Jan Danser

Prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren

Objectives Inhibition of (pro)renin receptor activation was demonstrated to inhibit or even abolish the development of end-organ damage in animal models. The new renin inhibitor, aliskiren, markedly increases the plasma concentration of the (pro)renin receptor ligands prorenin and renin in patients. The effects of prorenin and of renin inhibitors on the signal transduction cascade of the (pro)renin receptor are currently unknown. Results Our results indicate that renin and prorenin were equally potent in (pro)renin receptor activation by decreasing (pro)renin receptor mRNA, increasing phosphatidylinositol-3 kinase p85α mRNA and augmenting viable cell number, respectively. These effects of renin and prorenin are both abolished using small-interfering RNA against the (pro)renin receptor or its adaptor promyelocytic zinc finger protein. The renin inhibitor aliskiren did not inhibit the renin-induced or prorenin-induced activation of the (pro)renin receptor. Conclusion This is the first report demonstrating equal ligand activities of both, renin and prorenin, on the (pro)renin receptor - promyelocytic zinc finger protein–phosphatidylinositol-3 kinase–p85α pathway. The failure of aliskiren to inhibit the noncatalytic effects of renin and prorenin may be of clinical relevance considering the increase in plasma concentrations of (pro)renin under aliskiren treatment.

Bradykinin Potentiation by Angiotensin-(1-7) and ACE Inhibitors Correlates With ACE C- and N-Domain Blockade

Abstract—ACE inhibitors block B2 receptor desensitization, thereby potentiating bradykinin beyond blocking its hydrolysis. Angiotensin (Ang)-(1-7) also acts as an ACE inhibitor and, in addition, may stimulate bradykinin release via angiotensin II type 2 receptors. In this study we compared the bradykinin-potentiating effects of Ang-(1-7), quinaprilat, and captopril. Porcine coronary arteries, obtained from 32 pigs, were mounted in organ baths, preconstricted with prostaglandin F2&agr;, and exposed to quinaprilat, captopril, Ang-(1-7), and/or bradykinin. Bradykinin induced complete relaxation (pEC50=8.11±0.07, mean±SEM), whereas quinaprilat, captopril, and Ang-(1-7) alone were without effect. Quinaprilat shifted the bradykinin curve to the left in a biphasic manner: a 5-fold shift at concentrations that specifically block the C-domain (0.1 to 1 nmol/L) and a 10-fold shift at concentrations that block both domains. Captopril and Ang-(1-7) monophasically shifted the bradykinin curve to the left, by a factor of 10 and 5, respectively. A 5-fold shift was also observed when Ang-(1-7) was combined with 0.1 nmol/L quinaprilat. Repeated exposure of porcine coronary arteries to 0.1 &mgr;mol/L bradykinin induced B2 receptor desensitization. The addition of 10 &mgr;mol/L quinaprilat or Ang-(1-7) to the bath, at a time when bradykinin alone was no longer able to induce relaxation, fully restored the relaxant effects of bradykinin. Angiotensin II type 1 or 2 receptor blockade did not affect any of the observed effects of Ang-(1-7). In conclusion, Ang-(1-7), like quinaprilat and captopril, potentiates bradykinin by acting as an ACE inhibitor. Bradykinin potentiation is maximal when both the ACE C- and N-terminal domains are inhibited. The inhibitory effects of Ang-(1-7) are limited to the ACE C-domain, raising the possibility that Ang-(1-7) synergistically increases the blood pressure-lowering effects of N-domain-specific ACE inhibitors.

