Arachidonic acid (AA) is the precursor of prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LKs), and elicits renal vasoconstriction in the rat kidney (1). This vasoconstrictor effect of AA requires metabolism of the fatty acid by cyclooxygenase (COX) to a vasoconstrictor mediator (2), which has been identified as thromboxane A2(3) or endoperoxides (PGH2) (4). Strain differences in prostaglandin production (5,6) and vasoconstrictor responses (7,8) in vessel walls derived from spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats have been reported. This suggests that elevated synthesis of prostaglandins, including PGH2, might be involved in the pathogenesis of hypertension in SHRs. The possibility that augmented expression of COX-1 is responsible for the increased synthesis of prostaglandins and PGH2 is supported by studies showing that interleukin-2 increases COX-1 expression and endothelium-dependent contractions to arachidonic acid (9). Moreover, recent reports have shown increased expression of COX-1 in the aortas of SHRs (10,11), confirming that indeed increased expression of COX-1 is associated with elevated production of PGH2(10). In the kidney, changes in the prostaglandin synthesis have been associated with the development of hypertension (12) and increased vascular responsiveness to angiotensin II in hypertensive rats (13). We suggest that increased vascular responsiveness to AA in hypertensive animals is related to elevated production of endoperoxides due to increased expression of COX-1. This process may be involved in the development of increased peripheral vascular resistance. Thus, we propose to examine whether the effect of renal ischemia is associated with changes in AA metabolism in the contralateral kidney. We compared the local effect of AA on the renal vasculature of nonischemic kidney in rats with established hypertension and in normotensive rats, under control conditions and during inhibition of COX, TXA2 synthase, or blockade of endoperoxide receptors. We also evaluated COX activity and the expression of protein and mRNA of COX-1 in the renal tissue of sham-operated and aortic coarctation rats.
Arachidonic acid and indomethacin were purchased from Sigma Chemical Co (St. Louis, MO, U.S.A). 14C-arachidonic acid was purchased from Amersham International (Amersham, Buckinghamshire, U.K.). Prostaglandin E2 (PGE2) and SQ29548 were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). AA was stored as a 1 mg/ml stock solution and diluted as required on the day of the experiment. All other substances were freshly prepared before each experiment.
We used male albino Wistar rats (weighing 250-300 g) with sham surgery and aortic coarctation performed according to published procedures (14). In brief, after induction of anesthesia with diethyl ether, the abdominal aorta was exposed and partially ligated at a point below the right but above the left renal artery; reproducibility was achieved with a 19-gauge needle. Experiments were conducted 25-28 days after aortic coarctation or sham surgery. Before animals were killed, their blood pressure was measured by implanting a Tygon catheter (Baxter Diagnostics, McGram Park, IL, U.S.A.) in the carotid artery, and systolic blood pressure was recorded on a polygraph (Grass Medical Instruments, Quincy, MA, U.S.A.). Only when we found a difference ≥40 mm Hg between animals with aortic coarctation and sham operation were the isolated perfused kidney experiments performed.
Isolated perfused kidney of the rat
We used the rats 25-28 days after aortic coarctation or sham operation. Rats were anesthetized with 63 mg/kg intraperitoneal sodium pentobarbital. The right kidney was exposed by midline laparotomy, and the mesenteric and right renal arteries were cleared of surrounding tissue. Ties were loosely placed around these vessels and the vena cava. The right renal artery was then cannulated with a 19-gauge needle via the mesenteric artery to avoid interruption of the blood flow, and the vena cava was occluded and cut to provide an outlet for the perfusate. The right ureter also was cut, and the animal was killed by an intracardiac injection of 10 mg sodium pentobarbital. The kidney was removed, suspended in a water-jacketed bath at 37°C, and perfused at constant flow by means of a Watson-Marlow peristaltic pump (model 502S; New Brunswick Scientific, Edison, NJ, U.S.A.) with Kreb's solution at 37°C and gassed with O2/CO2 (95%:5%). The Krebs solution used had the following composition (mM); 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 NaH2PO4, 4.2 MgSO4, 25 NaHCO3, and 11.5 glucose, at pH 7.4. Flow was adjusted to obtain a basal perfusion pressure of 75-90 mm Hg. Mean flow rate of the perfusate was 9.0 ± 0.4 ml/min. Perfusion pressure was measured with a Harvard Transducer (model 377; Harvard Apparatus Company, Inc., South Natick, MA, U.S.A.) and recorded on a Sybron recorder (Baxter Diagnostics). Because flow was maintained at a constant rate, a change in the perfusion pressure represented a change in the resistance of the artery; an increase in perfusion pressure indicated vasoconstriction. The peristaltic pump produced a pulsatile pressure with maximal and minimal values, which were not electronically averaged. Changes in the perfusion pressure produced by AA were calculated by taking the mean of the pulsatile trace before the administration of AA and the mean of trace at the maximal perfusion pressure value after injection of AA. Data were expressed as changes (Δ) of the perfusion pressure in mm Hg.
