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J Am Soc Nephrol 15:1816-1825, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Antioxidant Intervention Prevents Renal Neovascularization in Hypercholesterolemic Pigs

Alejandro R. Chade*, Michael D. Bentley§, Xiangyang Zhu*, Martin Rodriguez-Porcel{dagger}, Sara Niemeyer§, Beatriz Amores-Arriaga*, Claudio Napoli, Erik L. Ritman{ddagger}, Amir Lerman{dagger} and Lilach O. Lerman*,{dagger}

Department of Internal Medicine (Divisions of *Nephrology and Hypertension, and {dagger}Cardiovascular Diseases), and {ddagger}Physiology and Biophysics, Mayo Clinic, Rochester, §Department of Biological Sciences, Minnesota State University, Mankato, Minnesota; and Department of Medicine and Clinical Pathology–Excellence Research Center on Cardiovascular Diseases, University of Naples, Italy.

Correspondence to Dr. Lilach O. Lerman, Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: 507-266-9376; Fax: 507-266-9316; E-mail: lerman.lilach{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Experimental hypercholesterolemia (HC) may lead to microvascular neovascularization, but the underlying pathogenic mechanism remains unclear. We tested the hypothesis that HC-induced intra-renal neovascularization is associated with inflammation and increased oxidative stress, and would be prevented by chronic antioxidant intervention. Kidneys were excised from pigs after a 12-wk normal (n = 10) or HC diet (n = 8), or HC diet supplemented daily with antioxidant vitamins C (1 g) and E (100 IU/kg) (HC + vitamins, n = 7). Renal cortical samples were then scanned three dimensionally with micro–computed tomography, and microvessels were counted in situ. Blood and tissue samples were removed for measurements of superoxide dismutase (SOD) activity, protein expression of the NADP(H)-oxidase subunits gp91phox, p47phox, and p67phox, vascular endothelial growth factor (VEGF) levels and mRNA, VEGF receptors (Flt-1 and Flk-1), the proinflammatory transcription factor NF{kappa}B, and the oxidized LDL receptor LOX-1. Microvascular spatial density was significantly elevated in HC compared with normal kidneys but preserved in HC + vitamins. Expression of gp91phox and p67phox was decreased in HC pigs after antioxidant intervention, and SOD improved. The increased renal expression of VEGF and Flk-1 in HC was blunted in HC + vitamins, as were the significant increases in LOX-1, NF{kappa}B, and interstitial fibrosis. This study shows that renal cortical neovascularization elicited by diet-induced HC is associated with renal inflammation, fibrosis, and upregulation of VEGF and its receptor Flk-1, likely mediated by increased endogenous oxidative stress. Chronic antioxidant supplementation may preserve the kidney in HC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vascular nephropathies are a steadily increasing cause of end-stage renal failure, and growing evidence indicates the role of hypercholesterolemia (HC) and atherosclerosis as important risk factors for renal disease progression (1,2). HC can impair both the function and structure of many types of vascular beds, and even at early stage, HC alters vasomotor regulation in both large vessels and microcirculation (3). In addition, we have previously shown that HC may induce both myocardial (4) and renal cortical neovascularization (5) and may accelerate renal vascular, glomerular, and tubular damage (6,7).

A prominent mechanism that may be involved in the deleterious effects of HC on the kidney is augmented formation of reactive oxygen species (ROS) and increased oxidative stress (3,7–9). ROS have been implicated in the pathogenesis of renal injury by liberation of vasoconstrictors, inactivation of nitric oxide, and direct cellular damage (10). The abundance of ROS in HC also facilitates oxidation of LDL (9) and upregulation of its receptor LOX-1 (11), thereby increasing both the availability and uptake of oxidized LDL (ox-LDL), a potent vasoconstrictor and cytotoxic substance (3). This may lead to a vicious cycle of oxidation and inflammation, because through LOX-1 (12) and other receptors ox-LDL further increases oxidative stress and induces a plethora of oxidation-sensitive transcriptional events, including the redox-sensitive transcription nuclear factor-{kappa}B (NF{kappa}B), which plays a major role in regulation of inflammation and cell proliferation (13). Furthermore, exposure to both ROS and ox-LDL may upregulate vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and Flk-1 (14,15), which are involved in the vessel response to injury and angiogenesis and are tightly controlled by the redox environment (16). New vessel formation under these circumstances may be an adaptive response to local tissue fibrosis and inflammation. Hence, increased oxidative stress may induce new vessel formation both directly and by virtue of ox-LDL and inflammation.

