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Abstract
Myogenic and angiotensin contractions of afferent arterioles generate reactive oxygen species. Resistance vessels express neutrophil oxidase-2 and -4. Angiotensin II activates p47phox/neutrophil oxidase-2, whereas it downregulates NOX-4. Therefore, we tested the hypothesis that p47phox enhances afferent arteriolar angiotensin contractions. Angiotensin II infusion in p47phox +/+ but not −/− mice increased renal cortical NADPH oxidase activity (7±1–12±1 [P<0.01] versus 5±1–7±1 103 · RLU · min−1 · μg protein−1 [P value not significant]), mean arterial pressure (77±2–91±2 [P<0.005] versus 74±2–77±1 mm Hg [P value not significant]), and renal vascular resistance (7.5±0.4–10.1±0.7 [P<0.01] versus 7.9±0.4–8.3±0.4 mm Hg/mL · min−1 · gram kidney weight−1 [P value not significant]). Afferent arterioles from p47phox −/− mice had a lesser myogenic response (3.1±0.4 versus 1.4±0.2 dynes · cm−1 · mm Hg−1; P<0.02) and a lesser (P<0.05) contraction to 10−6 M angiotensin II (diameter change +/+: 9.3±0.2–3.4±0.6 μm versus −/−: 9.9±0.6–7.5±0.4 μm). Angiotensin and increased perfusion pressure generated significantly (P<0.05) more reactive oxygen species in p47phox +/+ than −/− arterioles. Angiotensin II infusion increased the maximum responsiveness of afferent arterioles from p47phox +/+ mice to 10−6 M angiotensin II yet decreased the response in p47phox −/− mice. The angiotensin infusion increased the sensitivity to angiotensin II only in p47phox +/+ mice. We conclude that p47phox is required to enhance renal NADPH oxidase activity and basal afferent arteriolar myogenic and angiotensin II contractions and to switch afferent arteriolar tachyphylaxis to sensitization to angiotensin during a prolonged angiotensin infusion. These effects likely contribute to hypertension and renal vasoconstriction during infusion of angiotensin II.
Introduction
Blockade of angiotensin II (Ang II) reduced the blood pressure (BP) and renal vascular resistance (RVR) in many models of hypertension1 and in humans with essential or renovascular hypertension.2 Afferent arteriolar contractions with Ang II increase the RVR and reduce the transmission of arterial pressure into the kidney, which may protect the glomeruli from potential barotrauma.3
Ang II increases reactive oxygen species (ROS) in the afferent arteriole4–6 and the kidney,7,8 which increase the vascular contractility and the RVR.4–6,9–18 ROS are implicated in Ang II responses, because Tempol18 reduced the hypertension and the renal vasoconstriction of mice infused with Ang II at a slow pressor rate,7 which is considered a model of human essential hypertension.19
NADPH oxidase has been implicated in Ang II–induced increases in ROS.12,15,16,20,21 However, NADPH oxidase is a complex enzyme, and ≥2 neutrophil oxidases (NOX-2 and -4) are expressed in rodent microvessels with different regulation8,9,12,20 and activation by cytosolic subunits.20 NOX-2 is a prominent oxidase in small blood vessels and glomeruli where it interacts with p22phox in the membrane and p47phox, p67phox, p40phox, and Rac2 from the cytosol.12 Ang II reduced the expression of NOX-48 but increased the afferent arteriolar mRNA expression for p22phox4 and increased the vascular membrane association12,22–24 and c-Src–dependent phosphorylation and activation of p47phox,13 which assembled with p22phox, NOX-2, and other cytosolic subunits to form a functional membrane oxidase.12 Although knockout of p47phox −/− attenuated the increase in ROS9–11 and rate of rise of BP14,25 with pressor infusions of Ang II and attenuated large vessel myogenic responses,14 prolonged Ang II infusion at a slow pressor rate did not change p47phox expression in the kidney,8,26 and p47phox −/− mice had a maintained,25 or even increased,9,10,14,27 basal level of superoxide (O2·−) generation in blood vessels, kidneys, and vascular smooth muscle cells (VSMCs). Moreover, p47phox −/− mice had a normal basal BP.11,14,25,27 Thus, the role of p47phox in basal and Ang II–stimulated regulation of BP and RVR is not completely understood. We tested the hypothesis that p47phox is required for full afferent arteriolar contractions to Ang II and perfusion pressure (myogenic responses) and for increases in mean arterial pressure (MAP) and RVR in mice receiving a slow pressor (low-dose) infusion of Ang II by contrasting responses in p47phox +/+ versus −/− mice.
