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Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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To delineate the microvascular role of cyclooxygenase-2 (Cox-2) in modulating tubuloglomerular feedback (TGF) signals and to determine its relationship to neuronal nitric oxide synthase (nNOS), afferent (AA) and efferent (EA) arteriolar diameters of rat kidneys were assessed using the blood-perfused juxtamedullary nephron technique. The Cox-2 inhibitor NS-398 (10 µM) did not alter AA diameters in untreated kidneys but significantly constricted AAs by 17.0 ± 2.2% in kidneys treated with 10 mM acetazolamide, which enhances TGF-mediated AA constriction by increasing distal volume delivery. The NS-398-induced AA constriction was prevented after interruption of distal delivery by transection of the loops of Henle. The effect was selective for AAs since NS-398 did not influence EAs of untreated or acetazolamide-treated kidneys. Pretreatment with the nNOS inhibitor S-methyl-L-thiocitrulline (10 µM) prevented the NS-398-induced AA constriction observed during acetazolamide treatment. Although we previously demonstrated that acetazolamide treatment enhanced AA constrictor response to S-methyl-L-thiocitrulline, the enhancement by acetazolamide was inhibited by pretreatment with 10 µM NS-398 (16.4 ± 1.9 and 15.0 ± 0.5% with and without acetazolamide, respectively, P > 0.05). These results indicate that, during increased activation of TGF-dependent vasoconstrictor signals, Cox-2 generates vasodilatory metabolites in response to increased nNOS activity and thus participates in the counteracting modulation of TGF-mediated AA constriction.
tubuloglomerular feedback mechanism; renal microcirculation; macula densa; acetazolamide; nitric oxide; nitric oxide synthase
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE CYCLOOXYGENASE (Cox) pathway is a major route of arachidonic acid metabolism in the kidney (20). Cox exists as a constitutive isoform (Cox-1) and as an inducible isoform (Cox-2). Both Cox isoforms metabolize arachidonic acid to generate prostaglandins and thromboxanes, which play important roles in the regulation of renal vascular tone, glomerular filtration, and tubular reabsorptive mechanisms (20, 21). Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit Cox and thus decrease Cox-mediated generation of arachidonic acid metabolites. However, NSAIDs exert both deleterious effects as well as beneficial anti-inflammatory actions. It is currently believed that NSAIDs exert a variety of side effects as a consequence of inhibition of constitutive Cox-1 and that their beneficial effects result primarily from the inhibition of inducible Cox-2 (34). On the basis of this concept, new types of NSAIDs that selectively inhibit Cox-2 have been recently developed (9, 18).
Cox is a heme-containing enzyme, and its activation requires a hydroperoxide initiator (30). Nitric oxide (NO) is an oxidizing radical that interacts with heme-containing proteins (14). Therefore, NO can influence Cox activity. Studies have demonstrated that NO enhances prostaglandin production induced by various stimuli, including lipopolysaccharide, interleukins, and bradykinin in experimental settings such as cultured mouse macrophage cells (26), human fetal fibroblasts (26), rabbit hydronephrotic kidneys (27), rat mesangial cells (31), and in vivo rats (28). Overall, these results demonstrate that NO can activate or stimulate Cox activity. Nevertheless, the effect of NO on Cox activity in a specific tissue is not predictable since it has also been reported that NO inhibited bradykinin- and lipopolysaccharide-induced production of Cox metabolites in bovine aortic endothelial cells (19) and rat peritoneal macrophages (10). In addition, it has been shown that NO (17) and its coupling product, peroxynitrite (16), activate Cox-1 and Cox-2 activities in test tubes. NO also promotes the conversion of arachidonic acid to prostaglandins in tissues where Cox has not been induced. The stimulation of prostaglandin production by NO has been demonstrated in untreated rabbit kidneys (27), bovine coronary microvascular endothelial cells (7), and rat plasma (25). These studies suggest that NO also stimulates constitutive Cox activity, but the nature of the influence may be cell and tissue specific.
