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Abstract
We hypothesized that the downregulation of Cyp2c by tumor necrosis factor (TNF) α contributes to hypertension and renal injury in salt-sensitive angiotensin hypertension. Male Sprague-Dawley rats were fed a high-salt diet (8% NaCl), and osmotic minipumps were implanted to deliver angiotensin II for 14 days. Rats were divided into 3 groups: high salt, angiotensin high salt, and angiotensin high salt administered the TNF-α blocker, etanercept. Arterial pressure increased from 94±5 to 148±7 mm Hg during week 1 in the angiotensin high-salt group, whereas etanercept slowed blood pressure elevation during the first week in the treated group (90±2 to 109±6 mm Hg). After 2 weeks, arterial pressure increased to 156±11 mm Hg in the angiotensin high-salt group and 141±6 mm Hg in the etanercept-treated group. Albuminuria and proteinuria were significantly elevated in angiotensin high-salt rats and were reduced in the etanercept-treated rats. Urinary monocyte chemoattractant protein-1 excretion significantly increased in the angiotensin high-salt group (275±47 versus 81±19 ng/day) and was decreased in the etanercept-treated group (153±31 ng/day). Angiotensin high-salt rats also had a significant increase in renal monocyte/macrophage infiltration, and this was again attenuated by etanercept treatment. Renal expression of Cyp2c23 decreased, whereas renal epoxide hydrolase expression increased in angiotensin high-salt rats. Etanercept treatment increased Cyp2c23 expression and lowered epoxide hydrolase expression. These data suggest that TNF-α contributes to downregulation of Cyp2c23, blood pressure regulation, and renal injury in angiotensin high-salt hypertension.
Previous studies have shown that the renin–angiotensin system is important in the regulation of blood pressure, aldosterone release, and sodium reabsorption.1 Angiotensin II (Ang II) is a potent proinflammatory agent and mediates inflammatory cell chemotaxis and proliferation.2–4 Studies have also demonstrated that Ang II activation of the angiotensin type 1 receptor (AT1) results in an increase in the inflammatory cytokine tumor necrosis factor (TNF) α in a rat model of unilateral ureteral obstruction.5 Ang II also regulates the activation of nuclear factor κB and stimulates the production of chemokines, such as monocyte chemoattractant protein-1 (MCP-1), and cytokines, such as interleukin 6 and interleukin 8.4,6,7 Thus, Ang II is not only an important component in the regulation of blood pressure but is also involved in the inflammation that accompanies hypertension.
Epoxyeicosatrienoic acids (EETs) have been shown to contribute to vascular tone and improve endothelial function.8 In the Ang II model of salt-sensitive hypertension, renal EET production is inappropriately decreased. Cyp2c enzymes are the major EET-forming enzymes in the kidney,9 and EET production can be altered by changes in the expression and activity of these enzymes. Therefore, the downregulation of Cyp2c enzymes and any subsequent decrease in metabolites may limit any renal and cardiovascular protective effects of EETs. In addition to decreased epoxygenase output, the renal vasodilatory effects of EETs are also diminished when they are hydrated by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids.10 Recent reports have demonstrated a significant role for sEH in the long-term regulation of arterial blood pressure and inflammation.11–13
Epoxygenase metabolite and cytokine interactions have been demonstrated previously. However, a link to end-stage renal disease has not yet been well established. Cytokines have been shown to decrease cytochrome P450 expression in the liver and extrahepatic tissue.14,15 In endothelial cells, cytokines have also been shown to downregulate Cyp2c, as well as to reduce EET-mediated relaxation.16 Conversely, EETs are antiinflammatory in nature and possess the ability to inhibit TNF-α–elicited expression of vascular cell adhesion molecule 1 and to suppress cytokine activation.17 Nevertheless, a direct relationship between Cyp2c and TNF-α has not been completely elucidated. In the present study, we hypothesized that TNF-α is involved in the downregulation of the Cyp2c23 thereby reducing EET availability and contributing to blood pressure elevation and glomerular injury in Ang II salt-sensitive hypertension.
