). The present study was designed to determine whether these changes occur very early in gestation, and the extent to which maternal nutrient restriction may compromise the maternal RAS. Using sheep, we have investigated the effects of pregnancy per se, maternal nutrient restriction and later restoration of maternal diet on maternal body weight, plasma volume and plasma renin concentration (PRC), and angiotensinogen (Aogen) and arginine vasopressin (AVP) concentration. During the period of placental growth (i.e. 28-80 days gestation) ewes were fed either a nutrient-restricted (NR) diet or were well fed (WF). NR ewes consumed between 3.2 and 3.8 MJ day(-1) of metabolisable energy (ME) which is close to 60 % of requirements taking into account the ME required for both ewe maintenance and growth of the conceptus in order to produce a 4.5 kg lamb at term. WF ewes consumed 150 % of ME requirements. Restoration of maternal diet between 80 and 140 days gestation (i.e. fed to satiety and consuming between 8 and 10.9 MJ day(-1), which is close to 150 % of ME requirements) followed previous nutrient restriction. Between pre-conception and 28 days gestation, plasma volume increased in conjunction with a decline in PRC and Aogen concentration. During the period of nutrient restriction ewe body weight did not increase and plasma volume was lower in NR than WF ewes. During this time there was no effect of maternal nutrition on PRC; however, Aogen concentration was lower in the NR group. From 80 days gestation following the rise in food intake for previously NR ewes, greater increases in ewe body weight, plasma volume and PRC occurred up to term compared with ewes that were well fed throughout gestation. Plasma AVP concentration was not significantly affected by either maternal nutrition or gestational age. In conclusion, the stimulus of moderately severe maternal nutrient restriction evoked smaller rises in maternal weight, plasma volume and Aogen concentration than occurred in ewes that were well fed throughout gestation. Following the restoration of maternal diet after 80 days gestation, PRC gradually rose to peak at term. These adaptations in the maternal RAS during the critical period of placental growth may have long-term effects on fetal development.

Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation.

White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

Nine polymorphisms of angiotensinogen gene in the susceptibility to essential hypertension

Even if the importance of angiotensinogen (AGT) gene has been known in gene targeting animals and humans genetic studies, its precise mechanism and the interaction among AGT gene variants, plasma AGT concentration and risk for hypertension remain uncertain. We examined whether AGT gene variants predispose to hypertension via an increase of plasma AGT concentration. Plasma AGT concentration was estimated from plasma angiotensin I which was cleaved by an excess amount of human renin and measured by RIA. Using 9 AGT gene variants which included new polymorphisms (G-152A and T+31C), we examined the association with hypertension and with plasma concentration by a case-control study. Haplotype analysis revealed that G-6A, T+31C and M235T polymorphisms were in absolute linkage disequilibrium and were associated with hypertension but not with plasma AGT level. On the other hand, -1074t;T235 haplotype was associated with an increase of AGT level but not with hypertension. In the haplotype analysis, only H3 haplotype frequency, which contained G-6, T+31 and M235 alleles, was significantly increased in normotensive subjects, suggesting that this haplotype is associated with a hypotensive effect. According to combined haplotype analysis of diallele and microsatellite markers, it remains a possibility that M235T, T+31C, G-6A, A-20C and G-1074T polymorphisms may play an important role in increased risk for essential hypertension. Our results suggest that the positive association between AGT polymorphism and hypertension is not simply explained by an increase of plasma AGT concentration.