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(Hypertension. 2003;42:775.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
From the Vascular Biology Center (X.Z., D.M.P., J.D.I.), the Department of Physiology (J.D.I.), and the Department of Surgery (D.M.P.), Medical College of Georgia, Augusta; and the Division of Intramural Research (D.C.Z.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC.
Correspondence to John D. Imig, PhD, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500. E-mail jdimig{at}mail.\|[mgr ]\|g.edu
| Abstract |
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Key Words: endothelium-derived factors hypertension, sodium-dependent angiotensin II kidney vessels
| Introduction |
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Salt sensitivity is an important characteristic of a subgroup of humans with essential hypertension and other forms of salt-dependent hypertension that occurs in blacks, diabetics, patients treated with cyclosporine, or with aging.14,15 Previous studies in animal models of hypertension demonstrate that salt-sensitive hypertension develops after short-term exposure to angiotensin II.16,17 The mechanisms by which transient angiotensin IImediated blood pressure elevation can lead to persistent salt-sensitive hypertension have not been completely elucidated. Studies in rats suggest that a salt-inducible renal epoxygenase has antihypertensive properties, and kidney EET production is inappropriately low during the development of salt-sensitive hypertension.11,1820 Our previous study also showed that P450 epoxygenase protein levels were significantly decreased in angiotensin IIinfused animals fed a high-salt diet.21 The purpose of the present study was to determine if an inability to upregulate P450 epoxygenase enzymes is associated with persistent salt-sensitive hypertension after 2 weeks of exposure to angiotensin II.
| Methods |
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Measurement of Blood Pressure
Systolic blood pressure was measured in conscious rats by tail-cuff plethysmography to monitor the progression of hypertension.23 Blood pressure was measured between 9:00 AM and noon.
Mean arterial blood pressure was measured with the use of a radiotelemetry technique. Telemetry transmitters (Data Sciences) were implanted according to the manufacturers specifications into male Sprague-Dawley rats while under pentobarbital sodium anesthesia (65 mg/kg IP; Abbott Laboratories). After baseline measurements, rats were divided into 3 groups: HS, ANG/NS-NS, and ANG/HS-HS.
Urinary Albumin Levels in Angiotensin IIHypertensive Rats
Animals were housed in separate metabolic cages (Nalgene Corp) that efficiently separated urine from food and feces. Urine was collected in a conical tube containing 5 mg triphenylphosphine and cooled by dry ice. Samples were stored at -80°C until assayed for albumin. Urinary albumin levels were determined by an enzyme-linked immunosorbent assay (Exocell Inc).
Isolation of Renal Microvessels
Renal microvessels were isolated according to a method described previously.24 Briefly, the kidneys were infused with a physiological salt solution, and the renal microvessels were separated from the rest of the cortex with the aid of sequential sieving, a digestion period, and collection under a stereomicroscope. Renal microvessels collected from different groups were quickly frozen in liquid N2 and maintained at -80°C until assayed for protein or mRNA levels.
Immunoblot Analysis of CYP2C23, CYP2C11, and CYP2J Protein
Renal microvessels were harvested and microsomal fractions were prepared as previously described.23,24 Samples were separated by electrophoresis on 10% stacking Tris-glycine gels, and proteins were transferred electrophoretically to nitrocellulose membranes. The primary antibodies used were goat anti-rat CYP2C11 polyclonal antibody (1:1,000; Gentest), rabbit anti-rat CYP2C23 polyclonal antibody (1:5000; a generous gift from Dr Jorge Capdevila, Vanderbilt University), rabbit anti-human CYP2J2 polyclonal antibody (1:2000), and goat anti-rat CYP4A1 polyclonal antibody (1:2000; Gentest). The CYP2J2 antibody was made against a partially purified preparation of recombinant human CYP2J2 as described and has been shown to cross-react with all CYP2J isoforms.13 The blots were then washed in a PBS0.1% Tween 20 solution and incubated with the secondary antibody (anti-goat IgG 1:30 000 for CYP2C11 and CYP4A; anti-rabbit IgG, 1:100 000 for CYP2C23 and CYP2J2) conjugated to horseradish peroxidase for 1 hour at room temperature and washed. Detection was accomplished with the use of enhanced chemiluminescence (ECL, Amersham Corp), and blots were exposed to x-ray film (Hyperfilm-ECL, Amersham Corp). Band intensity was measured densitometrically, and the values were normalized to expression of ß-actin.