Arachidonic acid-induced renal vasoconstriction
Various doses of AA (1, 2, 4, and 8 μg) were administered randomly as a bolus into the perfusate lines, proximal to the kidneys, of both sham-operated and aortic coarctation rats, with each successive dose administered when perfusion pressure had returned to the basal value. After we had plotted this control dose-response curve to AA, the kidneys were then perfused with indomethacin (1 μg/ml) (15), SQ29548 (1 μM) (3), or UK38485 (1 μM) (16). Then 30 min after initiation of the perfusion of the inhibitor, the dose-response curve to AA was determined again. Two experiments were performed on the same day, that is, an AA dose-response curve experiment for the kidney from sham-operated rat and one aortic coarctation rat, alternating the order during the following days.
Thromboxane B2 assay
Prostaglandins were extracted from the perfusate by adding 0.5 ml of water/ethanol 1:4 and 10 μl of glacial acetic acid to 0.5 ml of perfusate. The mixture was well shaken, and the samples were incubated at room temperature for 5 min and centrifuged at 2,500 g for 5 min. The supernatant was applied to an amprep C18 minicolumn previously equilibrated with 2 volumes of 10% ethanol. The column was then washed with 1 volume of water followed by 1 volume of hexane, and prostaglandins were eluted with 2 × 0.75 ml of ethyl acetate. The samples were dried under a nitrogen stream, and thromboxane B2 was measured with a specific enzymatic immunoassay kit (Cayman Chemical, Ann Arbor, MI, U.S.A.) following the manufacturer's protocol. The concentration detected was ∼2.5 pg/ml.
Arachidonic acid metabolism was determined in microsomes from cortex, medulla, or papilla of kidneys from sham-operated or aortic coarctation rats as previously described (17). Renal tissue (cortex, medulla, papilla) was dissected under stereoscopic microscope and homogenized in 0.1 M Tris sucrose buffer, and centrifuged at 1,000 g for 5 min at 4°C in a Beckman centrifuge. The supernatant was centrifuged at 9,000 g for 10 min at 4°C in a Beckman ultracentrifuge, and the pellet discarded. Finally, the supernatant was centrifuged at 44,000 g for 1 h at 4°C, and the resultant pellet was resuspended in 5 ml 0.1 M phosphate buffer (PBS). Protein was measured by the Bradford method.
Cyclooxygenase activity was evaluated as previously described (17). In brief, 0.5 mg of microsomes of kidneys from sham-operated or aortic coarctation rats were incubated with 1.8 mM14C AA (55 mCi/mmol) and 10 mM reduced glutathione in 0.1 M PBS, pH 7.4 (final incubation volume, 1 ml), for 15 min at 37°C in a shaking water bath. Renal microsomes from sham-operated rats were incubated with indomethacin (0.2 mg/ml) to be used as a negative control. The reaction was terminated with 70 μl of 1 M citric acid (final pH, 3.5). AA metabolites were extracted twice with 3 ml of ethyl acetate and then evaporated under nitrogen. The residue dissolved in methanol was spotted on a silica gel G-60 plate together with unlabeled PGE2, TXB2, 6-oxo-PGF1α, AA, and developed in chloroform/methanol/acetic acid/water (90:8:1:1 by volume) to a high of 16 cm. The standards were visualized by brief exposure to iodine vapor. Autoradiography was performed at −70°C using Kodak X-Omat film. Prostaglandins were extracted from the plate and quantified by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's protocol. The detection limit was ∼2.5 pg/ml.
