The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin

Peptides 24 (2003) 1141–1147

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin John D. Imig a,b,∗ , Xueying Zhao a , Sheyla R. Orengo b , Susana Dipp c , Samir S. El-Dahr c a

c

Department of Physiology, Medical College of Georgia, Vascular Biology Center, Augusta, GA 30912-2500, USA b Department of Physiology, Tulane University Health Sciences Center, New Orleans, LA, USA Department of Pediatrics, Section of Pediatric Nephrology, Tulane University Health Sciences Center, New Orleans, LA, USA Received 9 May 2003; accepted 18 July 2003

Abstract Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R−/− mice by 40–50%. On the other hand, renal COX-1 levels were similar in B2R−/− and +/+ mice. Renal renin protein was 61% lower in B2R−/− compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R−/− mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R−/− mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261 ± 16 and 266 ± 15 fmol/g in B2R+/+ and B2R−/− mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R−/− mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression. © 2003 Elsevier Inc. All rights reserved. Keywords: Kallikrein–kinin system; Prostaglandins; Angiotensin; Kidney

1. Introduction Bradykinin, the enzymatic product of the kallikrein–kinin system exerts its actions via binding to and activation of the B1 or B2 receptor [6,22]. The renal and cardiovascular actions of bradykinin are primarily mediated through activation of the B2 receptor [6,22]. The bradykinin B2 receptor is a G-protein coupled receptor that mediates diverse renal and cardiovascular actions, such as vasodilation, sodium excretion, inflammatory responses and cellular growth [6,22]. Acute blockade of B2 receptors decreases renal blood flow and attenuates the natriuretic response to salt loading [28,31]. Likewise, B2 receptor deficient mice (B2R−/−) exposed to a high salt diet develop hypertension [1,7,15,23]. Two other systems known to be intimately involved in the ability of the kidney to regulate body fluid and electrolytes and maintain cardiovascular homeostasis are the renin–angiotensin system and cyclooxygenase (COX) pathway [18,19,30]. Thus, as would be expected, interactions between the renin–angiotensin system, bradykinin ∗

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0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.07.003

and COX-derived prostaglandins have been reported by a number of laboratories. The most prominent interaction described between the kallikrein–kinin system and the renin–angiotensin system is that angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin [5,12]. Bradykinin is degraded by ACE or kininase II to primarily inactive peptide fragments [5,12]. Evidence has supported the notion that the anti-hypertensive and cardio-protective action of ACE inhibitors is in part due to increasing bradykinin levels [5,12,35]. In addition, ACE inhibitors increase renal COX-2 and renin levels [9,10]. Along these lines, COX-2 inhibition or gene disruption suppresses renin synthesis and COX-2-derived prostaglandins stimulate kidney renin release [9,10,21,34]. Renal renin synthesis could be regulated through a bradykinin B2 receptor—COX-2 pathway since prostaglandin stimulation mediates many of the bradykinin B2 receptor cardiovascular and renal actions [6,22]. The aim of the present study was to determine the regulation of kidney COX-2 and renin gene expression in bradykinin B2 receptor gene disrupted mice (B2R−/−). We report here that renal renin protein and mRNA levels as well as levels of kidney angiotensin I are significantly reduced

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in mice lacking B2 receptors. Similarly, kidney COX-2 protein expression was significantly lower in B2R−/− mice. These data support the notion that the renin–angiotensin system, bradykinin and COX-derived prostaglandins act in a concerted manner to achieve cardiovascular homeostasis.

2. Materials and methods 2.1. Animals Targeted disruption of the B2R gene was accomplished by homologous recombination in embryonic stem cells as described by Borkowski et al. [2]. B2R−/− mice were provided by Drs. Hess and Chen (Merck Research laboratory, Rahway, NJ) and have since been bred on a C57BL/6J background. All mice were weaned at 3 weeks of age and fed a standard chow diet. Genotypes of mice were routinely determined by Southern analysis of genomic tail DNA. Animals were housed in a temperature- and light-controlled room and had access to water ad labium. The Tulane Advisory Committee for Animal Resources approved all experiments and the procedures followed were in accordance with institutional guidelines. 2.2. Processing of tissue Three to four month old B2R+/+ and B2R−/− mice were anesthetized with a combination of pentobarbital sodium (50 mg/kg i.p.) and ketamine (10 mg/kg i.p.) and the right carotid artery was cannulated for arterial blood sampling. Blood samples were collected into chilled tubes containing a mixed inhibitor solution (5 mM EDTA, 10 ␮M pepstatin, 20 ␮M enalaprilat, and 1.25 mM 1,10-phenanthroline). Blood samples were centrifuged at 4 ◦ C for 10 min and plasma separated, and immediately extracted and stored at −20 ◦ C until assayed as previously described [20]. For analysis of renal angiotensin peptide levels, kidneys were collected, weighed, immersed in cold methanol and homogenized immediately after harvesting. The kidney supernatants were dried in a vacuum centrifuge and reconstituted in a 50 mM sodium phosphate buffer (pH 7.4), containing 1 mM EDTA, 0.25 mM thimerosal, and 0.25% heat-inactivated bovine serum albumin. Samples were purified and stored as previously described [20]. 2.3. Kidney angiotensin I and angiotensin II levels and ACE activity

