Renal Redox-Sensitive Signaling, but Not Blood Pressure, Is Attenuated by Nox1 Knockout in Angiotensin II–Dependent Chronic Hypertension
We demonstrated previously that, in mice with chronic angiotensin II–dependent hypertension, gp91phox-containing NADPH oxidase is not involved in the development of high blood pressure, despite being important in redox signaling. Here we sought to determine whether a gp91phox homologue, Nox1, may be important in blood pressure elevation and activation of redox-sensitive pathways in a model in which the renin-angiotensin system is chronically upregulated. Nox1-deficient mice and transgenic mice expressing human renin (TTRhRen) were crossed, and 4 genotypes were generated: control, TTRhRen, Nox1-deficient, and TTRhRen Nox1-deficient. Blood pressure and oxidative stress (systemic and renal) were increased in TTRhRen mice (P<0.05). This was associated with increased NADPH oxidase activation. Nox1 deficiency had no effect on the development of hypertension in TTRhRen mice. Phosphorylation of c-Src, mitogen-activated protein kinases, and focal adhesion kinase was significantly increased 2- to 3-fold in kidneys from TTRhRen mice. Activation of c-Src, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and focal adhesion kinase but not of extracellular signal regulated kinase 1/2 or extracellular signal regulated kinase 5, was reduced in TTRhRen/Nox1-deficient mice (P<0.05). Expression of procollagen III was increased in TTRhRen and TTRhRen/Nox1-deficient mice versus control mice, whereas vascular cell adhesion molecule-1 was only increased in TTRhRen mice. Our findings demonstrate that, in Nox1-deficient TTRhRen mice, blood pressure is elevated despite reduced NADPH oxidase activation, decreased oxidative stress, and attenuated redox signaling. Our results suggest that Nox1-containing NADPH oxidase plays a key role in the modulation of systemic and renal oxidative stress and redox-dependent signaling but not in the elevation of blood pressure in a model of chronic angiotensin II–dependent hypertension.
In pathological conditions, angiotensin II (Ang II), through its vasoconstrictor, mitogenic, proinflammatory, and profibrotic actions, contributes to altered vascular tone, endothelial dysfunction, cardiovascular and renal remodeling, inflammation, and fibrosis, which are features of target organ damage in hypertension, atherosclerosis, vasculitis, and diabetes.1–4 The subcellular mechanisms and signaling pathways whereby Ang II mediates its effects are complex, but production of reactive oxygen species (ROS) and activation of redox-dependent signaling cascades are important.1–5
Redox-sensitive signaling results in numerous cellular responses, which, if uncontrolled, could contribute to hypertensive vascular and target-organ damage. In cardiac and renal tissue and in cultured vascular smooth muscle cells, we and others have shown that Ang II–mediated ROS generation modifies activity of tyrosine kinases, such as c-Src, Ras, JAK2, Pyk2, phosphatidylinositol 3-kinase, focal adhesion kinase (FAK), and epidermal growth factor receptor, as well as mitogen-activated protein kinases (MAPKs).2,6–8 ROS may also inhibit protein tyrosine phosphatase activity, further contributing to protein tyrosine kinase activation.9
Nonphagocytic reduced nicotinamide-adenine dinucleotide phosphate (NAD[P]H) oxidase, a major source of ROS in the cardiovascular and renal systems,10,11 is a multisubunit complex composed of 2 membrane-associated components, gp91phox (Nox2) and p22phox, 2 cytosolic components, p47phox and p67phox, and the small molecular weight protein rac-2.11,12 After the discovery of the first Nox2 homologue, Nox1, additional family members were rapidly identified and now include Nox3, Nox4, and Nox5.13,14 All of the vascular cell types, including endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts, as well as kidney fibroblasts, mesangial and tubular cells, and podocytes, express components of the prototypical NAD(P)H oxidase.11–15 In renal and cardiovascular cells Ang II is an important mediator of NAD(P)H oxidase-driven generation of ROS.15–18
We previously generated a transgenic mouse that produces human active renin in the liver under the control of the transthyretin promoter.19 These mice, termed TTRhRen, have slightly elevated plasma Ang II levels (1 to 2 times normal), which represent a better correlate of human hypertension than acute Ang II–infused models. They are also chronically hypertensive and have frank cardiac hypertrophy by 10 to 12 weeks of age.19 In TTRhRen mice, we demonstrated that pathophysiological outcomes of hypertension induced by Ang II through Ang II type 1 receptors are not affected by Nox2 knockout.20 Here, we sought to determine whether a homologue of Nox2, Nox1, plays a role in the activation of redox-sensitive pathways leading to the development of hypertension and renal damage in TTRhRen mice.
