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Cardiovascular Research 2006 71(3):596-605; doi:10.1016/j.cardiores.2006.05.020
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Copyright © 2006, European Society of Cardiology

Critical role for p47phox in renin–angiotensin system activation and blood pressure regulation

Karsten Grotea,*,1, Magdalene Ortmanna,1, Gustavo Salgueroa, Carola Doerriesa, Ulf Landmessera, Maren Luchtefelda, Ralf P. Brandesb, Wilfried Gwinnerc, Thomas Tschernigd, Ernst-Georg Brabante, Andreas Klosf, Arnd Schaefera, Helmut Drexlera and Bernhard Schieffera

aDepartment of Cardiology and Angiology, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
bInstitut of Cardiovascular Physiology, Clinical Center of the J.W. Goethe-University, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany
cDepartment of Nephrology, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
dDepartment of Anatomy, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
eDepartment of Endocrinology, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
fDepartment of Medical Microbiology, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

* Corresponding author. Tel.: +49 511 532 9584; fax: +49 511 532 3263. Email address: Grote.Karsten{at}MH-Hannover.DE

Received 30 November 2005; revised 4 May 2006; accepted 23 May 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Renin–angiotensin system (RAS) activation leads to increased production of NAD(P)H oxidase-derived reactive oxygen species (ROS), and both have been implicated in the initiation and progression of arterial hypertension, atherosclerosis, and cardiac hypertrophy. The cytosolic subunit p47phox is critically involved in agonist-induced NAD(P)H oxidase activation. Here, we investigated the role of p47phox in blood pressure control, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation, and renal oxidative stress under resting conditions.

Methods and results Mice deficient in p47phox (on C57BL/6 background) developed significantly higher systolic blood pressure levels compared to C57BL/6 wild-type animals (136.0±3.0 mmHg vs. 112.2±2.6, P<0.01, n=16) as measured by the tail cuff method from week 6 up to week 12 post partum. The increase in blood pressure in p47phox – / – mice was associated with an impaired endothelium-dependent relaxation (P<0.005 vs. wild-type, n=11). At the age of 12 weeks p47phox – / – mice showed increased plasma renin activity as analyzed by radioimmunoassay (14.5±1.8 ng/mL/h vs. 9.6±1.7 ng/mL/h, P<0.05, n=10) and enhanced angiotensin converting enzyme (ACE) activity in the kidney and aorta as measured by Hip–His–Leu cleavage (7.6±0.8 vs. 4.8±0.9 nmol/L His–Leu/mg protein, P<0.05, n=5) compared to wild-type mice. No differences in oxygen radical formation was determined in kidney samples by lucigenin- and luminol-enhanced chemiluminescence or by electron spin resonance spectroscopy. Consistently, treatment with the radical scavenger tempol did not lower blood pressure in p47phox – / – mice, whereas ACE and angiotensin II type I receptor inhibition normalized blood pressure.

Conclusion Deficiency of the NAD(P)H oxidase subunit p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner.

KEYWORDS NAD(P)H oxidase; p47phox; Reactive oxygen species; Blood pressure; Renin–angiotensin system


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Activation of the renin–angiotensin system (RAS) and resulting blood pressure elevation play a major role in the pathogenesis of hypertension, atherosclerosis and cardiac hypertrophy [1]. Components of the RAS are present in the vasculature and organs such as the heart and the kidney [2]. A major mechanism though which angiotensin II (Ang II) – the effector peptide of the RAS – induces pathological effects is by generation of reactive oxide species (ROS) [3]. Superoxide (O2–) acts as an important second messenger in the activation of different signaling pathways such as protein tyrosine phosphatases, protein tyrosine kinases, transcription factors, mitogen-activated protein kinases (MAPK), and ion channels [4]. Ang II-stimulated increases in ROS are involved in various cardiovascular diseases, e.g. in the development of endothelial dysfunction and cardiac hypertrophy [5,6]. In addition, enhanced vascular and renal NAD(P)H oxidase activity and augmented oxidative stress contributes to hypertension in many animal models [7].