The effects of oxidative stress might be modulated by antioxidants such as vitamin E and vitamin C, which inhibit LDL oxidation, leukocyte adhesion to endothelium, and endothelial dysfunction (17). In the kidney, vitamin E attenuates chronic injury associated with glomerulosclerosis (18) or aging (19), and prevents renal interstitial fibrosis associated with HC (20). We have previously shown that combined administration of vitamins E and C in experimental HC reduced LDL oxidizability and preserved renal endothelial function (9). However, the effects of antioxidant vitamins on renal neovascularization in HC have not been investigated. Micro–computed tomography (micro-CT) is a novel and powerful imaging technique that permits quantitative assessment of the three dimensional pattern of renal microvasculature structure in situ (5,21). Thus, the study presented here was designed to test the hypothesis that chronic antioxidant intervention would prevent HC-induced neovascularization in the kidney, in association with decreased expression of proinflammatory and growth factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was performed according to institutional guidelines for the care and use of laboratory animals. Twenty-five domestic, crossbred female pigs (60 to 70 kg) were studied after a 12 wk of either a normal (n = 10) or a 2% high-cholesterol diet (Harlan Teklad, Madison, WI; HC, n = 8), or a HC diet daily supplemented with 100 IU/kg of vitamin E and 1000 mg of vitamin C (HC + vitamins, n = 7). We have previously shown that this regimen effectively decreased oxidative stress (9,22–24). After completion of diet, the pigs were anesthetized with a combination of ketamine (0.2 mg/kg/min) and xylazine (0.03 mg/kg/min). A urine sample was collected through a suprapubic urinary catheter for determination of creatinine clearance and urine protein concentration by means of standard techniques. BP was measured by an intraarterial catheter placed in the left carotid artery. A blood sample was drawn from an ear vein cannula for determination of serum lipid levels, vitamin levels (HPLC) (24), and systemic superoxide dismutase (SOD) activity. Heparin (10,000 units) was then infused into the cannula, and the pigs euthanized with a lethal dose of pentobarbital sodium (Sleepaway, 100 mg/kg, Fort Dodge Laboratories). Immediately after the animals were euthanized, the left kidney was removed by a retroperitoneal incision and immersed in Krebs solution containing heparin (10 units/ml) to prevent drying. A lobe of tissue was removed from one end of the kidney and immersed in 10% buffered formalin (Sigma), and another was frozen in liquid nitrogen and stored at –80°C. In vitro studies were then performed. Expression of the CuZn and Mn SOD isoforms and the NAD(P)H-oxidase subunits gp91phox, p47phox, and p67phox were measured by Western blot and VEGF by real-time reverse transcriptase–PCR and ELISA. From renal tissue embedded in paraffin, 5-µm-thick midhilar cross-section slides were prepared for light microscopy (trichrome) and immunohistochemistry (NF{kappa}B and VEGF receptors, Flt-1 and Flk-1). In addition, a segmental artery perfusing the intact end of the kidney was cannulated, and a lobe of tissue was prepared for micro-CT.

