点击显示 收起
【摘要】 Reactive oxygen species (ROS) can modulate renal hemodynamics and function both directly, by leading to vasoconstriction, and indirectly, by inducing renal inflammation and tissue growth. The involvement of oxidative stress in the pathogenesis of renovascular disease (RVD) is increasingly recognized, but the relative contribution of long-term tissue injury to renal dysfunction remains unclear. We hypothesized that functional and structural alterations elicited by oxidative stress in RVD would be more effectively modulated by chronic than by acute antioxidant intervention. Renal hemodynamics and function were quantified in vivo in pigs using electron-beam computed tomography at baseline and after vasoactive challenge (ACh and sodium nitroprusside); after 12 wk of RVD (simulated by concurrent hypercholesterolemia and renal artery stenosis, n = 7); RVD acutely infused with the SOD-mimetic tempol (RVD+tempol, n = 7); RVD chronically supplemented with antioxidant vitamins C (1 g) and E (100 IU/kg; RVD+vitamins, n = 7); or control (normal, n = 7). Renal tissue was studied ex vivo using immunoblotting and immunohistochemistry. Basal renal blood flow (RBF) and glomerular filtration rate were similarly decreased in all RVD groups. ACh-stimulated RBF remained unchanged in RVD, increased in RVD+tempol, but further increased (similarly to normal) in RVD+vitamins ( P < 0.05 vs. RVD). Furthermore, RVD+vitamins also showed a decreased presence of superoxide anion, decreased NAD(P)H-oxidase and nitrotyrosine expression, increased endothelial nitric oxide synthase expression, and attenuated renal fibrosis. Chronic antioxidant intervention in early RVD improved renal hemodynamic responses more effectively than acute intervention, likely due to increased nitric oxide bioavailability and decreased structural injury. These suggest that chronic tissue changes play an important role in renal compromise mediated by oxidative stress in RVD.
【关键词】 oxidative stress renal function antioxidants tempol
RENOVASCULAR DISEASES (RVD) account for over one-third of all cases of end-stage renal disease ( 29 ). Renal artery stenosis (RAS; usually due to atherosclerosis) is one of the most common vascular nephropathies and may activate multiple and parallel mechanisms that lead to a progressive deterioration of renal function and renal tissue injury ( 6, 7, 40, 47 ). Our laboratory has previously shown that increased oxidative stress is likely to be involved in the mechanism of renal injury in RVD (6-8). Reactive oxygen species (ROS) can acutely modulate renal microvascular function, induce vasoconstriction ( 42 ), and contribute to the enhanced renal vascular tone and sensitivity to other vasoconstrictors, such as angiotensin II and endothelin-1. The superoxide anion can also react with nitric oxide (NO), decreasing its availability and resulting in formation of the prooxidant peroxynitrite, and can impair intrarenal vascular and glomerular function ( 35, 42 ). Furthermore, ROS interfere with renal oxygen usage for tubular sodium transport ( 53 ) and enhance tubuloglomerular feedback ( 36 ).
In addition, ROS are involved in multiple pathways that can lead to renal tissue injury, and chronic antioxidant intervention can slow the progression of renal insufficiency ( 10 ), underscoring the role of the oxidative stress pathway in renal disease ( 3, 32, 52 ). Our laboratory has previously shown that increased oxidative stress in a model of early RVD was associated not only with renal functional impairment but also with increased expression of proinflammatory and progrowth factors that augmented intrarenal inflammation and fibrosis ( 6, 7 ). These chronic structural changes may also impair renal hemodynamics and function ( 6, 7 ). Hence, increased oxidative stress can interfere with renal function by both acute and chronic mechanisms. However, the relative contributions of these pathways to renal dysfunction in RVD are as yet unclear.
Therefore, the current study was designed to investigate the effects of acute and chronic blockade of the oxidative stress pathway in RVD. We hypothesized that functional and structural alterations elicited by oxidative stress in RVD would be more effectively modulated by chronic than by acute antioxidant intervention. To evaluate this, renal hemodynamics and function were studied in RVD during an acute intrarenal infusion of the SOD mimetic tempol or after chronic supplementation with vitamins C and E. We used electron-beam computed tomography (EBCT), an ultrafast scanner that provides accurate and noninvasive quantifications of single-kidney volume, regional perfusion, blood flow, glomerular filtration rate (GFR), and segmental tubular function (5-8, 19, 23) of the intact RVD kidney in vivo distal to a stenosis. Moreover, subsequent in vitro studies further characterized the effects of antioxidant intervention on the stenotic kidney.
