Induction of Functional Bradykinin B1-Receptors in Normotensive Rats and Mice Under Chronic Angiotensin-Converting Enzyme Inhibitor Treatment
Background— The physiological effects of ACE inhibitors may act in part through a kinin-dependent mechanism. We investigated the effect of chronic ACE-inhibitor treatment on functional kinin B1- and B2-receptor expression, which are the molecular entities responsible for the biological effects of kinins.
Methods and Results— Rats were subjected to different 6-week treatments using various mixtures of the following agents: ACE inhibitor, angiotensin AT1-receptor antagonist, and B1- and B2-receptor antagonists. Chronic ACE inhibition induced both renal and vascular B1-receptor expression, whereas B2-receptor expression was not modified. Furthermore, with B1-receptor antagonists, it was shown that B1-receptor induction was involved in the hypotensive effect of ACE inhibition. Using microdissection, we prepared 10 different nephron segments and found ACE-inhibitor–induced expression of functional B1-receptors in all segments. ACE-inhibitor–induced B1-receptor induction involved homologous upregulation, because it was prevented by B1-receptor antagonist treatment. Finally, using B2-receptor knockout mice, we showed that ACE-inhibitor–induced B1-receptor expression was B2-receptor independent.
Conclusions— This study provides the first evidence that chronic ACE-inhibitor administration is associated with functional vascular and renal B1-receptor induction, which is involved in ACE-inhibitor–induced hypotension. The observed B1-receptor induction in the kidney might participate in the known renoprotective effects of ACE inhibition.
Received September 25, 2001; revision received November 12, 2001; accepted November 14, 2001.
It is now well recognized that the kallikrein kinin system acts at both the vasculature and in peripheral tissues, including heart, kidney, lung, and brain.1 Many tissues have the capacity to generate de novo kallikrein kinin system expression and therefore to locally produce bradykinin (BK). BK and its carboxypeptidase M product, des-Arg9[BK] (DBK), exert their biological effects by binding respectively to the G-protein–coupled B2- and B1-receptors.2 The main known difference between B2- and B1-receptors is that the B2-receptor is constitutively expressed, whereas the B1-receptor is weakly detectable under physiological conditions but strongly expressed in pathological states.3 Thus, until now, most studies have focused on the effects of B2-receptor activation, whereas the B1-receptor has received less attention.
In blood pressure (BP) regulation, it has been proposed that the kallikrein kinin system counterbalances the vasoconstrictor renin angiotensin system.2 The major link between these 2 systems is angiotensin-converting enzyme (ACE), because this enzyme is able to generate the vasoconstrictor peptide angiotensin II (Ang II) and to inactivate the vasodilator peptide BK.4 The vasodilator action of BK via activation of its B2-receptor has been well demonstrated.1,2⇓ Activation of the inflammation-induced B1-receptor also generally results in hypotension.5
ACE inhibitors are efficient therapeutic agents in the management of hypertension, heart failure, and diabetic nephropathy.6–8⇓⇓ An important point concerning ACE inhibition that should be taken into account is that ACE-inhibitor treatment not only decreases Ang II and increases BK concentrations but also favors the generation of the B1-receptor agonist DBK by the enzyme kininase I.3 Despite convincing data showing that ACE-inhibitor treatment increases the circulating BK concentration9 and potentiates the hemodynamic effects of exogenous BK administration,10 as well as that the short-term hypotensive effects of ACE inhibitors in normotensive and hypertensive subjects are BK dependent,7,11⇓ the respective roles of B1- and B2-receptors in these protective effects induced by ACE inhibitors have not been determined. The majority of available data report involvement of the B2-receptor in the protective effects of ACE inhibitors in cardiovascular pathologies12 and diabetic nephropathy.13,14⇓ To the best of our knowledge, the role of the B1-receptor in the effects of ACE inhibitors has not been studied.
Because the kidney is a major target organ in hypertensive and diabetic pathologies and could be considered a predictor of cardiovascular risk,15 the main objective of the present study was to analyze whether chronic ACE inhibition affects renal B1- and B2-receptor expression in rats and mice. The availability of specific BK-receptor antagonists and B2-receptor knockout mice (KOB2) allowed us to answer this question clearly. We report here for the first time that chronic ACE inhibition can induce functional renal B1-receptor expression in normotensive rats and mice, and we present evidence that the hypotensive effect of ACE inhibition involves B1-receptor activation. B2-receptor expression under these conditions was not modified.