A major source of cardiovascular and renal ROS is the nicotin-amide adenine dinucleotide phosphate (NAD[P]H) oxidase. Cardiovascular and renal cells express components of this multisubunit enzyme, including its cell membrane-associated subunits p22phox, gp91phox (Nox2), or Nox2 homologues Nox1 and Nox4, and cytosolic subunits, p47phox, p67phox, and p40phox [8,9]. The functional significance of each component may various in a cell type-specific manner and has not yet been fully elucidated.

In neutrophils, p47phox is critical for NAD(P)H oxidase activation and superoxide generation required for oxidative burst [10]. A pivotal role for p47phox in vascular smooth muscle cells (SMCs) in vitro has been shown for an appropriate subunit assembly at the cell membrane. This assembly is important for NAD(P)H oxidase activation and superoxide generation, e.g. after Ang II administration and mechanical stretch [11,12]. Intriguingly, previous in vitro studies in endothelial cells, vascular smooth muscle cells and aortic homogenates reported that p47phox was not essential for basal NAD(P)H oxidase-dependent superoxide generation. In fact, basal superoxide levels were noted to be slightly increased in p47phox-deficient mice [13–16].

An important role for p47phox in atherosclerosis was demonstrated by the finding that lesion progression was reduced in apolipoprotein E– / – mice lacking p47phox [17]. In p47phox-deficient mice, Ang II-dependent blood pressure elevation is suppressed [18] and in gp91phox-deficient mice, basal blood pressure is lower than in wild-type [19]. However, little is known about the role of p47phox in the regulation of basal blood pressure levels.

To address this issue, we determined whether p47phox-containing NAD(P)H oxidases play a role in the regulation of basal blood pressure. Therefore, we investigated p47phox deficient mice with regards to blood pressure, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation and renal oxidative stress.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Mice
The p47phox – / – mice, lacking the cytosolic subunit p47phox of the NAD(P)H oxidase were a generous gift from Steven Holland [20] and were backcrossed to the C57BL/6 background for 12 generations. C57BL/6 (wild-type) mice (obtained from Charles River, Sulzbach, Germany) were used as controls. For some experiments litter mates of F2 intercross progeny (p47phox – / –xC57BL/6) were used. Colonies were maintained at the animal facility of the Medical School Hannover. Mice were housed under specific pathogen-reduced conditions receiving autoclaved chow and water at libitum containing antibiotics (Co-trimoxazole; 30 mg/kg). Male mice at the age of 6 to 12 weeks were used in this study. All experiments were approved by the district government Hannover (Az. 02/568). Lack of p47phox was confirmed by PCR genotyping of tail DNA using specific primers. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.2 Histology of lungs
Mice were sacrificed and the lungs were removed. The right lungs were fixed in 4% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin.

2.3 Determination of plasma endotoxin by chromogenic Limulus test
Endotoxin activity of wild-type and p47phox – / – plasma samples was determined by a quantitative kinetic assay based on the reactivity of Gram-negative endotoxin with Limulus amebocyte lysate (LAL), by a chromogenic LAL test (Charles River, Sulzbach, Germany). The assay was carried out as recommended by the manufacturer. The standard endotoxin used in this test was prepared from E. coli O113:H10:K(–). Endotoxin-free water (Aqua B. Braun, Melsungen, Germany) was used for sample dilution.

2.4 Blood pressure measurements
Systolic blood pressure from wild-type and p47phox – / – mice was monitored by the tail cuff technique with the aid of a computerized system (BP2000 Blood Pressure Analysis System, Visitech System, Apex, NC, USA). Measurements were performed at the same time of day in mice from week 6 up to week 12 post partum with previous 3 days of training. On each day of blood pressure determination, 10 measurements were obtained and averaged for each mouse.

2.5 Organ chamber experiments
Experiments were performed using mouse aortic rings from wild-type and p47phox – / – mice preconstricted with phenylephrine to 80% of the contraction elicited by KCl (80 mmol/L). Endothelium-dependent relaxation to acetylcholine (ACh) was recorded.