METHODS
The Institutional Animal Care and Use Committee approved all procedures. Twenty-eight domestic pigs (55-65 kg) were studied after 12 wk of observation. In twenty-one pigs, a local-irritant coil was placed in the main renal artery at baseline and induced gradual development of unilateral RAS, as previously described ( 6, 7, 23 ). The degree of RAS was subsequently measured by quantitative renal angiography ( 6, 23 ). A PhysioTel telemetry system (Data Sciences) was implanted at baseline in the left femoral artery, and blood pressure measurements were obtained in all animals. Mean arterial pressure (MAP) was recorded at 5-min intervals and averaged for each 24-h period. Levels reported were those obtained for 2 days before each in vivo study ( 7, 8 ). A high-cholesterol diet (2% cholesterol and 15% lard, TD-93296, Harlan-Teklad, Madison, WI) (5-7, 9) was used to simulate early atherosclerosis (RVD, n = 7). Seven RVD animals were orally supplemented with daily doses of vitamin C (1,000 mg) and E (100 IU/kg; RVD+vitamins, n = 7) ( 46 ), which provides effective blockade of oxidative stress ( 38, 39, 46 ). An additional group of RVD pigs were acutely infused intrarenally with the SOD mimetic tempol (RVD+tempol, n = 7, 225 µmol·kg -1 ·min -1 ) during the in vivo studies, following previous established protocols ( 43 ). The dose was titrated to the maximal dose that did not decrease blood pressure in our model. The remaining seven pigs were fed with a normal diet and used as controls (normal, n = 7).
On the day of the studies, each animal was anesthetized with intramuscular telazol (5 mg/kg) and xylazine (2 mg/kg), intubated, and mechanically ventilated with room air. Anesthesia was maintained with a mixture of ketamine (0.2 mg·kg -1 ·min -1 ) and xylazine (0.03 mg·kg -1 ·min -1 ) in normal saline, administered via an ear vein cannula (0.05 ml·kg -1 ·min -1 ). Blood samples were collected from the inferior vena cava for measurement of vitamin levels (high-performance liquid chromatography), plasma renin activity (PRA; radioimmunoassay), SOD activity, and serum creatinine (spectrophotometry). Under sterile conditions and fluoroscopic guidance, an 8F arterial guide was inserted in the left carotid artery and then advanced and positioned in the descending aorta. Through it, a low-profile tracker catheter ( 20 ) was positioned in the midsection of the stenotic main renal artery, proximal to the stenosis. Tempol infusion was initiated in RVD+tempol animals 15 min before the beginning of the EBCT studies, and because of its short half-life ( 48 ) it was continuously infused throughout the study. In vivo EBCT flow studies were then performed as previously detailed ( 5, 6, 9, 22 ), for assessment of basal regional renal perfusion, RBF, GFR, and tubular function and repeated during suprarenal infusion of ACh (5 µg·kg -1 ·min -1 ) and sodium nitroprusside (SNP; 6 nM·kg -1 ·min -1 ) to test endothelium-dependent and -independent responses, respectively. Blood pressure was monitored during each acute experiment using an intra-arterial vascular sheath in the carotid artery.
After completion of all studies, the pigs were euthanized with a lethal intravenous injection of Sleepaway (100 mg/kg iv, Fort Dodge Laboratories, Fort Dodge, IA). Kidneys were removed using a retroperitoneal incision, immediately shock-frozen in liquid nitrogen, and stored at -80°C, or preserved in formalin ( 5, 7, 8 ). In vitro studies were then performed to characterize oxidative stress, nitric oxide synthase (NOS) isoform expression, and renal morphology. SOD activity was quantified in renal tissue and plasma using spectrophotometry. Superoxide anion was investigated in renal tissue using dihydroethidium (DHE) staining and fluorescence microscopy. Protein expression of the NAD(P)H-oxidase p47 phox and p67 phox subunits, CuZn-SOD, nitrotyrosine (as a footprint for peroxynitrite formation in vivo), and endothelial NOS (eNOS) were measured using Western blotting. In addition, using deparaffinized 5-µm-thick midhilar cross sections, renal morphology was evaluated using trichrome.
EBCT-derived data analysis. Manually traced regions of interest were selected in EBCT images in the aorta, renal cortex, medulla, and papilla, and their densities were sampled. Time-density curves were generated and fitted with extended -variate curve fits, and the area enclosed under each segment of the curve and its first moment were calculated using the curve-fitting parameters ( 19 ). These were used to calculate renal regional perfusion (ml·min -1 ·g tissue -1 ), intratubular fluid concentration (ITC), which is an index of segmental tubular fluid reabsorption and tubular function, single-kidney GFR, and RBF, using previously validated methods ( 6, 8, 9, 19, 22 ).
SOD assay. Total Mn and CuZn SOD isoform activities were measured in renal tissue using a SOD Kit (R&D Systems, Minneapolis, MN), as previously detailed ( 8 ). For Mn SOD activity, 100 µl of potassium cyanide (KCN, 60 mmol), which blocks CuZn SOD activity, were added to the mixture in parallel experiments.
