Bruin, R.J.A. de

Amino-terminal pro-brain natriuretic peptide (NT-proBNP) is a biomarker of cardiac filling pressures in pre-eclampsia (Article)

2010-11-01T00:00:00Z

Objective: To evaluate if amino-terminal pro-brain natriuretic peptide (NT-proBNP) plasma levels reflect intracardiac filling pressures in pre-eclamptic patients. Study design: In a cross-sectional study we investigated 22 untreated critically ill pre-eclamptic women between 22 and 34 weeks gestation. All patients underwent intra-arterial blood pressure and central hemodynamic measurements and NT-proBNP was determined in stored plasma. Baseline characteristics, plasma NT-proBNP concentrations and relevant laboratory variables were investigated for correlations with hemodynamic values using Spearman's rank correlation test. Results: No significant correlations were demonstrated between NT-proBNP concentrations and variables associated with the severity of the pre-eclampsia. We found significant positive correlations between NT-proBNP and diastolic pulmonary pressure (r = 0.59; p = 0.005) and pulmonary capillary wedge pressure (PCWP) (r = 0.51; p = 0.015). Multiple linear regression analysis showed that the association between NT-proBNP and PCWP was not affected by creatinine level. Conclusion: NT-proBNP is a biomarker of left ventricular cardiac filling pressures in untreated pre-eclamptic patients.

Cardiac renin levels are not influenced by the amount of resident mast cells (Article)

2009-08-01T00:00:00Z

To investigate whether mast cells release renin in the heart, we studied renin and prorenin synthesis by such cells, using the human mast cell lines human mastocytoma 1 and LAD2, as well as fresh mast cells from mastocytosis patients. We also quantified the contribution of mast cells to cardiac renin levels in control and infarcted rat hearts. Human mastocytoma 1 cells contained and released angiotensin I-generating activity, and the inhibition of this activity by the renin inhibitor aliskiren was comparable to that of recombinant human renin. Prorenin activation with trypsin increased angiotensin I-generating activity in the medium only, suggesting release but not storage of prorenin. The adenylyl cyclase activator forskolin, the cAMP analogue 8-db-cAMP, and the degranulator compound 48/80 increased renin release without affecting prorenin. Angiotensin II blocked the forskolin-induced renin release. Angiotensin I-generating activity was undetectable in LAD2 cells and fresh mast cells. Nonperfused rat hearts contained angiotensin I-generating activity, and aliskiren blocked < 70% of this activity. A 30-minute buffer perfusion washed away >70% of the aliskiren-inhibitable angiotensin I-generating activity. Prolonged buffer perfusion or compound 48/80 did not decrease cardiac angiotensin I-generating activity further or induce angiotensin I-generating activity release in the perfusion buffer. Results in infarcted hearts were identical, despite the increased mast cell number in such hearts. In conclusion, human mastocytoma 1 cells release renin and prorenin, and the regulation of this release resembles that of renal renin. However, this is not a uniform property of all mast cells. Mast cells appear an unlikely source of renin in the heart, both under normal and pathophysiological conditions.

Aliskiren accumulates in renin secretory granules and binds plasma prorenin (Article)