Immunoblotting for cyclooxygenase-1
Western blot for COX-1 was performed as described (18). Kidneys from sham-operated or aortic coarctation rats were dissected (cortex, medulla, papilla). Microsomes were prepared as described in the previous section; 100 μg of microsomal protein was mixed with loading buffer (glycerol 59% vol/vol; Tris-Cl/pH 6.5; SDS 1% wt/vol, bromophenol blue 0.1% wt/vol; 2-mercaptoethanol). The mixture was heated to 100°C for 2-3 min, and the proteins were separated on two 10% SDS/PAGE gels under reducing conditions and transferred to Hybond-P (Amersham) transfer membranes. The blots were blocked for 40 min with TBS containing 5% nonfat dry milk and 0.5% Tween-20. A monoclonal antibody raised against COX 1 (Cayman Chemical) was applied to a gel at a dilution 1:1,000 for 1 h. After washing, visualization was achieved using peroxidase-labeled goat antirabbit antibody and enhanced chemiluminescence technique (ECL; Amersham). The autoradiography was scanned with a densitometer system (KODAK EDAS 120 system). Values of each band were expressed in arbitrary units (AUs). All samples were run simultaneously to determine the quantity of each protein and eliminate assay-to-assay variation.
RNA isolation and RT-PCR
Rats were subjected to kidney extraction under pentobarbital anesthesia. Rapidly kidneys were extracted and frozen under liquid nitrogen. Tissue was homogenized using Trizol reagent (Gibco Inc.) in an Ultra Turrax 25 homogenizer. Total RNA (2 μg) was converted to cDNA using a SuperScript II kit from Gibco. PCR conditions were optimized such that only the desired product was produced (18). PCR was performed using a Gene Cycler (BIO RAD) thermocycler. Initial denaturation was done at 94°C for 5 min followed by 35 cycles of amplification. Each cycle consisted of 1 min of denaturation at 94°C, 1 min of annealing at 53°C, and 2 min for enzymatic primer extension at 72°C. After the final cycle, the temperature was held at 72°C for 7 min to allow reannealing of the amplified products. PCR products were then size-fractionated through 1% agarose gel, and the bands were visualized using ethidium bromide. Gels were analyzed with a densitometer system (KODAK EDAS 120 system). Values of each band were expressed in AUs. Rat COX-1 primers were designed according to the published (19) sequences of rat COX 1. The sequences of the rat COX 1 primers were 5′-TAA GTA CCA GGT GCT GGA TGG-3′ (sense, bases 772-792) and 5′-GGT TTC CCC TAT AAG GAT GAG G-3′ (antisense, bases 1,036-1,015). The PCR product was 265 bp in size. Rat GAPDH, a constitutive expressed gene, was chosen as a control gene. The primer sequences were 5′-ACC ACA GTC CAT GCC ATC AC-3′ (sense, bases 562-581) and 5′-TCC ACC ACC CTG TTG CTG TA-3′ (antisense, bases 1,013-1,032). The PCR product was 452 bp in size.
All results are expressed as mean ± SEM. Multiple comparisons were done by one-way analysis of variance (ANOVA) test. Differences were analyzed using Student's t test or Dunnett's test. Differences were significant when p was <0.05.