In addition, SOD activity was also measured in plasma using the Cayman Chemical SOD Assay Kit (Cayman Chemical, Ann Arbor, MI) following vendor instructions. Briefly, blood anticoagulated with EDTA was centrifuged twice at 4°C, and the supernatant was collected. The standards and samples were placed in a sample plate and assayed in duplicate. Reaction was initiated by adding 20 µl of diluted xanthine oxidase to all wells, and the plate was then incubated on a shaker at room temperature for 20 min. The absorbance of each standard and samples was read at 450 nm using a plate reader. SOD activity was calculated from the linear regression of the standard curve by substituting the linearized rate for each sample. One unit was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.
Western blotting. Standard blotting protocols were followed, as previously described ( 5, 7 ). Briefly, frozen renal tissue (including both cortex and medulla) was pulverized and homogenized at 4°C in chilled protein extraction buffer. The homogenate was incubated in buffer for 1 h at 4°C, and the homogenized lysates were then centrifuged for 15 min at 14,000 rpm. The supernatant was removed, and the protein concentration was determined by spectrophotometry using the Coomassie Plus Protein Assay (Pierce, Rockford, IL). The lysate was then diluted 1:4 in 1 x PAGE sample buffer, sonicated, and heated at 95°C to denature the proteins. The lysate was then loaded onto a gel and subsequently run using standard Western blotting protocols with specific antibodies against the NAD(P)H oxidase subunits p47 phox and p67 phox (1:200, Santa Cruz Biotechnology, Santa Cruz, CA); CuZn-SOD (1:1,000, Santa Cruz Biotechnology); nitrotyrosine residues (1:500, Cayman, Ann Arbor, MI); eNOS (1:250, BD Transduction Laboratories, Lexington, KY); and actin (1:500, Sigma-Aldrich, St. Louis, MO), which was used as a loading control. The membrane was exposed for 5 min to a chemiluminescent developing system (for polyclonal antibodies: SuperSignal West Pico Chemiluminescent Substrate, Pierce; for monoclonal antibodies: ECL Western Blotting Detection reagents, Amershan Biosciences) and then finally exposed to X-ray film (Kodak, NY), which was subsequently developed, and intensities of the protein bands were determined using densitometry.
Superoxide anion. In addition, in situ production of superoxide anion was measured in 30-µm frozen kidney sections using the oxidative fluorescent dye dihydroethidium (DHE). Cytosolic DHE exhibits blue fluorescence, but once oxidized by superoxide to ethidium bromide it intercalates within the cell's DNA, staining its nucleus a fluorescent red (excitation at 488 nm, emission 610 nm). Serial sections were equilibrated under identical conditions for 30 min at 37°C in Krebs-HEPES buffer. Then, fresh buffer containing DHE (2 µmol/l) was applied onto each section, coverslipped, incubated for 30 min in a light-protected humidified chamber at 37°C ( 15 ), and then evaluated under fluorescence microscopy.
Histology. Midhilar cross sections of the ischemic kidney (1/animal) were examined using a computer-aided image-analysis program (MetaMorph, Meta Imaging Series 4.6). In each representative slide, immunostaining was semiautomatically quantified in 15-20 fields by the computer program, expressed as a percentage of staining of total surface area, and the results from all fields were averaged (5-8, 55).
Statistical analysis. Results are means ± SE. Comparisons within groups were performed using a paired Student's t -test, and among groups using ANOVA, with the Bonferroni correction for multiple comparisons, followed by an unpaired Student's t -test. Statistical significance was accepted for P
RESULTS
Lipid profile, MAP, and plasma creatinine were similarly and significantly elevated in all RVD groups compared with normal animals ( Table 1 ), whereas the degree of stenosis and PRA was not different among the groups ( Table 1 ) ( 6, 7, 22 ). Serum levels of vitamin C and E were significantly increased in the RVD+vitamins group ( Table 1 ).
Table 1. Systemic and single-kidney characteristics in normal, renovascular disease, and renovascular disease treated animals
Basal renal hemodynamics and function. Basal renal volume, RBF, GFR, and cortical perfusion were similarly and significantly decreased in all RVD compared with normal animals ( Table 1 ), whereas medullary perfusion was similar among the groups. Compared with normal animals, ITC was significantly lower along the nephron of RVD from the loop of Henle and distally, suggesting impaired tubular fluid reabsorption. Although ITC is normally increased in the stenotic kidney ( 19 ), RVD is characterized by distinctive tubular injury and decreased ITC ( 6 ). This impairment was slightly improved in RVD+vitamins and RVD+tempol animals, because ITC tended to increase compared with RVD in the loop of Henle (both in RVD+vitamins and RVD+tempol) and the distal tubule (in RVD+vitamins), although it remained attenuated in the collecting duct ( Table. 1 ).
