点击显示 收起
【摘要】 Oxidant stress is an important contributor to renal dysfunction and hypertension. We have previously demonstrated that regulation of renal oxygen consumption by nitric oxide (NO) is impaired in the kidney of spontaneously hypertensive rats (SHR) due to increased superoxide production. We further explored the mechanisms of enhanced oxidant stress in the kidney of SHR. Suppression of cortical oxygen consumption by bradykinin (BK) or enalaprilat (Enal), which act through stimulation of endogenous NO, was impaired in SHR (BK: -14.1 ± 1.2%; Enal: -15.5 ± 1.2%) and was restored by addition of apocynin, an inhibitor of assembly of the NAD(P)H oxidase complex (BK: -21.0 ± 0.6%; Enal: -25.3 ± 1.4%), suggesting this as the source of enhanced superoxide production. Addition of an angiotensin type 1 receptor blocker, losartan, also restored responsiveness to control levels (BK: -22.0 ± 1.1%; Enal: -23.6 ± 1.3%), suggesting that ANG II is responsible for enhanced oxidase activity. A similar defect in responsiveness to BK and Enal could be induced in Wistar-Kyoto kidneys by ANG II and was reversed by a superoxide scavenger (tempol), apocynin or losartan. Immunoblotting of cortical samples demonstrated enhanced expression of endothelial NO synthase (eNOS 1.9 x ) and NAD(P)H oxidase components (gp91 phox 1.6 x and Rac-1 4.5 x ). Expression of SOD-1 and -2 were unchanged, but SOD-3 was significantly decreased in SHR (0.5 x ). Thus NO bioavailability is impaired in SHR owing to an ANG II-mediated increase in superoxide production in association with enhanced expression of NAD(P)H oxidase components, despite increased expression of eNOS. Loss of SOD-3, an important superoxide scavenger, may also contribute to enhanced oxidant stress.
【关键词】 nitric oxide oxygen radicals renal physiology
ENHANCED LEVELS OF OXYGEN radicals, leading to a state of oxidant stress, have been shown to be present in many disease states characterized by endothelial dysfunction and vascular injury, including diabetes, hypertension, and chronic renal insufficiency, both in animal models and in humans ( 14, 15, 22, 26 - 28, 37 ). The rapid interaction of superoxide with nitric oxide (NO) results in the production of peroxynitrite, another harmful radical, and the destruction of NO, decreasing NO bioavailability in situations of oxidant stress ( 10, 25 ). Decreased availability of NO, resulting in vasoconstriction and loss of endothelium-mediated vasodilation, has been documented in models of hypertension and in human vessels ( 12, 20, 28, 35 ). In particular, in the spontaneously hypertensive rat (SHR) model, the contribution of oxidant stress to the development of hypertension has been suggested in studies demonstrating amelioration or reversal of hypertension by the use of oxygen radical scavengers ( 27, 28, 37 ), and we have demonstrated a decrease in NO bioavailability due to superoxide in the kidneys of SHR ( 2 ). Elevated blood pressure, in and of itself, may also contribute to superoxide production via activation of vascular NAD(P)H oxidase ( 31 ), leading to a vicious cycle that perpetuates hypertension.
Levels of oxidant stress are determined by the rates of oxygen radical production and scavenging. The discovery of homologs of the phagocyte NADPH oxidase that are expressed in blood vessels and organs such as the kidney has allowed further characterization and a better understanding of the sources of oxygen radicals other than white blood cells ( 8, 9, 19, 29 ). ANG II has been found to be a major stimulus for oxygen radical production via stimulation of these oxidases, defining another mechanism for its injurious effects ( 19, 23 ). Possibly of equal importance in the control of oxidant stress and nitric oxide bioavailability are oxygen radical scavenging mechanisms, such as the superoxide dismutases, which also play an important role in ameliorating hypertension ( 4, 17 ).
We have previously shown that there is impaired regulation of renal O 2 consumption in SHR in response to stimulators of NO production ( 2 ). Experiments with the superoxide scavenger tempol suggested that increased superoxide levels lead to decreased NO bioavailability in these animals ( 2 ). Other studies have suggested vascular and renal oxidases as sources of enhanced oxidant stress in hypertension and other disease states ( 9, 19, 23 ). We hypothesized that the enhanced oxidant stress in these animals, leading to decreased NO and possibly potentiating hypertension, was due to enhanced oxygen radical production by the NAD(P)H oxidase complex or decreased scavenging of superoxide by endogenous systems. We also speculated that ANG II played a role in the development of oxidant stress. The studies presented here tested these possibilities in isolated kidney tissue from SHR.