2.6 Echocardiographic measurements
Echocardiography studies were performed under light anesthesia (100 mg/kg ketamine, 1.25 mg/kg xylazine, and 0.6 mg/kg atropine i.p.) and spontaneous respiration using an ultrasound system (ATL5000 CV) with a linear 15-MHz, high-frequency transducer as described previously [21]. The observer was blinded for the experimental group assignment.

2.7 Plasma renin activity
Plasma renin activities from wild-type and p47phox – / – mice were measured by radioimmunoassay for the end product angiotensin I using the commercially available GammaCoat Plasma Renin Activity iodine-125 radioimmunoassay kit (DiaSorin, Dietzenbach, Germany). In brief, the test is based on the estimation of the renin-dependent rate of angiotensin I formation under optimal conditions which is subsequently measured by a radioimmunoassay.

2.8 Tissue ACE activity measurements
Tissue from the kidney and aorta from wild-type and p47phox – / – mice was homogenized in ice-cold phosphate-buffered saline containing 0.5% Triton-X-100 and protease inhibitor cocktail (Sigma–Aldrich, Taufkirchen, Germany), pH 7.4). Homogenates were centrifuged (14,000 rpm for 10 min), and supernatants were used for fluorimetric determination of ACE activity. 10 µL of the supernatants were incubated with 240 µL assay buffer containing 5 mmol/L Hip–His–Leu (Sigma–Aldrich, Taufkirchen, Germany) in 0.1 mol/L phosphate buffer with 0.3 mol/L NaCl, pH 8.0, for 30 min at 37 °C. The reaction was halted by the addition of 0.6 mL of 0.1 N NaOH. After addition of 50 µL o-phthaldialdehyde (20 mg/mL) in methanol and an incubation period of 10 min at room temperature the reaction was stopped by the addition of 100 µL 0.8 N HCl. The product, His–Leu, was measured at 390 nm excitation and 520 nm emission using a fluoroscope (Fluoroscan, Labsystems, Helsinki, Finland). To correct for the intrinsic tissue fluorescence, blanks were measured after the addition of NaOH to the samples. Turnover of Hip–His–Leu with recombinant ACE enzyme (Sigma–Aldrich, Taufkirchen, Germany) was used as positive control. All experiments were performed in duplicates. ACE activity was related to tissue protein measured with the Bradford method.

2.9 Semiquantitative RT-PCR
Total RNA from wild-type and p47phox – / – mice tissue samples was isolated using TriFast-Reagent (peqLAB, Erlangen, Germany). RNA was reverse transcribed using Superscript reverse transcriptase (Invitrogen, Karlsruhe, Germany), oligo(dT) primers and dNTPs. The products were amplified using Taq DNA polymerase (Invitrogen, Karlsruhe, Germany). PCR for was carried out for angiotensinogen (25 cycles), for renin (34 cycles), for ACE (34 cycles), for AT1 receptor (27 cycles), for AT2 receptor (38 cycles), for bradykinin B1 receptor (35 cycles) and for bradykinin B2 receptor (35 cycles) and normalized to 18S rRNA (18 cycles). Oligonucleotides were obtained from MWG Biotech (Ebersberg, Germany). Sequences are available upon request. PCR products were separated by 1% agarose gel electrophoresis and quantified densitometrically using a Gel Doc image analysis system (Bio-Rad).

2.10 Measurement of reactive oxygen species (ROS)
ROS-generation in SMCs from wild-type and p47phox – / – mice was measured as hydrogen peroxide formation generated in equimolar amounts from superoxide. Radicals were monitored by fluoroscopy (Fluoroscan, Labsystems, Helsinki, Finland) using DCFH-DA (2',7'dichlorofluorescin diacetate) to DCF conversion [22]. SMCs were incubated with DCFH-DA (5 µmol/L) and DCF fluorescence was monitored for the indicated time.