Methods
Animal protocols were approved by the Medical College of Georgia Animal Care and Use Committee. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg IP) and implanted with radiotelemetry transmitters (Data Sciences, Inc) according to the manufacturer’s specifications.18 After steady basal blood pressure recording, rats were again anesthetized with sodium pentobarbital (50 mg/kg IP) and implanted with osmotic minipumps that were used to deliver Ang II at a dose of 65 ng/min for 14 days. All of the rats (n=27) were fed an 8% NaCl diet (HS) throughout the experiment and were divided into 3 groups: HS, ANG/HS, and ANG/HS treated with the soluble TNF-α receptor, etanercept (1.25 mg/kg per day via SC osmotic minipump for 2 weeks, Immunex Corp). Rats were placed in metabolic cages to facilitate 24-hour urine collection at the end of the experiment.
Biochemical Assays
Urinary protein excretion was determined with a standard Bradford assay.19 Urinary MCP-1 and albumin excretion were measured using commercially available immunoassay kits (BD Biosciences and Exocell, Inc, respectively).
Vascular Homogenization for Protein Preparations
Renal microvessels were dissected quickly in ice-cold homogenization buffer in the presence of protease inhibitors and frozen in liquid nitrogen for determination of Cyp2c23 and sEH protein expression using Western blotting as described previously.9 The primary antibodies used were Cyp2c23 rabbit anti-rat Cyp2c23 (1:5000; Dr. Capdevila, Vanderbilt University, Nashville, TN) and rabbit anti-mouse sEH (1:2000; Dr. Hammock, University of California at Davis, Davis, CA), respectively. Goat anti-rabbit IgG-horseradish peroxidase was used as a secondary antibody for Cyp2c23 (1:100 000) and sEH (1:40 000). Band intensity was measured densitometrically, and the values were normalized to β-actin.
Isolation of Glomeruli for Real-Time PCR
Glomeruli were isolated from rat kidneys by a modified procedure as described previously.20 Total RNA was isolated from glomeruli using ultra-pure Trizol reagent according to the procedure described by the manufacturer (GIBCO-BRL). Concentration of total RNA was determined by measuring absorbance at 260 nm. A blend of oligonucleotide and random hexanucleotide primers were used for the RT of equal amounts of total RNA (2 μg) using the iScript cDNA synthesis kit in accordance with the manufacturer’s instructions (Bio-Rad Laboratories). Oligonucleotide primer pairs and TaqMan probes were designed from the published cDNA sequences of Cyp2c23, sEH, and GAPDH using Beacon Designer software (Premier Biosoft International). GAPDH was used as an internal standard, and TaqMan real-time RT-PCR was used to quantify mRNA expression relative to a control. The amplification was performed using iQ Supermix with the iCycler iQ Real-Time Detection System according to the manufacturer’s instructions (Bio-Rad Laboratories). A standard curve was generated for each primer pair and probe set for the purpose of determining PCR efficiency. Each sample was run in triplicate, and the mean threshold cycle was used to calculate relative mRNA expression using the comparative mean threshold cycle method. TaqMan probes were dual labeled with a 5′ fluorophore and a 3′ quencher and sequences were used as follows: Cyp2c23 probe 5′-AGG CAC CGA GAC AAC CAG CAC CAC-3′; Cyp2c23 forward 5′-TGG CTG TCT GTG GGT CTA ACT-3′; Cyp2c23 reverse 5′-AAT CAC ACG GTC AAG TTC CTC AT-3′; sEH probe 5′-TTC CAC ACC AGC ACA CCA GCC CAG-3′; sEH forward 5′-CAG AAT AAA CTG GGA ATC CCT CAA-3′; sEH reverse 5′-CTC ACT CTC TCA GGG TGG AAG-3′; GAPDH probe 5′-ACT CCA CGA CAT ACT CAG CAC CAG CA-3′; GAPDH forward 5′-CAC GGC AAG TTC AAC GGC-3′; and GAPDH reverse 5′-GGT GGT GAA GAC GCC AGT A-3′.