The study was approved by the animal ethics committee of the Clinical Research Institute of Montreal and the University of Ottawa. The generation of the TTRhRen mice was described previously.19 Briefly, the human prorenin cDNA was expressed primarily in the liver of transgenic mice under control of a 3-kb region of the transthyretin gene promoter. To generate active human renin, a cleavage site for the ubiquitous protease furin was inserted at the juncture of the prosegment and the active renin molecule, resulting in prosegment removal by endogenous proteases in the secretory pathway of expressing cells. TTRhRen mice are chronically hypertensive and exhibit frank cardiac hypertrophy by 10 to 12 weeks of age. The TTRhRen-A3 line was originally derived in the FVB strain of mice, but was transferred by back-crossing into the C57BL/6J strain for this study. The mice used for breeding in the experiments described were from the F5 back-cross generation and are referred to as TTRhRen. Nox1–deficient mice were generated by replacing exon 3 to 6 of the Nox1 gene in the wild-type allele with a pGK-neo cassette, as described previously.21 Because Nox1 gene is on the X chromosome, heterozygous females were crossed with C57BL/6 males to obtain Nox1-deficient mice (Nox1−/Y). Four genotypes of mice were generated: control, TTRhRen transgenic (TTRhRen), Nox1-deficient (Nox1−/Y), and TTRhRen transgenic Nox1-deficient (TTRhRen/Nox1−/Y). The genotype of cross-bred mice was verified by PCR analysis of tail DNA biopsy specimens, and 8 to 15 mice were genotyped.
Measurement of Blood Pressure
Systolic blood pressure was measured by tail-cuff plethysmography (model BP-2000, Visitech Systems) as described previously.20 In preliminary studies, we compared blood pressure readings by tail cuff and telemetry and did not find significant differences. Accordingly, in the present study, we used the tail cuff method. Mice were trained to the apparatus for 7 continuous days, and measurements were recorded for the last 3 days, as we detailed.20
Plasma Measurement of Thiobarbituric Acid-Reacting Substances
Blood was collected in EDTA, centrifuged, and plasma levels of thiobarbituric acid-reacting substances (TBARS) were measured by a colorimetric method. Briefly, plasma was mixed with 2% butylated hydroxytoluene and quintanilla reagent (26 mmol/L of thiobarbituric acid and 15% trichloroacetic acid). The mixture reaction was boiled for 15 minutes. Thereafter, the reaction mixture was cooled and centrifuged at 3000g for 10 minutes. The soluble phase was measured with a spectrophotometer at a wavelength of 535 nm. In parallel, malondialdehyde standards were diluted in the range of 0 to 4 μmol/L. TBARS values were expressed in nanomoles per milliliter of malondialdehyde equivalents.