Superoxide generation in kidney homogenates was measured from wild-type and p47phox – / – mice containing 10 µg protein using a lucigenin (bis-N'methylacridinium nitrate)-enhanced chemiluminescence (CL) technique [23]. Hydroperoxide generation in the same samples was measured using a peroxidase-coupled luminol-enhanced CL technique [23]. Light emissions were detected in a luminometer (Biolumat LB 9505; Berthold, Wildbad, Germany). The final concentrations of lucigenin and luminol used were 0.23 mM and 0.15 mM, respectively, dissolved in hepes bicarbonate buffer (99 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 20 mM Na–Hepes and 10 mM Glucose). Chemiluminescence was recorded and averaged over a 5 min period. The specific CL signal was expressed as counts per minute minus the average background activity.

2.11 Measurement of NAD(P)H and xanthine oxidase activity by electron spin resonance (ESR) spectroscopy
Activity of the NAD(P)H and the xanthine oxidase was determined in kidney and aorta tissue samples from wild-type and p47phox – / – mice (10 µg protein) by ESR spectroscopy as described previously [24] by using the spin trap 1-hydroxy-3-carboxypyrrolidine and a MiniScope ESR spectrometer (Magnettech, Berlin, Germany). The intensity of ESR spectra was quantified after subtraction of the ESR signal of samples without NAD(P)H and xanthine, respectively.

2.12 Western blot
Renal protein extracts (50 µg) from wild-type and p47phox – / – mice were separated by denaturing SDS (10%) PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences). Transferred proteins were probed with a mouse monoclonal eNOS antibody (1:1000, BD Bioscience, Pharmingen, Germany) and visualized using a horseradish peroxidase conjugated secondary anti-mouse antibody (1:3.500, Amersham Biosciences) and ECL solution. Equal protein loading was verified by reprobing the membrane with a mouse monoclonal GAPDH antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA).

2.13 Tempol, ACE-inhibitor and AT1-inhibitor administration
8 week old wild-type and p47phox – / – mice were treated with the radical scavenger tempol (4-hydroxy-Tempo, Sigma–Aldrich, Taufkirchen, Germany) with a dosage of 15 mg/kg body weight i.p. for 21 days [25], with the ACE-inhibitor quinapril (Pfizer, Karlsruhe, Germany) or the AT1-receptor antagonist telmisartan (Boehringer Ingelheim, Germany) both with a dosage of 10 mg/kg body weight by gavage for 5 days [26,27]. Systolic blood pressure was monitored daily by the tail cuff technique as described above.

2.14 Statistical analysis
Results are expressed as the mean±SEM. Comparisons were made by the unpaired t test, 1-way ANOVA, or a repeated-measures ANOVA as appropriate. Differences were considered statistically significant at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Infectious state of p47phox – / – mice
To exclude infections due to targeted disruption of the p47phox gene physical health of p47phox – / – mice was examined. Sections of lung tissue from 12 week old p47phox – / – mice maintained under antibiotic prophylaxis (Co-trimoxazole; 30 mg/kg) were free of granuloma (Fig. 1A and B). Determination of plasma endotoxin activity at the same time revealed no significant differences between p47phox – / – and wild-type mice (0.137±0.022 EU/mL vs. 0.156±0.032 EU/mL, P=0.16, n=10). A screening for pathogens was without pathological findings in p47phox – / – mice (health monitoring report). In contrast, a small colony (n=6) maintained for control studies without antibiotic prophylaxis showed lung granuloma formation after 12 weeks (Fig. 1C). Similar observations were made in 47phox – / – mice older than 20 weeks despite adequate antibiotic prophylaxis (data not shown). The NAD(P)H oxidase agonist arachidonic acid (5 µmol/L) [28] induced a fast ROS formation in SMCs from wild-type mice measured as DCF fluorescence. Contrary, agonist-induced ROS formation is completely blunted in SMCs from p47phox – / – mice proofing the functionality of p47phox deletion (Fig. 2).