Micro-CT
A saline-filled cannula was ligated in a segmental artery perfusing the intact end of the kidney, and infusion of 0.9% saline (containing 10 units/ml heparin) was initiated at 10 ml/min (Syringe Infusion Pump 22, Harvard Apparatus, Holliston, MA) under physiologic perfusion pressure (100 mmHg). After 10 to 15 min, the saline infusion was replaced with infusion (0.8 ml/min) of an intravascular contrast agent, which was a freshly mixed radiopaque silicone polymer containing lead chromate (Microfil MV122; Flow Tech, Carver, MA). This infusion was continued until the polymer drained freely from the segmental vein. After complete polymerization, a lobe of the polymer-filled tissue was trimmed from the kidney, placed in 10% buffered formalin, glycerinated, and encased in paraffin. The paraffin encasement served to physically stabilize the lobe for scanning and prevented air exposure during the scan. The kidney samples were scanned at 0.5-degree increments with a micro-CT scanner as described previously (5,25). After the scan, three-dimensional volume images were reconstructed, consisted of cubic voxels of 20 µm on a side, and were displayed at 40 µm cubic voxels for subsequent analysis.

Image Analysis
Image analysis was carried out by the ANALYZE (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN) software package, which provides tools to compute, display and analyze reconstructed volume images. For analysis of the cortex, the three-dimensional tomographic images were oriented so that the z-axis was parallel to the radial vessels.

Cortical thickness was determined, and the tomographic cortex was divided into inner, middle, and outer thirds, starting at the juxtamedullary cortex. A 40-µm-thick region of interest with a cross-sectional area of 16 mm2 (100 x 100 voxels) was subsequently analyzed at each zone. Blood vessels of different diameters (80 µm and larger, at 1-voxel increments) were then counted in the sample region and expressed as number of vessels per square centimeter (5). In addition, radial vessels and their branches were three-dimensionally isolated by a "connectivity" software (which allows for tomographic isolation of a vessel), and the vascular ramifications were examined in each group. To evaluate the elongation and tortuosity of each vessel, the three-dimensional vascular path length (the actual length of the vessel) and linear length (the shortest distance from base to tip of the vessel) was determined from the base of the main vessel at the corticomedullary junction to its tip at the superficial cortex. The vessel elongation was determined by Tree Analysis software, and the elongation factor was analyzed by dividing the path length by the linear length (26)

SOD Assay
SOD activity was measured in plasma with the Cayman Chemical Superoxide Dismutase Assay kit (Cayman Chemicals, Ann Arbor, MI) following the vendor’s instructions, as described previously (24,27).

Western Blotting
Standard Western blotting protocols were followed, and intensities of the protein bands were determined by densitometry, as described previously (23,28,29). Specific antibodies against the NAD(P)H-oxidase subunits gp91phox, p47phox, and p67phox (Santa Cruz Biotechnology, Santa Cruz, CA; 1:200 for all), CuZn-SOD (Santa Cruz; 1:500), and Mn-SOD (Stressgen Biotechnologies, Victoria, Canada; 1:5000) were used.

Total RNA Isolation and cDNA Synthesis
Total RNA was isolated from kidney by TriZol (Invitrogen) method. cDNA was synthesized with Invitrogen SuperScrip first-strand synthesis kit as recently described (26).

Real-Time Quantitative PCR for VEGF
To investigate the expression of VEGF mRNA, real-time PCR (DNA engine OPTICON, MJ Research) was performed with SYBR Green JumpStart Taq ReadyMix kit (Sigma). Briefly, 12.5 µl SYBR Green JumpStart Taq ReadyMix 0.25 µl internal reference, 0.5 µl primer 5', 0.5 µl primer 3', 1 µl cDNA, and 10.25 µl DEPC water reached 25 µl final reaction volume. The porcine gene specific sequence of VEGF primer used was upper 5'-ACC AAG GCC AGC ACA TAG GAG AGA-3' and lower 5'-CTC GCT CTA TCT TTC TTT GGT CTG-3'. The temperature profile included denaturation at 95°C for 3 min, followed 45 cycles of denaturation at 95°C for 40 s, 60 s at 60°C annealing and elongation with optics on for fluorescence monitoring. The relative amount of VEGF mRNA, normalized to an internal control GAPDH and relative to a calibrator (Normal), was calculated by 2–{Delta}{Delta}CT (26). The sequence of GAPDH primer is upper 5'-GGG CAT GAA CCA TGA GAA GT-3' and lower 5'- GTC TTC TGG GTG GCA GTG AT-3'. Real-time quantitative PCR results were quantified and expressed as percentage change in copy numbers compared with normal group.