Detection of Renal NADPH Oxidase Activity by Enhanced Lucigenin Chemiluminescence
The lucigenin-derived chemiluminescence assay was used to determine NAD(P)H oxidase activity in renal tissue homogenates 10% (wt/vol) prepared in phosphate buffer (20 mmol/L of KH2PO4, 1 mmol/L of EGTA, and protease inhibitors [pH 7.4]) with a glass-to-glass homogenizer. The reaction was started by the addition of NAD(P)H (0.1 mmol/L) to the suspension (250 μL of final volume) containing sample (50 μL), lucigenin (5 μmol/L), and assay buffer (50 mmol/L of KH2PO4, 1 mmol/L of EGTA, and 150 mmol/L of sucrose [pH 7.4]). Luminescence was measured every 1.8 seconds for 3 minutes in a luminometer (Orion Luminometer, Berthold detection systems). Buffer blank was subtracted from each reading. Activity was expressed as arbitrary units per milligram of protein. Protein concentrations were determined with protein assay reagent (Bio-Rad Laboratories).
Detection of Renal H2O2 Concentration
H2O2 concentration was measured using Amplex red (Molecular Probes). Amplex red is a fluorogenic substrate with very low background fluorescence; it reacts with H2O2 with a 1:1 stoichiometry to produce highly fluorescent resorufin. Briefly, Amplex red reagent (50 μmol/L) and horseradish peroxidase type II (0.1 U/mL) were added to renal tissue or a standard curve with known concentrations of H2O2. The samples were incubated for 30 minutes in 96-well microplates in the dark at room temperature, and the signal was analyzed according to manufacturer instruction. H2O2 concentrations of renal tissue were calculated on the basis the H2O2 standard curve.
Kidneys were used as follows. Frozen tissue was homogenized in lysis buffer A (50 mmol/L of Tris/HCl [pH 7.4], 5 mmol/L of EGTA, 2 mmol/L of EDTA, 0.1 mmol/L of PMSF, 1 mmol/L of pepstatin A, 1 mmol/L of leupeptin, and 1 mmol/L of aprotinin). Half of the homogenate was used for total fraction protein analysis. The remaining portion was used for membrane and cytosolic separation. Homogenates were centrifuged at 500g for 10 minutes, the nuclei-rich pellet discarded, and the supernatant fluid recentrifuged at 100 000g for 1 hour at 4°C. The supernatant fluid (cytosolic fraction) was removed, whereas the pellet, containing the particulate fraction, was resuspended in lysis buffer A containing 1% Triton X-100 and recentrifuged at 100 000g for 1 hour. The resultant supernatant fluid (now membrane-enriched fraction) was used. Total or fractionated proteins were separated by electrophoresis on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween for 1 hour at 24°C. Membranes were then incubated with specific antibodies overnight at 4°C. Phospho-antibodies were as follows: anti–c-Src (Tyr418), anti-p38MAPK (Thr180/Tyr182), anti-extracellular signal regulated kinase (ERK) 5 (Thr218/Tyr220), anti–stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK; Thr183/Tyr185), anti-ERK 1/2 (Thr202/Tyr204), and anti-FAK (Tyr576/577; Cell Signaling). The respective nonphospho-antibodies (1:2000) were also used: c-Src (Biosource), p38 MAPK, ERK 5, SAPK/JNK, ERK 1/2, and FAK (Cell Signaling). An antibody characterized previously was used to specifically recognize the NAD(P)H oxidase subunit, p47phox, and the cytoplasmic-associated subunit (clone 43.27).22 Anti- vascular cell adhesion molecule (VCAM)-1, anti-procollagen I, and anti-procollagen III were from Santa Cruz Biotechnology. After incubation with secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography, and quantified densitometrically. β-Actin and GAPDH were used as housekeeping proteins.
Data are presented as means±SEMs. Groups were compared using 1-way ANOVA or Student’s t test, as appropriate. Tukey correction was used to compensate for multiple testing procedures. P<0.05 was significant.
Systolic Blood Pressure
Consistent with our previous report, systolic blood pressure was significantly elevated in TTRhRen transgenic mice (117.5±4 mm Hg) versus control littermates (101.2±3 mm Hg; P<0.05).20 TTRhRen/Nox1−/Y mice also exhibited significantly elevated blood pressure (122.4±3 mm Hg) as compared with control counterparts (104.0±3 mm Hg), with the magnitude of increase being similar to that observed in TTRhRen mice. Systolic blood pressure was not different between Nox1−/Y and control mice.