Bradykinin potentiation by ACE inhibitors: a matter of metabolism

Studies in isolated cells overexpressing ACE and bradykinin type 2 (B2) receptors suggest that ACE inhibitors potentiate bradykinin by inhibiting B2 receptor desensitization, via a mechanism involving protein kinase C (PKC) and phosphatases. Here we investigated, in intact porcine coronary arteries, endothelial ACE/B2 receptor ‘crosstalk’ as well as bradykinin potentiation through neutral endopeptidase (NEP) inhibition. NEP inhibition with phosphoramidon did not affect the bradykinin concentration‐response curve (CRC), nor did combined NEP/ACE inhibition with omapatrilat exert a further leftward shift on top of the ≈10 fold leftward shift of the bradykinin CRC observed with ACE inhibition alone. In arteries that, following repeated exposure to 0.1 μM bradykinin, no longer responded to bradykinin (‘desensitized’ arteries), the ACE inhibitors quinaprilat and angiotensin‐(1‐7) both induced complete relaxation, without affecting the organ bath fluid levels of bradykinin. This phenomenon was unaffected by inhibition of PKC or phosphatases (with calphostin C and okadaic acid, respectively). When using bradykinin analogues that were either completely or largely ACE‐resistant ([Phe8Ψ(CH2‐NH)Arg9]‐bradykinin and [ΔPhe5]‐bradykinin, respectively), the ACE inhibitor‐induced shift of the bradykinin CRC was absent, and its ability to reverse desensitization was absent or significantly reduced, respectively. Caveolar disruption with filipin did not affect the quinaprilat‐induced effects. Filipin did however reduce the bradykinin‐induced relaxation by ≈25–30%, thereby confirming that B2 receptor‐endothelial NO synthase (eNOS) interaction occurs in caveolae. In conclusion, in porcine arteries, in contrast to transfected cells, bradykinin potentiation by ACE inhibitors is a metabolic process, that can only be explained on the basis of ACE‐B2 receptor co‐localization on the endothelial cell membrane. NEP does not appear to affect the bradykinin levels in close proximity to B2 receptors, and the ACE inhibitor‐induced bradykinin potentiation precedes B2 receptor coupling to eNOS in caveolae.

L-NAME-resistant bradykinin-induced relaxation in porcine coronary arteries is NO-dependent: effect of ACE inhibition

NO synthase (NOS) inhibitors partially block bradykinin (BK)‐mediated vasorelaxation. Here we investigated whether this is due to incomplete NOS inhibition and/or NO release from storage sites. We also studied the mechanism behind ACE inhibitor‐mediated BK potentiation. Porcine coronary arteries (PCAs) were mounted in organ baths, preconstricted, and exposed to BK or the ACE‐resistant BK analogue Hyp3‐Tyr(Me)8‐BK (HT‐BK) with or without the NOS inhibitor L‐NAME (100 μM), the NO scavenger hydroxocobalamin (200 μM), the Ca2+‐dependent K+‐channel blockers charybdotoxin+apamin (both 100 nM), or the ACE inhibitor quinaprilat (10 μM). BK and HT‐BK dose‐dependently relaxed preconstricted vessels (pEC50 8.0±0.1 and 8.5±0.2, respectively). pEC50s were &10 fold higher with quinaprilat, and &10 fold lower with L‐NAME or charybdotoxin+apamin. Complete blockade was obtained with hydroxocobalamin or L‐NAME+ charybdotoxin+apamin. Repeated exposure to 100 nM BK or HT‐BK, to deplete NO storage sites, produced progressively smaller vasorelaxant responses. With L‐NAME, the decrease in response occurred much more rapidly. L‐Arginine (10 mM) reversed the effect of L‐NAME. Adding quinaprilat to the bath following repeated exposure (with or without L‐NAME), at the time BK and HT‐BK no longer induced relaxation, fully restored vasorelaxation, while quinaprilat alone had no effect. Quinaprilat also relaxed vessels that, due to pretreatment with hydroxocobalamin or L‐NAME+charybdotoxin+apamin, previously had not responded to BK. In conclusion, L‐NAME‐resistant BK‐induced relaxation in PCAs depends on NO from storage sites, and is mediated via stimulation of guanylyl cyclase and/or Ca2+‐dependent K+‐channels. ACE inhibitors potentiate BK independent of their effect on BK metabolism.

Mechanism of hypertension and proteinuria during angiogenesis inhibition: Evolving role of endothelin-1

Angiogenesis inhibition by blocking vascular endothelial growth factor (VEGF)-mediated signalling with monoclonal antibodies or tyrosine kinase inhibitors has become an established treatment of various forms of cancer. This treatment is frequently associated with the development of hypertension and proteinuria. As VEGF increases the expression and the activity of nitric oxide synthase in endothelial cells, a decrease in the bioavailability of nitric oxide has been proposed as a key mechanism leading to hypertension during angiogenesis inhibition. However, results of clinical and experimental studies exploring this possibility are conflicting. Rarefaction, that is a structural decrease of microcirculatory vessels, has been reported during antiangiogenic treatment, but evidence that it plays a role in development of hypertension is lacking. Elevated circulating and urinary levels of endothelin-1 have been observed in clinical and experimental studies with angiogenesis inhibitors. Furthermore, the observation that endothelin receptor blockers can prevent or revert the rise in blood pressure during angiogenesis inhibition and attenuate proteinuria provides strong evidence that an activated endothelin-signalling pathway is a final common mediator of angiogenesis inhibition-induced rise in blood pressure and renal toxicity.