ported [20], there is cross-reactivity with angiotensin-(1–10) and angiotensin-(2–10) for the angiotensin I anti-sera and angiotensin-(1–8) and angiotensin-(2–8) exhibit similar potencies for the angiotensin II anti-sera. Results are reported in femtomoles/gram kidney weight or femtomoles/milliliter plasma. The sensitivities of the angiotensin I and angiotensin II assays were 1.8 ± 0.7 and 1.3 ± 0.5 fmol, respectively during 90% maximal binding. For the angiotensin I and angiotensin II assays, the specific binding was 54 ± 2 and 52±3%, respectively, with a non-specific binding of 2.4±0.5 and 2.7 ± 0.8%, respectively. ACE was extracted from kidney by homogenization in tris(hydroxymethyl)aminomethane (Tris) buffer (0.1 M, pH 7.4), and its activity in the kidney supernatant was determined by fluorimetric measurement of the enzymatic cleavage of hippurate from hippuryl-histidyl-leucine. By use of the control conditions as previously described [16], there was no detectable breakdown of histidyl-leucine product by renal peptidase enzymes. Renal ACE activity is reported as nmol per minute per milligram protein. 2.4. Kidney COX-2 and renin–angiotensin system protein levels Kidneys were harvested and processed as previously described. Samples were separated by electrophoresis on a 10% stacking Tris-glycine gel, and proteins were transferred electrophoretically to a PVDF membrane. The primary antibodies used were polyclonal rabbit anti-murine COX-2 anti-serum (1:000; Cayman, Ann Arbor, MI), polyclonal goat anti-murine COX-1 anti-serum (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit antihuman AT1 antiserum (1:200, N-10, sc-1173, Santa Cruz), angiotensinogen (Ao) (1/4000; anti-rat sheep polyclonal antibody, gift from Dr. C. Sernia) [11], polyclonal rabbit anti-human renin anti-serum, which cross-reacts with mouse pro- and active renin (1:4000, gift from D. Catanzaro) [4] and a mouse monoclonal ␤-actin antibody (1/5000, Sigma). The blots were then washed in a PBS-0.1% Tween 20 and incubated with the secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature and washed. Detection was accomplished using enhanced chemiluminescence Western blotting (ECL, Amersham Corp.), and blots were exposed to X-ray film (Hyperfilm-ECL, Amersham Corp.). Band intensity was measured densitometrically and the values were factored for ␤-actin. 2.5. RNA hybridization analysis of renin gene expression

Angiotensin I and angiotensin II levels were measured by radioimmunoassay. The reconstituted samples were incubated with anti-angiotensin I or anti-angiotensin II anti-sera and 125 I-labeled angiotensin I or angiotensin II for 48 h at 4 ◦ C. Bound and free angiotensin peptides were separated by dextran coated charcoal and the supernatants were counted by a gamma counter for 3 min. As previously re-

Total kidney RNA was extracted as previously described and RNA was measured spectrophotometrically at 260 nm. RNA samples were resolved by gel electrophoresis in 1% agarose containing 2.2 M formaldehyde. After vacuum blotting into a positively charged nylon membrane and UV cross-linking, the integrity of RNA was assessed by visu-