Evaluation of Oxidative Stress
Systemic oxidative stress was evaluated by measuring concentrations of plasma TBARS (Figure 1A). Plasma TBARS levels were significantly increased (P<0.05) in TTRhRen transgenic mice compared with control mice. TTRhRen/Nox1−/Y mice did not display an increase in TBARS levels. To evaluate the functional impact of Nox1 deficiency, NAD(P)H oxidase activity was measured in renal tissue. Figure 1B shows that lucigenin-derived luminescence was significantly higher (P<0.05) in TTRhRen mice. Activation of NAD(P)H oxidase was not increased in TTRhRen/Nox1−/Y mice compared with controls. As shown in Figure 1C, p47phox translocation, critically involved in triggering activation of NADPH oxidase, was significantly increased in renal tissues from TTRhRen mice, as demonstrated by the increased ratio of p47phox in the particulate:soluble fractions. This was not apparent in Nox1-deficient mice. To further assess the renal redox state, we measured renal H2O2 levels in the different groups. As shown in Figure 2, H2O2 concentration was significantly elevated in the TTRhRen group compared with controls. In the double transgenic mice, renal H2O2 levels were significantly reduced compared with TTRhRen mice.
Nox1-Dependent c-Src Phosphorylation in TTRhRen Mice
Western blot analysis demonstrated that phosphorylation of c-Src was increased in kidneys from TTRhRen mice (Figure 3). c-Src phosphorylation was not significantly altered in Nox1−/Y and TTRhRen/Nox1−/Y mice.
Differential Regulation of MAPK Phosphorylation by Nox1 Deficiency in TTRhRen Mice
The effect of Nox1 deficiency on MAPK activation was assessed in TTRhRen mice. As shown in Figure 4A, kidneys from TTRhRen mice displayed increased p38MAPK phosphorylation as compared with controls, whereas TTRhRen/Nox1−/Y mice had similar levels of p38MAPK phosphorylation compared with Nox1−/Y mice. SAPK/JNK phosphorylation was also increased in TTRhRen mice, and no changes were observed in TTRhRen/Nox1−/Y mice (Figure 4B). On the other hand, phosphorylation of ERK 1/2 (Figure 4C) and ERK 5 (Figure 4D) was increased in kidneys from both TTRhRen and TTRhRen/Nox1−/Y mice.
FAK Phosphorylation and VCAM-1 Expression in TTRhRen and TTRhRen/Nox1−/Y Mice
FAK phosphorylation, critically involved in integrin signaling, cell-cell interaction, and inflammation, was increased in TTRhRen mice compared with control mice. Nox1 deficiency attenuated renal FAK phosphorylation (Figure 5A). VCAM-1 expression was also increased in TTRhRen mice, and this effect was not observed in TTRhRen/Nox1−/Y mice (Figure 5B).
Renal Expression of Procollagens I and III in TTRhRen and TTRhRen/Nox1−/Y Mice
To assess whether Nox1 influences profibrotic processes in TTRhRen mice, we investigated procollagen content in kidneys from the different groups. Procollagen I expression was not altered in TTRhRen nor in TTRhRen/Nox1−/Y mice (Figure 6A). Procollagen III expression was similarly increased in TTRhRen and TTRhRen/Nox1−/Y mice as compared with control and Nox1−/Y mice (Figure 6B).
Major findings from the present study demonstrate that in mice in which the renin-angiotensin system is chronically upregulated, Nox-1 deficiency blunts activation of NAD(P)H oxidase, reduces systemic and renal oxidative stress, and attenuates phosphorylation of redox-sensitive signaling molecules and expression of proinflammatory mediators in the kidney. These effects are not associated with reduction of blood pressure or with renal collagen content. Our results suggest that Nox1-containing NAD(P)H oxidase plays a key role in the modulation of specific redox signaling pathways, particularly those linked to inflammation, but not in the development of hypertension in a model in which the endogenous renin-angiotensin system is chronically upregulated.