Figure 1
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  		Fig. 1 Histology of lungs from 12 week old p47phox – / – and wild-type mice. A. Wild-type maintained with antibiotic prophylaxis. B. p47phox – / – maintained with antibiotic prophylaxis. C. p47phox – / – maintained without antibiotic prophylaxis. Sections from the right lungs were fixed in 4% formalin, embedded in paraffin and stained with hematoxylin/eosin. Data are representative of 3 different animals in each group. G=granuloma.

 

Figure 2
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  		Fig. 2 Agonist-induced ROS production in SMCs from p47phox – / –and wild-type mice. Time-dependency of DCF fluorescence as a measure of superoxide generation was determined in SMCs following arachidonic acid (5 µmol/L) stimulation. A representative graph of three independent experiments is shown.

 
3.2 Effect of p47phox deficiency on blood pressure, endothelial function and cardiac hypertrophy
Blood pressure monitoring in wild-type mice from week 6 up to week 12 revealed normotensive pressure as expected (112.2±2.6 mmHg, n=16). Intriguingly, p47phox – / – mice of the same age showed significantly elevated blood pressure levels (136.0±3.0 mmHg, P<0.001 vs. wild-type, n=16) during the observation period (Fig. 3A). Likewise, significant differences in blood pressure are also present in 6 week old litter mates (p47phox – / –: 131.7±2.6 mmHg, WT: 109.2±5.4 mmHg, P<0.01, n=6–7) proving the genetic influence of the p47phox gene on blood pressure. Notably, blood pressure levels of p47phox – / – mice slowly decreased by age between week 20 and 60 post partum (Fig. 3B). Focusing on younger animals in the following we studied vascular relaxation in aortic rings from 12 week old wild-type and p47phox – / – mice. Endothelium-dependent relaxation to acetylcholine was significantly attenuated in p47phox – / – mice (P<0.005 vs. wild-type, n=11) (Fig. 3C). Cardiac parameters were determined by heart weight/body weight ratio and by echocardiography. p47phox – / – mice did not show cardiac hypertrophy or altered left ventricular function at the end of the observation period 12 weeks post partum. Determination of heart weight/body weight ratio, interventricular septum thickness, left ventricular (LV) fractional shortening, LV ejection fraction and LV end-diastolic/systolic diameter revealed no significant differences between wild-type and p47phox – / – mice (Table 1).


Figure 3
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  		Fig. 3 A. Blood pressure and endothelium-dependent relaxation in p47phox – / – and wild-type mice. B. Blood pressure monitoring in p47phox – / – and wild-type mice between week 6 and week 12. Systolic blood pressure was monitored by tail cuff technique three times per week. *P<0.001 vs. wild-type, n=16 A. Age-dependent development of systolic blood pressure in p47phox – / – and wild-type mice between week 20 and week 60. **P<0.01 vs. wild-type, *P<0.05 vs. wild-type, n=3–5 per group C. Endothelium-dependent relaxation to acetylcholine (ACh) in aortic rings from 12 week old p47phox – / – and wild-type mice. *P<0.01 p47phox – / – vs. wild-type, n=11.

 

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  		Table 1 Determination of cardiac parameters of 12 week old p47phox – / – and wild-type mice by heart weight/body weight ratio and by echocardiography (LV=left ventricular)

 
3.3 Role of p47phox deficiency for the renin–angiotensin system
Analysis of plasma samples showed elevated renin activity in 12 week old p47phox – / – mice compared to wild-type mice of the same age (14.5±1.8 ng/mL/h vs. 9.6±1.7 ng/mL/h, P<0.05, n=10) (Fig. 4A). In addition, ACE activity in tissue samples of 12 week old p47phox – / – mice was found to be significantly enhanced in the kidney (24.9±3.9 vs. 20.7±4.1 nmol/L His–Leu/mg protein, P<0.05, n=5) and aorta (7.6±0.8 vs. 4.8±0.9 nmol/L His–Leu/mg protein, p<0.05, n=5) compared to coeval wild-type mice as measured by Hip–His–Leu turnover (Fig. 4B). The tissue-specific mRNA expression of RAS components and the receptors for bradykinin was investigated by semiquantitative RT-PCR normalized to the expression of 18S rRNA. No differences in mRNA expression were found for angiotensinogen in the liver, renin in the kidney, ACE in the kidney and aorta and AT1 receptor, AT2 receptor, bradykinin B1 receptor and bradykinin B2 receptor in the kidney between p47phox – / – and wild-type mice at the age of 12 weeks (P=n.s., n=6–8) (Table 2).