Response to ACh and SNP. Infusion of ACh and SNP was not associated with a persistent change in blood pressure, as our laboratory has previously shown ( 6 ). In normal animals, ACh significantly increased RBF and GFR (to 747.9 ± 92.7 and 102.7 ± 7.4 ml/min, respectively, P
Fig. 1. Changes (%) in renal blood flow (RBF) and glomerular filtration rate (GFR) in response to ACh in normal, renovascular disease (RVD), RVD+tempol, and RVD+vitamins pigs. RBF response was particularly augmented after chronic antioxidant intervention, whereas GFR responses were significant in both treated groups but slightly greater in RVD+tempol. * P < 0.05 vs. baseline. P < 0.063 vs. baseline. # P = 0.089 vs. RVD+vitamins.
In response to SNP, normal animals showed a significant increase in RBF (to 728.1 ± 45.9 ml/min, P = 0.04) and cortical and medullary perfusion (to 5.2 ± 0.4 and 3.6 ± 0.39 ml·min -1 ·g -1, respectively, P < 0.04 for both), whereas in treated and untreated RVD animals these remained unchanged.
Redox status, renal protein expression, and morphology. Plasma endogenous SOD activity, as well as renal SOD activity (both total and its isoforms, Table 1 ) and protein expression ( Fig. 2 A ), was similarly and significantly decreased in both treated and untreated RVD compared with normal animals. However, the increased protein expression of NAD(P)H oxidase (p47 phox and p67 phox ) observed in RVD and RVD+tempol was normalized in RVD+vitamins ( Fig. 2, B and C ) and was consistently accompanied by diminished superoxide presence in renal tissue, as indicated by decreased red and increased blue fluorescence ( Fig. 2 D ). This clearly showed an overall decreased abundance of superoxide in vitamin-treated RVD animals. Furthermore, the elevated immunoreactivity of nitrotyrosine residues observed in RVD and RVD+tempol animals was normalized in RVD+vitamins ( Fig. 3 A ). Protein expression of eNOS in renal endothelial cells was similar to normal in both RVD and RVD+tempol. However, it showed a trend toward an increase in RVD+vitamins ( Fig. 3 B, P = 0.08 vs. RVD and RVD+tempol), suggesting increased generation of NO. Overall, these imply attenuation in both superoxide generation and reaction with NO, and consequently, decreased generation of peroxynitrite and increased availability of NO in RVD+vitamins animals. Moreover, RVD+vitamins showed a decrease in trichrome staining ( P < 0.05 vs. RVD and RVD+tempol), suggesting a significant decrease in renal fibrosis ( Fig. 4 ), although it remained greater than normal ( P < 0.001 vs. normal).
Fig. 2. Representative immunoblots and quantification of renal expression (cortex and medulla) of CuZn-SOD ( A ), NAD(P)H subunits p47 phox ( B ) and p67 phox ( C ), and in situ production of superoxide anion ( D ) in normal, RVD, RVD+tempol, and RVD+vitamins groups. Results of p47 phox quantification were similar to that of p67 phox and are not shown. Chronic antioxidant intervention induced a substantial decrease in superoxide generation. * P < 0.05 vs. normal.
Fig. 3. Representative immunoblots and quantification of renal expression (cortex and medulla) of nitrotyrosine ( A ) and endothelial nitric oxide synthase (eNOS; B ) in normal, RVD, RVD+tempol, and RVD+vitamins groups. Chronic antioxidant intervention tended to increase in eNOS and significantly decreased nitrotyrosine, suggesting potentially augmented nitric oxide bioavailability in RVD+vitamins. * P < 0.05 vs. normal. # P = 0.08 vs. normal, RVD, and RVD+tempol.
Fig. 4. Representative staining for trichrome in normal ( A ), RVD ( B ), RVD+tempol ( C ), and RVD+vitamins ( D ) groups. Long-term blockade of the oxidative stress pathway led to a substantial decrease in perivascular and tubulointerstitial fibrosis compared with RVD and RVD+tempol. Magnification: x 40.
DISCUSSION
This study demonstrates that both acute and chronic antioxidant intervention in experimental early RVD improve RBF and GFR responses to endothelium-dependent challenge. However, RBF response to ACh was greater in RVD animals chronically supplemented with antioxidants and was accompanied by greater attenuation of superoxide production, possibly increased availability of NO, and a decrease in renal fibrosis. Thus the impact of increased oxidative stress on the hemodynamics and function of the stenotic kidney in RVD is amplified by chronic mechanisms that promote renal structural injury.