Methods
Experimental Design
Male p47phox +/+ and −/− littermate mice aged 10 to 14 weeks were bred from +/− founders and backcrossed ≥8 times to the C57BL/6 background.14 Because these mice are prone to infection, we followed the advice of our veterinarians that they be individually housed with precautions to minimize infection and with trimethoprim/sulfamethoxazole added to the drinking water for 7 days, followed by 3 days without antibiotics, as in a previous study.27 They were fed a normal mouse chow with a regular salt content of 0.4%.
Groups of p47phox +/+ and −/− mice (n=6–7) were anesthetized with isofluorane (1% to 2% in O2) 2 weeks before experiments. Radiotelemeters were inserted into a carotid artery.26 Basal recordings of MAP were made for 4 days, after which mice were anesthetized with isofluorane for insertion of osmotic minipumps (ALZET Corp, DURECT Corp, Cupertino, CA) to deliver Ang II (400 ng−1 · kg−1 · min−1) or vehicle subcutaneously for 2 weeks.7,28 Thereafter, the mice were euthanized and the kidney cortex was harvested. Cell membranes were separated to measure membrane-bound NADPH oxidase activity from lucigenin-enhanced chemiluminescence after the addition of 200 μmol · L−1 of NADPH, as described.26 Other mice (n=5–7 per group) were anesthetized with thiobarbital (Inactin 50 mg−1 · kg−1) and ketamine (40 mg−1 · kg−1) and prepared for renal clearance studies26 12 to 14 days after Ang II or vehicle. The glomerular filtration rate (GFR) was the clearance of [3H]inulin, and the renal plasma flow (RPF) was the clearance [14C]paraaminohippurate corrected for renal extraction. Renal blood flow (RBF) was RPF factored by 1 hematocrit, and RVR was MAP factored by renal blood flow.
Afferent Arteriolar Responses
After 12 to 14 days of Ang II or vehicle infusions, mice were anesthetized, the kidneys removed, and an afferent arteriole dissected and perfused.6 Contractions to bath addition of Ang II (10−12 to 10−6m) were recorded. The myogenic responses were studied in other arterioles during ≈20-mm Hg increases in renal perfusion pressure.29,30 The slope of active wall tension (difference between Ca2+-free and physiological solution) against perfusion pressure defined the myogenic response. Only 1 arteriole was used per animal. The inner luminal diameter and medial thickness were measured at 60-mm Hg perfusion pressure to compute the media:lumen ratio. The ROS generated in the afferent arterioles during incubation with 10−6m Ang II or during increases in renal perfusion pressure from 40 to 80 mm Hg were assessed from the ratio of ethidium:dihydroethidium fluorescence, as described and validated.6,30
Statistical Analysis
Values are presented as mean±SEM. Repeated-measures ANOVA was used to test concentration-dependent changes in MAP or arteriolar diameter. A 2×2 ANOVA with interaction was used to assess the effects of genotype, Ang II, and effects of genotype on the response to Ang II (interaction). Post hoc comparisons were performed using a Fisher test. Differences were considered to be statistically significant if P<0.05.
Ethics
The experiments were approved by the Georgetown University Animal Care and Use Committee. They conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Results
The mice were healthy and had similar body and kidney weight (Table S1, available in the online Data Supplement at http://hyper.ahajournals.org). Afferent arterioles from p47phox +/+ mice had readily detectable levels of p47phox mRNA, whereas it was absent from arterioles of p47phox −/− mice (Figure S1).
The basal levels of telemetric MAP were not different between strains, whereas the MAP measured during the daytime (Figure 1A) or nighttime (Figure 1B) increased after 2 to 3 days of Ang II infusion in p47phox +/+ mice but did not change with Ang II in p47phox −/− mice. The basal values of NADPH oxidase, MAP, renal blood flow, and RVR of vehicle-infused mice under anesthesia were similar in the 2 strains (Figure 2), but the GFR was lower in p47phox −/− mice (P<0.05). Two weeks of Ang II infusion in p47phox +/+ mice increased the NADPH oxidase (100±5–400±50 mm Hg; P<0.0001), the MAP (77±2–91±2 mm Hg; P<0.005), and the RVR (7.5±0.4–10.1±0.7 mm Hg/mL · min−1 · gram kidney weight−1; P<0.01). However, Ang II infusion in p47phox −/− mice did not change these variables. The basal afferent arteriolar diameter and the media:lumen ratio were similar in both strains and were unaffected by Ang II infusion (Table S2A).
Mean±SEM values for telemetric mean arterial pressure of p47phox +/+ (solid squares and continuous lines; n=6) and p47phox −/− (open circles and broken lines n=7) mice before and during angiotensin II (400 ng · kg−1 · min−1 SC) during the daytime (asleep) in A and the nighttime (awake) in B. Comparing groups: *P<0.05.