Twelve groups (n=7 animals per group) of male Sprague-Dawley rats (weight 205 to 212 g) were used. They received a normal sodium diet (UAR A.40, 104 mmol of Na+ per kilogram). Food and water intake were monitored daily. The ACE inhibitor ramipril (Hoechst) and AT1-receptor antagonist irbesartan (Sanofi) were added to drinking water, whereas both B1- and B2-receptor antagonists were injected daily by a single subcutaneous injection for 6 weeks. All experiments were conducted as stated in the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press, Washington, DC) and were approved by a local animal care and use committee. At the end of the experimental period, mean arterial BP was monitored as described previously.16 Under our experimental conditions, we verified that anesthesia with inactine did not produce a depressor effect and that direct mean BP determined under anesthesia was correlated to the tail-cuff systolic BP. Body weight, ACE activity, and blood glucose level were also determined. ACE activity in plasma was measured as described previously.17 Blood glucose was determined with the kit Glu 1108 (Test Strips, One Touch, Lifescan).
KOB2 mice were generously provided by Dr Fred Hess (Merck & Co Inc, Rahway, NJ).18 KOB2 mice were originally on a mixed genetic background (J129sv X C57Bl/6J). We have backcrossed (10 times) the KOB2 mice to C57Bl/6J and therefore used C57Bl/6J as control mice. The mice were housed in a pathogen-free environment (SPF). Each group contained 10 mice, and ramipril was given in drinking water at 10 mg · kg−1 · d−1. Measurements of BP were performed in unanesthetized mice by the tail-cuff plethysmography method.
Microdissection of Rat Nephron Segments
Microdissection was performed as previously described by our laboratory.19 The following nephron segments were isolated: glomeruli, efferent arteriole, proximal convoluted tubule, proximal straight tubule, medullary thin descending limb, inner medullary thin limb, medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts. Glomerular and tubular surfaces were measured as described previously.19
mRNA Expression Analysis
Total RNA was extracted with the RNeasy kit (Qiagen). RNase protection was performed on 200 μg of total RNA from kidney tissue, carotid artery, aorta, and left ventricle with the RPAIII kit (Ambion). Semiquantitative B1- and B2-receptor and GAPDH mRNA expression in microdissected nephron segments (1 glomerulus, surface 480±80 μm2, or a total surface of 480±89 μm2 of each renal tubule segment) was analyzed and semiquantified by reverse transcription–polymerase chain reaction (RT-PCR) followed by Southern blot analysis as described previously.19
Quantitative Competitive PCR
Quantitative competitive PCR (QC-PCR) was performed with an internal competitive template of 304 bp according to a previously described protocol.20 QC-PCR contained 1 μL of 500, 100, 50, 10, 5, and 1 fg and 500 ag of a synthetic DNA competitor. The primer sequences for the mouse B1-receptor were as follows: upstream primer, 5′-CGGAAGCCTGGGATCTGCTGTG-3′ (nucleotides 173 to 194, Genbank U47281); downstream primer, 5′-CCAGCAACCTGTAGCGGTCC-3′ (nucleotides 507 to 526). PCR products were analyzed on 2% agarose gels stained with ethidium bromide and quantified by densitometric analysis. B1-receptor mRNA concentration was calculated by determining where the logarithmic ratio of endogenous B1-receptor mRNA expression (amplified 354 bp) and competitor expression (304 bp) was equal. PCR-amplified products were sequenced to confirm that the PCR bands corresponded to B1-receptor mRNA.