Reverse TranscriptionPolymerase Chain Reaction
Total RNA was prepared from isolated renal microvessels with the use of an ultra-pure TRIzol reagent according to the procedure described by the manufacturer (Gibco-BRL). Random hexanucleotide primers were used for reverse transcription (RT) of equal amounts of RNA. Oligonucleotide primers were designed from the published cDNA sequences of CYP2C11, CYP2C23, and GAPDH. GAPDH was used as an internal standard. The sequences of the CYP2C11 primers are sense 5'-CAA TCC GCA GTC TGA GTT-3' and antisense 5'-TGC TGA GAA TGG CAT AAA-3'. The sequences of the CYP2C23 primers are sense 5'-TCA CTA CGC TTG CTC TTG-3' and antisense 5'-TAT CCC TTA TGG GTA TCT TC-3'. The sequences of the GAPDH primers are sense 5'-AAT GCA TCC TGC ACC ACC AA-3' and antisense 5'-GTA GCC ATA TTC ATT GTC ATA-3'. The expected sizes of the amplified CYP2C11, CYP2C23, and GAPDH polymerase chain reaction (PCR) products are 463, 325, and 515 base pairs, respectively. RT-PCR was performed as previously described.25 After amplification, 15 µL of each PCR reaction mixture was electrophoresed on a 1.5% agarose gel with ethidium bromide (0.5 µg/mL). The gel was scanned with ultraviolet illumination by means of Digital Imaging and Analysis (Alpha Innotech Corporation).
Statistics
All data are presented as mean±SEM. Physiological parameter comparisons between groups were made through the use of ANOVA combined with a Newman-Keuls multiple range test. An unpaired 2-tailed t test was applied to compare the P450 protein levels. A value of P<0.05 was considered statistically significant.
| Results |
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As shown in Table 2, infusion of angiotensin for 14 days resulted in hypertension in association with a marked increase in urine albumin excretion. The increase in urinary albumin level was significantly greater in the ANG/HS-HS group than in the NS group, HS group, and ANG/NS-NS group. These data suggests that the combination of angiotensin II and a high salt diet greatly exacerbates the albumin excretion during the 2-week period. Two weeks after ending angiotensin infusion, the urinary albumin levels were not different among the groups (Table 2). These data suggest that the primary determinant of increased urinary albumin excretion is an arterial blood pressure >150 mm Hg.
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A previous study showed that levels of P450 epoxygenase enzymes were significantly decreased in angiotensin IIinfused rats given a high salt diet.21 To determine if an inability to upregulate the CYP2C and CYP2J isoforms was associated with the persistent hypertension after exposure to angiotensin II, we evaluated epoxygenase mRNA and protein expression in renal microvessels. Figure 2 and Figure 3 show representative Western blots and densitometric analysis of CYP2C11, CYP2C23, and CYP2J protein in renal microvessels. A high salt diet alone for 4 weeks significantly increased levels of CYP2C11 and CYP2C23 protein in renal microvessels. Two weeks after angiotensin withdrawal, CYP2C23 and CYP2C11 protein levels were not different in the ANG/HS-NS group (203±22 and 62±18, respectively) compared with the HS group (177±15 and 56±7, respectively). In contrast, the levels of CYP2C23 and CYP2C11 protein were reduced in renal microvessels in ANG/HS-HS animals (64±2 and 21±4, respectively) compared with the NS animals (113±11 and 37±6, respectively). In addition, CYP2J protein level was also significantly reduced in the ANG/HS-HS group (73±6) compared with the NS group (95±6). Thus, renal microvascular CYP2C11 and CYP2C23 protein levels decreased by 50% to 60% and CYP2J protein levels decreased by 23% in the ANG/HS-HS group versus the NS group. In contrast, at the end of the 4-week period, CYP4A protein expression was unaltered in the 6 groups (data not shown).
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Figure 4 shows P450 epoxygenase mRNA levels in renal microvessels at the end of the 4-week period in the various groups. CYP2C23 and CYP2C11 renal microvessel mRNA levels were increased in HS animals. No significant differences were found in ANG/HS-NS animals compared with HS rats, but CYP2C23 and CYP2C11 mRNA were lower in the ANG/HS-HS group (69±19 and 43±5, respectively) compared with the NS (103±27 and 51±1, respectively), HS (153±15 and 71±1, respectively), and ANG/HS-NS (165±7 and 64±3, respectively) groups.