VEGF Immunoabsorbent Assay
Tissue concentration of VEGF-165 was measured in homogenized renal tissue with an ELISA kit (Quantikine, R&D Systems, Minneapolis, MN), with plates precoated with a monoclonal antibody specific to human VEGF-165. Standards and samples were pipetted to 200 µl per well. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific to VEGF was added to the wells. After a wash, a substrate solution was added to the wells and color developed in proportion to the amount of the growth factor bound in the initial step. The color development was stopped, and the optical density of each well was determined with a microplate reader at 450 nm (26).

Immunohistochemistry for LOX-1 was performed in frozen renal tissue with monoclonal primary antibodies against LOX-1 (1:280) (29,30), followed by the Vectastain-Elite ABC Kit (Vector Laboratories, Burlingame, CA), following vendor’s instructions.

Immunohistochemistry for NF{kappa}B, and Flt-1 and Flk-1 was performed on deparaffinized renal tissue. Primary antibodies utilized were either monoclonal against p65 NF{kappa}B (Santa Cruz; 1:50), which measured total protein expression of both the active and inactive forms, or polyclonal against Flt-1 and Flk-1 (Santa Cruz; 1:20). The secondary antibody, IgG Envision Plus (Dako), was followed by staining with the Vector NovaRED substrate kit (Vector), and slides were counterstained with hematoxylin (29,30).

Histology
All histology slides, including corticomedullary cross sections of the kidneys (1 per animal) stained with trichrome, were examined by a computer-aided image analysis program (MetaMorph, Meta Imaging Series 4.6). In each representative slide, staining in renal cortex was semiautomatically quantified in 15 to 20 fields by the computer program, expressed as percentage of staining of total surface area, and the results from all fields averaged (29). In addition, glomerular score (percentage of sclerotic glomeruli) and arteriolar media-to-lumen ratio were assessed by means of standard techniques (7,28).

Statistical Analyses
Results are expressed as mean ± SEM. ANOVA was used to detect differences in values among the normal, HC, and HC + vitamins groups. If significant differences were found, the unpaired t test was used to detect specific differences. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight, mean arterial pressure, creatinine clearance, and urine protein excretion were similar among the groups. With the exception of triglycerides, lipid levels were similarly and significantly elevated in both HC groups compared with normal animals, as were vitamins C and E levels in HC + vitamins compared with the other groups (Table 1).


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  		Table 1. General characteristics (mean ± SEM) of normal (n = 10), high cholesterol (HC, n = 8), and HC pigs supplemented with antioxidant vitamins C and E (HC + Vit, n = 7)a
 
Vascular Architecture
Cortical vessels that were clearly distinguishable by micro-CT and could be counted ranged from large (diameters greater than 360 µm) to small (diameters 80 µm) radial vessels. Several branching orders of radial vessels extended from the arcuate vessels peripherally. The cortical vessel density was significantly greater in the HC compared with the normal kidneys (Table 2 and Figure 1, a and b, P < 0.01), most evidently in the small vessels (80 to 120 µm) of the inner cortex. However, vitamin supplementation in HC significantly reduced vascular density in the kidneys (Table 2 and Figure 1, a and b, P = NS versus normal), and the appearance of tomographically isolated vessels suggested that this reduction resulted from fewer microvascular sprouts (Figure 1c). There were no significant differences among the groups in vascular three-dimensional path length, linear length, or the elongation factor (Table 2).