Oxygen radicals may lead to renal vasoconstriction and endothelial dysfunction, may enhance tubuloglomerular feedback, and may modulate NO-dependent actions in the normal and sick kidney ( 16, 42, 53 ). Augmented formation of ROS in common pathological situations such as atherosclerosis and hypertension is often mediated by activation of the renin-angiotensin system and, consequently, NAD(P)H oxidase ( 16, 34, 35 ). Despite the fact that systemic PRA was not elevated in RVD animals in the current study, the intrarenal renin-angiotensin system was likely activated within the stenotic kidney. The signaling cascade for ROS generation through activation of NAD(P)H oxidase by angiotensin II seems to involve a feed-forward mechanism that permits ongoing prolonged production of ROS ( 16 ). ROS play an important role in many cardiovascular and renal diseases and modulate several factors involved in vascular dysfunction, inflammation, cell proliferation, and tissue growth. Indeed, the ability to inhibit these pathogenic mechanisms using antioxidants and oxygen-radical scavengers ( 8, 21 ) provides further evidence for the importance of ROS in regulating these mechanisms.
Our laboratory has previously shown that increased oxidative stress and upregulation of inflammatory and profibrotic factors in RVD resulted in considerable structural injury and was associated with marked impairment of renal hemodynamics, function, and responses to challenge ( 6, 7 ). The current study extended our previous observations and explored the contribution of increased oxidative stress to short- and long-term regulation of renal hemodynamics in RVD. We interfered with the oxidative stress pathway in RVD using either intrarenal infusion of the short-acting membrane-permeable SOD mimetic tempol ( 43, 53 ) or chronic antioxidant intervention using long-acting vitamins C and E. These agents can improve endothelial function and increase NO availability by scavenging ROS or by reducing ROS production ( 28, 50 ). The superoxide anion has high affinity for NO, and their rapid reaction is 3 times faster than the reaction of superoxide with SODs and 10,000 times faster than its reaction with antioxidant enzymes ( 12, 13 ). Tempol is a specific and potent short-acting ( 48 ) superoxide radical scavenger, which does not act as a catalase mimetic or alter hydrogen peroxide concentration and does not bind NO or produce superoxide anion ( 28 ). On the other hand, although vitamins C and E have lower affinity to ROS, they are long acting ( 2, 18 ) and can protect LDL from oxidation, reduce superoxide anion production and contribute to its scavenging, and modulate cytokines release and cell proliferation through inhibition of ROS-sensitive signaling pathways ( 25, 27, 50 ). Therefore, the use of these different antioxidant strategies allows comparing the immediate and long-term effects of oxidative stress in RVD.
The current study shows that basal renal hemodynamics and function remained attenuated in treated RVD compared with normal animals and were not different than in untreated RVD animals, likely because the severity of stenosis and renal atrophy was similar in all the RVD groups. In addition, ROS other than superoxide, such as singlet oxygen or hydroxyl radicals ( 17 ), might not have been modulated by the antioxidants used in this study ( 49 ). Nevertheless, we observed improved RBF response to ACh in RVD+tempol animals, resulting from decreased interaction with superoxide ( 43 ) and, consequently, increased bioavailability of NO ( 44, 51 ). A decrease in renal sympathetic nerve activity by tempol may have also played a role in this renovascular improvement ( 45 ). In addition, the vasodilatory effects of this active SOD analog might have greater effects in the afferent than the efferent arteriole ( 44, 51 ), which may give rise to the distinct effect on GFR observed in this and other studies ( 53 ). Moreover, superoxide scavenging may also increase GFR by diminishing the tubuloglomerular feedback mechanism ( 36, 54 ). On the other hand, the improvement in renal hemodynamic response to the endothelium-dependent vasodilator ACh was greater in RVD+vitamins animals. Several factors may help explain this differential response. In addition to potential scavenging of ROS, RVD+vitamins animals showed normalization of NAD(P)H oxidase expression, which remained elevated in the RVD+tempol group. NAD(P)H oxidase is the major source of vascular and tissue superoxide anion, and its p47 phox and p67 phox subunits are located in renal endothelial, mesangial, vascular smooth muscle, and adventitial cells ( 14 ). Furthermore, the substantial decrease in superoxide anion abundance in situ in the chronically supplemented animals provides compelling support to decreased formation of this potent vasoconstrictor in the RVD+vitamins group. The short-term nature of tempol infusion in this study is probably the reason that this effect was not observed in tempol-treated animals. Neither tempol nor vitamins modified endogenous SOD activity in this study, underscoring the pivotal contribution of the attenuation in ROS generation to the effects of chronic antioxidant intervention. Therefore, antioxidant vitamins may have resulted in a long-term effective decrease in ROS abundance, mainly by decreasing ROS formation in RVD.