MATERIALS AND METHODS
Reagents. Bradykinin, enalaprilat, S -nitroso- N -acetylpenicillamine (SNAP), N -nitro- L -arginine methyl ester ( L -NAME), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (tempol), sodium succinate, and sodium cyanide were purchased from Sigma (St. Louis, MO). 4-Hydroxy-3-methoxyacetophenone (apocynin) was purchased from Fluka (Sigma). Losartan (DuP 753) was a gift from DuPont Pharmaceutical (Wilmington, DE). Affinity-purified monoclonal mouse antibodies to endothelial nitric oxide synthase (eNOS/NOS type III), manganese containing SOD (MnSOD; SOD-2), and gp91 phox were purchased from BD Transduction Laboratories (San Diego, CA). Affinity-purified polyclonal antibody (from rabbit) to Rac-1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody (from sheep) to Cu/Zn SOD (SOD-1) was purchased from Calbiochem (La Jolla, CA). Affinity-purified polyclonal antibody (from rabbit) to extracellular SOD (ecSOD; SOD-3) was a gift from Dr. Wei Wang (Univ. of Colorado Health Sciences Center, Denver, CO). Donkey anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase were purchased from Sigma.
Animals. Male SHR and Wistar-Kyoto rats (WKY) were purchased from Taconic Farms (Germantown, NY) at 10 wk of age and studied after 1 wk of acclimatization. Rats were maintained on a standard rat chow with 0.4% sodium content (Laboratory Rodent Diet, Richmond, IN) and allowed free access to food and water until the day of study. After death, the left kidneys were removed, decapsulated, and weighed. Tissue samples from the right kidney cortex were snap-frozen in liquid nitrogen and stored at -80°C for measurement of eNOS, SOD-1, -2, and -3, gp91 phox, and Rac-1 levels (see below). The protocols used conform to the Guiding Principles for the Care and Use of Laboratory Animals of the American Physiological Society and the National Institutes of Health.
Preparation of kidney tissue slices and measurement of O 2 consumption. Thin slices of renal cortex ( 1 mm, weight 10-20 mg) were prepared and incubated in Krebs bicarbonate solution containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.5 CaCl 2, 25 NaHCO 3, 1.2 KH 2 PO 4, 1.1 MgSO 4, and 5.6 glucose, pH 7.4, bubbled with 21% O 2 -5% CO 2 -74% N 2 at 37°C for 2 h. At the end of incubation, each piece of tissue was placed in a stirred chamber with 3 ml of air-saturated Krebs bicarbonate solution containing 10 mmol/l HEPES and 5.6 mmol/l glucose (pH 7.4). The chamber was sealed with a Clark-type platinum O 2 electrode (Yellow Springs Instruments, Yellow Springs, OH). O 2 consumption was measured polarographically using an O 2 monitor (model YSI 5300) connected to a linear chart recorder (model 1202, Barnstead/Thermolyne, Dubuque, IA). Dose-response curves of the effect of different agonists on kidney O 2 consumption were then measured. Succinate (10 -3 mol/l) and then sodium cyanide (10 -3 mol/l) were added at the end of each experiment to confirm that changes in O 2 consumption originated from mitochondrial respiration.
Renal cortical O 2 consumption was calculated as the rate of decrease in O 2 concentration, assuming an initial O 2 concentration of 224 nmol/ml (calculated from O 2 solubility at 37°C and 1 atm), and is expressed as nanomoles O 2 consumed per minute per gram of tissue. O 2 consumption due to the electrode is <5% of that observed in the presence of tissue. The effects of drugs used on O 2 consumption are expressed as percent change from baseline O 2 consumption. Baseline O 2 consumption was measured in the cortex in the absence and presence of L -NAME (10 -3 mol/l) in each group.
Effect of agonists of NO production on O 2 consumption. Bradykinin or enalaprilat at concentrations of 10 -7 -10 -4 mol/l was added in a cumulative concentration-dependent manner. They were used to measure the effects of stimulation of endogenous NO production on renal O 2 uptake. The response to these drugs was also examined after preincubation with the NOS inhibitor L -NAME (10 -3 mol/l) to verify the role of NO production by NOS in the regulation of O 2 uptake. Each drug was assayed using tissue from six or seven rats in the presence or absence of L -NAME. Changes in O 2 consumption in response to bradykinin and enalaprilat were also assessed in the presence of the superoxide dismutase mimetic tempol (10 -3 mol/l), the ANG II type I receptor (AT 1 R)-antagonist losartan (10 -6 mol/l), and the NAD(P)H oxidase inhibitor apocynin (10 -5 mol/l) using tissue from SHR and WKY rats ( n = 6 for each). In separate experiments, tissue from WKY rats was preincubated with ANG II (10 -8 mol/l) before the addition of agonists and inhibitors ( n = 6).
Effect of NO donor on O 2 consumption. SNAP at concentrations of 10 -7 -10 -4 mol/l was added in a cumulative concentration-dependent manner to assess the effects of exogenous NO on renal cortical O 2 uptake. The response to SNAP was also examined after preincubation with L -NAME (10 -3 mol/l). Each condition was tested in six or seven rats from each group.