Production and Measurement of Prostaglandin E2 Production by Rat Microdissected Tubules
An equal surface of each renal tubule segment was transferred to a reaction tube with 55 μL of solution 1 (135 mmol/L NaCl, 1 mmol/L Na2SO4, 1.2 mmol/L MgSO4, 5 mmol/L KCl, 2 mmol/L CaCl2, 5.5 mmol/L glucose, and 5 mmol/L HEPES pH 7.4) containing the drug to be tested at 0.2 μmol/L, followed by rapid volume adjustment with solution 1 to 110 μL to give a final drug concentration of 0.1 μmol/L. Then, tubes were transferred rapidly to a 37°C water bath for 10 minutes. The incubation was stopped by freezing at −80°C until prostaglandin E2 (PGE2) measurements were performed. After they were thawed, the samples were centrifuged, and 60 μL of supernatant was used to measure the PGE2 concentration with enzyme immunoassay kits (EIA, Cayman Chemical). Pellet protein concentration was measured by the dye-blue-binding method (BioRad Laboratories) after solubilization for 1 hour with 1N NaOH.
Data are presented as mean±SEM. Statistical analyses were performed with SPSS software (Statistical Package for the Social Sciences, SPSS Inc). ANOVA (2-way ANOVA analysis, repeated measurements) with a post hoc Tukey α-test was performed for comparison between the different groups. P<0.05 was considered statistically significant.
Efficiency of Pharmacological Treatments
The 6-week treatment with ACE inhibitor resulted in a complete inhibition of circulating ACE activity that was not changed in the presence of B1- or B2-antagonists (Figure 1). No significant changes were found in blood glucose (mean value for all groups 0.75±0.09 g/L) and body weight (mean value for all groups 342±15 g). No effect on the histological structure of the kidneys was observed (no sign of hypertrophy or of glomerular or tubulointerstitial alterations; data not shown).
Both B1- and B2-Receptors Are Involved in Chronic ACE-Inhibition–Induced Hypotension in Rats
Figure 2 shows BP measured at the end of the different 6-week treatments. As expected, chronic treatment with ACE inhibitors induced a substantial decrease in BP (−47±10 mm Hg). No additional effect of ACE inhibitor plus AT1-receptor antagonist was found. As expected, treatment with the AT1-receptor antagonist alone decreased BP. Interestingly, the ACE-inhibitor–induced decrease in BP was partly reduced (≈50%) in the 2 groups of rats treated with 2 different B1-receptor antagonists, des-Arg9-Leu8[BK] and R715, as well as in the group treated with the B2-antagonist HOE140. Furthermore, the hypotensive effect of ACE inhibition was completely prevented with an equimolar mixture of both B1- and B2-receptor antagonists. Administration of B1- or B2-receptor antagonists alone was without effect on BP. These results suggest that both B1- and B2-receptors are involved in the hypotensive effects of ACE inhibitors.
Quantitative B1-Receptor mRNA Analysis in Whole Kidney, Carotid Artery, Aorta, and Left Ventricle
To verify whether chronic ACE inhibition in these rats modified kinin receptor mRNA expression, we performed RNase protection analysis in whole kidney (Figure 3A) and in some cardiovascular tissues (Figure 3B). Six weeks of ACE-inhibitor treatment induced significant renal B1-receptor mRNA expression compared with control rats. Concomitant B1-receptor antagonist treatment strongly decreased this ACE-inhibitor–induced B1-receptor mRNA induction.
In contrast, B2-receptor and AT1-receptor antagonist treatment were without effect on ACE-inhibitor–induced B1-receptor mRNA expression. Combined B1- and B2-receptor antagonist treatment in the presence of ACE inhibitor prevented B1-receptor mRNA induction. Separate treatment with the B1- or B2-receptor antagonists was without effect on B1-receptor mRNA expression. The different treatments were without effect on GAPDH mRNA expression. Parallel RNase protection analysis of B2-receptor expression showed that the different treatments were without effect on B2-receptor mRNA expression levels (data not shown). This B1-receptor mRNA induction was not restricted to the kidney level, because significant B1-receptor mRNA expression was observed in 3 different cardiovascular tissues (Figure 3B) after ACE-inhibitor treatment that was not observed in control rats (not shown).