Furthermore, vitamins C and E may have potentially increased NO bioavailability by upregulating eNOS ( 37, 50 ) an effect that was not observed with short-term tempol infusion. In addition to improving RBF response to ACh, suggestive evidence for increased NO bioavailability by chronic antioxidant intervention in the stenotic kidney was the decreased presence of nitrotyrosine residues, considered to be the footprint of peroxynitrite formation, which reflects interaction between superoxide and NO. Peroxynitrite is also a prooxidant cytotoxic metabolite capable of causing lipid peroxidation and cell damage ( 31 ). Although peroxynitrite formation may decrease using chronic tempol administration ( 11 ), this was not observed after the acute infusion performed in the present study. Furthermore, increased local and systemic oxidative stress can facilitate oxidation of LDL, a cytotoxic vasoconstrictor agent that promotes generation of ROS ( 1 ), downregulates eNOS ( 24 ), and augments functional and structural damage in the ischemic kidney. Hence, vitamins C and E may also improve RBF responses and limit tissue oxidative damage by protecting LDL from oxidation ( 4, 25 ). A decrease in both nitrotyrosine and lipid peroxidation may have interrupted the vicious cycle of oxidative stress and thus attenuated renal tissue injury in RVD+vitamins animals. In addition, chronic antioxidant intervention may modulate proinflammatory factors involved in renal injury ( 6, 8 ), which may account for the greater improvement in tubular function observed in RVD+vitamins animals in this study. Therefore, chronic modulation of the oxidative stress pathway may preserve renal function by several parallel mechanisms and thereby more effectively than short-term superoxide scavenging.
Previous studies have reported inconsistent effects of chronic antioxidant intervention on blood pressure ( 8, 30, 53 ), which probably depends on the model or the regimen used. In the current study, the protocols used for acute or chronic blockade of the oxidative stress pathway did not affect blood pressure in our model, and decreased renal perfusion pressure likely did not influence our results. Previous animal studies have used chronic but short-term administration of tempol, either through infusion by osmotic minipumps ( 53 ) or by oral ingestion through the drinking water ( 33 ) for 15 days. However, for technical reasons, this approach would not be practical for a 12-wk chronic treatment in a large animal like the pig. Moreover, the specific roles of oxidative and nitrisative radicals other than superoxide remain to be explored. The attenuated response to SNP compared with ACh in both RVD-treated groups might also be due to increased activity of additional vasoconstrictors (e.g., endothelin-1) that the low systemic dose of this NO donor that we used may have been insufficient to negate. Notably, the dose of SNP was limited by the systemic effects of this vasodilator and was titrated so as to prevent a decrease in blood pressure. In addition, attenuated sensitivity to NO and smooth muscle cell dysfunction cannot be excluded, either. It is possible that increased expression of eNOS (potentially suggesting increased NO bioavailability) was necessary to normalize renovascular responses to challenge with ACh and resulted in greater availability of NO than that provided by SNP. In parallel, ACh may possess alternative vasodilator properties by directly stimulating other downstream pathways, such as cGMP ( 26 ) or prostaglandins ( 41 ), that were modulated by antioxidants.
In summary, this study demonstrates that chronic antioxidant intervention in early RVD resulted in greater improvement in RBF and perfusion responses to challenge, likely as a result of prolonged restoration of NO availability, and decreased structural injury compared with acute intervention. These findings suggest that chronic effects of oxidative stress play an important part in the pathogenesis of renal injury in RVD.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grant HL-63282 and by the American Heart Association.
【参考文献】
Asmis R and Begley JG. Oxidized LDL promotes peroxide-mediated mitochondrial dysfunction and cell death in human macrophages: a caspase-3-independent pathway. Circ Res 92: E20-E29, 2003.
Bjorneboe A, Bjorneboe GE, and Drevon CA. Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats. Biochim Biophys Acta 921: 175-181, 1987.
Boaz M, Smetana S, Weinstein T, Matas Z, Gafter U, Iaina A, Knecht A, Weissgarten Y, Brunner D, Fainaru M, and Green MS. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomised placebo-controlled trial. Lancet 356: 1213-1218, 2000.
Carr AC, McCall MR, and Frei B. Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection. Arterioscler Thromb Vasc Biol 20: 1716-1723, 2000.
Chade AR, Best PJ, Rodriguez-Porcel M, Herrmann J, Zhu X, Sawamura T, Napoli C, Lerman A, and Lerman LO. Endothelin-1 receptor blockade prevents renal injury in experimental hypercholesterolemia. Kidney Int 64: 962-969, 2003.
Chade AR, Rodriguez-Porcel M, Grande JP, Krier JD, Lerman A, Romero JC, Napoli C, and Lerman LO. Distinct renal injury in early atherosclerosis and renovascular disease. Circulation 106: 1165-1171, 2002.
Chade AR, Rodriguez-Porcel M, Grande JP, Zhu X, Sica V, Napoli C, Sawamura T, Textor SC, Lerman A, and Lerman LO. Mechanisms of renal structural alterations in combined hypercholesterolemia and renal artery stenosis. Arterioscler Thromb Vasc Biol 23: 1295-1301, 2003.