Figure 4
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  		Fig. 4 Renin and ACE activity in p47phox – / – and wild-type mice. A. Measurement of plasma renin activity of 12 week old p47phox – / – and wild-type mice by radioimmunoassay. *P<0.05 p47phox – / – vs. wild-type, n=10. B. ACE activity of kidney and aorta tissue samples from 12 week old p47phox – / – and wild-type mice measured as Hip–His–Leu turnover. *P<0.05 p47phox – / – vs. wild-type, n=5–10.

 

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  		Table 2 Tissue-specific mRNA expression of RAS components and the bradykinin B1/B2 receptors in p47phox – / – and wild-type mice

 
3.4 Impact of p47phox deficiency on renal ROS production
Kidney samples from 12 week old p47phox – / – mice were investigated with regards to their ROS levels and compared to wild-type mice of the same age. Surprisingly, they did not show any differences in superoxide or in hydroperoxide formation (P=n.s., n=5) measured by lucigenin- and luminol-enhanced chemiluminescence, respectively (Fig. 5A and B). Accordingly, there was no significant difference in renal NAD(P)H and xanthine oxidase activity in these samples (P=n.s., n=6) determined by ESR spectroscopy (data not shown). In addition, renal samples from both groups exhibited comparable eNOS protein expression levels (n=8) investigated by Western blot (data not shown).


Figure 5
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  		Fig. 5 Renal ROS production in p47phox – / – and wild-type mice. A. Lucigenin-enhanced chemiluminescence as a measure of superoxide production was determined in kidney samples of 12 week old p47phox – / – and wild-type mice. P=n.s., n=5. B. Luminol-enhanced chemiluminescence as a measure of hydroperoxide production was determined in kidney samples of 12 week old p47phox – / – and wild-type mice. P=n.s., n=5.

 
3.5 Effects of tempol administration, ACE- and AT1-receptor blockage on blood pressure in p47phox – / – mice
The effect of antioxidative treatment on the blood pressure of 8 week old p47phox – / – mice and wild-type mice was investigated using the radical scavenger tempol (4-hydroxy-Tempo) with a daily dose of 15 mg/kg body weight. Importantly, elevated systolic blood pressure levels in p47phox – / – mice could not be rescued by 21 days of tempol treatment (p47phox – / – mice: 141.3±8.8 mmHg, wild-type mice: 109.8±5.4 mmHg, P<0.001 vs. wild-type, n=6) (Fig. 6A). RAS blockage demonstrated that blood pressure elevation in p47phox – / – mice was RAS-dependent. Administration of the ACE-inhibitor quinapril and the AT1-receptor-inhibitor telmisartan in 8 week old p47phox – / – mice with a twice daily dose of 10 mg/kg body weight applied by gavage reduced blood pressure to values of identically treated wild-type mice (Fig. 6B and C).


