1Hypertension and 2Cardiovascular Diseases, Department of Internal Medicine, and 3Department of Diagnostic Radiology, Mayo Clinic, Rochester, Minnesota 55905
Submitted 31 October 2003 ; accepted in final form 8 January 2004
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ABSTRACT |
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oxidative stress; renal function; antioxidants; tempol
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 (58, 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.
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METHODS |
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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·kg1·min1) and xylazine (0.03 mg·kg1·min1) in normal saline, administered via an ear vein cannula (0.05 ml·kg1·min1). 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·kg1·min1) and sodium nitroprusside (SNP; 6 nM·kg1·min1) 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 p47phox and p67phox 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·min1·g tissue1), 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 1x 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 p47phox and p67phox (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 1520 fields by the computer program, expressed as a percentage of staining of total surface area, and the results from all fields were averaged (58, 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 0.05.
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RESULTS |
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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 0.003 for both) and cortical and medullary perfusion (to 5.9 ± 0.6 and 4.3 ± 0.3 ml·min1·g1, respectively, P
0.02 each). This response was blunted in RVD, in which ACh did not further increase RBF, GFR, or any regional perfusion compared with baseline (Fig. 1A). However, responses in treated animals were improved. RVD+tempol showed a strong trend toward an increase in RBF (to 429.9.9 ± 110.6 ml/min, P = 0.063) and cortical perfusion (to 4.0 ± 0.9 ml·min1·g1, P = 0.07) and a substantial increase in GFR (to 65.4 ± 16.6 ml/min, P = 0.001) in response to ACh, whereas in RVD+vitamins ACh significantly increased single-kidney RBF (to 318.5 ± 73.2 ml/min, P = 0.01), GFR (to 48.3 ± 16.9 ml/min, P = 0.03), and cortical perfusion (to 4.8 ± 0.6 ml·min1·g1, P = 0.02). The increase in RBF responses to ACh was greater in RVD animals treated with chronic antioxidant supplementation compared with the other RVD groups (Fig. 1A). On the other hand, GFR responses were normalized in RVD+vitamins and were slightly greater in RVD+tempol compared with RVD+vitamins (Fig. 1B). Medullary perfusion remained unchanged among the experimental groups.
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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. 2A), 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 (p47phox and p67phox) 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. 2D). 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. 3A). 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. 3B, 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).
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DISCUSSION |
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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 p47phox and p67phox 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.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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