2008-12-01T00:00:00Z

The vascular effects of aliskiren last longer than expected based on its half life, and this renin inhibitor has been reported to cause a greater renin rise than other renin-angiotensin system blockers. To investigate whether aliskiren accumulation in secretory granules contributes to these phenomena, renin-synthesizing mast cells were incubated with aliskiren, washed, and exposed to forskolin in medium without aliskiren (0.1 to 1000 nmol/L). (Pro)renin concentrations were measured by renin- and prorenin-specific immunoradiometric assays, and renin activity was measured by enzyme-kinetic assay. Without aliskiren, the culture medium predominantly contained prorenin, the cells exclusively stored renin, and forskolin doubled renin release. Aliskiren dose-dependently bound to (pro)renin in the medium and cell lysates and did not alter the effect of forskolin. The aliskiren concentrations required to bind prorenin were 1 to 2 orders of magnitude higher than those needed to bind renin. Blockade of cell lysate renin activity ranged from 27±15% to 79±5%, and these percentages were identical for the renin that was released by forskolin, indicating that they represented the same renin pool, ie, the renin storage granules. Comparison of renin and prorenin measurements in blood samples obtained from human volunteers treated with aliskiren, both before and after prorenin activation, revealed that ≤30% of prorenin was detected in renin-specific assays. In conclusion, aliskiren accumulates in renin granules, thus allowing long-lasting renin-angiotensin system blockade beyond the half-life of this drug. Aliskiren also binds to prorenin. This allows its detection as renin, and might explain, in part, the renin rise during renin inhibition.

Aliskiren-binding increases the half life of renin and prorenin in rat aortic vascular smooth muscle cells (Article)

2008-06-01T00:00:00Z

Renin inhibition with aliskiren has been reported to cause a greater rise in renin than other types of renin-angiotensin system blockade, thereby potentially leading to angiotensin generation or stimulation of the human (pro)renin receptor (h(P)RR). Here we studied whether this rise in renin is attributable to an aliskiren-induced change in the prorenin conformation, allowing its detection in renin assays, or a change in renin/prorenin clearance. We also investigated whether aliskiren affects (pro)renin binding to its receptors, using rat aortic vascular smooth muscle cells (VSMCs) overexpressing the h(P)RR. Methods and Results-A 48-hour incubation with aliskiren at 40C converted the prorenin conformation from "closed" to "open," thus allowing its recognition in active site-directed renin assays. VSMCs accumulated (pro)renin through binding to mannose 6-phosphate receptors (M6PRs) and h(P)RRs. Aliskiren did not affect binding at 40C. At 370C, aliskiren increased (pro)renin accumulation up to 40-fold, and M6PR blockade prevented this. Aliskiren increased the intracellular half life of prorenin 2 to 3 times. Conclusion-Aliskiren allows the detection of prorenin as renin, and decreases renin/prorenin clearance. Both phenomena may contribute to the "renin" surge during aliskiren treatment, but because they depend on aliskiren binding, they will not result in angiotensin generation. Aliskiren does not affect (pro)renin binding to its receptors.

Different contributions of the angiotensin-converting enzyme C-domain and N-domain in subjects with the angiotensin-converting enzyme II and DD genotype (Article)

2008-04-01T00:00:00Z

BACKGROUND: Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism-related differences in ACE concentration do not result in differences in angiotensin levels. METHODS AND RESULTS: To investigate whether this relates to differences in the contribution of the ACE C-domain and N-domain, we quantified, using the C-domain-selective inhibitors quinaprilat and RXPA380, and the N-domain-selective inhibitor RXP407, the contribution of both domains to the metabolism of angiotensin I, bradykinin, the C-domain-selective substrate Mca-BK(1-8), and the N-domain-selective substrate Mca-Ala in serum of IIs, DDs, and 'hyperACE' subjects (i.e., subjects with increased ACE due to enhanced shedding). During incubation with angiotensin I, the highest angiotensin II levels were observed in sera with the highest ACE activity. This confirms that ACE is rate-limiting with regard to angiotensin II generation. C-domain-selective concentrations of quinaprilat fully blocked angiotensin I-II conversion in DDs, whereas additional N-domain blockade was required to fully block conversion in IIs. Both domains contributed to bradykinin hydrolysis in all subjects, and the inhibition profile of RXP407 when using Mca-Ala was identical in IIs and DDs. In contrast, the RXPA380 concentrations required to block C-domain activity when using Mca-BK (1-8) were three-fold higher in IIs than DDs. CONCLUSION: The contributions of the C-domain and N-domain differ between DDs and IIs, and RXPA380 is the first inhibitor capable of distinguishing D-allele ACE from I-allele ACE. The lack of angiotensin II accumulation in DDs in vivo is not because of the often quoted concept that ACE is a nonrate-limiting enzyme. It may relate to the fact that in IIs both the N-domain and C- domain generate angiotensin II, whereas in DDs only the C-domain converts angiotensin I.

