A.H. Jan Danser

Angiotensin-Converting Enzyme in the Human Heart Effect of the Deletion/Insertion Polymorphism

BACKGROUND An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with differences in the plasma levels of ACE as well as with myocardial infarction, cardiomyopathy, left ventricular hypertrophy, and coronary artery disease. METHODS AND RESULTS We determined the cardiac ACE activity and the ACE genotype in 71 subjects who died of noncardiac disorders. Cardiac ACE activity was significantly higher (P < .01) in subjects with the ACE DD genotype (12.7 +/- 1.9 mU/g wet wt) compared with subjects with the ID (8.7 +/- 0.8 mU/g) and the II (9.1 +/- 1.0 mU/g) genotypes. This difference was independent of sex, age, and the time required for tissue collection. CONCLUSIONS Cardiac ACE activity is highest in subjects with the DD genotype. Elevated cardiac ACE activity in these subjects may result in increased cardiac angiotensin II levels, and this may be a mechanism underlying the reported association between the ACE deletion polymorphism and the increased risk for several cardiovascular disorders.

Effects of Estrogen Replacement Therapy on the Renin-Angiotensin System in Postmenopausal Women

BACKGROUND Oral estrogen replacement therapy (ERT) is known to stimulate the synthesis of angiotensinogen. The effects of such therapy on renin, ACE, and aldosterone are less clear. This seems noteworthy, however, since further activation of the system could be disadvantageous to postmenopausal women who replace estrogen in the context of heart failure, coronary artery disease, or hypertension. METHODS AND RESULTS Estrogen status and components of the renin-angiotensin system were examined in a population-based sample of postmenopausal women and age-matched men. Renin was quantified immunoradiometrically, ie, independent of substrate abundance; aldosterone, angiotensinogen, and ACE activity were determined by standard methods. Renin levels were lower in women with ERT (n = 107; 12.0 +/- 0.7 mU/L) compared with women without ERT (n = 223; 16.6 +/- 0.9 mU/L; P = .001) or men (n = 342, 20.5 +/- 1.5 mU/L, P < .0001). In contrast, angiotensinogen was higher in women with ERT (1.36 +/- 0.08 mg/L) compared with women without ERT (1.03 +/- 0.02 mg/L; P < .0001) or compared with men (0.97 +/- 0.01 mg/L; P < .0001). Renin suppression was seen with either oral or transdermal estrogen replacement (-30% and -31%, respectively; both P < .001). In contrast, the increase of angiotensinogen was limited to women taking oral estrogens (+58%, P < .001). Multivariate analysis revealed that these estrogen effects were independent of age, body mass index, blood pressure, and/or antihypertensive medication. Finally, only marginal differences between groups were observed for serum ACE activity and aldosterone. CONCLUSIONS Aside from a well-documented induction of angiotensinogen, ERT is related to a substantial suppression of renin, a phenomenon that might have received little attention because of widely used indirect measurements of the hormone.

Effects of Aliskiren on Blood Pressure, Albuminuria, and (Pro)Renin Receptor Expression in Diabetic TG(mRen-2)27 Rats

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-&bgr; and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 &mgr;mol/L to 10 &mgr;mol/L) did not inhibit binding of 125I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor.

Aliskiren, a Human Renin Inhibitor, Ameliorates Cardiac and Renal Damage in Double-Transgenic Rats

We tested the hypothesis that the renin inhibitor aliskiren ameliorates organ damage in rats transgenic for human renin and angiotensinogen genes (double transgenic rat [dTGR]). Six-week-old dTGR were matched by albuminuria (2 mg per day) and divided into 5 groups. Untreated dTGR were compared with aliskiren (3 and 0.3 mg/kg per day)-treated and valsartan (Val; 10 and 1 mg/kg per day)-treated rats. Treatment was from week 6 through week 9. At week 6, all groups had elevated systolic blood pressure (BP). Untreated dTGR showed increased BP (202±4 mm Hg), serum creatinine, and albuminuria (34±5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115±6 and 139±5 mm Hg) and albuminuria (0.4±0.1 and 1.6±0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148±4 mm Hg) and albuminuria (2.1±0.7 mg per day), low-dose Val reduced BP (182±3 mm Hg) and albuminuria (24±3.8 mg per day) to a lesser extent. Mortality was 100% in untreated dTGR and 26% in Val (1 mg/kg per day) treated rats, whereas in all other groups, survival was 100%. dTGR treated with low-dose Val had cardiac hypertrophy (4.4±0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and β-myosin heavy chain mRNA, albuminuria, fibrosis, and cell infiltration were also increased. In contrast, both aliskiren doses and the high-dose Val lowered BP to a similar extent and more effectively than low-dose Val. We conclude that in dTGR, equieffective antihypertensive doses of Val or aliskiren attenuated end-organ damage. Thus, renin inhibition compares favorably to angiotensin receptor blockade in reversing organ damage in dTGR.

Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II

Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.