Recent studies have demonstrated that Cox-2 is constitutively expressed in the kidney (11, 15, 35) and is primarily localized to the thick ascending limb and macula densa cells of rats (11, 35) and to human intraglomerular podocytes (15). These immunohistochemical findings suggest that Cox-2 may participate in the regulation of renal perfusion and glomerular hemodynamics. Interestingly, neuronal (n) nitric oxide synthase (NOS) is also present in the macula densa cells of rat kidneys (2, 36). We have recently shown that selective nNOS inhibition with S-methyl-L-thiocitrulline (L-SMTC; see Ref. 8) significantly decreases afferent arteriolar diameters and that increased volume delivery to the distal nephron significantly enhances the L-SMTC-induced afferent arteriolar constriction (13). These findings suggested that nNOS-derived NO provides vasodilatory influences on afferent arterioles and thus modulates tubuloglomerular feedback (TGF) responsiveness (33). It is possible that Cox-2 may also contribute to the regulation of the glomerular hemodynamics by its influence on the TGF mechanism and that it may interact with the nNOS-mediated effects.
The aim of the present study was to delineate the role of intrarenal Cox-2 in the regulation of microvascular responses and to determine its relationship to the nNOS-activated mechanisms. Afferent and efferent arteriolar diameter responses to the selective Cox-2 inhibitor, NS-398 (9, 18), and L-SMTC were examined under normal conditions and during increased volume delivery to the macula densa segment. The in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy was utilized to provide direct assessment of renal microvascular diameters under conditions of normal and enhanced activity of the TGF mechanism (6). Kidneys were treated with the carbonic anhydrase inhibitor acetazolamide to inhibit net proximal tubule reabsorption rate and thus increase volume delivery to the macula densa segment, leading to enhancement of TGF-mediated vasoconstrictor effects (13).
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METHODS |
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Abstract
Introduction
Methods
Results
Discussion
References
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Assessment of Afferent and Efferent Arteriolar Diameters
The experiments were performed in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee. Afferent and efferent arteriolar responsiveness was assessed in vitro using the blood-perfused juxtamedullary nephron technique combined with videomicroscopy, as previously described (5, 6). Each experiment used two male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 350-400 g, with one rat serving as the blood donor and the second rat as the kidney donor. Rats had free access to water and standard rat chow (Ralston-Purina, St. Louis, MO) before the experiments. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a cannula was inserted into the left carotid artery of the blood donor. Donor blood was collected into a heparinized (500 units) syringe via the carotid arterial cannula and centrifuged to separate the plasma and cellular fractions. The buffy coat was removed and discarded. Plasma oncotic pressure was adjusted to 18 mmHg by the addition of bovine serum albumin (Sigma Chemical, St. Louis, MO). After sequential passage of the plasma through 5- and 0.22-µm filters (Gelman Sciences, Ann Arbor, MI), erythrocytes were added to achieve a hematocrit of 33%. This reconstituted blood was passed through a 5-µm nylon mesh and thereafter stirred continuously in a closed reservoir that was pressurized with a 95% O2-5% CO2 gas mixture.The right kidney of the kidney donor was perfused through a cannula inserted into the superior mesenteric artery and advanced into the right renal artery. The perfusate was a Tyrode solution (pH 7.4) containing 5.1% bovine serum albumin and a mixture of L-amino acids as previously described (5). The kidney was excised and sectioned longitudinally, retaining the papilla intact with the perfused dorsal two-thirds of the organ. The papilla was reflected to expose the pelvic mucosa and tissue covering the inner cortical surface. Overlying tissue was removed to expose the tubules, glomeruli, and related vasculature of the juxtamedullary nephrons. The arterial supply of the exposed microvasculature was isolated by ligating the larger branches of the renal artery with fine suture (nylon black monofilament, 10-0; Vanguard Surgical System, Houston, TX).
After the dissection was completed, the Tyrode perfusate was replaced with the reconstituted blood. Perfusion pressure was monitored by a pressure cannula centered in the tip of the perfusion cannula. Renal perfusion pressure was regulated by adjusting the rate of gas inflow into the blood reservoir and maintained at 100 mmHg throughout the study. The inner cortical surface of the kidney was continuously superfused with a warmed (37°C) Tyrode solution containing 1% bovine serum albumin.
