(Hypertension. 1999;33:424-428.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
From the Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, DC.
Correspondence to Christine G. Schnackenberg, PhD, Georgetown University Medical Center, Division of Nephrology and Hypertension, Bldg D, Room 385, 4000 Reservoir Rd NW, Washington, DC 20007.
| Abstract |
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(8-ISO) is formed
nonenzymatically from the attack of superoxide radical on
arachidonic acid. Therefore, 8-ISO is a marker of
oxidative stress in vivo. We have recently shown that short-term
administration of the membrane-permeable, metal-independent superoxide
dismutase mimetic tempol (4-hydroxy-2, 2, 6, 6-tetramethyl
piperidinoxyl) normalizes blood pressure in spontaneously
hypertensive rats (SHR). The present study was designed to test
whether prolonged administration of tempol ameliorates oxidative stress
and hypertension in SHR. In control SHR (n=8), mean
arterial pressure and heart rate were increased and renal
blood flow and glomerular filtration rate were reduced
compared with control Wistar-Kyoto rats (WKY) (n=7). Twenty-four-hour
renal excretion of 8-ISO was significantly increased in SHR compared
with WKY. Two weeks of tempol administration in the drinking water
(1 mmol/L) to SHR (n=8) decreased mean arterial
pressure by 18% (162±8 to 134±6 mm Hg,
P<0.05), increased glomerular filtration
rate by 17% (1.6±0.2 to 1.9±0.3 mL/min), and decreased renal
excretion of 8-ISO by 39% (9.8±0.7 to 6.0±0.7 ng/24 hours,
P<0.05). In contrast, tempol administration to WKY
(n=6) had no significant effect on mean arterial pressure
(115±5 versus 118±8 mm Hg), glomerular filtration
rate (3.0±0.4 versus 2.5±0.5 mL/min), or renal excretion of 8-ISO
(7.9±0.4 versus 6.8±0.7 ng/24 hours). In conclusion, the SHR is a
model of hypertension and renal vasoconstriction associated with
oxidative stress. Because long-term administration of a superoxide
scavenger reduces blood pressure and oxidative stress in vivo, this
study suggests a role for oxygen radicals in the maintenance of
hypertension in SHR.
Key Words: oxidative stress isoprostanes oxygen radicals superoxide dismutase
| Introduction |
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F2-isoprostanes are a family of
prostaglandin (PG) F2-like compounds
that are formed from the nonenzymatic reaction of
arachidonic acid and oxygen radicals in vivo and in
vitro.15 Because of their unique synthesis pathway,
F2-isoprostanes are recently proposed markers of
oxidative stress. For example, tissue and plasma levels of
F2-isoprostanes are increased in rats with
oxidative stress caused by carbon tetrachloride.16 Among
the several PGF2-like compounds,
8-iso-PGF2
(8-ISO) is the major urinary
metabolite of F2-isoprostanes17 and
is markedly elevated in the urine of rats after renal
ischemia/reperfusion.18
Tempol (4-hydroxy-2, 2, 6, 6-tetramethyl piperidinoxyl) is a stable, membrane-permeable, metal-independent superoxide dismutase mimetic. Tempol is a small molecular weight cyclic nitroxide that has been used as a spin trap for superoxide19 20 and reduces superoxide-related injury in ischemia/reperfusion,21 inflammation,22 and radiation.23 We have recently reported that short-term infusion or 7-day intraperitoneal administration of tempol normalizes blood pressure in the SHR.8 The aim of the present study was to assess the oxidative stress of adult SHR from the measurement of the renal excretion of 8-ISO and to determine whether long-term tempol administration ameliorates the hypertension and oxidative stress in SHR. We measured mean arterial pressure and renal hemodynamic and excretory function in WKY and SHR under normal conditions and after 2 weeks of oral tempol administration.
| Methods |
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Thereafter, WKY and SHR were anesthetized with thiobutabarbital (100 mg/kg IP, Inactin, Research Biochemicals International) and maintained at 37°C on a servo-controlled heated rodent operating table. A tracheostomy was performed with polyethylene PE-240 tubing and the left jugular vein and carotid artery were cannulated with PE-50 tubing. A 1% albumin solution in 0.154 mol/L NaCl was infused at 2 mL/h IV, to maintain a euvolemic state. A midline incision was made and the left renal artery was isolated. A blood flow probe was placed around the renal artery and connected to a transit-time blood flowmeter (1RB, Transonic Systems, Inc). We have shown previously that this method of measuring real-time changes in renal blood flow (RBF) is valid in the rat.24 Mean arterial pressure (MAP) and heart rate were recorded continuously from the carotid artery using a Statham pressure transducer (model P23, Gould Instruments) and MACLab data acquisition software. Glomerular filtration rate (GFR) was determined from the clearance of [3H]-inulin. After surgery and a 60-minute equilibration period, MAP, heart rate, GFR, and RBF were measured for 30 minutes and the data were averaged.