Elevated Blood Pressure and Heart Rate in Human Renin Receptor Transgenic Rats

Recently, a receptor for renin was described that may be important for vascular uptake and activation of (pro)renin, thus leading to local generation of angiotensin II. To assess the in vivo relevance of this protein, we generated transgenic rats overexpressing the human renin receptor gene in smooth muscle tissue, under the control of a 16-kb fragment of the mouse smooth muscle myosin heavy chain gene [TGR(SMMHC-HRR)]. Four lines of transgenic animals were obtained. The correct pattern of expression of the transgene was confirmed by RNase protection assay and in situ hybridization. TGR(SMMHC-HRR) rats are fertile and develop normally. After 6 months of age, transgenic rats develop a cardiovascular phenotype with an elevated systolic blood pressure (137.8±5 versus 118.9±3.7 mm Hg; P=0.008), and an augmentation in heart rate (349.1±7.7 versus 303.1±16.16 bpm; P=0.023) in TGR(SMMHC-HRR) and controls, respectively. These alterations are progressively increasing with aging. Although kidney function and plasma renin were normal in TGR(SMMHC-HRR), an increase in plasma aldosterone [TGR(SMMHC-HRR) 428±64.9 versus 207.3±73.24 pg/mL in control; P=0.02] and in aldosterone/renin ratio [TGR(SMMHC-HRR) 8.04±2.2 versus 2.8±0.55 in control; P=0.03] was observed. This suggests that renin receptor overexpression has resulted in increased intraadrenal angiotensin II, thereby provoking enhanced aldosterone generation in the absence of changes in plasma renin. The rise in aldosterone may underlie, at least in part, the observed cardiovascular phenotype of TGR(SMMHC-HRR).

Determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans

Background Plasma renin concentrations are an important factor in cardiovascular risk profiling. Objective To investigate the effects of sex, medication, and anthropometric factors that may contribute to the interindividual variation in the plasma concentrations of renin and its precursor prorenin. Design and methods Prorenin and renin levels in 327 men and 383 women, aged 52–69 years, who participated in a 1994 reexamination of a previous population survey in Bavaria, were measured by immunoradiometric assay. Results Prorenin and renin levels in men were significantly higher than those in women, those in women without estrogen replacement therapy were significantly higher than those in women with estrogen replacement therapy, and those in diabetics were significantly higher than those in nondiabetics. Prorenin level was correlated negatively to blood pressure and positively to age and the use of diuretics; it was normal in subjects using angiotensin converting enzyme inhibitors and β-adrenergic antagonists (β-blockers). Renin level was correlated negatively to atrial natriuretic peptide level and the use of b-blockers, and it was elevated above normal levels in subjects using angiotensin converting enzyme inhibitors and diuretics as well as in subjects who had previously suffered myocardial infarction. After exclusion of data for women being administered estrogen replacement therapy, multivariate analysis revealed that sex (P < 0.001), age (P < 0.02), blood pressure (P < 0.002), diabetes (P < 0.05), and the use of angiotensin converting enzyme inhibitors (P < 0.002), β-blockers (P < 0.001), and diuretics (P < 0.05) were independent determinants of plasma prorenin. Plasma renin was independently related to atrial natriuretic peptide level (P < 0.01) and the use of angiotensin converting enzyme inhibitors (P < 0.001), b-blockers (P < 0.001), and diuretics (P < 0.05). Conclusions These data demonstrate that there is a sexual dimorphism of prorenin levels in humans, suggesting that sex hormones affect the regulation of the renin gene. Data confirm previous reports of elevated prorenin levels in diabetics and older subjects, as well as of lower than normal prorenin levels in subjects with hypertension in smaller populations. Our findings may help to clarify the potential (patho)physiologic functions of prorenin and to identify the factors that influence the constitutive secretion and intracellular processing of this prohormone.

Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor

Objective Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation. Methods and results Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37°C with human renin or prorenin. Incubation at 37°C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist ‘handle region peptide’, which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation. Conclusions H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.

Prorenin and Renin-Induced Extracellular Signal-Regulated Kinase 1/2 Activation in Monocytes Is Not Blocked by Aliskiren or the Handle-Region Peptide

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the “handle-region” peptide (HRP). We tested whether human prorenin and renin induce extracellular signal–regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of 125I-renin or 125I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate–labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

Angiotensin Production by the Heart A Quantitative Study in Pigs With the Use of Radiolabeled Angiotensin Infusions

BACKGROUND Beneficial effects of ACE inhibitors on the heart may be mediated by decreased cardiac angiotensin II (Ang II) production. METHODS AND RESULTS To determine whether cardiac Ang I and Ang II are produced in situ or derived from the circulation, we infused 125I-labeled Ang I or II into pigs (25 to 30 kg) and measured 125I-Ang I and II as well as endogenous Ang I and II in cardiac tissue and blood plasma. In untreated pigs, the tissue Ang II concentration (per gram wet weight) in different parts of the heart was 5 times the concentration (per milliliter) in plasma, and the tissue Ang I concentration was 75% of the plasma Ang I concentration. Tissue 125I-Ang II during 125I-Ang II infusion was 75% of 125I-Ang II in arterial plasma, whereas tissue 125I-Ang I during 125I-Ang I infusion was <4% of 125I-Ang I in arterial plasma. After treatment with the ACE inhibitor captopril (25 mg twice daily), Ang II fell in plasma but not in tissue, and Ang I and renin rose both in plasma and tissue, whereas angiotensinogen did not change in plasma and fell in tissue. Tissue 125I-Ang II derived by conversion from arterially delivered 125I-Ang I fell from 23% to <2% of 125I-Ang I in arterial plasma. CONCLUSIONS Most of the cardiac Ang II appears to be produced at tissue sites by conversion of in situ-synthesized rather than blood-derived Ang I. Our study also indicates that under certain experimental conditions, the heart can maintain its Ang II production, whereas the production of circulating Ang II is effectively suppressed.