Immunoblotting of proteins. Samples of renal cortex were pulverized in liquid nitrogen and homogenized in 5 vol of lysis buffer (0.05 M Tris·HCl, pH 7.2, 1 mM EDTA, 0.01 M dithiothreitol, 1 mg/ml PMSF, 100 µg/ml leupeptin, 100 µg/ml soybean trypsin inhibitor, and 20 µg/ml aprotinin, volume = 5 x tissue weight) followed by sonication for 1 min at 4°C. Lysates were centrifuged at 10,000 g for 10 min at 4°C and stored at -80°C until use. Protein content of supernatants was measured using a Bio-Rad protein assay (Bio-Rad Laboratories).
Samples of tissue lysate (100 µg of protein) were loaded into individual lanes, subjected to electrophoresis on 8 or 15% polyacrylamide gels, and electrophoretically transferred from the gels to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) using a semidry transfer cell (Bio-Rad). After at least 1 h of blocking with 5% milk/PBS, membranes were incubated with antibodies to eNOS, SOD-1, SOD-2, SOD-3, gp91 phox, or Rac-1 in 1% milk/PBS at 4°C overnight. After being washed, membranes were incubated with horseradish peroxidase-conjugated second antibodies to mouse, rabbit, or sheep IgG in 1% milk/PBS at room temperature for 2 h. Membranes were also probed with antibody to -actin (Novus Biologicals, Littleton, CO) to correct for differences in protein loading. Sites of antibody-antigen reaction were observed using Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) before exposure to X-ray film (Kodak, Rochester, NY).
The relative intensities of bands in autoradiograms were determined on an AlphaImager 2000 documentation and analysis system (Alpha Innotech, San Leandro, CA) followed by analysis using image software. Band intensity for each measured protein was divided by the intensity of the corresponding -actin band to correct for any differences in protein loading.
Statistical analysis. All data are expressed as means ± SE. Statistical analysis of baseline O 2 consumption and densities of protein bands was performed using Students t -test. Changes in O 2 consumption caused by drug treatment were analyzed using two-way ANOVA followed by multiple comparisons using the Tukey test (Sigma-Stat, SPSS-Science, Chicago, IL). Statistical significance was achieved at P < 0.05.
RESULTS
Baseline renal cortical O 2 consumption. Baseline renal cortical O 2 consumption was similar in the two groups, in agreement with our previously published results ( 2 ): WKY: 1,211 ± 87 nmol O 2 ·min -1 ·g -1, n = 6; SHR: 1,195 ± 117 nmol O 2 ·min -1 ·g -1, n = 7, P 0.05. Addition of the NOS inhibitor L -NAME (10 -3 mol/l) did not significantly alter O 2 consumption (WKY: 1,235 ± 126 nmol O 2 ·min -1 ·g -1, n = 6; SHR: 1,243 ± 90 nmol O 2 ·min -1 ·g -1, n = 7, P 0.05).
Effect of bradykinin and enalaprilat on renal cortical O 2 consumption. Cumulative doses of bradykinin (10 -7 -10 -4 mol/l) produced significant, concentration-dependent decreases in renal cortical O 2 consumption in WKY and SHR rats (WKY: from -1.3 ± 0.9 to -25.3 ± 2.3%, n = 6; SHR: from -0.3 ± 0.3 to -14.1 ± 1.2%, n = 7). The depression of renal cortical O 2 consumption by bradykinin was significantly less in SHR than WKY rats ( P < 0.05), confirming previous observations ( 2 ) ( Fig. 1 A ). Similarly, the angiotensin-converting enzyme inhibitor enalaprilat (10 -7 -10 -4 mol/l), which also stimulates endogenous NO production, caused concentration-dependent decreases in renal cortical O 2 consumption in WKY and SHR rats (WKY: from -1.0 ± 0.6 to -25.6 ± 1.4%, n = 6; SHR: from 0 ± 0 to -15.5 ± 1.2%, n = 7), with a significantly lower effect in SHR than WKY rats ( P < 0.05) ( Fig. 1 B ), confirming previous observations. Addition of L -NAME significantly attenuated the effects of bradykinin and enalaprilat only in WKY rats, suggesting diminished production of NO by NOS in SHR (data not shown). The addition of tempol restored responsiveness to inhibition of O 2 consumption in SHR to levels seen in WKY rats, confirming that destruction of NO by superoxide accounts for decreased NO bioavailability in SHR, as we have previously suggested (data not shown) ( 2 ).
Fig. 1. Effect of cumulative doses of bradykinin ( A ) or enalaprilat ( B ) on renal cortical O 2 consumption in Wistar-Kyoto (WKY; ) and spontaneously hypertensive rats (SHR; ). Both drugs caused significant dose-dependent decreases in O 2 consumption that were significantly less in SHR. * P < 0.05 vs. WKY rats.