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  		Table 2. Morphological evaluation and protein expression (mean ± SEM) in kidneys of normal (n = 10), high cholesterol (HC, n = 8), and HC pigs supplemented with antioxidant vitamins C and E (HC + Vit, n = 7)a
 


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  		Figure 1. Representative renal microvascular density (a, b) and tomographically isolated vessels (c) in normal, hypercholesterolemia (HC) and HC + vitamins pigs. Antioxidant vitamin intervention normalized the increased vascular density and sprouting in HC kidneys. *P < 0.05 versus normal.

 
Redox Status
The systemic SOD activity was significantly decreased in HC compared with normal, but normalized in HC + vitamins (Figure 2a). Although renal tissue expression of the CuZn isoform was similarly attenuated in both HC and HC + vitamins compared with normal (Figure 2b), the increased in renal MnSOD expression (Figure 2c) and SOD activity in HC + vitamins suggested that antioxidant intervention improved the endogenous SOD-mediated superoxide anion scavenging. In addition, protein expression of the NAD(P)H-oxidase subunits gp91phox and p67phox was increased in HC (P = 0.01 and 0.07 versus normal, respectively), but normalized in HC + vitamins (Figure 3, a and b), suggesting decreased potential for generation of superoxide. The expression of p47phox was similar among the groups (Figure 3c).



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  		Figure 2. Systemic superoxide dismutase (SOD) activity (a) and renal expression of CuZn SOD (b) and MnSOD (c) in normal, hypercholesterolemia (HC) and HC + vitamins. Antioxidant intervention normalized SOD activity and improved renal expression of MnSOD, suggesting enhanced superoxide anion scavenging. *P < 0.05 versus normal, {dagger}P < 0.05 versus HC.

 


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  		Figure 3. Renal expression of the NAD(P)H-oxidase subunits gp91phox (a), p67phox (b), and p47phox (c) in normal, hypercholesterolemia (HC) and HC + vitamins. Antioxidant intervention decreased gp91phox and p67phox expression in HC, suggesting a decrease in superoxide anion production. *P < 0.05 versus normal, #P = 0.07 versus normal.

 
Inflammation and Fibrosis
Although no glomerulosclerosis was observed in any group, trichrome staining revealed a significant increase in inflammation, and perivascular and tubulointerstitial fibrosis in the HC kidneys compared with normal, as well as increased media-to-lumen ratio in intrarenal vessels. These were all markedly attenuated in HC + vitamins, although trichrome staining remained greater than normal kidneys (Table 2, Figure 4a). A similar pattern was observed in the protein expression of the proinflammatory NF{kappa}B, which was mostly expressed at the proximal tubules and glomerular compartment. The attenuated (albeit not normalized) expression in HC + vitamins suggested decreased intrarenal inflammation compared with HC (Table 2, Figure 4b). Moreover, LOX-1 expression, which was increased in vascular endothelial cells of HC pigs compared with normal (suggesting increased potential for ox-LDL uptake), was normalized after vitamin supplementation (Table 2, Figure 4c).



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  		Figure 4. Representative renal staining for trichrome (a) and expression of proinflammatory nuclear factor kappa-B (NF{kappa}B) (b) and the specific oxidized LDL receptor, LOX-1 (c), in normal, hypercholesterolemia (HC) and HC + vitamins. Note the presence of inflammatory infiltrates in the HC kidneys (arrow). Vitamins significantly decreased the elevated expression of both tubular and glomerular NF{kappa}B and vascular LOX-1 in HC (arrows), and substantially attenuated renal inflammation and fibrosis. Original magnification, x20.

 
The cortical levels of VEGF protein were significantly elevated in HC pigs in comparison to normal, and were slightly decreased in HC + vitamins, in which they were not significantly different from either normal or HC (Table 2). Furthermore, VEGF mRNA was markedly increased in HC and significantly attenuated after antioxidant intervention in HC (Figure 5a). In addition, immunostaining for Flk-1, which was apparent in vascular endothelial cells of large vessels and in renal tubules, was significantly greater in HC than in normal and normalized in HC + vitamins kidneys (Table 2, Figure 5b). Immunostaining for Flt-1 was not different among the groups (Table 2, Figure 5c).