Chade AR, Rodriguez-Porcel M, Herrmann J, Krier JD, Zhu X, Lerman A, and Lerman LO. Beneficial effects of antioxidant vitamins on the stenotic kidney. Hypertension 42: 605-612, 2003.
Chade AR, Rodriguez-Porcel M, Rippentrop SJ, Lerman A, and Lerman LO. Angiotensin II AT 1 receptor blockade improves renal perfusion in hypercholesterolemia. Am J Hypertens 16: 111-115, 2003.
Chen J, He J, Ogden LG, Batuman V, and Whelton PK. Relationship of serum antioxidant vitamins to serum creatinine in the US population. Am J Kidney Dis 39: 460-468, 2002.
Cuzzocrea S, McDonald MC, Mazzon E, Filipe HM, Centorrino T, Lepore V, Terranova ML, Ciccolo A, Caputi AP, and Thiemermann C. Beneficial effects of tempol, a membrane-permeable radical scavenger, on the multiple organ failure induced by zymosan in the rat. Crit Care Med 29: 102-111, 2001.
Fukai T, Folz RJ, Landmesser U, and Harrison DG. Extracellular superoxide dismutase and cardiovascular disease. Cardiovasc Res 55: 239-249, 2002.
Griendling KK and FitzGerald GA. Oxidative stress and cardiovascular injury. Part II: animal and human studies. Circulation 108: 2034-2040, 2003.
Griendling KK, Sorescu D, and Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86: 494-501, 2000.
Guzik TJ, West NE, Pillai R, Taggart DP, and Channon KM. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39: 1088-1094, 2002.
Harrison D, Griendling KK, Landmesser U, Hornig B, and Drexler H. Role of oxidative stress in atherosclerosis. Am J Cardiol 91: 7A-11A, 2003.
Harrison DG and Ohara Y. Physiologic consequences of increased vascular oxidant stresses in hypercholesterolemia and atherosclerosis: implications for impaired vasomotion. Am J Cardiol 75: 75B-81B, 1995.
Kipp DE and Rivers JM. Comparison of isotope dilution and excretion methods for determining the half-life of ascorbic acid in the guinea pig. J Nutr 114: 1386-1395, 1984.
Krier JD, Ritman EL, Bajzer Z, Romero JC, Lerman A, and Lerman LO. Noninvasive measurement of concurrent single-kidney perfusion, glomerular filtration, and tubular function. Am J Physiol Renal Physiol 281: F630-F638, 2001.
Krier JD, Rodriguez-Porcel M, Best PJ, Romero JC, Lerman A, and Lerman LO. Vascular responses in vivo to 8-epi PGF 2 in normal and hypercholesterolemic pigs. Am J Physiol Regul Integr Comp Physiol 283: R303-R308, 2002.
Landmesser U, Spiekermann S, Dikalov S, Tatge H, Wilke R, Kohler C, Harrison DG, Hornig B, and Drexler H. Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase. Circulation 106: 3073-3078, 2002.
Lerman LO, Nath KA, Rodriguez-Porcel M, Krier JD, Schwartz RS, Napoli C, and Romero JC. Increased oxidative stress in experimental renovascular hypertension. Hypertension 37: 541-546, 2001.
Lerman LO, Schwartz RS, Grande JP, Sheedy PF, and Romero JC. Noninvasive evaluation of a novel swine model of renal artery stenosis. J Am Soc Nephrol 10: 1455-1465, 1999.
Liao JK, Shin WS, Lee WY, and Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem 270: 319-324, 1995.
Libby P and Aikawa M. Vitamin C, collagen, and cracks in the plaque. Circulation 105: 1396-1398, 2002.
Lincoln TM and Keely SL. Effects of acetylcholine and nitroprusside on cGMP-dependent protein kinase in the perfused rat heart. J Cyclic Nucleotide Res 6: 83-91, 1980.
Meydani M. Vitamin E and atherosclerosis: beyond prevention of LDL oxidation. J Nutr 131: 366S-368S, 2001.
Mitchell JB, Samuni A, Krishna MC, DeGraff WG, Ahn MS, Samuni U, and Russo A. Biologically active metal-independent superoxide dismutase mimics. Biochemistry 29: 2802-2807, 1990.
National Institutes of Health. US Renal Data System Annual Data Report. Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2002.
Ness AR, Chee D, and Elliott P. Vitamin C and blood pressure-an overview. J Hum Hypertens 11: 343-350, 1997.
Noiri E, Nakao A, Uchida K, Tsukahara H, Ohno M, Fujita T, Brodsky S, and Goligorsky MS. Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol 281: F948-F957, 2001.