Figure 6
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  		Fig. 6 Effects of tempol treatment, ACE-and AT1-receptor blockage on blood pressure in p47phox – / – and wild-type mice. A. Systolic blood pressure in 8 week old p47phox – / – and wild-type mice after the treatment with the radical scavenger tempol (15 mg/kg body weight per day, i.p.). *P<0.05 p47phox – / – vs. wild-type, n=6. Arrow indicates time of tempol administration. B. Systolic blood pressure in 8 week old p47phox – / – and wild-type mice after the treatment with the ACE-antagonist quinapril (2 x 10 mg/kg body weight per day, by gavage). *P<0.05 p47phox – / – vs. wild-type, n=8. Arrow indicates time of quinapril administration. C. Systolic blood pressure in 8 week old p47phox – / – and wild-type mice after the treatment with the AT1-receptor antagonist telmisartan (2 x 10 mg/kg body weight per day, by gavage). *P<0.05 p47phox – / – vs. wild-type, n=8. Arrow indicates time of telmisartan administration.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrates that p47phox deficiency leads to RAS activation and blood pressure elevation in a ROS-independent manner. Elevated blood pressure in p47phox – / – mice is driven by an activated RAS without involving increased renal ROS formation.

Targeted disruption of the p47phox gene locus is a model of the human chronic granulomatous disease, an immune deficiency resulting from impaired phagocyte activity [20]. The disease is characterized by recurrent life-threatening bacterial and fungal infections and tissue granuloma formation. Identical to the human disease p47phox – / – mice housed without specific pathogen-reduced conditions and antibiotic prophylaxis developed lethal infections and granulomatous inflammation, especially in the lungs. To exclude any kind of infections we carefully investigated the p47phox – / – mice with regards to lung granuloma, plasma endotoxin activity and pathogens in the observation period of our studies. Importantly, we did not find any hint pointing to increased infections in p47phox – / – mice in the observation period between week 6 and 12 suggesting a non-pathogen related cause for the present observations. Notably, since older p47phox – / – mice showed lung granuloma formation in spite of antibiotic prophylaxis all studies were performed in young animals.

Despite RAS activation and blood pressure elevation we did not observe cardiac hypertrophy in p47phox – / – mice. Determination of heart weight/body weight ratio and echocardiography revealed no differences in comparison to wild-type hearts thus no other cardiac hypertrophy markers such as myocyte cross-section area or atrial natriuretic peptide expression were assessed. Our observations in p47phox – / – mice were supported by the finding that a different NAD(P)H oxidase knock-out model – gp91phox – / – – is protected against cardiac hypertrophy induced by Ang II [29]. Moreover, in p47phox – / – mice we did not find different tissue mRNA expression levels of components of the RAS, including the AT2 receptor and additionally the bradykinin B1 and B2 receptor suggesting that RAS activation in p47phox – / – mice is not attributed to altered gene expression.

The vasculature and the kidney are rich sources of NAD(P)H oxidase-derived ROS which are critically involved in the formation of oxidative stress induced by an activated RAS. Focussing on the kidney which is the exclusive location of renin synthesis and a major source of ACE synthesis enhanced renal oxidative stress plays an important role in the regulation of blood pressure. NAD(P)H oxidases are expressed in renal vasculature, interstitium, juxtaglomerular apparatus, and the distal nephron [30]. Enhanced NAD(P)H oxidase activity and augmented oxidative stress contributed to hypertension in many animal models, including Ang II infusion, renovascular hypertension, the Dahl salt-sensitive (S) model of hypertension, the deoxycorticosterone acetate (DOCA) salt model of hypertension, and obesity-related hypertension. In most of these models, inhibition of oxidative stress lowered blood pressure [7]. For instance, administration of the Dahl S rats with tempol, a superoxide mimetic, decreased renal superoxide production and systolic blood pressure [31]. Likewise, treatment of the DOCA salt rats with apocynin, a NAD(P)H oxidase inhibitor, decreased superoxide production and systolic blood pressure [32]. In addition, administration of the synthetic peptide gp91ds-tat that inhibits p47phox association with gp91phox/Nox2 blocked superoxide production and blood pressure elevation in Ang II-induced hypertension in mice [33].