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  		Figure 5. Renal vascular endothelial growth factor (VEGF) mRNA expression assessed by real-time quantitative PCR (a) and immunohistochemistry for VEGF receptors Flk-1 (b) and Flt-1 (c) in normal, hypercholesterolemia (HC) and HC + vitamins. HC distinctly increased VEGF mRNA and tubular expression of Flk-1, which were normalized after chronic antioxidant supplementation. Flt-1 expression was not different among the groups. Original magnification, x40. *P < 0.05 versus normal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that the augmented microvascular density in renal cortex of HC kidneys was normalized after chronic blockade of the endogenous oxidative stress pathway with antioxidant vitamins. Furthermore, chronic antioxidant intervention significantly decreased intrarenal inflammation and fibrosis, suggesting a potential role for antioxidant intervention in preserving the kidneys in the early stages of atherosclerosis.

HC can induce functional vascular changes in both the heart (22) and the kidneys (7,31,32) before appearance of overt atherosclerotic lesions. We have previously shown that these were associated with increased spatial density of microvessels (4,5). Furthermore, we have suggested a crucial role for oxidation-sensitive mechanisms in modulating several aspects of renal damage in HC (7,9). However, the role of increased oxidative stress in modulating neovascularization was unclear, and it remained unknown whether the latter could be mitigated by antioxidant intervention.

Various mechanisms may be responsible for the changes in the spatial density of blood vessels observed in this study. HC can stimulate the release of various cytokines and growth factors that modulate angiogenesis (3). We have previously observed that increased neovascularization was associated with augmented VEGF immunostaining in HC kidneys (5). In addition, we have also shown that HC increased the levels of the PGF-2{alpha} isoprostanes, markers of oxidative stress in vivo, and that these were decreased by our antioxidant regimen (22,26,28). The study presented here extends our previous observations and implies that increased oxidative stress in HC may play a role in upregulation of VEGF message and protein levels, as well as in renal cortical neovascularization.

To test the origin of the increased oxidative stress, we explore the renal protein expression of the NAD(P)H-oxidase system, which is a major source of vascular and tissue superoxide anion. This enzyme is composed of several subunits, of which we studied the gp91phox (plasma membrane mainly of neutrophils and vascular cells), p47phox, and p67phox (cytosolic of nonphagocytic cells) subunits, which are located in vascular smooth muscle, endothelial, mesangial, and adventitial cells (33). Indeed, p67phox (which is essential for electron flow from NAD(P)H to O2 to form superoxide (34)) and gp91phox were both increased in HC kidneys, potentially indicating both inflammatory, vascular, and renal cell sources for increased superoxide generation. Of note, diverse expression of these subunits may result from their variable predominance in different types of renal cells, which cannot be easily distinguished with our Western blot techniques. In parallel, HC can activate other sources of ROS such as xanthine oxidase (35,36). Increased potential for superoxide generation was accompanied by attenuated activity and expression of SOD, suggesting an overall abundance of superoxide anion, which may have consequently increased VEGF expression (37).

Furthermore, HC kidneys also showed increased immunoreactivity of the VEGF receptor Flk-1. Both Flt-1 and Flk-1 are tyrosine kinase receptors found in endothelial cells and monocytes, and are involved in both physiologic and pathologic vasculogenesis and angiogenesis (38). VEGF receptors can also be expressed in tubular epithelial cells, and the enhanced expression might provide survival benefit in situations such as renal ischemia and toxic injury (39). In addition, recent evidence indicates that VEGF plays an important role in endothelial cell proliferation and capillary repair in the glomeruli, mediated mainly through the Flk-1 receptor (40). Indeed, the differential expression of VEGF receptors during HC supports the notion that Flk-1 may have a greater influence on the spatial density of vasculature than Flt-1. Notably, normalized p67phox and gp91phox expression as well as improved SOD activity and expression, vascular density, and Flk-1 expression were observed in HC + vitamins, suggesting improved milieu during chronic antioxidant intervention. Interestingly, the increase in total SOD activity was achieved by upregulation of MnSOD rather than CuZnSOD, which had been decreased in HC. Indeed, disparate changes in the expression and function of this system may be noted in disease states and likely reflect the diverse roles of this scavenger system (41).

