Gool, J.M. van

New renin inhibitor VTP-27999 alters renin immunoreactivity and does not unfold prorenin (Article)

2013-05-01T00:00:00Z

Renin inhibitors like aliskiren not only block renin but also bind prorenin, thereby inducing a conformational change (like the change induced by acid) allowing its recognition in a renin-specific assay. Consequently, aliskiren can be used to measure prorenin. VTP-27999 is a new renin inhibitor with an aliskiren-like IC50 and t1/2, and a much higher bioavailability. This study addressed (pro)renin changes during treatment of volunteers with VTP-27999 or aliskiren. Both drugs increased renin immunoreactivity. Treatment of plasma samples from aliskiren-treated subjects with excess aliskiren yielded higher renin immunoreactivity levels, confirming the presence of prorenin. Unexpectedly, this approach did not work in VTP-27999-treated subjects, although an assay detecting the prosegment revealed that their blood still contained prorenin. Subsequent in vitro analysis showed that VTP-27999 increased renin immunoreactivity for a given amount of renin by ≥30% but did not unfold prorenin. Yet, it did bind to acid-activated, intact prorenin and then again increased immunoreactivity in a renin assay. However, no such increase in immunoreactivity was seen when measuring acid-activated prorenin bound to VTP-27999 with a prosegment-directed assay. The VTP-27999-induced rises in renin immunoreactivity could be competitively prevented by aliskiren, and antibody displacement studies revealed a higher affinity of the active site-directed antibodies in the presence of VTP-27999. In conclusion, VTP-27999 increases renin immunoreactivity in renin immunoassays because it affects the affinity of the active site-directed antibody. Combined with its lack of effect on prorenin, these data show that VTP-27999 differs from aliskiren. The clinical relevance of these results needs to be established.

Urinary renin, but not angiotensinogen or aldosterone, reflects the renal renin-angiotensin-aldosterone system activity and the efficacy of renin-angiotensin-aldosterone system blockade in the kidney (Article)

2011-11-01T00:00:00Z

Objective: To study which renin-angiotensin-aldosterone system (RAAS) component best reflects renal RAAS activity. Methods and Results: We measured urinary and plasma renin, prorenin, angiotensinogen, aldosterone, albumin and creatinine in 101 diabetic and nondiabetic patients with or without hypertension. Plasma prorenin was elevated in diabetic patients. Urinary prorenin was undetectable. Urinary albumin and renin were higher in diabetic patients. Men had higher plasma renin/prorenin levels, and lower plasma angiotensinogen levels than women. Plasma creatinine and albumin were also higher in men. Urinary RAAS components showed no sexual dimorphism, whereas urinary creatinine and albumin were higher in men. Angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor blockers increased plasma renin and decreased plasma angiotensinogen, without altering plasma aldosterone. In contrast, in urine, these drugs decreased renin and aldosterone without affecting angiotensinogen. When analyzing all patients together, urinary angiotensinogen excretion closely mimicked that of albumin, whereas urinary angiotensinogen and albumin levels both were 0.05% or less of their concomitant plasma levels. This may reflect the identical glomerular filtration and tubular handling of both proteins, which have a comparable molecular weight. In contrast, urinary renin excretion did not correlate with urinary albumin excretion, and the urinary/plasma concentration ratio of renin was more than 200 times the ratio of albumin, despite its comparable molecular weight. Urinary aldosterone excretion closely followed urinary creatinine excretion. Conclusion: The increased urinary renin levels in diabetes and the decreased urinary renin levels following RAAS blockade, occurring independently of changes in plasma renin, reflect the activated renal RAAS in diabetes and the success of RAAS blockade in the kidney, respectively. Urinary renin, therefore, more closely reflects renal RAAS activity than urinary angiotensinogen or aldosterone.

Evaluation of a direct prorenin assay making use of a monoclonal antibody directed against residues 32-39 of the prosegment (Article)

2011-11-01T00:00:00Z

Background: Prorenin is an early marker of microvascular complications in diabetes. However, it can only be measured indirectly (following its conversion to renin), with a renin immunoradiometric assay (IRMA). Unfortunately, treatment with a renin inhibitor interferes with this assay, because renin inhibitors induce a conformational change in prorenin, thereby allowing its detection as renin. Methods: We evaluated Molecular Innovation's new direct prorenin ELISA, which makes use of an antibody that recognizes an epitope near prorenin's putative cleavage site (RL), thus no longer requiring prorenin activation. Plasma samples of 41 diabetic individuals treated with aliskiren (renin inhibitor) or irbesartan were tested. Semi-purified recombinant prorenin was used as standard, because the ELISA standard yielded approximately 10-fold lower values in the renin IRMA following its conversion to renin. Results: The ELISA detected prorenin levels that were identical to those determined by the IRMA in untreated and irbesartan-treated individuals. Yet, it yielded higher prorenin levels in aliskiren-treated individuals. Aliskiren, at levels reached in plasma during treatment, did not interfere with the ELISA, but allowed the detection of up to 20-30% of prorenin as renin in the IRMA, thereby resulting in a significant overestimation of renin and an underestimation of prorenin. The ELISA rendered results within 2 h and did not require a pretreatment period of several days to convert prorenin to renin. Conclusion: The new direct assay allows rapid prorenin detection, is not hampered by aliskiren when used at clinically relevant doses, and might be used to identify diabetic patients developing retinopathy and/or nephropathy.