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 (Article)

2007-12-01T00:00:00Z

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.

Validation of a new automated renin assay. (Article)

2004-11-01T00:00:00Z

BACKGROUND: Measurement of plasma renin is important for the treatment of patients with congenital adrenal hyperplasia (CAH) and in the evaluation of patients with suspected hyperaldosteronism. Immunologic assays for plasma renin offer easier implementation and standardization than enzyme-kinetic assays for plasma renin activity, but their sensitivity and specificity have been questioned. We studied a renin immunochemiluminescence assay on an automated platform. METHODS: Renin was measured by an enzymatic assay, by IRMA, and by the new Nichols Advantage Specialty System immunochemiluminometric assay (ICMA), in plasmas from unselected individuals from our outpatient departments and in samples from patients with selected diagnoses. RESULTS: The detection limit in the ICMA was 0.1 mU/L. The recovery was >90%, and the imprecision (CV) was generally <9%. Mean (SD) concentrations measured by ICMA were 32 (21)% lower than those measured by IRMA. Renin concentrations as measured by ICMA were identical in serum and EDTA-, heparin-, and citrate-anticoagulated plasmas. Prolonged incubation of whole blood at room temperature before centrifugation did not affect renin concentrations. The central 95% interval for 80 healthy adults was 6-85.5 mU/L. Plasma renin as assessed by ICMA in patients with primary hyperaldosteronism was <0.2 mU/L. CONCLUSIONS: The performance characteristics of the new renin ICMA allow its use for patients with CAH and for the diagnosis of mineralocorticoid hypertension. In view of the variability of renin concentrations, use for other forms of hypertension or physiologic research calls for the development of uniform sampling protocols.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin (Article)

2001-01-01T00:00:00Z

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors (Article)

2001-01-01T00:00:00Z

ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance.

Plasma renin and prorenin and renin gene variation in patients with insulin-dependent diabetes mellitus and nephropathy (Article)

1999-01-01T00:00:00Z

BACKGROUND: The most striking abnormality in the renin angiotensin system in diabetic nephropathy (DN) is increased plasma prorenin. Renin is thought to be low or normal in DN. In spite of altered (pro)renin regulation the renin gene has not been studied for contribution to the development of DN. METHODS: We studied plasma renin, prorenin, and four polymorphic markers of the renin gene in 199 patients with IDDM and DN, and in 192 normoalbuminuric IDDM controls matched for age, sex, and duration of diabetes. Plasma renin and total renin were measured by immunoradiometric assays. Genotyping was PCR-based. RESULTS: Plasma renin was increased in patients with nephropathy (median (range), 26.3 (5.2-243.3) vs 18.3 (4.2-373.5) microU/ml in the normoalbuminuric group, P<0.0001). Prorenin levels were elevated out of proportion to renin levels in nephropathic patients (789 (88-5481) vs 302 (36-2226) microU/ml, P<0.0001). Proliferative retinopathy had an additive effect on plasma prorenin, but not on renin. DN was associated with a BglI RFLP in the first intron of the renin gene (bb-genotype: n=106 vs 82 in DN and normoalbuminuric patients respectively, P=0.037), but not with three other polymorphisms in the renin gene. A trend for association of higher prorenin levels with the DN-associated allele of this renin polymorphism was observed in a subgroup of patients with DN (bb vs Bb+BB, P=0.07). CONCLUSIONS: The results indicate that in DN there is an increase in both renin and prorenin levels. A renin gene polymorphism may contribute weakly to DN. Although speculative, one of the renin gene alleles could lead to increased renin gene expression, leading to higher renin and prorenin levels. These may play a role in the pathogenesis of DN.