Administration of AA increased the renal perfusion pressure of isolated perfused kidney from sham-operated and aortic coarctation rats in a dose-dependent manner (Fig. 1). However, the increment in the perfusion pressure was higher in the kidneys from rats with aortic coarctation; 8 μg of AA produced maximal increments of the perfusion pressure of 44 ± 7 and 113 ± 8 mm Hg (p < 0.05) for sham-operated and aortic coarctation rats, respectively. Indomethacin (1 μM) abolished the AA-induced increase in renal perfusion pressure in the kidneys from sham-operated and aortic coarctation rats (Fig. 2A and B). The maximal responses to AA (8 μg) in the absence of indomethacin were 55 ± 10 and 105 ± 5 mm Hg in kidneys from sham-operated and aortic coarctation rats, respectively. With indomethacin, AA (8 μg) increased the renal perfusion pressure to 12 ± 7 and 13 ± 5 mm Hg in kidneys from sham-operated and aortic coarctation rats, respectively (p < 0.05). When the participation of vasoconstrictor prostaglandins in the AA vasoconstrictor effects was evaluated, we found that the thromboxane/endoperoxide receptor antagonist SQ29548 (1 μM) prevented AA-induced increase of the perfusion pressure of kidneys from sham-operated and aortic coarctation rats (Fig. 3A and B). The maximal responses to AA (8 μg) without SQ29548 were 35 ± 7 and 85 ± 6 mm Hg in the kidneys from sham-operated and aortic coarctation rats, respectively (p < 0.05). With SQ29548, AA (8 μg) increased renal perfusion pressure to 10 ± 4 and 15 ± 3 mm Hg in the kidneys from sham-operated and aortic coarctation rats, respectively (p < 0.05). The thromboxane synthase inhibitor UK38485 (1 μM) did not modify AA-induced increments in the renal perfusion pressure in kidneys from sham-operated or aortic coarctation rats. Thus, maximal responses to AA (8 μg) in the absence of UK38485 were 47 ± 5 and 98 ± 5 mm Hg in sham-operated and aortic coarctation rats, respectively. In the presence of UK38485, AA (8 μg) increased perfusion pressure to 52 ± 5 and 105 ± 7 mm Hg in the kidneys from sham-operated and aortic coarctation rats, respectively. Thromboxane A2 renal excretion decreased from a basal value of 195 ± 35 to 17 ± 7 pg/ml/min in sham-operated rats kidneys and decreased from 225 ± 45 to 20 ± 5 pg/ml/min in the aortic coarctation rat kidneys, after perfusion with UK38485. PGE2 increased renal perfusion pressure of isolated perfused kidneys from sham-operated or aortic coarctation rats in a dose-dependent manner. Comparison of the PGE2 dose-response curves in the kidneys from sham-operated and aortic coarctation rats did not show differences. To evaluate whether the changes in the AA responses were related to damage of the kidneys, we performed a control dose-response curve and allowed the kidney to stabilize for 45 min (time that we used to allow the effect of the different inhibitors used). Then we repeated the dose-response curve. Under these experimental conditions, we did not find any differences between the first and second dose-response curves.
Comparison of COX activity in sham-operated and aortic coarctation rat kidneys showed that production of prostaglandins in the kidneys from hypertensive rats was higher than that of kidneys from sham-operated rats. Thus, COX activity was 4.3, 3.6, and 1.7 times higher in the renal tissue from aortic coarctation rats in the cortex, medulla, and papilla, respectively (Fig. 4). Immunoblotting with COX-1 specific antiserum demonstrated that the expression of COX-1 immunoreactive protein in microsomes prepared from cortex, medulla, and papilla tissue from aortic coarctation rats was higher than that of sham-operated rats (Fig. 5). The rate of COX-1 to α-smooth muscle actin was 0.268 ± 0.01, 0.516 ± 0.014, and 0.7 ± 0.01 AU in cortex, medulla, and papilla of sham-operated rats kidneys, compared with 0.586 ± 0.017, 0.746 ± 0.022, and 1.16 ± 0.012 AU in the corresponding portions of aortic coarctation rat kidneys. COX-1 mRNA expression in the renal tissue from aortic coarctation rats was significantly increased compared with COX-1 mRNA expression in renal tissue of sham-operated rats (Fig. 6). The ratio of COX-1 and GAPDH mRNA was 0.39 ± 0.02 AU in the renal tissue from sham-operated rats compared with 0.77 ± 0.02 in that of aortic coarctation rats.