Effect of apocynin on renal cortical O 2 consumption. Because scavenging of superoxide with tempol had restored the ability of bradykinin and enalaprilat to suppress renal cortical O 2 consumption in SHR, we explored the source of increased superoxide production in these animals using apocynin, an inhibitor of NAD(P)H oxidase assembly. Bradykinin and enalaprilat both decreased O 2 consumption significantly more in WKY rats than in SHR ( Fig. 2 ). Addition of apocynin significantly augmented the suppression of O 2 consumption by bradykinin ( Fig. 2 A ) and enalaprilat ( Fig. 2 B ) in SHR rats to a level that was not different from that in WKY rats (bradykinin+apocynin in WKY: from -0.0 ± 0.0 to -22.6 ± 1.7%, n = 6, SHR: from -0.6 ± 0.6 to -21.0 ± 0.6%, n = 7, P 0.05; enalaprilat+apocynin in WKY: from -1.2 ± 0.7 to -24.6 ± 1.2%, n = 6, SHR: from -0.9 ± 0.7 to -25.3 ± 1.4%, n = 7, P 0.05).
Fig. 2. Effect of inhibition of NAD(P)H oxidase with apocynin on inhibition of renal cortical O 2 consumption by cumulative doses of bradykinin ( A ) or enalaprilat ( B ). The response to both drugs was significantly less in SHR ( ) than in WKY ( ) rats. Addition of apocynin had no effect in WKY rats ( ) but restored responsiveness in SHR ( ). * P < 0.05 vs. SHR with apocynin.
Effect of angiotensin blockade on renal cortical O 2 consumption. Because ANG II has been implicated in increases in superoxide production, we explored the effect of blocking ANG II action with the AT 1 -R antagonist losartan. Renal cortical tissue was incubated with losartan (10 -6 mol/l) before performance of dose-response curves. Inhibition of renal cortical O 2 consumption by bradykinin and enalaprilat was significantly augmented by losartan in SHR but not WKY rats ( Fig. 3 ). In the presence of losartan, the response in SHR was not significantly different from that in WKY animals (bradykinin+losartan in WKY: from -1.4 ± 0.7 to -23.5 ± 1.6%, n = 6, SHR: from 0 ± 0 to -22.0 ± 1.1%, n = 7, P 0.05; enalaprilat+losartan in WKY: from -2.0 ± 0.7 to -24.6 ± 1.5%, n = 6, SHR: from -0.7 ± 0.5 to -23.6 ± 1.3%, n = 7, P 0.05).
Fig. 3. Effect of blockade of the AT 1 receptor with losartan on inhibition of renal cortical O 2 consumption by cumulative doses of bradykinin ( A ) or enalaprilat ( B ). The response to both drugs was significantly less in SHR ( ) than in WKY ( ) rats. Losartan had no effect in WKY ( ) but restored responsiveness in SHR ( ). * P < 0.05 vs. SHR with losartan.
Effect of NO donor SNAP on renal cortical O 2 consumption. Administration of cumulative doses of the NO donor SNAP (10 -7 -10 -4 mol/l) decreased renal cortical O 2 consumption similarly in WKY and SHR, demonstrating no inherent differences between the strains in responsiveness of O 2 consumption to exogenous NO ( Fig. 4 ). Addition of tempol (data not shown), apocynin, losartan, or L -NAME did not affect responsiveness to SNAP.
Fig. 4. Effect of exogenous nitric oxide (NO) administered as S -nitroso- N -acetylpenicillamine (SNAP) on changes in renal cortical O 2 consumption in WKY ( A ) rats and SHR ( B ). Similar degrees of inhibition were seen in both groups. Addition of apocynin ( ), losartan ( ), or N -nitro- L -arginine methyl ester ( L -NAME; ) did not significantly alter results obtained in control tissue ( ).
Effect of ANG II on renal cortical O 2 consumption. Because experiments with losartan suggested ANG II as a cause of increased superoxide production in SHR, we explored the effect of ANG II on renal cortical O 2 consumption in WKY rats. Preincubation of renal cortical tissue from WKY rats with ANG II (10 -8 mol/l) significantly suppressed responsiveness to inhibition of renal cortical O 2 consumption by bradykinin (bradykinin: from 0 ± 0 to -23.5 ± 1.0%, n = 6; bradykinin+ANG II: from 0 ± 0 to -10.7 ± 3.0%, n = 6, P < 0.05) ( Fig. 5 A ). Scavenging of superoxide with tempol, inhibition of NAD(P)H oxidase with apocynin, or blockade of the AT 1 R with losartan all restored the responsiveness to inhibition of renal cortical O 2 consumption by bradykinin toward the effect seen in control WKY despite the presence of ANG II ( Fig. 5 B ).