Evaluation of Kidney Monocyte Infiltration
In a separate group of animals, kidneys were isolated, perfused with 10% formalin solution to fix the kidney tissue, and cut into sections for immunohistochemistry as described previously.21 Kidney slices were incubated with a primary antibody that recognizes monocytes/macrophages (mouse anti-rat CD68). Sections were then incubated with anti-mouse secondary conjugated to horseradish peroxidase and visualized using diaminobenzamine chromogen. CD68-positive cells were counted in cortex and medulla by an evaluator who had no knowledge of the treatment groups.
Statistical Analysis
All of the data are presented as mean±SEM. Between-group comparisons were made using 1-way ANOVA for randomized samples in conjunction with Tukey’s post hoc test to identify significant difference between groups. Differences were considered significant at P<0.05. The mRNA and protein expression data were analyzed by unpaired t test. Analyses were performed using GraphPad Prism Version 4.0 software.
Results
ANG/HS treatment significantly increased mean arterial pressure (MAP) compared with rats fed an HS diet. Etanercept began to lower MAP on day 4 of Ang II infusion and significantly attenuated the increase in MAP from day 6 through day 11 (Figure 1A). Afterward, from day 12 through day 14, MAP was not different between the ANG/HS and etanercept-treated group. Heart rate was initially reduced, apparently because of the increase in MAP, but then began to increase after day 9 of Ang II infusion (Figure 1B). The initial decrease in heart rate was identical in ANG/HS rats and ANG/HS rats given etanercept treatment. However, heart rate in the etanercept group was not restored to the same level as that of the ANG/HS group.
Figure 1. Average 12 hours MAP (A) and heart rate (B) in ANG/HS rats with or without etanercept treatment for 2 weeks. Values are means±SE (n=5 per group).
Proteinuria after 2 weeks of Ang II infusion was used as an indicator of renal damage (Figure 2A). Urinary protein excretion was 5-fold higher in ANG/HS hypertensive rats than in control HS rats. This increase was significantly attenuated with etanercept treatment. Albuminuria was also used as an indicator of kidney damage (Figure 2B). Similar to protein excretion, urinary albumin excretion was elevated 80-fold in ANG/HS hypertensive rats compared with HS controls, and etanercept lowered urinary albumin excretion in ANG/HS rats.
Figure 2. Urinary protein excretion (A) and albumin excretion (B) in ANG/HS rats with or without etanercept treatment for 2 weeks (n=9 animals per group). Values are means±SE. *P<0.05 vs HS and †P<0.05 vs ANG/HS.
Monocyte/macrophage infiltration was determined and used as an indicator of inflammation in the kidney. ANG/HS hypertensive rats showed a significant increase in CD68-positive staining, and etanercept treatment lowered macrophage infiltration in ANG/HS rats (Figure 3B). Urinary MCP-1 excretion was increased 3-fold in ANG/HS hypertensive rats compared with HS rats. Etanercept treatment also reduced MCP-1 excretion in ANG/HS hypertensive rats (Figure 3A).
Figure 3. (A) Urinary MCP-1 excretion in HS rats and ANG/HS rats with or without etanercept treatment (n=9). (B) Average number of CD68 positive cells (monocytes/macrophages) per 1 mm2 in the kidney (cortex and medulla) of HS and ANG/HS rats with or without etanercept treatment (n=4 per group). Values are means±SE. *P<0.05 vs HS and †P<0.05 vs ANG/HS. (C) Representative image of monocyte/macrophage staining in the glomeruli of HS and ANG/HS rats with or without etanercept treatment.