Nowzari FB, Davidson SD, Eshghi M, Mallouh C, Tazaki H, and Konno S. Adverse effects of oxidative stress on renal cells and its prevention by antioxidants. Mol Urol 4: 15-19, 2000.
Ortiz MC, Manriquez MC, Romero JC, and Juncos LA. Antioxidants block angiotensin II-induced increases in blood pressure and endothelin. Hypertension 38: 655-659, 2001.
Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, and Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97: 1916-1923, 1996.
Reckelhoff JF and Romero JC. Role of oxidative stress in angiotensin-induced hypertension. Am J Physiol Regul Integr Comp Physiol 284: R893-R912, 2003.
Ren Y, Carretero OA, and Garvin JL. Mechanism by which superoxide potentiates tubuloglomerular feedback. Hypertension 39: 624-628, 2002.
Rodriguez J, Grau A, Eguinoa E, Nespereira B, Perez-Ilzarbe M, Arias R, Belzunce M, Paramo J, and Martinez-Caro D. Dietary supplementation with vitamins C and E prevents downregulation of endothelial NOS expression in hypercholesterolemia in vivo and in vitro. Atherosclerosis 165: 33, 2002.
Rodriguez-Porcel M, Lerman A, Best PJ, Krier JD, Napoli C, and Lerman LO. Hypercholesterolemia impairs myocardial perfusion and permeability: role of oxidative stress and endogenous scavenging activity. J Am Coll Cardiol 37: 608-615, 2001.
Rodriguez-Porcel M, Lerman LO, Holmes DR Jr, Richardson D, Napoli C, and Lerman A. Chronic antioxidant supplementation attenuates nuclear factor- B activation and preserves endothelial function in hypercholesterolemic pigs. Cardiovasc Res 53: 1010-1018, 2002.
Safian RD and Textor SC. Renal-artery stenosis. N Engl J Med 344: 431-442, 2001.
Salom MG, Lahera V, and Romero JC. Role of prostaglandins and endothelium-derived relaxing factor on the renal response to acetylcholine. Am J Physiol Renal Fluid Electrolyte Physiol 260: F145-F149, 1991.
Schnackenberg CG. Physiological and pathophysiological 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. The SOD mimetic tempol restores vasodilation in afferent arterioles of experimental diabetes. Kidney Int 59: 1859-1864, 2001.
Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, Kohno M, and Abe Y. Renal sympathetic nerve responses to tempol in spontaneously hypertensive rats. Hypertension 41: 266-273, 2003.
Stulak JM, Lerman A, Porcel MR, Caccitolo JA, Romero JC, Schaff HV, Napoli C, and Lerman LO. Renal vascular function in hypercholesterolemia is preserved by chronic antioxidant supplementation. J Am Soc Nephrol 12: 1882-1891, 2001.
Textor SC and Wilcox CS. Renal artery stenosis: a common, treatable cause of renal failure? Annu Rev Med 52: 421-442, 2001.
Ueda A, Nagase S, Yokoyama H, Tada M, Noda H, Ohya H, Kamada H, Hirayama A, and Koyama A. Importance of renal mitochondria in the reduction of TEMPOL, a nitroxide radical. Mol Cell Biochem 244: 119-124, 2003.
Ueda J, Saito N, Shimazu Y, and Ozawa T. A comparison of scavenging abilities of antioxidants against hydroxyl radicals. Arch Biochem Biophys 333: 377-384, 1996.
Ulker S, McKeown PP, and Bayraktutan U. Vitamins reverse endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities. Hypertension 41: 534-539, 2003.
Wang D, Chen Y, Chabrashvili T, Aslam S, Borrego Conde LJ, Umans JG, and Wilcox CS. Role of oxidative stress in endothelial dysfunction and enhanced responses to angiotensin II of afferent arterioles from rabbits infused with angiotensin II. J Am Soc Nephrol 14: 2783-2789, 2003.
Wardle EN. Antioxidants in the prevention of renal disease. Ren Fail 21: 581-591, 1999.
Welch WJ, Mendonca M, Aslam S, and Wilcox CS. Roles of oxidative stress and AT 1 receptors in renal hemodynamics and oxygenation in the postclipped 2K,1C kidney. Hypertension 41: 692-696, 2003.
Wilcox CS and Welch WJ. Interaction between nitric oxide and oxygen radicals in regulation of tubuloglomerular feedback. Acta Physiol Scand 168: 119-124, 2000.
Wilson SH, Chade AR, Feldstein A, Sawamura T, Napoli C, Lerman A, and Lerman LO. Lipid-lowering independent effects of simvastatin on the kidney in experimental hypercholesterolemia. Nephrol Dial Transplant 18: 703-709, 2003.
作者单位：1 Hypertension and 2 Cardiovascular Diseases, Department of Internal Medicine, and 3 Department of Diagnostic Radiology, Mayo Clinic, Rochester, Minnesota 55905