Recent studies from several groups have demonstrated an essential role for p47phox in the activation of vascular NAD(P)H oxidase by several agonists [13–15,18,33,34]. At the same time it is obvious that p47phox is not essential for NAD(P)H oxidase-dependent ROS production in the absence of agonists [13–15]. Importantly, it was previously reported that p47phox seems to have an inhibitory role under basal conditions, such that vessels from p47phox deficient mice have slightly increased ROS production, important for endothelium-dependent vascular relaxation and the activation of the MAPK cascade [16]. Similarly, we observed an impaired endothelium-dependent vascular relaxation in p47phox – / – mice. However, in contrast to the vasculature we did not observe alterations in renal oxidative stress involved in RAS activation. Neither ROS levels nor NAD(P)H oxidase activity, xanthine oxidase activity or eNOS expression were enhanced in the kidney of p47phox – / –. Thus, the increase in serum renin activity and renal ACE activity and subsequent elevation of blood pressure cannot be attributed to increased oxidative stress in the kidney.

Ang II administration caused an up to 3-fold increase of vascular superoxide production in wild-type mice which was completely blunted in p47phox – / – mice. In addition, the blood pressure response to Ang II was markedly attenuated in these mice, demonstrating an important role of p47phox-containing NAD(P)H oxidase for Ang II-dependent blood pressure elevation [18]. Potentially, p47phox-dependent RAS activation observed in our study caused in part by impaired responsiveness to Ang II. Interestingly, p47phox – / – mice at the age of 6 to 8 months used in the study of Landmesser et al. showed normal basal blood pressure levels prior to Ang II infusion. These findings are in concert with our observations because we observed a slow decrease in blood pressure by age in p47phox – / – mice starting around week 25 of life time. There is one more work on blood pressure in p47phox – / – mice observing comparable levels under basal conditions but without specifying the age of the mice used in this study [13]. Thus, blood pressure elevation appears to be confined to younger animals suggesting an age-dependent adaptation process counterbalancing blood pressure elevation caused by p47phox deficiency. The underlying mechanism seems to be very complex since older p47phox – / – mice exhibited lung granuloma formation despite antibiotic prophylaxis. Note, pathogen defense in p47phox – / – mice is impaired due to diminished oxidative burst. It's tempting to speculate that older p47phox – / – mice develop due to increased infections septic symptoms finally lead to vascular relaxation and blood pressure normalization.

Based on our observations we postulate a link between p47phox and RAS activation leading to blood pressure elevation. Recent studies reported that the Dahl S rat model of hypertension is associated with augmented renal oxidative stress and renal angiotensinogen expression and Tempol treatment decreased renal oxidative stress, renal angiotensinogen expression and blood pressure [35,36]. Obviously, the influence of p47phox on RAS activation is completely different in our model; first p47phox – / – mice did not show enhanced renal oxidative stress and second we did not detect increased angiotensinogen expression in the liver of p47phox – / – mice. But, we measured enhanced renin and ACE activity in our model. This points to a crucial role of p47phox – beyond its involvement in the NAD(P)H oxidase assembly – in RAS activation and blood pressure regulation. Yeast two-hybrid screens yielded the signaling protein TRAF4 and the NF-{kappa}B family member RelA as a functional binding partner for p47phox [37,38] In this regard, RelA has been shown to be important for RAS activation [39]. To further investigate the underlying mechanism specific genetically models of p47phox deficiency are necessary. Such as, tissue-specific conditional knock-outs for p47phox, i.e. in the vasculature, kidney or liver.

There is an ongoing discussion to use radical scavengers in general and specific NAD(P)H oxidase inhibitors in particular to treat patients with hypertension. At least in the case of p47phox great caution is needed because p47phox failure itself led to blood pressure elevation.

In summary, the present study indicates that the NAD(P)H oxidase subunit p47phox plays a pivotal role in the control of blood pressure. Deficiency of p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner. Further studies are necessary to elucidate the underlying molecular mechanism linking p47phox to RAS activation and blood pressure elevation.


   Acknowledgements
 
This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich SFB 566/B9 and Schie 386/7-2. We are indebted to Dr. Elvan Akin for the technical assistance in establishing non-invasive blood pressure measurements. We thank Tanja Sander and Silke Pretzer for the excellent technical assistance.


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 34 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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