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| Discussion |
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CYP2C isoforms are considered the major renal arachidonic acid epoxygenases in the kidney. In particular, CYP2C23 is highly expressed in rat kidney, where it is involved in regulating renal EET biosynthesis.11 The CYP2C11 isoform is expressed in rat kidney at much lower levels than CYP2C23. Increased expression of CYP2C enzymes and increased formation of EETs in the kidney occurs in response to increased salt intake.1011,18 These salt-inducible renal P450 epoxygenases have antihypertensive properties and contribute importantly to the maintenance of body fluid and electrolyte composition. Our previous study confirmed that kidney P450 epoxygenase protein levels are elevated in salt-loaded animals; however, this increase in P450 epoxygenase enzymes was abolished in angiotensin IIinfused animals fed a high-salt diet.21 These findings suggest that an inability to increase CYP2C protein levels is associated with the salt-sensitivity of angiotensin IIdependent hypertension. The current study showed that CYP2C11 and CYP2C23 protein levels were reduced in the ANG/HS-HS group 2 weeks after removal of angiotensin administration. Consistent with this data, renal microvascular CYP2C11 and CYP2C23 mRNA levels were increased in rats fed a high salt diet and decreased in ANG/HS-HS animals. The decreased levels of P450 epoxygenase enzymes were associated with a sustained hypertension after terminating angiotensin infusion. Taken together, these data suggest that inappropriate regulation of P450 epoxygenases plays a role in the development and maintenance of salt-sensitive hypertension.
The CYP2J subfamily also contributes to the formation of EETs in human and rodent kidneys.12,2729 In the past several years, multiple CYP2J enzymes have been isolated and characterized as arachidonic acid epoxygenases.12,27 Human CYP2J2, rat CYP2J3, and mouse CYP2J5 isoforms all preferentially catalyze epoxidation of arachidonic acid at the 14,15 position.27 It is important to point out that the catalytic activity of the CYP2J isoforms is severalfold less than that of the CYP2C enzymes.28 In addition, CYP2J3 biosynthesizes 19-HETE, which has been shown to inhibit voltage-gated calcium channels and is a potent vasodilator.29 Recent studies have shown that CYP2J immunoreactive protein is increased in the spontaneously hypertensive rat kidney, and this is associated with increased EET formation both in vitro and in vivo.13 Thus, several lines of evidence suggest that CYP2J proteins may play a role in renal arachidonic acid metabolism and blood pressure regulation. Previous findings demonstrate that CYP2J enzymes are expressed in renal microvessels, and neither a high salt diet alone or chronic angiotensin II infusion alone changes the CYP2J protein level in renal cortex or microvessels.21 Interestingly, the combination of angiotensin II infusion and a high salt diet significantly decreased renal microvascular CYP2J protein levels. In our current study, 2 weeks after the removal of angiotensin II infusion, CYP2J protein levels remained lower in the ANG/HS-HS group compared with the NS group. This finding suggests that renal microvascular CYP2J downregulation may also play a role in the maintenance of salt-sensitive hypertension after angiotensin II infusion.
Overall, the current study demonstrates that kidney P450 epoxygenase mRNA and protein levels are decreased in ANG/HS-HS animals 2 weeks after stopping angiotensin II infusion and that this decrease in P450 epoxygenase enzymes is associated with a persistent salt-sensitive hypertension. These studies support the postulate that EET regulation is associated with the salt-sensitive hypertension that accompanies and follows angiotensin II infusion.
Perspectives
Studies conducted over the past decade have provided convincing evidence that the renal P450 epoxygenase enzymes play a critical role in the renal response to a chronic salt overload.10,11,18,19,21,30 Epoxygenase enzymes are upregulated in response to increased salt intake and the vascular and tubular actions of EETs appear essential for the proper maintenance of fluid and electrolyte homeostasis and arterial blood pressure in this setting. In the present study, we demonstrate an impaired regulation of renal microvascular CYP2C11, CYP2C23, and CYP2J enzymes and persistent salt-sensitive hypertension after a 2-week exposure to a high salt diet and angiotensin II infusion. These findings are consistent with other animal models of salt-sensitive hypertension that also lack proper regulation of renal epoxygenase enzymes. Obviously, the association of renal P450 epoxygenase regulation and salt-sensitive hypertension needs to be investigated in human subjects before this pathway can be deemed a viable therapeutic target for salt-sensitive hypertension.
| Acknowledgments |
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Received May 12, 2003; first decision June 10, 2003; accepted June 27, 2003.
| References |
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-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride. J Clin Invest. 1999; 104: 11311137.[Medline]
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