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alization of 28S and 18S ribosomal RNA by UV shadowing of the membrane at 254 nm. Northern blot hybridization was performed as previously described [14]. The blots were hybridized to a random-primed 32 P-labeled full-length rat renin cDNA. The integrity of RNA and equal RNA loading were assessed by ethidium bromide staining of the RNA gel. 2.6. RT–PCR analysis of COX-1 and COX-2 expression Total RNA was prepared from mouse kidney using an ultra-pure TRIzol reagent according to the procedure described by the manufacturer (Gibco-BRL, Grand Island, NY). Random hexanucleotide primers were used for reverse transcription (RT) of equal amounts of RNA. Oligonucleotide primers were designed from the published cDNA sequences of COX-1, COX-2, and GAPDH. GAPDH was used as an internal control. The sequences of the COX-1 primers are sense 5 -GTG CGG TCC AAC CTT ATC-3 and antisense 5 -CAA CCC AAA CAC CTC CTG-3 . The sequences of the COX-2 primers are sense 5 -TGT GCG ACA TAC TCA AGC-3 and antisense 5 -TAT CCC TTA TGG GTA TCT TC-3 . The sequences of the GAPDH primers are sense 5 -AAT GCA TCC TGC ACC ACC AA-3 and antisense 5 -GTA GCC ATA TTC ATT GTC ATA-3 . The expected sizes of the amplified COX-1, COX-2 and GAPDH polymerase chain reaction (PCR) products are 528, 693, and 515 base pairs, respectively. RT–PCR was performed and after amplification, 15 ␮l of each PCR reaction mixture were electrophoresed through a 1.5% agarose gel with ethidium bromide (0.5 ␮g/ml). The gel was scanned with ultraviolet illumination using Digital Imaging and Analysis (Alpha Innotech Corp.).

Fig. 1. Top, Western blot analysis of renin in mouse kidneys. The blot is representative of 43 kDa renin bands from 4 to 5 animals per experimental group. Bottom, Densitometeric values for kidney protein levels factored for ␤-actin (40 ␮g protein/lane; B2R+/+ n = 8, B2R−/− n = 8). Values are mean ± S.E.M. (∗ ) Significant difference from B2R+/+.

reduced more than half in the B2R−/− mice compared to the B2R+/+ mice (Fig. 3). In contrast, kidney levels of angiotensin II were not altered in mice lacking the B2R. We determined renal Ao protein levels, AT1 receptor protein levels and ACE activity to determine the impact of B2R gene disruption on these aspects of the renin–angiotensin system. Ao and AT1 receptor protein levels were unaltered

2.7. Statistics Comparisons among the groups were performed by Student t test. P < 0.05 was considered statistically significant. All data are reported as mean ± S.E.M. 3. Results 3.1. Renal renin and angiotensin I are decreased in B2R−/− mice The effect of B2R gene disruption on renal renin protein levels and mRNA expression was evaluated. Western blots and densitometric analysis of renal renin protein levels is presented in Fig. 1. Kidney renin protein levels were 3-fold higher in B2R+/+ than B2R−/− mice. Likewise, renin mRNA levels were significantly higher in the B2R+/+ compared to the B2R−/− mice (Fig. 2). Renal angiotensin I and angiotensin II levels were also determined in B2R gene disrupted mice and compared to mice homozygous for the B2R. In agreement with the renin protein levels and mRNA expression, kidney angiotensin I levels were

Fig. 2. Top, Northern blot analysis of renin mRNA in mouse kidneys. The blot shows a 1.4 kb band from 4 animals per experimental group. Bottom, densitometeric values for kidney renin mRNA levels (20 ␮g RNA/lane; B2R+/+ n = 4, B2R−/− n = 4). Values are mean±S.E.M. (∗ ) Significant difference from B2R+/+.

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detectable COX-2 signal, whereas phorbol 12-myristate 13-acetate (PMA) treated RAW264.7 cells had a strong 72 kDa immunoreactive signal for COX-2 (data not shown). As can be seen in the right panel of Fig. 5, renal COX-2 protein levels were 40% higher in B2R+/+ mice compared to B2R−/− mice. Additionally, COX-1 and COX-2 mRNA levels were determined in kidneys from B2R+/+ and B2R−/− mice. Consistent with the protein expression results, COX-2 but not COX-1 expression was decreased in mice lacking the B2 receptor (Fig. 6). Fig. 3. Kidney angiotensin I (ANG I, left panel) and angiotensin II (ANG II, right panel) levels in B2R+/+ and B2R−/− mice. Values are mean ± S.E.M. (B2R+/+ n = 5, B2R−/− n = 5). (∗ ) Significant difference from B2R+/+.

in B2R−/− mice compared to B2R+/+ mice (Fig. 4). Unlike the decrease in kidney renin enzyme levels in B2R−/− mice, renal ACE activity was not altered in mice lacking B2R and there was a trend for increased ACE activity in mice lacking the B2R. 3.2. Kidney COX-2 protein and mRNA levels are decreased in B2R−/− mice We assessed COX-1 and COX-2 protein levels to determine the impact of the B2R on the regulation of kidney cyclooxygenases. COX-1 protein levels were not different between B2R+/+ and B2R−/− mice (Fig. 5, left panel). The COX-2 antibody was assessed in the RAW264.7 mouse macrophage cell line. Control RAW264.7 cells lacked a