Fig. 5. Effect of ANG II on inhibition of renal cortical O 2 consumption by bradykinin in WKY rats. A : treatment with ANG II ( ) significantly decreased the response to bradykinin compared with control ( ). * P < 0.05 vs. control. B : scavenging of oxygen radicals with tempol ( ), inhibition of NAD(P)H oxidase with apocynin ( ), or blockade of the AT 1 receptor with losartan ( ) all restored responsiveness to bradykinin compared with addition of ANG II alone ( ). * P < 0.05 vs. addition of inhibitors.
Levels of renal cortical proteins (NOS, SOD, gp91 phox, Rac-1). Expression of several proteins involved in NO production and metabolism of oxygen radicals were measured in samples of renal cortex from WKY and SHR rats ( Fig. 6 ). Despite evidence of decreased NO bioavailability in SHR, levels of eNOS protein in renal cortex of SHR are significantly increased compared with WKY rats ( SHR: 3.02 ± 0.53, n = 7; WKY: 1.58 ± 0.07, n = 6, P < 0.05). A similar, albeit smaller, increase was seen in medullary eNOS (data not shown). Levels of SOD-1 and SOD-2 were similar in both groups of rats (SOD-1: SHR: 0.44 ± 0.08, n = 7; WKY: 0.59 ± 0.08, n = 7, P 0.05; SOD-2: SHR: 0.42 ± 0.12, n = 6; WKY: 0.42 ± 0.05, n = 6, P 0.05), but levels of extracellular SOD (SOD-3) were significantly reduced in SHR (SHR: 0.32 ± 0.04, n = 7; WKY: 0.62 ± 0.08, n = 7, P < 0.01). Two components of the NAD(P)H oxidase complex, gp91 phox and Rac-1, are both significantly increased in SHR rats (gp91 phox : SHR: 3.13 ± 0.49, n = 7; WKY: 2.0 ± 0.09, n = 7, P < 0.05; Rac-1: SHR 1.38 ± 0.28, n = 7; WKY: 0.31 ± 0.05, P < 0.01).
Fig. 6. Levels of protein expression in renal cortex determined by immunoblotting using antibodies to the indicated proteins. eNOS, endothelial nitric oxide synthase. Intensities of the measured bands divided by the intensity of the -actin band are expressed as a ratio on the ordinate. Each bar represents the means ± SE of measurements in 6-7 animals. * P < 0.05. SHR, open bars; WKY, hatched bars.
DISCUSSION
The studies presented here confirm our previous work in the SHR kidney ( 2 ), namely, of decreased NO bioavailability, reflected as an impairment of regulation of renal O 2 consumption, and again suggest excess superoxide production as the cause. We have used the regulation of renal oxygen consumption by NO as a direct measure of a NO effect in renal cortex in vitro in the absence of glomerular filtration, thus demonstrating a basic change in renal metabolism in SHR independent of the workload of sodium transport. Decreased NO bioavailability occurs despite increased expression of eNOS, the major NOS isoform responsible for regulation of renal oxygen consumption by NO ( 1 ). The data also suggest that the major source of superoxide is the NAD(P)H oxidase complex and that ANG II plays an important role in the stimulation of superoxide production. Finally, we document that in addition to increased expression of proteins involved in the assembly of the NAD(P)H oxidase complex in the kidney, there is loss of an important antioxidant defense, extracellular SOD (SOD-3). The end result of these processes is an increase in the level of oxidant stress mediated mainly through an effect of ANG II.
Enhanced production of superoxide has been demonstrated in numerous models of hypertension, including those due to partial nephrectomy, lead-induced hypertension, salt-sensitive hypertension, and the SHR ( 2, 14, 20, 21, 26 - 28, 35, 37 ). Evidence of enhanced oxidative stress has been obtained in humans with renovascular hypertension and appears to decrease after angioplasty ( 15, 22 ). Interestingly, patients with essential hypertension did not have enhanced excretion of a marker of in vivo lipid peroxidation despite a similar degree of hypertension ( 22 ). Segments of internal mammary artery and saphenous veins removed from patients undergoing coronary artery revascularization, most of whom were hypertensive, displayed NADH- and NAD(P)H-enhanced superoxide production, which was reversed by addition of apocynin or SOD ( 12 ). Suppression of superoxide production resulted in increased NO bioavailability and improved endothelium-mediated vasorelaxation via NO. Similarly, Guzik et al. ( 11 ) demonstrated superoxide production in all three vessel layers (adventitia, media, endothelium) but also noted that NO production scavenges superoxide, suggesting a much more complex interaction within the vessel wall.