Glomerular Cyp2c23 mRNA expression was decreased in ANG/HS hypertensive rats compared with HS rats (ANG/HS: 0.48±0.1 versus HS: 1.11±0.27 comparative Ct [2ďdCt]). Etanercept treatment increased Cyp2c23 mRNA expression to levels similar to HS rats (0.84±0.2 2ďdCt). We also detected an &6-fold increase in glomerular sEH mRNA expression in ANG/HS hypertensive rats compared with HS rats, and, in this case, etanercept treatment lowered sEH mRNA expression (ANG/HS: 10±5 versus HS: 1.7±0.8 versus ANG/HS/etanercept: 3.4±1.4 2ďdCt). Consistent with the glomerular mRNA expression data, Western blot analysis revealed a significant decrease in renal microvascular Cyp2c23 and an increase in sEH protein expression in ANG/HS rats when compared with rats on the HS diet alone (Figure 4). Etanercept treatment prevented the decrease in Cyp2c23 and the increase in sEH protein levels produced by ANG/HS.
Figure 4. Renal microvascular Cyp2c23 (A) and sEH (B) protein expression normalized to β-actin expression in ANG/HS rats with or without etanercept treatment (n=9 per group). Values are means±SE. *P<0.05 vs HS and †P<0.05 vs ANG/HS.
Discussion
Recent studies suggest that inflammation is a key component in the pathogenesis of hypertension and cardiovascular disease. Therefore, targeting the inflammatory response may be a valuable strategy for lowering the incidence of hypertension and subsequent cardiovascular complications. In the present study, we examined the role of TNF-α in salt-sensitive Ang II hypertension. TNF-α blockade slowed the progression of hypertension and renal damage as evidenced by reduced urinary albumin and protein excretion. Blocking TNF-α also reduced urinary MCP-1 excretion and renal macrophage infiltration in ANG/HS rats. Furthermore, mRNA and protein expression of the epoxygenase Cyp2c23 decreased, whereas sEH mRNA and protein expression increased in hypertensive rats, presumably lowering EET bioavailability. TNF-α blockade resulted in increased Cyp2c23 and decreased sEH in ANG/HS. Together, these data suggest that TNF-α plays a central inflammatory role in the pathogenesis of salt-sensitive hypertension and its associated renal damage.
In agreement with our previously published data,8,18 Ang II infusion in rats fed a high-salt diet significantly increased blood pressure and urinary albumin and protein excretion. Etanercept treatment reduced urinary excretion of albumin and protein in ANG/HS rats. This result was consistent with the findings of Muller et al22 in which etanercept treatment attenuated albuminuria in double-transgenic rats harboring both human renin and angiotensinogen. Muller et al22 measured tail-cuff systolic blood pressure at the end of the etanercept treatment period and reported that the decrease in albuminuria was independent of blood pressure. In the present study, we continuously measured blood pressure by telemetry throughout the etanercept treatment period and found that etanerecept slowed the elevation of blood pressure. This blood pressure–reducing effect was not maintained to the end of the treatment period. Thus, the data in the present study provide evidence that etanercept treatment protects the kidney from damage, and this effect could be, at least in part, because of the ability of etanercept to slow the onset of hypertension.
Aside from promoting the inflammatory response, TNF-α has a broad spectrum of biological activity including vasodilation, increased vascular permeability, and platelet activation.23 Etanercept binds to TNF-α and prevents it from interacting with TNF-α receptors.24 Thus, blocking TNF-α may prove useful in reducing the incidence of cardiovascular diseases associated with hypertension. Studies have also shown that Ang II is capable of inducing inflammation through both blood pressure–dependent and –independent mechanisms and may be partly responsible for vascular inflammation associated with hypertension.25 Additionally, Ang II receptor blockers have been shown to prevent Ang II-induced vascular inflammation in a high-renin model of hypertension.25–27 One of the important aspects of inflammation is macrophage infiltration, which is normally a defense mechanism against bacterial infection. Vascular tissues, however, can sustain damage from inflammation and the resulting macrophage infiltration.27 Ang II augments the production of the inflammatory cytokines and chemokines.27 MCP-1 is a chemokine involved in the recruitment of monocytes during inflammation and is induced in vascular endothelial and smooth muscle cells in response to Ang II.28 In the present study, we found increased macrophage infiltration in the kidney of ANG/HS rats and a corresponding rise in protein, albumin, and MCP-1 excretion. By blocking TNF-α, macrophage infiltration and urinary MCP-1 excretion in ANG/HS were reduced along with markers of kidney damage. These data suggest that TNF-α is involved in the inflammatory response and renal damage in ANG/HS hypertension.