Chronic ACE Inhibition Induces B1-Receptor mRNA Expression in Microdissected Nephron Segments
To identify more precisely the renal location of the induced B1-receptor mRNA after ACE inhibition, we performed semiquantitative B1-receptor mRNA expression analysis using an RT-PCR/Southern blot approach on microdissected nephron segments. Although no renal B1-receptor mRNA could be detected along the nephron (Figure 4A) under our control conditions, ACE inhibition induced significant B1-receptor mRNA expression in all nephron segments studied (Figure 4C). Controls (Figure 4B and D) for contaminating genomic DNA amplification and relative quantification with GAPDH were performed as described previously.19 As evaluated by this method, high B1-receptor mRNA expression was observed in the efferent arteriole, glomeruli, medullary thin descending limb, inner medullary thin limb, and distal tubule; moderate expression was found in the proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb, and cortical collecting duct; and low expression was observed in the outer medullary collecting duct after ACE inhibition. As previously reported,19 expression of B2-receptor mRNA was observed in all nephron segments of control rats, and ACE inhibition was without significant effect in B2-receptor mRNA in the different nephron segments (data not shown).
ACE Inhibition Results in Induction of Functional B1-Receptors Along the Nephron
To verify whether induction of B1-receptor mRNA by ACE inhibitors is effectively translated into functional B1-receptors, the ability of the B1-receptor agonist DBK to stimulate PGE2 secretion was examined on microdissected nephron segments (Figure 5). B1-receptor stimulation had no effect on PGE2 secretion in microdissected nephrons obtained from untreated rats compared with basal PGE2 production. In contrast, a significant increase in PGE2 secretion after B1-receptor stimulation was observed in the efferent arteriole, glomeruli, proximal convoluted tubule, proximal straight tubule, medullary thin descending limb, inner medullary thin limb, distal tubule, cortical collecting duct, and outer medullary collecting duct from ACE-inhibitor–treated animals. No effect of B1-receptor stimulation on PGE2 production was observed in the medullary thick ascending limb. B1-receptor–induced stimulation of PGE2 secretion was inhibited in the presence of the B1-receptor antagonist des-Arg9-Leu8[BK], whereas this antagonist alone was without effect on PGE2 secretion (data not shown).
Chronic ACE Inhibition Induces B1-Receptor mRNA Expression in KOB2 Mice
The results displayed in Figures 2 and 3⇑ suggest that induction of the B1-receptor by ACE inhibitors can occur independently of B2-receptor activation. This hypothesis was tested by treating KOB2 and wild-type mice for 6 weeks with the ACE inhibitor ramipril. Under physiological conditions, there was no difference in BP between control and KOB2 mice, but ACE inhibition induced a similar significant decrease in BP of both control and KOB2 mice (Figure 6A). When QC-PCR was used, no renal B1-receptor mRNA expression was detectable in wild-type mice, whereas basal B1-receptor mRNA expression was found in KOB2 mice (Figure 6B). ACE inhibition induced renal B1-receptor mRNA expression in both wild-type and KOB2 mice, although this was significantly higher in KOB2 than in wild-type mice (Figure 6B). B1-receptor mRNA induction by ACE inhibition, therefore, does occur independently of the B2-receptor.
The present study provides evidence for the first time that chronic ACE inhibition can induce functional renal and vascular kinin B1-receptors. Furthermore, these newly synthesized B1-receptors are involved in the hypotensive action of ACE inhibition. No effect of chronic ACE inhibition was observed on B2-receptor expression.
In this article, evidence for B1-receptor induction by chronic ACE inhibition was presented on the level of B1-receptor mRNA expression, on the basis of renal B1-receptor activity (PGE2 production), and on the basis of the ability of 2 different B1-receptor antagonists to partially reverse ACE-inhibitor–induced hypotension. The ability of B1-receptor antagonists to reverse ACE-inhibitor–induced hypotension correlates with the often-observed B1-agonist–induced hypotension under inflammatory conditions.5 Depending on the animal species and the vascular bed studied, the mechanism underlying this B1-receptor–induced vasodilator effect involves either prostaglandins or nitric oxide.3,5⇓ Studies on the short-term effect of ACE inhibition on B1-receptor expression in rabbits gave conflicting results. In Nwator and Whalley,21 ACE inhibition (3 to 20 hours IV) resulted in an increased response to the B1-agonist, whereas under similar conditions, another laboratory3 observed no effect on B1-receptor expression (mRNA, hemodynamic response to B1-agonist administration).