4. Discussion The regulation of the renin–angiotensin system by COX-2 derived prostaglandins has been extensively studied [18,19,21,30,34,35]. Likewise, interactions between the kallikrein–kinin system and the renin–angiotensin system have been recognized for many years [5,12]. Cardiovascular and fluid and electrolyte homeostasis are regulated by bradykinin, angiotensin and prostaglandins and disruption of this fine balance can be detrimental to kidney and cardiovascular function [1,7,8,18,19,26]. In this regard, targeted disruption of the cyclooxygenase pathway, renin–angiotensin system or kallikrein–kinin system results in an inability to properly regulate cardiovascular function. Hypotension and increased renal blood flow and glomerular filtration rate has been demonstrated in mice over-expressing the human B2 receptor [33]. Although renal plasma flow and glomerular filtration rate are unaltered in B2R−/− mice, decreased renal nitrate excretion and glomerular capillary

Fig. 4. Top, Western blot analysis of angiotensinogen and AT1 receptor in mouse kidneys. The blot is representative of 52 kDa Ao (left panel) and 46 kDa AT1 receptor (right panel) bands from 4 to 7 animals per experimental group. Bottom, densitometeric values for kidney Ao protein levels (left panel, 40 ␮g protein/lane; B2R+/+ n = 4, B2R−/− n = 5) and AT1 receptor protein levels (center panel, 40 ␮g protein/lane; B2R+/+ n = 7, B2R−/− n = 7) factored for ␤-actin. The bottom right panel depicts ACE activity (B2R+/+ n = 6, B2R−/− n = 7). Values are mean ± S.E.M.

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Fig. 5. Top, Western blot analysis of COX-1 and COX-2 in mouse kidneys. The blot is representative of 72 kDa COX-1 (left panel) and 72 kDa COX-2 receptor (right panel) bands from 4 to 5 animals per experimental group. Bottom, densitometeric values for kidney COX-1 protein levels (left panel, 20 ␮g protein/lane; B2R+/+ n = 8, B2R−/− n = 8) and COX-2 receptor protein levels (right panel, 40 ␮g protein/lane; B2R+/+ n = 8, B2R−/− n = 8) factored for ␤-actin. Values are mean ± S.E.M. (∗ ) Significant difference from B2R+/+.

surface area could be one reason for the inability of these mice to properly handle a chronic salt load [8,29]. Along these lines, studies have also demonstrated that mice lacking the B2 receptor can develop salt-sensitive hypertension and that the cardioprotective effects of ACE inhibitors and AT1 receptor antagonists are attenuated in B2R−/− mice [1,7,15,23,35]. Interestingly, developmental expression of B2 receptors, COX-2 and renin are similar and are regu-

lated in a concerted fashion [13,17,18,36]. B2 receptors, COX-2 and renin gene expression are high at birth then decline with maturation [13,17,18,36]. Since recent evidence suggest that COX-2 derived prostaglandins regulate kidney renin release [18,19,30] and that bradykinin B2 receptor activation increases COX-2 in various cell types [25,27], we postulated that renal renin synthesis could be controlled through a B2 receptor—COX-2 pathway. Our findings that

Fig. 6. Top, RT–PCR analysis of COX-1 and COX-2 mRNA in mouse kidneys. The blot shows the expected 528 and 693 bp bands for COX-1 and COX-2, respectively. Bottom, densitometeric values for kidney COX-1 and COX-2 mRNA levels (B2R+/+ n = 4, B2R−/− n = 4). Values are mean ± S.E.M. (∗ ) Significant difference from B2R+/+.