To explore the mechanisms by which etanercept reduced blood pressure, renal damage, and inflammation in salt-sensitive ANG hypertension, we examined the expression of enzymes that regulate EET levels in the glomeruli and renal microvessels of ANG/HS-treated rats. We found that Ang II infusion with an HS diet elicited a decrease in glomerular Cyp2c23 mRNA expression and an increase in sEH mRNA expression, whereas etanercept treatment corrected these changes. Western blotting revealed comparable changes in protein expression of Cyp2c23 and sEH in the renal microvessels of ANG/HS rats, again with reversal of these trends by etanercept treatment. Downregulation of renal CYP450 epoxygenase enzymes has been shown to be associated with hypertension and end-organ damage in double-transgenic rats and Ang II hypertension.18,29 In particular, Cyp2c23 is highly expressed in rat kidney, where it contributes to renal EET biosynthesis.9 EETs are involved in the regulation of vascular tone and have been shown to possess antiinflammatory, antiproliferative, antimigratory, and antithrombotic properties.10 A connection between inflammatory cytokines and Cyp2c enzymes has been established. Cytokines have been shown to downregulate Cyp2c enzymes and reduce EET-mediated relaxation.16 Node et al30 demonstrated that 11,12-EET inhibited TNF-α–elicited expression of vascular cell adhesion molecule 1 and activation of nuclear factor κB suggesting that EETs possess antiinflammatory properties in addition to their vasodilatory actions. Regulation of EETs by the sEH enzyme also contributes to vascular and inflammatory responses.10 Our laboratory has shown previously that sEH inhibition attenuates afferent arteriolar constriction to Ang II in hypertension.13 sEH inhibition also lowered blood pressure and ameliorated renal damage associated with Ang II hypertension.13 A recent study has demonstrated that sEH inhibitors decreased plasma levels of proinflammatory cytokines in lipopolysaccharide-treated mice.31 Collectively, the data in the present study suggest that the downregulation of Cyp2c23 and the increase in sEH expression are likely to be associated with the activation of TNF-α signaling in ANG/HS hypertension.
Perspectives
In conclusion, our data indicate that TNF-α is involved in the progression of hypertension and renal damage in salt-sensitive Ang II hypertension, and these effects may be, in part, attributed to decreased EET bioavailability through the downregulation of Cyp2c23 and the upregulation of sEH. In ANG/HS hypertensive rats, we observed that etanercept slowed the progression of hypertension and decreased renal damage. These results demonstrate a clear association between inflammation and the development of hypertension. Overall, these results suggest that reduction of the TNF-α–mediated inflammatory response may be a potential therapeutic strategy for treatment of hypertension and related end-organ dysfunction. Future studies will be needed to determine the specific mechanisms by which TNF-α regulates epoxygenase enzymes in salt-sensitive hypertension.
Acknowledgments
We express our appreciation for the expert technical assistance provided by Hiram Ocasio, Eric Merkley, Janet Hobbs, and Laura Townsend. These studies were supported by grants from the National Heart Lung and Blood Institute (HL59699 and HL074167), American Heart Association Established Investigator Awards (to J.D.I. and D.M.P.) and postdoctoral fellowships from Georgia National Kidney Foundation and Southeast Affiliate of the American Heart Association (to A.A.E.).
- Received September 30, 2005.
- Revision received October 26, 2005.
- Accepted November 22, 2005.
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- Tumor Necrosis Factor α Blockade Increases Renal Cyp2c23 Expression and Slows the Progression of Renal Damage in Salt-Sensitive Hypertension