Urinary renin and angiotensinogen in type 2 diabetes: added value beyond urinary albumin?

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

Drug Mechanisms to Help in Managing Resistant Hypertension in Obesity

Obesity is a major risk factor for the development of hypertension. Because the prevalence of obesity is increasing worldwide, the prevalence of obesity hypertension is also increasing. Importantly, hypertension in obesity is commonly complicated by dyslipidemia and type 2 diabetes mellitus and hence imposes a high cardiovascular disease risk. Furthermore, obesity is strongly associated with resistant hypertension. Activation of the sympathetic nervous system and the renin-angiotensin system, leading to renal sodium and water retention, links obesity with hypertension. There is also evidence for the release of factors by visceral adipose tissue promoting excessive aldosterone production, and a more central role of aldosterone in obesity hypertension is emerging. Randomized studies evaluating the effect of different classes of antihypertensive agents in obesity hypertension are scarce, short-lasting, and small. Considering the emerging role of aldosterone in the pathogenesis of obesity hypertension, mineralocorticoid receptor antagonism may play a more central role in the pharmacologic treatment of obesity hypertension in the near future.

Negative inotropic effect of bradykinin in porcine isolated atrial trabeculae: Role of nitric oxide

Objectives To investigate whether bradykinin affects cardiac contractility independently of its effects on coronary flow and noradrenaline release, and whether such inotropic effects, if present, are mediated via nitric oxide (NO). Methods Right atrial trabeculae were obtained from 35 pigs, suspended in organ baths and attached to isometric transducers. Resting tension was set at approximately 750 mg and tissues were paced at 1.5 Hz. Tissue viability was checked by constructing a concentration response curve (CRC) to noradrenaline. Next, CRCs were constructed to bradykinin, either under baseline conditions or after pre-stimulation with the positive inotropic agent forskolin (1 or 10 μmol/l), in the absence or presence of the bradykinin type 2 (B2) receptor antagonist d-Arg [Hyp3-Thi5, d-Tic7, Oic8]-bradykinin (Hoe 140) (1 μmol/l), the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) (100 μmol/l) and/or the NO scavenger hydroxocobalamin (200 μmol/l). Results Bradykinin exerted a negative inotropic effect, both with and without forskolin pre-stimulation, reducing contractility by maximally 22 ± 3.6% (mean ± SEM) and 23 ± 3.6%, respectively (pEC50 8.37 ± 0.23 and 8.62 ± 0.22, respectively). l-NAME reduced this effect in pre-stimulated, but not in unstimulated, trabeculae. Hoe 140 and hydroxocobalamin fully blocked the inotropic effect of bradykinin. Conclusions Bradykinin induces a modest negative inotropic effect in porcine atrial trabeculae that is mediated via B2 receptors and NO. The inconsistent results obtained with l-NAME suggest that it depends on NO synthesized de novo and/or NO from storage sites.

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H+-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H+-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H+-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H+-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H+-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H+-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H+-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H+-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H+-ATPase activity in intercalated cells.

Role of endothelin in preeclampsia and hypertension following antiangiogenesis treatment.

Purpose of reviewPreeclampsia is a systemic, pregnancy-related disorder featuring hypertension and proteinuria arising from placental overproduction of soluble FMS-like tyrosine kinase-1, resulting in an antiangiogenic state because of the inhibition of the vascular endothelial growth factor (VEGF) family. Similarly, antiangiogenetic treatment aimed at targeting VEGF in patients with cancer is associated with a preeclampsia-like syndrome. In this study we discuss the pathophysiological role of an activated endothelin system in both conditions. Recent findingsIn different experimental forms of preeclampsia, in clinical preeclampsia, and in cancer patients on antiangiogenic treatment, activation of the endothelin axis invariably occurs and this activation is directly related to the circulating level of sFlt-1 or the intensity of antiangiogenic treatment. Administration of endothelin receptor A-selective or dual endothelin receptor antagonists can prevent or largely attenuate the hypertension and proteinuria in experimental forms of preeclampsia, as well as in rats exposed to receptor tyrosine-kinase inhibitors targeting VEGF-signaling, supporting the concept that activation of the endothelin axis plays a key role in the manifestations of these disorders. SummaryActivation of the endothelin axis has now emerged as a crucial player in the manifestations of preeclampsia and following antiangiogenic treatment. As a consequence, blockade of the endothelin system may be considered as a treatment option both in preeclampsia and in antiangiogenesis-induced hypertension and renal toxicity in patients with cancer.