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mice lacking B2 receptors had decreased kidney COX-2 and renin protein and mRNA levels supports the notion that bradykinin regulation of COX-2 participates in the steady-state control of renin expression. In the present study, we assessed the kidney renin–angiotensin system in B2 receptor gene disrupted mice. B2R−/− mice had decreased renin protein and mRNA levels that were associated with decreased kidney angiotensin I levels compared to B2R+/+ mice. Even though renal renin levels are decreased, kidney angiotensinogen, AT1 receptor and angiotensin II levels were not different between B2R+/+ and B2R−/− mice. We interpret the sustained intrarenal levels of angiotensin II in the face of low angiotensin I as a reflection of maintained ACE activity and/or increased internalization of angiotensin II into the intracellular compartment. Alternatively, B2R−/− mice may have adapted by activation of renin-independent pathways. The finding of decreased renal renin is contradictory to studies demonstrating increased renin expression in B2R−/− mice and unaltered renin levels in mice overexpressing the human B2 receptor [32,33]. The reasons for these discrepancies are not known but may be related to the genetic background of the genetically manipulated B2R mice, which has an important influence on the number of renin genes. In any case, the present results confirm and extend our previous observation that renin mRNA levels are decreased and AT1 receptor levels unaltered in B2R−/− mice [7]. Interestingly, B2R-deficient mice on a high salt diet are unable to decrease renal renin mRNA levels but are capable of suppressing kidney angiotensin II levels [7]. Taken together, these studies suggest that renin or ACE independent generation of kidney angiotensin II may be important in the B2R−/− mice. We have previously demonstrated ACE and renin independent generation of angiotensin II by an unidentified serine protease enzyme in the rat kidney [36]. Evaluation of renin or ACE independent pathways of angiotensin generation in mice lacking B2 receptors will undoubtedly be the focus of future studies. ACE inhibition studies have provided indirect evidence that a bradykinin—COX-2 pathway could regulate renal renin synthesis in vivo. Bradykinin, COX-2 and renin levels are increased in animals treated with ACE inhibitors [5,12,18]. In the present study, our findings demonstrate that renal COX-2 but not COX-1 levels are decreased in mice lacking the B2 receptor. This finding of bradykinin regulation of COX-2 is consistent with reports in other cell types. Bradykinin has been demonstrated to increase IL-8 generation in airway epithelial cells via stimulation of COX-2 [27]. In human airway epithelial cells, pulmonary artery smooth muscle cells and gingival fibroblasts bradykinin increases COX-2 gene expression and promoter activity [3,25,27]. PGE2 appears to be the COX-2 metabolite that mediates the biological response [3,25]. Along these lines, recent reports have provided evidence that support the postulate that the prostaglandin receptors involved in renal renin production are the EP4 and/or the IP receptor [10].

Prostaglandin stimulation of renal renin expression and production involves the activation of adenylate cyclase and an increase in juxtaglomerular cell cAMP levels [18,19]. Three prostaglandin receptors, EP2 , EP4 , and IP, could possibly mediate activation of kidney renin secretion. The fact that regulation of renal renin production is not altered in EP2 −/− mice [10] suggest that EP2 receptors are not essential for PGE2 regulation of kidney renin production. Although many studies have demonstrated a very strong relationship between COX-2 regulation and renal renin secretion, there is evidence to suggest that, under certain experimental conditions that COX-2 does not regulate renal renin expression. For example, acute renal artery stenosis results in upregulation of kidney COX-2 and renin expression; however, the COX-2 inhibitor, celecoxib, did not block the increase in renin [24]. The strongest evidence for COX-2 regulation of renal renin has been derived from the COX-2−/− mice [9,10,34]. One recent study found that the increase in renal renin mediated in response to the ACE inhibitor, captopril, was absent in COX-2−/− but not COX-1−/− or EP2 −/− mice [10]. Interestingly, a role for bradykinin in this pathway can be inferred because ACE inhibition increases bradykinin levels. The finding in the present study that mice lacking the bradykinin B2 receptor have decreased kidney COX-2 and renin expression provides the initial evidence implicating the bradykinin B2 receptor in COX-2 mediated regulation of renal renin. In summary, B2 receptor deficient mice have decreased kidney COX-2 protein levels that are associated with decreased renin protein and mRNA expression and kidney angiotensin I levels. The finding that kidney angiotensin II, AT1 receptor levels and ACE activity indicate that feedback regulation by angiotensin II via AT1 receptors or ACE regulation of bradykinin levels were not responsible for the decreased renin levels observed in B2R−/− mice. Taken as a whole, the present study gives credence to the hypothesis that B2 receptor regulation of COX-2 participates in the steady-state control of renin gene regulation. Acknowledgments This study was supported by the National Institutes of Health grants DK56264 (S.S.E.-D.) and HL-59699 (J.D.I.). Dr. Xueying Zhao is supported by an American Heart Association Southeast Affiliate Postdoctoral Fellowship. We are grateful to Fred Hess and Howard Chen (Merck Research Laboratories) for providing the B2R null mice. We thank Drs. Dan Catanzaro (Cornell Medical School) and Conrad Sernia (Qld., Australia) for the generous gifts of renin and angiotensinogen antibodies, respectively. References [1] Alfie ME, Sigmon DH, Pomposiello SI, Carretero OA. Effect of high salt intake in mutant mice lacking bradykinin-B2 receptors. Hypertension 1997;29:483–7.

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