Previous studies reported a pivotal role of Nox1-containing NAD(P)H oxidase and ROS in hypertension induced by short-term (2 weeks) Ang II infusion.22 However, the function of Nox1 in the pathogenesis of slowly developing chronic Ang II–induced hypertension is unknown. To address this we examined Nox1-deficient TTRhRen mice (TTRhRen/Nox−/Y). Unlike acute studies of Ang II–induced hypertension, our data indicate that Nox1 is not involved in chronic Ang II–dependent blood pressure elevation, because blood pressure was similar in TTRhRen and TTRhRen/Nox1−/Y mice. In the present study we used the tail-cuff method rather than telemetry to measure blood pressure. Although it could be argued that telemetry measurements might have detected subtle differences between groups, there is obviously no reduction in blood pressure in TTRhRen/Nox−/Y versus TTRhRen mice, as we would have expected. In fact, systolic blood pressure was slightly higher in Nox1-deficient TTRhRen versus Nox1-intact mice. Accordingly, we believe that the tail-cuff readings accurately represent true blood pressures in the different groups.
Many studies demonstrated an increase in NAD(P)H oxidase activity in the vessel wall and kidney of hypertensive animals.1,3,4,23,24 Inhibition of NAD(P)H oxidase with apocynin25 and treatment with antioxidants26,27 have been associated with decreased oxidative stress and normalization of blood pressure in hypertensive models. Findings from our study dissociate oxidative stress and NADPH oxidase from blood pressure, because TTRhRen/Nox1−/Y mice were hypertensive even in the presence of reduced NADPH oxidase activity and decreased ROS levels. Whether NADPH oxidase–derived ROS production was increased in cardiovascular tissue, which could contribute to blood pressure elevation in the double transgenic mice, is unclear. However, the fact that levels of plasma TBARS were reduced in these mice suggest that Nox1 deficiency and decreased ROS formation are probably global phenomena.
Despite no influence on blood pressure, Nox1 deficiency had significant effects on redox-sensitive Ang II–dependent signaling in TTRhRen mice. The predominant signaling events in response to Ang II seem to involve not only the generation of ROS but also activation of the nonreceptor tyrosine kinase c-Src. c-Src plays an important role in Ca2+ mobilization and activation of other downstream proteins including MAPKs, FAK, Pyk2, and paxillin.28–30 In addition, c-Src appears to be both upstream and downstream of NAD(P)H oxidase.30–32 In the present study, we show that enhanced NAD(P)H oxidase activity and increased ROS generation observed in TTRhRen mice were blunted by Nox1 knockout. Moreover, this effect was associated with reduced c-Src phosphorylation in kidneys from TTRhRen/Nox1−/Y mice. These data suggest that, at least in kidneys, c-Src is a downstream target of NAD(P)H oxidase-mediated generation of ROS. Accumulating in vivo and in vitro evidence supports the notion that Ang II may, through c-Src–dependent mechanisms, cause cardiovascular and renal damage.30 These pathologies are associated with increased cellular protein synthesis, gene expression, and growth, which all depend on MAPK activation.
MAPKs are key regulatory proteins that control the cellular response to growth, apoptosis, and stress signals.33 Four main mammalian families of MAPKs, including ERK 1/2, JNKs (also termed SAPKs), p38MAPKs, and big MAPK-1 or ERK 5, have been identified. Although the redox sensitivity of ERK 1/2 remains controversial,34 it has been consistently found that Ang II–induced activation of p38MAPK and SAPK/JNK is dependent on ROS generation in various cell types.34 In our study, phosphorylation of p38MAPK, SAPK/JNK, and FAK was increased in TTRhRen mice, and this effect was blunted in kidneys from TTRhRen/Nox1−/Y mice. Recent studies demonstrated that Ang II stimulates phosphorylation of these kinases, which play an important role in renal and vascular inflammation.33,35 In support of this we found that VCAM-1 expression, a proinflammatory mediator and important in renal damage,36 was significantly increased in kidneys from TTRhRen mice and that in Nox1-deficient TTRhRen mice, VCAM-1 content was reduced, in line with that of c-Src, p38MAPK, JNK, and FAK. These findings suggest that Nox1-driven ROS generation influences specific redox-sensitive MAPK pathways, which influence inflammatory responses in the kidney.