We have found abnormally increased reactivity to AA in the renal vasculature in hypertensive rats. AA effects on renal perfusion pressure of isolated kidneys were prevented by either COX inhibition or endoperoxide/thromboxane-receptor blockade. This suggests that COX-dependent vasoconstrictor products, probably endoperoxides, were the mediators of AA-induced vasoconstriction. These observations are supported by previous studies that showed that AA or acetylcholine-induced vasoconstrictor effects were attributable to constrictor actions of vascular prostanoids that activated the TXA2/endoperoxide receptor (13,20-22). To investigate the differences in AA vasoconstrictor renal responses in aortic coarctation rats compared with control rats, we examined the ability of exogenous PGE2 to increase renal perfusion pressure. PGE2 as well as U-46619 (13) produced similar increments in perfusion pressure in kidneys from control and aortic coarctation rats. Thus, the differences in the AA-induced renal vasoconstriction could not be attributed to changes in the characteristics of the prostaglandin receptor or to changes in postreceptor transducing signal. The data suggest that the increased vasoconstrictor response to AA in the kidneys from aortic coarctation rats may be the result of increased synthesis of a vasoconstrictor prostanoid. Indeed, our results showed increased COX activity in the renal tissue of hypertensive rats, and in previous studies where we have reported increased renal excretion of vasoconstrictor prostaglandins in aortic coarctation rats (13). Because we investigated the activity of COX in vitro, where AA and cofactors were supplemented adequately, changes in the COX activity could not be explained in terms of changes in the availability of the substrate (AA) or the cofactors. Thus, increase in the content of COX could be proposed to explain the increased COX activity. Indeed, we demonstrated augmented expression of COX-1 protein and mRNA. COX-1 is constitutively expressed in most tissues but at different levels in various cell types (23), so COX-1 induction would not be predicted. However, in the last decade, several authors have demonstrated induction of COX-1 gene expression in nonvascular tissue (24-26) and in cultured endothelial cells (27,28). Moreover, increased vascular reactivity to AA has been associated with increased expression of COX-1 in vitamin E-deprived rats (20). Furthermore, an increased expression of COX-1 has been shown in vascular diseases like portal hypertension (29) or systemic hypertension (10,11). The results in the present study are in agreement with these findings, although our data do not allow us to define the mechanism by which COX-1 mRNA is induced in hypertensive rats. Because the experimental model we used requires time to develop the hypertension and reports of a previous study showing that induction of COX-1 in spontaneously hypertensive rats begins at 20 weeks of age (11), it is reasonable to conclude that induction of COX-1 gene is not genetically acquired. Therefore, presence of an endogenous inducer of COX-1 expression may be the mechanism responsible for augmented expression of COX-1 in the renal tissue of hypertensive rats. The presence of the endogenous inducer hypothesis is further supported by the observation of Yanagisawa et al. (30) that increased renal COX-1 may be associated with increased levels of angiotensin II.
The functional consequences of an exaggerated expression of COX-1 and increased release of the vasoconstrictor PGH2 in the vasculature in the hypertensive rats may explain impaired endothelium-dependent relaxation to acetylcholine in spontaneously hypertensive rats (7,31) and the vascular dysfunction of patients with essential hypertension (32). This suggests that prostaglandins may be participating in the maintenance of the prohypertensive mechanisms that sustain elevated peripheral vascular resistance rather than in the mechanisms that initiate the development of hypertension.
In summary, the present study demonstrates that in hypertension induced by aortic coarctation, the increased AA vasoconstrictor responses are modulated by the release of a COX-dependent vasoconstrictor that acts on the TXA2/PGH2 receptor and is associated with increased COX-1 activity due to increased expression of COX-1 mRNA. This study indicates that in the phase of established hypertension, increased expression of COX-1 modulates the vascular actions of vasodilators or vasoconstrictors that require the release of COX products to mediate their vascular actions, thus participating in the maintenance of increased peripheral vascular resistance.
Acknowledgment: This study was supported by a National Institute of Health grant (B.E., Fogarty International Collaborative Award) and CONACyT Mexico (31048-M). We thank Leonor Zuniga for his excellent technical help.
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