The tissue was transilluminated on the fixed stage of a Leitz Laborlux-12 microscope (Midland, Canada), equipped with a water-immersion objective (×40; Zeiss). Video images of the microvessels were transferred by a Newvicon camera (model NC-67M; Dage-MTI, Michigan City, IN) through an image enhancer (MFJ-1452; MFJ Enterprises, Starkville, MS) to a video monitor (Conrac Display Systems, Covina, CA). The video signal was recorded on videotape for later analysis (Super VHS Videocassette recorder; Panasonic, Secaucus, NJ). Afferent and efferent arteriolar inside diameters were measured at 12-s intervals using a calibrated digital image-shearing monitor (Instrumentation for Physiology and Medicine, San Diego, CA). Measurement sites along the microvasculature were selected to achieve the maximum clarity of the vascular walls at a location as close to the glomerulus as possible. Afferent arteriolar diameter was measured at sites 75-120 µm upstream from the glomerulus, and efferent arteriolar diameter was measured before the first branch at sites within 75 µm of the glomerulus. A 10-min equilibration period was allowed before the initiation of each experimental protocol. The average diameter during the final 2 min of each 5-min treatment period was utilized for statistical analysis of steady-state responses.
Experimental Protocols
Effect of Cox-2 inhibition on afferent and efferent arteriolar diameters. Afferent and efferent arteriolar responsiveness to increasing concentrations (0.01, 0.1, 1, and 10 µM) of the selective Cox-2 inhibitor NS-398 (Cayman Chemical, Ann Arbor, MI) were assessed under normal conditions and during increased volume delivery past the macula densa segment. Experiments were performed using two groups: untreated kidneys and kidneys treated with acetazolamide (Sigma Chemical). To increase volume delivery past the macula densa, 10 mM acetazolamide was added to the blood perfusate after the initial 5-min control period. After a 5-min stabilization period, afferent and efferent arteriolar responsiveness to increasing concentrations of NS-398 were determined. Previous studies have shown that acetazolamide inhibits proximal tubular reabsorption and thus increases distal volume and sodium delivery. We have previously demonstrated that this dose of acetazolamide leads to enhanced TGF constrictor signals that decrease afferent arteriolar diameter (13).Effect of papillectomy on afferent arteriolar responsiveness to Cox-2 inhibition in acetazolamide-treated kidneys. The juxtamedullary nephrons visualized in this preparation give rise to long loops of Henle that extend into the papilla before looping back to the distal tubule and past the macula densa (6). In this preparation, the papilla is reflected and is easily accessible. Transection of the loops of Henle by acute papillectomy interrupts the flow of tubular fluid to the distal nephron, including the macula densa segment, and thus minimizes TGF-dependent vasoconstrictor influences on microvascular function (13). After assessment of arteriolar diameters in acetazolamide-treated kidneys, papillectomy was performed by cleanly severing the papilla near the corticomedullary junction by a single cut thus preventing damage to the adjacent tissue. After a 5-min stabilization period, afferent arteriolar responsiveness to the same concentrations of NS-398 was determined.
Effect of nNOS inhibition on afferent arteriolar responsiveness to Cox-2 inhibition in acetazolamide-treated kidneys. The response to NS-398 was examined in the presence of the selective nNOS inhibitor L-SMTC (10 µM; Alexis, San Diego, CA) in afferent arterioles of acetazolamide-treated kidneys harvested from a separate group of rats. We chose L-SMTC over 7-nitroindazole because it is much more selective for nNOS. When compared with endothelial NOS, L-SMTC is 17-fold more selective for nNOS, and 7-nitroindazole is only 5-fold more selective (8). Accordingly, this dose (10 µM) of L-SMTC did not influence acetylcholine-induced vasodilation of juxtamedullary afferent or efferent arterioles (13). After 5 min of pretreatment with acetazolamide, the superfusate was changed to one containing 10 µM L-SMTC. After another 5-min stabilization period, afferent arteriolar responses to NS-398 (0.01-10 µM) were determined. In addition, afferent arteriolar responsiveness to 1 and 10 nM angiotensin II (ANG II; Novabiochem, San Diego, CA) was assessed to examine whether or not vascular reactivity is altered by the combination of acetazolamide plus L-SMTC.