In addition to a direct effect of ROS, neovascularization might be enhanced in HC by increased ox-LDL uptake by its vascular endothelial receptor, LOX-1 (42). VEGF upregulation by ox-LDL (43) may conceivably be mediated by LOX-1, whose gene expression is in turn tightly regulated by the redox state (11). Both ox-LDL and LOX-1 have also been implicated in endothelial dysfunction and atherosclerosis, likely by upregulating NAD(P)H oxidase (mainly gp91phox (44)) as well as in glomerulosclerosis (45). Additionally, one of the pathophysiological consequences of ox-LDL binding to LOX-1 is activation of the proinflammatory NF{kappa}B (12). Experimental HC is known to invoke renal inflammation and fibrosis (5,6,20), and we have shown that these were associated with renal upregulation in both the expression and activity of NF{kappa}B (7,28). NF{kappa}B is a transcription factor present in virtually all cell types and involved in inflammation and cell proliferation (13), and might have actively contributed to augmented vascular density, inflammation, and fibrosis in the HC kidneys (13,46). Remarkably, animals supplemented with antioxidant vitamins C and E showed markedly decreased LOX-1, NF{kappa}B total protein expression, and inflammation, which may have resulted from blockade of the oxidative stress pathway (47,48). This was accompanied by a substantial attenuation in renal fibrosis, which might have resulted from the modulating actions of vitamins on lipid peroxidation and profibrotic activity involved in renal tissue damage (23,24,49,50). Notably, vitamin C contributes to the recycling of vitamin E (3), thus the combined intervention used in the study presented here likely maximize the beneficial effects. Furthermore, antioxidant intervention also improves renal endothelial function in HC (9). Therefore, fewer episodes of renal ischemia may be another mechanism that blunted VEGF, NF{kappa}B, and LOX-1 expression, and diminished renal fibrosis and neovascularization.

Indeed, rather than the result of angiogenesis, the increase in spatial vascular density in this study might have been related to microvascular vasodilation in response to changes in renal perfusion or to some other stimulus. Alteration in renal perfusion may also be responsible for the appearance of new vessels or collateral arterial circulation. However, Stulak et al. (9) have shown that basal renal perfusion in vitamin-supplemented HC pigs was similar to normal. Alternatively, changes in the extravascular space may have artifactually altered the vascular density, but this is improbable because cortical thickness was not affected by HC in the current or in previous studies (31). Furthermore, in the study presented here we did not observe changes in vascular elongation or tortuosity, which would likely accompany changes in the extravascular space. The reason for the relatively high urinary protein levels, which was observed in all of the experimental groups, remains unclear and may be related to species differences.

In conclusion, the study presented here shows that the increase in cortical vascular density with HC diet was attenuated after antioxidant vitamin intervention, with corresponding decreases in the cortical tissue levels of VEGF and immunostaining for the VEGF receptor, Flk-1. Likewise, interstitial fibrosis and inflammation accompanying the HC diet were attenuated with vitamin supplementation. These results support a role for antioxidant strategies for preserving the kidney in the early stage of atherosclerosis.


   Acknowledgments
 
This study was supported in part by NIH grants HL77131, HL-63282, HL69840, and EB00305, and the AHA. We thank the following people for their technical assistance: Denise A Reyes, Steven M. Jorgensen, Patricia E. Beighley, and Julie M. Patterson.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 11, 2004. Accepted for publication April 19, 2004.




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