Determination of 8-Iso Prostaglandin F2
Urinary 8-ISO was extracted, purified, and measured according to
methods previously established using an enzyme immunoassay kit (Cayman
Chemical). Briefly, urine was spiked with
[3H]-8-ISO, treated with ethanol followed by
15% potassium hydroxide, incubated for 1 hour at 40°C, and acidified
to pH 4.0 with hydrochloric acid. The sample was extracted using a
polyboronic acid column, eluted with ethyl acetate containing 1%
methanol, and evaporated under nitrogen. 8-ISO was assayed using
competitive binding with mouse anti-rabbit IgG monoclonal antibody in a
96-well plate. Concentration of the reaction product was determined
from its absorbency at 412 nm using a standard curve. Samples were
assayed in duplicate and corrected for individual recovery of
[3H]-8-ISO. The recovery averaged 76% (n=12).
The limits of sensitivity of the assay are 1 to 3 pg/mL and the
intraassay coefficient of variation is 8% (n=6). Samples were diluted
to fall in the middle portion of the linear standard curve (10 to 100
pg/mL). To validate the collection method for measurement of 8-ISO in
urine, a second set of urine was collected from control SHR (n=5) and
WKY (n=5) in metabolic cages into containers containing 10
µL of 0.01% butylated hydroxytoluene as suggested by Roberts and
Morrow15 and assayed as described above. The results
showed a similar increase (30%) in renal excretion of 8-ISO in control
SHR compared with control WKY as described for the control groups
reported below.
Statistics
All values shown are mean ± standard error.
Analysis of variance was used to test the overall effect of
tempol. Unpaired comparisons using Student's t test were
used to determine significance between specific groups.
P<0.05 was considered statistically significant.
| Results |
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MAP in WKY and SHR is represented in Figure 1
. Under normal conditions, MAP in SHR
was increased by 41% compared with WKY (SHR: 162±8 versus WKY:
115±5 mm Hg, P<0.001). After 2 weeks of tempol
administration, MAP was reduced in SHR to a value that was not
significantly different from WKY (SHR: 134±6 versus WKY: 118±7
mm Hg). MAP in SHR given tempol was significantly lower by 18%
compared with normal SHR. Analysis of variance showed that
tempol specifically and significantly (P<0.05) decreased
MAP in SHR. Heart rate was significantly (P<0.001) elevated
in SHR (420±6 bpm) compared with WKY (374±9 bpm) during control
conditions and was not changed by tempol (SHR: 414±9 versus WKY:
373±8 bpm).
|
The Table
depicts renal
hemodynamic and excretory function during control
conditions and after 2 weeks of tempol administration in the drinking
water. Under control conditions, the RBF of SHR was decreased by 34%
(SHR: 5.8±0.6 versus WKY: 8.8±0.7 mL/min, P<0.01), the
GFR was decreased by 47% (SHR: 1.6±0.2 versus WKY: 3.0±0.4 mL/min,
P<0.05), and the renal vascular resistance was
increased by 117% (SHR: 29.4±2.7 versus WKY: 13.5±1.0
mm Hg · mL-1 ·
min-1, P<0.001). After 2 weeks of
tempol administration there were no significant changes in renal
hemodynamics in SHR, although there were tendencies
toward a fall in renal vascular resistance (16%) and a rise in GFR
(17%), such that there was no longer a significant difference in GFR
between SHR and WKY. Tempol had no marked effects on renal
hemodynamics in WKY. Renal excretory function was not
significantly different between WKY and SHR during control conditions
or tempol administration. In rats of similar weight and age, we find
that urinary excretion of creatinine is 8.3±0.4 mg/24
hours in control WKY (n=6) and 7.7±0.6 mg/24 hours
creatinine in control SHR (n=6).
|
Figure 2
illustrates the renal excretion
of 8-ISO in WKY and SHR. In control rats, renal excretion of 8-ISO was
elevated by 24% in SHR compared with WKY (9.8±0.7 versus 7.9±0.4
ng/24 hours, P<0.05). After 2 weeks of tempol
administration in the drinking water of SHR, the renal excretion of
8-ISO was significantly (P<0.05) reduced by 39% (6.0±0.7
ng/24 hours). However, renal excretion of 8-ISO was not significantly
affected in WKY rats given tempol in the drinking water (6.8±0.7 ng/24
hours). There was no significant difference in renal excretion of 8-ISO
between WKY and SHR after 2 weeks of tempol administration.