Evidence that scavenging of NO by superoxide contributes to hypertension comes from experiments demonstrating increased NO bioavailability and decreased blood pressure after manipulations that decrease oxygen radical levels. Administration of superoxide scavengers ameliorates hypertension in SHR and salt-sensitive hypertension ( 21, 27, 28, 37 ). Production of superoxide in the renal cortex and medulla in Dahl salt-sensitive rats was decreased by tempol in association with a drop in blood pressure ( 21 ). Life-long supplementation with antioxidants also delays the onset of hypertension, as well as ameliorating its severity, in SHR ( 42 ), along with decreasing markers of oxidative stress. Directly decreasing production of superoxide with a highly specific inhibitor of vascular NAD(P)H oxidase decreases blood pressure in a mouse model of hypertension induced by ANG II infusion ( 24 ). Finally, scavenging of superoxide by SOD leads to decreases in blood pressure. This is demonstrated by significantly increased blood pressure in SOD-3-deficient mice with one clipped kidney or after infusion with ANG II compared with controls. ( 17 ). Infusion of recombinant SOD-3 into ANG II-treated mice rapidly lowered blood pressure ( 17 ). Transfection of SHR with the gene for human SOD-3 similarly lowered blood pressure ( 4 ). This was accompanied by improvement of acetylcholine-mediated vascular relaxation, suggesting increased availability of endothelium-generated NO.
There are several sources of increased superoxide production potentially implicated in vascular pathology, including xanthine oxidase, cytochrome P -450, uncoupled NOS, and the NAD(P)H oxidases ( 19 ). A kidney specific oxidase (Renox or NOX-4) has been described but is also found in vascular smooth muscle cells ( 8, 13, 29 ). Previous work in the SHR has documented overexpression of several components of the NAD(P)H oxidase complex through measurement of mRNA and/or protein levels by immunoblotting ( 3, 30, 42 ). Chabrashvili et al. ( 3 ) found expression of the p22 phox, p47 phox, and p67 phox subunits of the NAD(P)H complex in the renal vasculature and elements of the distal tubule (starting at the thick ascending limb) in the kidney of SHR with significantly increased levels of p47- and p67 phox in SHR compared with WKY animals. Zhan et al. ( 42 ) described increased levels of gp91 phox, the catalytic subunit of the complex, and p22 phox in the renal cortex of SHR. In the latter studies, the feeding of antioxidants not only reduced hypertension and oxidative stress but also decreased the level of expression of these subunits. Our work confirms overexpression of gp91 phox and the small G protein Rac-1 that is necessary for activation of the oxidase. In thoracic aortas of SHR, increased levels of p22 phox were also detected ( 30 ). Overexpression of these components, along with the beneficial effects of apocynin, an inhibitor of assembly of the complex, on the responsiveness of O 2 consumption to stimulators of NO production, suggests that the NAD(P)H complex is an important source of oxygen radicals in SHR.
Another potential source of superoxide is uncoupled NOS. When levels of arginine or tetrahydrobiopterin (BH 4 ) are low, NOS activation may favor production of superoxide rather than NO ( 32, 41 ). Evidence of enhanced oxygen radical production by NOS has been found in aortas from young, prehypertensive SHR ( 5 ), and administration of BH 4 to SHR animals suppresses the development of hypertension as well as decreases superoxide production by aortic segments ( 16 ). Levels of NOS isoforms in the kidney have been reported to be elevated in SHR, including inducible NOS, neuronal NOS, and eNOS ( 33, 40 ). We have confirmed elevated eNOS expression in the renal cortex of SHR but have been unable to detect either inducible NOS or neuronal NOS in cortical samples. While we have not directly studied the possibility that uncoupled NOS in the renal cortex leads to the observed increase in oxidant stress, the ability of apocynin to completely reverse the defect seen in SHR suggests that NAD(P)H oxidase is the most important source of superoxide in the kidney.
The ability of the AT 1 R antagonist losartan to restore the effect of bradykinin and enalaprilat on the regulation of renal O 2 consumption suggests a role of ANG II in mediating enhanced oxidant stress in SHR. This is further supported by the observation of induction of a similar defect in kidney from WKY rats by incubation with ANG II, a defect again reversed by superoxide scavenging with tempol or inhibition of NAD(P)H oxidase assembly with apocynin. ANG II is known to stimulate both activity and expression of NAD(P)H oxidase ( 9, 13, 19 ), which in turn reduces NO availability through scavenging. O 2 utilization in the kidney of SHR for sodium transport has been shown to be less efficient than in WKY rats ( 38 ), an observation consistent with our finding of impaired regulation of renal O 2 consumption in SHR and decreased NO bioavailability ( 2 ). This defect is largely reversed by the AT 1 R blocker candesartan in vivo ( 39 ), suggesting that activation of the AT 1 R mediates the increased O 2 consumption. Our data further support a role in vitro for an AT 1 R-mediated increase in renal oxidant stress, leading to decreased NO bioavailability in the SHR.