Activation of ERK, important in mitogenesis and hypertrophy,37 also plays a role in signaling associated with collagen synthesis in cardiovascular and renal tissue.5 Inhibition of this pathway in Ang II–mediated renal damage ameliorates renal function and reverses fibrotic injury.38 In our model, increased ERK 1/2 phosphorylation was unaffected by Nox1 knockout. We also show that procollagen III expression was increased in kidneys from TTRhRen, and this was not affected in TTRhRen/Nox1−/Y mice. Our data suggest that ERK 1/2 might be involved in Ang II–mediated collagen deposition in kidneys in TTRhRen mice through Nox1-independent mechanisms. These findings support our earlier in vitro studies, where we demonstrated that Ang II induces potent activation of ERK 1/2 through ROS-independent pathways.39 Taken together, it seems that Nox1/ROS effects on proinflammatory (p38MAPK, JNK, FAK, and VCAM-1) signaling pathways are more important than those on profibrotic pathways (ERK 1/2 procollagen), at least in the kidney in the context of chronic Ang II–dependent hypertension.
We considered the possibility that Nox1 homologues, gp91phox and Nox 4, may be upregulated in TTRhRen/Nox1−/Y mice, which could compensate for Nox1 deficiency. However, neither gp91phox nor Nox4 expression was altered in vascular, cardiac, or renal tissue of TTRhRen/Nox1−/Y (data not shown), suggesting that systems other than gp91phox/Nox1/Nox4-containing NADPH oxidase–mediated ROS production contribute to hypertension in this model.
In summary, our data demonstrate that increased systolic blood pressure in a model of chronic Ang II–dependent hypertension is not affected by decreased activity of NADPH oxidase and can occur in the presence of reduced oxidative stress. However, decreased Nox1-mediated ROS generation was able to attenuate activation of redox-sensitive signaling pathways, particularly those related to inflammation. Our findings highlight the complexities relating to interactions between the renin-angiotensin system and ROS-generating systems and suggest that redox-dependent blood pressure–elevating mechanisms differ in acute and chronic settings of Ang II upregulation.
ROS, derived primarily from nonphagocytic NAD(P)H oxidase, have been demonstrated to play a major role in the pathogenesis of Ang II–dependent hypertension. However, most previous studies were performed in acute models of Ang II–mediated hypertension. We demonstrated previously that, in mice with chronic Ang II-dependent hypertension, Nox2-containing NADPH oxidase was not implicated in the development of high blood pressure, despite being involved in redox signaling. Considering that Nox2 was not important, we questioned here whether a Nox2 homologue, Nox1, may be playing a role. Our results clearly demonstrate that, in the absence of Nox1, global oxidative stress and renal redox signaling pathways are blunted, with no effect on blood pressure. These data extend our previous studies and confirm that Nox2 and Nox1, although important in ROS generation and redox signaling, are not critically involved in chronic forms of Ang II–dependent hypertension. Such findings raise the possibility that long-term hypertension may involve mechanisms other than oxidative stress.
We thank Dr Chihiro Yabe-Nishimura, Kyoto Prefectural University of Medicine, Kyoto, Japan, for kindly providing the Nox−/Y mice.
Sources of Funding
This study was supported by grants from the Canadian Institutes of Health Research (to R.M.T. and T.R.). R.M.T. is funded by a Canada Research Chair of the Canadian Institutes of Health Research and through the Canadian Foundation for Innovation.
- Received October 12, 2007.
- Revision received November 4, 2007.
- Accepted December 11, 2007.
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