Analysis of variance showed that tempol specifically and
significantly (P<0.01) decreased renal excretion of 8-ISO
in SHR.
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| Discussion |
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One of the stable products when ROS attack lipids is 8-ISO. 8-ISO is generated from arachidonic acid in phospholipids and subsequently released in free form.15 Investigators have shown that 8-ISO is formed both in vitro30 and in vivo.31 Because 8-ISO is a direct, enzyme-independent, stable product of ROS, measurement of 8-ISO has been used as a marker of oxidative stress in vivo.15 Elevated renal excretion of 8-ISO has been reported in humans with scleroderma15 and preeclamptic toxemia of pregnancy32 and in rats with cyclosporin-induced nephrotoxicity,33 rhabdomyolysis,34 bile duct ligation,35 and renal ischemia/reperfusion injury.18
Measurement of the rate of excretion of 8-ISO for assessing total endogenous 8-ISO is advantageous over measurement of plasma 8-ISO for two reasons. First, this eliminates the problem of ex vivo generation of 8-ISO because the amount of lipid in urine is negligible. Second, 24-hour urinary measurement of 8-ISO presumably provides an integrated assessment of 8-ISO production with time.
The rate of excretion of 8-ISO in conscious WKY was similar to that previously reported for conscious Sprague Dawley rats.35 The finding that the SHR has an elevated rate of renal excretion of 8-ISO indicates that it is a model of oxidative stress in vivo. Prolonged tempol administration reduced renal excretion of 8-ISO significantly, consistent with reports that antioxidant therapy in rat models of oxidative stress associated with cyclosporin nephrotoxicity33 and bile duct ligation35 reduces renal excretion of 8-ISO. In the present study, the marked decrease in renal excretion of 8-ISO could not be attributed to a decrease in renal function because tempol had no significant effect on either renal hemodynamic or excretory function. In fact, 2-week tempol administration tended to improve GFR in SHR.
Previous in vivo and in vitro studies suggest that systemic vessels of SHR have increased oxidative stress, although the source of ROS remains unclear. The only previous in vivo studies in SHR show that mesenteric vessels generate oxygen radicals11 12 through xanthine oxidase.11 Earlier studies established that glomeruli generate hydrogen peroxide under normal conditions36 and can upregulate the activities of superoxide dismutase and catalase during oxidative stress.37 Overproduction of an ROS or dysregulation of antioxidants in glomeruli, other vascular beds, or tissues of SHR could contribute to the oxidative stress in this hypertensive model. We and others have shown that renal nitric oxide synthase (NOS) gene and protein expression is higher in SHR compared with WKY,38 39 but that nitric oxide generation in blood vessels is limited, perhaps because of a deficiency in the NOS cofactor tetrahydrobiopterin. Tetrahydrobiopterin deficiency enhances the formation of superoxide from NOS.27 28 In fact, addition of tetrahydrobiopterin or superoxide dismutase to the isolated aorta of SHR simultaneously reduces superoxide and increases nitric oxide production.14 These data suggest that NOS may be an important source of ROS in SHR.
Finally, 8-ISO is not only a marker of oxidative stress in vivo, but is also a vasoconstrictor. Receptors for 8-ISO have been located in rat aortic smooth muscle cells,40 retinal vascular smooth muscle cells,41 and renal arterial smooth muscle cells.18 Intrarenal infusion of 8-ISO reduces GFR and RBF in rats, in part, through activation of thromboxane A2 receptors.18 Whether elevated levels of 8-ISO in the SHR contribute to the hypertension and renal vasoconstriction remains to be determined fully. The present data show, however, that 2 weeks of antioxidant treatment with tempol decreased renal excretion of 8-ISO and blood pressure significantly but did not improve renal hemodynamics significantly, suggesting that 8-ISO may not be the primary mediator of the hypertension and renal vasoconstriction in SHR.
In conclusion, this study provides evidence that the SHR is a model of oxidative stress in vivo. The finding that a superoxide dismutase mimetic reduces blood pressure and oxidative stress in vivo suggests that oxygen radicals may be important in the long-term regulation of blood pressure in SHR. Although there are limitations in extrapolating data from the anesthetized state to the conscious state, this study provides a rational basis for a novel form of antihypertensive therapy based on tempol or similar agents. Such therapy may correct the complications created by hypertension associated with oxidative stress.
| Acknowledgments |
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Received September 17, 1998; first decision October 12, 1998; accepted October 23, 1998.
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