Of interest, we also found that levels of extracellular SOD were reduced by 50% in SHR, potentially contributing further to oxidant stress in these animals. Studies in mice lacking either SOD-3 or Cu/Zn SOD (SOD-2) have suggested that deficiency of either of these enzymes can lead to increased superoxide levels and loss of NO, leading to decreased endothelium-mediated vasodilatation ( 6, 17 ), although basal blood pressure was not elevated in either group of deficient animals. Under stress, induced by clipping of one kidney or ANG II infusion, blood pressure was higher in SOD-3-deficient animals ( 17 ). Overexpression of human SOD-3 in SHR also leads to lowering of blood pressure ( 4 ), further supporting the importance of superoxide scavenging mechanisms in responding to oxidant stress. Another study of SOD expression in the aorta of SHR found increased levels of SOD-1 and SOD-2, whereas SOD-3 was not measured ( 30 ). These animals were, however, much older than the ones studied here, and these results might represent a change with aging or different regulation of SODs in the vessel wall vs. the kidney. Because expression of SOD-3 in mice is regulated by NO, the loss of SOD-3 in SHR may be a secondary, albeit aggravating, factor ( 7 ). In other models with enhanced oxidant stress, namely nephrectomy, salt loading, and lead-induced hypertension, renal expression of SOD-1 has been found to be decreased or increased, whereas SOD-2 was decreased or unchanged ( 18, 34, 36 ). Thus a general pattern of response of SOD expression in situations of oxidant stress is not apparent.
In summary, we have further confirmed the role of enhanced intrarenal superoxide production in limiting NO availability in the kidney in the SHR model of hypertension. We have demonstrated that enhanced expression and activation of elements of the NAD(P)H complex occur in these animals, contributing to oxidant stress, along with loss of an important counterregulatory element, SOD-3. Loss of NO occurs despite enhanced expression of eNOS, which in itself could be a response to lower levels of NO. Finally, an important role of stimulation of the AT 1 R by ANG II in the enhanced oxidant stress that occurs in these animals has been demonstrated.
GRANTS
These studies were supported by a grant from the Westchester Artificial Kidney Foundation.
ACKNOWLEDGMENTS
The authors thank Dr. Thomas Hintze for generous advice and assistance.
【参考文献】
Adler S, Huang H, Loke KE, Xu X, Tada H, Laumas A, and Hintze TH. Endothelial nitric oxide synthase plays an essential role in regulation of renal oxygen consumption by nitric oxide. Am J Physiol Renal Physiol 280: F838-F843, 2001.
Adler S, Huang H, and Hintze TH. Impaired regulation of renal oxygen consumption in spontaneously hypertensive rats. J Am Soc Nephrol 13: 1788-1794, 2002.
Chabrashvilli T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, and Wilcox CS. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension 39: 269-274, 2002.
Chu Y, Iida S, Lund DD, Weiss RM, DiBona GF, Watanabe Y, Faraci FM, and Heistad DD. Gene transfer of extracellular superoxide dismutase reduces arterial pressure in spontaneously hypertensive rats role of heparin-binding domain. Circ Res 92: 461-468, 2003.
Cosentino F, patton S, DUscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T, and Luscher TF. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest 101: 1530-1537, 1998.
Didion SP, Ryan MJ, Didion LA, Fegan PE, Sigmund CD, and Faraci FM. Increased superoxide and vascular dysfunction in CuZnSOD-deficient mice. Circ Res 91: 938-944, 2002.
Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, and Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. Clin Invest 105: 1631-1639, 2000.
Geiszt M, Kopp JB, Varnai P, and Leto TL. Identification of Renox, an NAD(P)H oxidase in kidney. PNAS 97: 8010-8014, 2000.
Griendling KK, Sorescu D, and Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology, and disease. Circ Res 86: 494-501, 2000.
Gryglewski RJ, Palmer RMJ, and Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454-456, 1986.
Guzik TJ, West NEJ, Pillai R, Taggart DP, and Channon KM. Nitric oxide modulates superoxide release and peroxynitrite formation in human lood vessels. Hypertension 39: 1088-1094, 2002.
Hamilton CA, Brosnan MJ, Al-Benna S, Berg G, and Dominiczak AF. NAD(P)H oxidase inhibition improves endothelial function in rat, and human blood vessels. Hypertension 40: 755-762, 2002.
Hanna IR, Taniyama Y, Szocs K, Rocic P, and Griendling KK. NAD(P)H oxidase-derived reactive oxygen species as mediators of angiotensin II signaling. Antioxidants Redox Signaling 4: 899-914, 2002.
Hasdan G, Benchetrit S, Rashid G, Green J, Bernheim J, and Rathaus M. Endothelial dysfunction and hypertension in nephrectomized rats are medicated by vascular superoxide. Kidney Int 61: 586-590, 2002.
Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Oshima T, and Chayama K. Endothelial function and oxidative stress in renovascular hypertension. N Engl J Med 346: 1954-1962, 2002.
Hong H, Hsiao G, Cheng T, and Yen M. Supplementation with tetrahydropterin suppresses the development of hypertension in spontaneously hypertensive rats. Hypertension 38: 1044-1048, 2001.