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.

Selective angiotensin-converting enzyme C-domain inhibition is sufficient to prevent angiotensin I-induced vasoconstriction. (Article)

2005-01-01T00:00:00Z

Somatic angiotensin-converting enzyme (ACE) contains 2 domains (C-domain and N-domain) capable of hydrolyzing angiotensin I (Ang I) and bradykinin. Here we investigated the effect of the selective C-domain and N-domain inhibitors RXPA380 and RXP407 on Ang I-induced vasoconstriction of porcine femoral arteries (PFAs) and bradykinin-induced vasodilation of preconstricted porcine coronary microarteries (PCMAs). Ang I concentration-dependently constricted PFAs. RXPA380, at concentrations >1 mumol/L, shifted the Ang I concentration-response curve (CRC) 10-fold to the right. This was comparable to the maximal shift observed with the ACE inhibitors (ACEi) quinaprilat and captopril. RXP407 did not affect Ang I at concentrations < or =0.1 mmol/L. Bradykinin concentration-dependently relaxed PCMAs. RXPA380 (10 micromol/L) and RXP407 (0.1 mmol/L) potentiated bradykinin, both inducing a leftward shift of the bradykinin CRC that equaled approximately 50% of the maximal shift observed with quinaprilat. Ang I added to blood plasma disappeared with a half life (t(1/2)) of 42+/-3 minutes. Quinaprilat increased the t(1/2) approximately 4-fold, indicating that 71+/-6% of Ang I metabolism was attributable to ACE. RXPA380 (10 micromol/L) and RXP407 (0.1 mmol/L) increased the t(1/2) approximately 2-fold, thereby suggesting that both domains contribute to conversion in plasma. In conclusion, tissue Ang I-II conversion depends exclusively on the ACE C-domain, whereas both domains contribute to conversion by soluble ACE and to bradykinin degradation at tissue sites. Because tissue ACE (and not plasma ACE) determines the hypertensive effects of Ang I, these data not only explain why N-domain inhibition does not affect Ang I-induced vasoconstriction in vivo but also why ACEi exert blood pressure-independent effects at low (C-domain-blocking) doses.

Angiotensin II type 2 receptors and cardiac hypertrophy in women with hypertrophic cardiomyopathy (Article)

2001-01-01T00:00:00Z

The development of left ventricular hypertrophy in subjects with hypertrophic cardiomyopathy (HCM) is variable, suggesting a role for modifying factors such as angiotensin II. Angiotensin II mediates both trophic and antitrophic effects, via angiotensin II type 1 (AT(1)-R) and angiotensin II type 2 (AT(2)-R) receptors, respectively. Here we investigated the effect of the AT(2)-R gene A/C(3123) polymorphism, located in the 3' untranslated region of exon 3, on left ventricular mass index (LVMI) in 103 genetically independent subjects with HCM (age, 12 to 81 years). LVMI and interventricular septum thickness were determined by 2D echocardiography. Extent of hypertrophy was quantified by a point score (Wigle score). Plasma prorenin, renin, and ACE were determined by immunoradiometric or fluorometric assays, and genotyping was performed by polymerase chain reaction. In men, no associations between AT(2)-R genotype and any of the measured parameters were observed, whereas in women, LVMI decreased with the number of C alleles (211+/-19, 201+/-18, and 152+/-10 g/m(2) in women with the AA, AC, and CC genotype, respectively; P=0.015). Similar C allele-related decreases in women were observed for interventricular septum thickness (P=0.13), Wigle score (P=0.05), plasma renin (P=0.03), and plasma prorenin (P=0.26). Multiple regression analysis revealed that the AT(2)-R C allele-related effect on LVMI (beta=-30.7+/-11.1, P=0.010) occurred independently of plasma renin, the AT(1)-R gene A/C(1166) polymorphism, or the ACE gene I/D polymorphism. In conclusion, AT(2)-Rs modulate cardiac hypertrophy in women with HCM, independently of the circulating renin-angiotensin system. These data support the contention that AT(2)-Rs mediate antitrophic effects in humans.

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.