Mean±SEM values (n=6–9 per group) for mice studied under anesthesia showing renal cortical NADPH oxidase activity (A), arterial pressure (B), glomerular filtration rate (C), renal blood flow (D), and renal vascular resistance (E) in groups of p47phox +/+ and −/− mice given a vehicle (open boxes) or Ang II (400 · kg−1 · min−1 SC) for 12 days. Compared with equivalent p47phox +/+ mice: a, P<0.05.
Afferent arterioles isolated from vehicle-infused mice of both strains had graded contractions with Ang II (Figure 3). After vehicle infusion, afferent arterioles from p47phox −/− mice, compared with p47phox +/+ mice, had a 40% reduced maximum response (supplemental Table S2B). Two weeks of Ang II infusion in p47phox +/+ mice increased the Ang II sensitivity, whether assessed from the lowest Ang II concentration to cause a contraction (from 10−8m during vehicle to 10−11m with Ang II infusion; Figure 3B) or from the ED50 (Table S2B). In contrast, 2 weeks of Ang II infusion in p47phox −/− mice actually reduced the maximum response to 10−6m Ang II (Figure 3 and Table S2B) without changing the Ang II sensitivity.
Mean±SEM values (n=4–7) for afferent arteriolar diameter (A) and changes in diameter from baseline (B) during bath addition of angiotensin II to p47phox +/+ mice infused with vehicle (solid circles and continuous lines) or angiotensin II (open circles and broken lines) or p47phox −/− mice infused with vehicle (solid triangles and continuous lines) or angiotensin II (open triangles and broken lines). Significance of difference between groups: *P<0.05. Significance of difference from vehicle: a, P<0.05.
An increase in perfusion pressure of afferent arterioles >40 mm Hg from vehicle-infused p47phox +/+ mice led to a graded contraction (Figure 4A and 4B). There were no changes in passive wall tension assessed in the absence of calcium but an increase in active wall tension (Figure 4C). This myogenic response was reduced by 48% in arterioles from p47phox −/− mice (Figure 4 and Table S2A). Ang II infusion did not change myogenic responses is either strain. Ang II (10−6m) or increasing perfusion pressure (40–80 mm Hg) both increased afferent arteriolar ROS more in afferent arterioles from p47phox +/+ than −/− mice (Figure 5).
Mean±SEM values (n=4–7) for afferent arteriolar diameter (A), change in diameter (B) and active wall tension (C) during increases in renal perfusion pressure. Data are shown for vessels from p47phox +/+ mice infused with vehicle (solid circles and continuous lines) or angiotensin II (open circles and broken lines) or p47phox −/− mice infused with vehicle (solid triangles and continuous lines) or angiotensin II (open triangles and broken lines). Significance of difference between groups: *P<0.05.
Mean±SEM values (n=5–7) for increases in ethidium: dihydroethidium ratio of afferent arterioles from p47phox +/+ mice (solid black) and p47phox −/− mice (open box). Data are shown for changes with 10−6m angiotensin II or increased perfusion from 40 to 80 mm Hg.
Discussion
The main new findings were the p47phox −/− mice had a normal basal renal cortical NADPH oxidase activity, MAP, and RVR under anesthesia and a normal conscious MAP during the daytime or nighttime but a lower GFR. However, unlike the p47phox +/+ mice, the NADPH oxidase activity, MAP, and RVR of p47phox −/− mice failed to increase during a slow pressor infusion of Ang II. Afferent arterioles from p47phox −/− mice had a 38% reduced maximum responsiveness to Ang II and a 48% reduced myogenic response. Ang II and increased perfusion pressure increased afferent arteriolar ROS, but these responses were reduced in p47phox −/− mice. Although Ang II infusion enhanced the maximum Ang II responsiveness of afferent arterioles from p47phox +/+ mice by 26% and enhanced their sensitivity 1000-fold, Ang II infusion in p47phox −/− mice actually reduced the maximum response of their arterioles to Ang II by 26% and did not change their sensitivity.
The normal basal NADPH oxidase activity in the kidney cortex from p47phox −/− mice confirms a previous study.27 Basal O2·− generation from cultured VSMCs9,11 and from aortas of p47phox −/− mice has been unchanged14 or increased.10,27 Apparently, p47phox is not required for basal O2·− generation. The source of the p47phox-independent basal O2·− generation in this study was not established but likely was from the NOX-4 component of NADPH oxidase, because it does not require p47phox for activity, and in VSMCs, NOX-4 is the major source of basal ROS but does not contribute to Ang II–stimulated ROS generation.31
Grote et al27 reported a higher basal MAP in young p47phox −/− mice that they attributed to an enhanced aortic angiotensin-converting enzyme activity, although angiotensin-converting enzyme gene expression in the kidney was unchanged. However, the absence of any rise in MAP with Ang II infusion in the p47phox −/− mice in our study suggests that excessive Ang II generation by angiotensin-converting enzyme likely did not account for altered responses of p47phox −/− mice in our study. Moreover, we did not confirm the difference in basal MAP between p47phox +/+ and −/− mice.