Jung O, Marklund SL, Geiger H, Pedrazzini T, Busse R, and Brandes RP. Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability in vivo and ex vivo evidence from ecSOD-deficient mice. Circ Res 93: 622-629, 2003.
Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, Welch WJ, and Wilcox CS. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol 14: 2775-2782, 2003.
Lassegue B and Clempus RE. Vacular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 285: R277-R297, 2003.
McIntyre M, Bohr DF, and Dominiczak AF. Endothelial function in hypertension. The role of superoxide anion. Hypertension 34: 539-545, 1999.
Meng S, Cason GW, Gannon AW, Racusen LC, and Manning RD. Oxidative stress in Dahl salt-sensitive hypertension. Hypertension 41:1346-1352, 2003.
Minuz P, Patrignani P, Gaino S, Degan M, Menapace L, Tommasoli R, Seta F, Capone M, Tacconelli S, Palatresi S, Bencini C, DelVecchio C, Mansueto G, Arosio E, Santonastaso CL, Lechi A, Morganti A, and Patrono C. Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease. Circulation 106: 2800-2805, 2002.
Reckelhoff JF and Romero JC. Role of oxidative stress in angiotensin- induced hypertension. Am J Physiol Regul Integr Comp Physiol 284: R893-R912, 2003.
Rey FE, Cifuentes ME, Kiarash A, Quinn MT, and Pagano PJ. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O 2 - and systolic blood pressure in mice. Circ Res 89: 408-414, 2001.
Rubanyi GM and Vanhoutte PM. Superoxide anions, and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 250: H822-H827, 1986.
Schnackenberg CG. Physiological and pathological roles of oxygen radicals in the renal microvasculature. Am J Physiol Regul Integr Comp Physiol 282: R335-R342, 2002.
Schnackenberg CG, Welch WJ, and Wilcox CS. Normalization of blood pressure, and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic. Role of nitric oxide. Hypertension 32: 59-64, 1998.
Schnackenberg CG and Wilcox CS. Two-week administration of tempol attenuates both hypertension, and renal excretion of 8-Iso prostaglandin F 2. Hypertension 33: 424-428, 1999.
Shiose A, Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M, Sakaki Y, and Sumimoto H. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem 276: 1417-1423, 2001.
Ulker S, Mcmaster D, Mckeown PP, and Bayraktutan U. Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation. Cardiovasc Res 59: 488-500, 2003.
Ungvari Z, Csiszar A, Huang A, Kaminiski PM, Wolin MS, and Koller A. High pressure induces superoxide production in isolated arteries via protein kinase c-dependent activation of NAD(P)H oxidase. Circulation 108: 1253-1258, 2003.
Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BSS, Karoui H, Tordo P, and Pritchard KA. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci USA 95: 9220-9225, 1998.
Vaziri N, Ni Z, and Oveisi F. Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31: 1248-1254, 1998.
Vaziri ND, Dicus M, Ho ND, Boroujerdi-Rad L, and Sindhu RK. Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency. Kidney Int 63: 179-185, 2003.
Vaziri ND, Liang K, and Ding Y. Increased nitric oxide inactivation by reactive oxygen species in lead-induced hypertension. Kidney Int 56: 1492-1498, 1999.
Vaziri ND, Lin C, Farmand F, and Sindhu RK. Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension. Kidney Int 63: 186-194, 2003.
Vaziri ND, Ni Z, Oveisi F, and Trnavsky-Hobbs DL. Effect of antioxidant therapy on blood pressure, and NO synthase expression in hypertensive rats. Hypertension 36: 957-964, 2000.
Welch WJ, Baumgartl H, Lubbers D, and Wilcox CS. Nephron P O 2 and renal oxygen usage in the hypertensive rat kidney. Kidney Int 59: 230-237, 2001.
Welch WJ, Baumgartl H, Lubbers D, and Wilcox CS. Renal oxygenation defects in the spontaneously hypertensive rat: role of AT 1 receptors. Kidney Int 63: 202-208, 2003.
Welch WJ, Tojo A, Lee J, Kang DG, Schnackenberg CG, and Wilcox CS. Nitric oxide synthase in the JGA of the SHR: expression and role in tubuloglomercular feedback. Am J Physiol Renal Physiol 277: F130-F138, 1999.
Xia Y, Tsai A, Berka V, and Zweier JL. Superoxide generation from endothelial nitric-oxide synthase. J Biol Chem 273: 25804-25808, 1998.
Zhan C, Sindhu RK, and Vaziri ND. Up-regulation of kidney NAD(P)H oxidase and calcineurin in SHR: reversal by lifelong antioxidant supplementation. Kidney Int 65: 219-227, 2004.
作者单位：Division of Nephrology, Department of Medicine, and Department of Physiology, New York Medical College, Valhalla, New York 10595