Simultaneous chronic AT1-receptor antagonism and ACE inhibition did not have an additive effect on BP, but chronic AT1-receptor antagonism lowered BP when used alone, thereby demonstrating the effectiveness of the treatment and the drug under our experimental conditions. In general, long-term combined ACE inhibitor and AT1-receptor antagonist treatment under pathological conditions in humans and in experimental pathologies in animals is more efficient to reduce BP than treatment with one of these drugs alone (congestive heart failure22; essential hypertension23; patients with hypertension, microalbuminuria, and non–insulin-dependent diabetes24; and spontaneously hypertensive rats25). In contrast, as observed in the present study on normotensive rats, 4 weeks of combined treatment with ACE inhibitor and AT1-receptor antagonist in normotensive patients with diabetic nephropathy or glomerulonephritis was without further effect on BP, whereas the glomerular filtration rate was efficiently increased in this “add-on” treatment.26 It thus seems that long-term ACE inhibitor and AT1-receptor antagonist treatment are more efficient in reducing BP than separate drug treatment in several pathologies but not under normotensive conditions. In contrast to the absence of an add-on effect on BP in chronic combined ACE inhibitor and AT1-receptor antagonist treatment under normotensive conditions, a single dose of both drugs was found to have an additive effect on BP in sodium-depleted (ie, renin angiotensin system activation) healthy normotensive humans and lasted up to 6 hours.27 An observation in the present study that might be related to the absence of an add-on effect is that ACE inhibition in the presence of both a B1- and B2-receptor antagonist returned BP to the value of nontreated animals. This suggests the absence or loss of the role of Ang II blockade in chronic ACE-inhibitor–induced hypotension, possibly explaining the absence of an add-on effect.
The most important observation of the present study is the ACE inhibitor–induced functional BK B1-receptor expression. This induction could be mediated by stimulation of preexisting B1-receptors by increased B1-agonist formation due to chronic ACE-inhibitor treatment,9 which confirms, in vivo, previous studies that the B1-agonist can induce the expression of its own receptor.28,29⇓ The induction is probably not due to an inflammatory reaction, at least in the kidney, because histomorphometric kidney analysis did not show signs of such a reaction after chronic ACE inhibition. ACE-inhibition–induced B1-receptor induction is not mediated by the B2-receptor, because we observed that ACE inhibition induced significant B1-receptor expression in KOB2 mice. Interestingly, using QC-PCR, we observed basal B1-receptor expression in the kidney of nontreated KOB2 mice. The B2-receptor might thus negatively control B1-receptor expression. Our observation using a quantitative method confirms a recent study in which nonquantitative RT-PCR analysis also showed basal B1-receptor expression in KOB2 mice.30
A large number of studies have described the protective cardiovascular and renal effects of ACE inhibitors, which are increasingly attributed, at least in part, to kinin potentiation.8,12–14⇓⇓⇓ The beneficial effects of ACE inhibitors are not solely due to the reduction of BP, and because ACE inhibitors exert tissue protective effects in diabetic nephropathy independently of their vasodepressive effects,13,14⇓ we sought to determine whether chronic ACE inhibition had an effect on renal BK receptor expression. Although there was no effect on renal B2-receptor expression, functional B1-receptors were induced in all nephron segments studied. What could be the role of renal B1-receptor induction under physiological or pathological conditions? It has been reported that activation of renal B1-receptors increases renal vascular resistance, which suggests that the B1-receptor might be important in the maintenance of renal vasoconstriction in pathologies that lead to renal failure.16,31⇓ Finally, a protective role for the B1-receptor in the development of end-stage renal diseases has been proposed recently with the use of polymorphism analysis.32
In addition to the well-known involvement of the B1-receptor in inflammation and hyperalgesia, because of which B1-receptor antagonists may be clinically useful as anti-inflammatory and analgesic drugs,3 other pathologies (mainly, cardiovascular and some renal pathologies) associated with cell enlargement and proliferation might benefit from B1-receptor activation. Indeed, recent studies have reported the involvement of the B1-receptor in the inhibition of neointima formation.33 In addition to the evidence for a role of the B1-receptor in ACE-inhibitor–induced hypotension, ACE-inhibition–induced renal B1-receptor expression could thus participate in the other “beneficial effects” attributed to ACE inhibitors.