Unexpectedly, the p47phox −/− mice had lower GFRs in the basal state and during Ang II infusion. This contrasts with the effects of Tempol to increase the GFR of mice during a slow pressor infusion of Ang II.7 Because both Tempol7 and p47phox knockout prevented Ang II–induced oxidative stress, it suggests that the effects of p47phox −/− on the GFR may be dissociated from its antioxidant actions. We detected prominent expression of p47phox in the podocytes of rats,32 where it could regulate the GFR, although the mechanism is unclear. There were no differences in basal MAP between p47phox +/+ and −/− mice to explain the lower basal GFR in the p47phox −/− mice.
Previous studies in dogs and rats have reported that Ang II infusion did not change33 or diminish the myogenic components of renal autoregulation.34–37 This is the first direct study in isolated afferent arterioles. We detected no effect of 2 weeks of Ang II infusion on myogenic responses. This difference from the previous studies may relate to interactions between myogenic responses and tubuloglomerular feedback that were conducted in whole kidneys, because tubuloglomerular feedback responses are enhanced by Ang II infusion,38 and this can impair myogenic responses.39 The present study removed any confounding effects of tubuloglomerular feedback by studying isolated arterioles.
Our finding that Ang II infusion increased the Ang II responsiveness of afferent arterioles from p47phox +/+ mice confirms previous studies in rabbits,4 where the enhanced response was accompanied by a significant increase in the mRNA expression for p22phox but a significant reduction in mRNA expression for angiotensin type I receptors in the afferent arterioles. This suggests that enhanced responsiveness to Ang II in afferent arterioles of rodents infused with Ang II is attributed to activation of NADPH oxidase by p22phox and phosphorylated p47phox that enhances ROS generation. In the absence of this enhancing effect of ROS, the effects of Ang II to downregulate Ang type 1 receptors may become apparent as tachyphylaxis and an impaired response to Ang II.
Because oxidative stress determined BP in many rodent models,18 our findings of an unchanged basal level of NADPH oxidase activity in the kidneys may explain the unchanged basal MAP and RVR in p47phox −/− mice. Ang II infusion increased the excretion of lipid peroxidation products,7 ROS generation in kidneys7 and in afferent arterioles of normal mice.6 Indeed, NADPH oxidase is a major source of O2·− in VSMCs,15 kidneys of rats infused with Ang II at a slow pressor rate,8 and the NADPH oxidase activity increased 4-fold with Ang II infusion in p47phox +/+ mice in this study. Because the NADPH oxidase activity was unchanged in the renal cortex of p47phox −/− mice infused with Ang II, we concluded that p47phox was required for NADPH oxidase activation in the kidneys by Ang II. This extends previous reports of a reduced generation of O2·− in VSMCs or aortas from p47phox −/− mice during stimulation with arachidonic acid,27 Ang II,10,11,14,25 platelet-derived growth factor, or phorbol myristate acetate.11 In another study, p47phox −/− mice had a preserved, albeit delayed, increase in MAP in response to a direct pressor infusion of Ang II.14 This suggests that O2·− derived from p47phox-dependent NADPH oxidase sets the sensitivity of the BP response to Ang II, whereas other actions of Ang II contribute to its maximal response. This is consistent with the substantial increase in sensitivity of afferent arterioles from p47phox +/+ mice to Ang II produced by an Ang II infusion, which was entirely lacking in those from p47phox −/− mice.
Perspectives
Studies from gene-deleted mice have highlighted the importance of ROS in the regulation of BP. Superoxide dismutase 1−/− mice had an enhanced slow pressor response to Ang II, their afferent arterioles generated more ROS, and they had a 10 000-fold increased sensitivity to Ang II.6 The present studies demonstrated the essential role of p47phox in O2·− generation and in the hypertension and renal vasoconstriction with a low-dose infusion of Ang II. Oxidative stress underlies many adverse cardiovascular and renal effects of Ang II.17,18,40 Renal vasoconstriction is implicated in the development of hypertension.41 Thus, p47phox is an attractive target to prevent hypertension and some of its complications.
Sources of Funding
This study was supported by research grants to C.S.W. and W.J.W. from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-036079 and DK-049870); and to C.S.W., W.J.W., A.W., and K.S. from the National Heart, Lung, and Blood Institute (HL-68686); and by funds from the George E. Schreiner Chair of Nephrology.
Disclosures
None.
Acknowledgments
We thank Glenda Baker for preparing and editing the article.
- Received October 3, 2011.
- Revision received October 28, 2011.
- Accepted November 22, 2011.
- © 2012 American Heart Association, Inc.
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