↵*Drs Marin-Castaño and Schanstra contributed equally to this article.
- ↵Margolius HS. Kallikreins and kinins. Hypertension. 1995; 26: 221–229.
- ↵Marceau F, Hess JF, Bachvarov DR. The B1-receptors for kinins. Pharmacol Rev. 1998; 50: 357–386.
- ↵Erdös EG. Angiotensin I converting enzyme and the changes in our concepts through the years. Hypertension. 1990; 16: 363–370.
- ↵McLean PG, Peretti M, Ahluwalia A. Kinin B1-receptors and the cardiovascular system: regulation of expression and function. Cardiovascular Res. 2000; 48: 194–210.
- ↵Campbell DJ, Kladis A, Duncan AM. Effects of converting enzyme inhibitors on angiotensin and bradykinin peptides. Hypertension. 1994; 23: 439–449.
- ↵Brown NJ, Vaughan DE. Angiotensin-converting enzyme inhibitors. Circulation. 1998; 97: 1411–1420.
- ↵Squire IB, O’Kane KPJ, Anderson N, et al. Bradykinin B2-receptor antagonism attenuates blood pressure response to acute angiotensin-converting enzyme inhibition in normal men. Hypertension. 2000; 36: 132–136.
- ↵Remme WJ. Bradykinin-mediated cardiovascular protective actions of ACE-inhibitors. Drugs. 1997; 54 (suppl 5): 59–70.
- ↵Geiger H. Are angiotensin II receptor blockers superior to angiotensin converting enzyme inhibitors with regard to their renoprotective effect? Nephrol Dial Transplant. 1997; 12: 640–642.
- ↵Schanstra JP, Marin-Castano EM, Praddaude F, et al. Bradykinin B1 receptor-mediated changes in renal hemodynamics during endotoxin-induced inflammation. J Am Soc Nephrol. 2000; 11: 1208–1215.
- ↵Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol. 1971; 20: 1637–1648.
- ↵Borkowski JA, Ranson RW, Seabrook GR, et al. Targeted disruption of a B2 bradykinin receptor gene in mice eliminates bradykinin action in smooth muscle and neurons. J Biol Chem. 1995; 270: 13706–13710.
- ↵Celi FS, Zenilman ME, Shuldiner AR. A rapid versatile method to synthesize internal standards for competitive PCR. Nucleic Acid Res. 1993; 21: 1047.
- ↵Mogensen CE, Neldam S, Tikkanen I, et al. Randomised controlled trial of dual blockade of renin-angiotensin system in patients with hypertension, microalbuminuria, and non-insulin dependent diabetes: the Candesartan And Lisinopril Microalbuminuria (CALM) study. BMJ. 2000; 321: 1440–1444.
- ↵Azizi M, Chatellier G, Guyene TT, et al. Additive effects of combined angiotensin-converting-enzyme inhibition and angiotensin II antagonism on blood pressure and renin release in sodium-depleted normotensives. Circulation. 1995; 92: 825–834.
- ↵Phagoo SB, Poole S, Leeb-Lundberg LM. Autoregulation of bradykinin receptors: agonists in the presence of interleukin-1β shift the repertoire of receptor subtypes from B2 to B1 in human lung fibroblasts. Mol Pharmacol. 1999; 56: 325–333.
- ↵Duka I, Kintsurashvili E, Gavras I, et al. Vasoactive potential of the B1 bradykinin receptor in normotension and hypertension. Circ Res. 2001; 88: 275–281.
- ↵Bachvarov DR, Landry M, Pelletier I, et al. Characterization of two polymorphic sites in the human kinin B1-receptor gene: altered frequency of an allele in patients with a history of end-stage renal failure. J Am Soc Nephrol. 1998; 9: 598–604.
- ↵Agata J, Miao RQ, Yayama K, et al. Bradykinin B1-receptor mediates inhibition of neointima formation in rat artery after balloon angioplasty. Hypertension. 2000; 36: 364–370.