Effect of Cox-2 inhibition on enhanced afferent arteriolar responsiveness to nNOS inhibition by acetazolamide treatment. We have previously investigated afferent and efferent arteriolar responsiveness to increasing concentrations of L-SMTC (0.1, 1, and 10 µM) in untreated kidneys and acetazolamide-treated kidneys. Those studies revealed that acetazolamide treatment predominantly enhanced afferent arteriolar responsiveness to L-SMTC (13). In the present study, we examined the effect of acetazolamide treatment on afferent arteriolar responsiveness to L-SMTC during Cox-2 inhibition with NS-398. Afferent arteriolar responsiveness to the same concentrations of L-SMTC was assessed using kidneys treated with NS-398 alone or in combination with acetazolamide treatment. In acetazolamide plus NS-398-treated kidneys, 10 µM NS-398 was added to the superfusate after a 5-min stabilization period after the addition of 10 mM acetazolamide to the blood perfusate. After another 5-min stabilization period, afferent arteriolar responsiveness to L-SMTC (0.1-10 µM) was determined.
Statistical Analysis
Analyses of changes in basal diameters during acetazolamide treatment alone and combined with papillectomy or L-SMTC were performed using a paired t-test with Bonferroni's correction, in which a value of P < 0.05 plus Bonferroni's correction was considered as statistically significant. Differences within and between treatment groups in microvascular diameter responsiveness to increasing concentrations of NS-398 and L-SMTC were determined using the analysis of variance for repeated measures combined with Newman-Keuls post hoc test. A probability value of P < 0.05 was considered statistically significant. Data are presented as the means ± SE.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of Cox-2 Inhibition on Afferent and Efferent Arteriolar Diameter in Untreated and Acetazolamide-Treated Kidneys
Figure 1 illustrates the afferent arteriolar responses to increasing concentrations of the Cox-2 inhibitor NS-398 in untreated kidneys and kidneys treated with acetazolamide. In untreated kidneys, basal afferent arteriolar diameter averaged 23.2 ± 1.2 µm (n = 6) and was not influenced by treatment with NS-398 (0.01-10 µM). Prolonged exposure of afferent arterioles to 10 µM NS-398 for 15 min did not result in any significant changes in vessel caliber. As previously shown (13), afferent arterioles constricted in response to the increased distal nephron volume delivery after treatment with acetazolamide. In paired studies, acetazolamide treatment significantly decreased basal afferent arteriolar diameter by 6.4 ± 1.3% from 20.7 ± 0.6 to 19.1 ± 0.6 µm (n = 9). In addition, NS-398 significantly decreased afferent arteriolar diameters in a dose-dependent manner in kidneys treated with acetazolamide.
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The effects of NS-398 were selective for the afferent arterioles because the efferent arterioles from untreated kidneys or kidneys treated with acetazolamide did not respond to concentrations of NS-398 up to 10 µM. In untreated kidneys, basal efferent arteriolar diameter averaged 22.7 ± 1.8 µm (n = 5) and was not altered by administration of NS-398. Efferent arteriolar diameter averaged 22.5 ± 1.8, 22.7 ± 1.7, 22.8 ± 1.6, and 22.9 ± 1.8 µm for 0.01, 0.1, 1, and 10 µM NS-398, respectively. In another series of kidneys, basal efferent arteriolar diameter after acetazolamide treatment averaged 18.9 ± 1.9 µm (n = 5), a value similar to that measured before acetazolamide treatment (19.0 ± 1.9 µm). During acetazolamide treatment, efferent arteriolar diameter remained unchanged at all NS-398 concentrations tested (18.9 ± 1.9, 18.9 ± 1.9, 19.0 ± 2.0, and 19.1 ± 1.9 µm for 0.01, 0.1, 1, and 10 µM NS-398, respectively). Also, prolonged exposure of efferent arterioles to 10 µM NS-398 for 15 min did not result in any significant changes in vessel caliber.
Effect of Papillectomy on Afferent Arteriolar Responsiveness to Cox-2 Inhibition in Acetazolamide-Treated Kidneys
The changes in basal diameter during acetazolamide treatment and papillectomy were similar to those reported previously (13). As shown in Fig. 2, basal afferent arteriolar diameter averaged 23.4 ± 1.2 µm (n = 6), and acetazolamide treatment significantly decreased afferent arteriolar diameter by 6.9 ± 1.0% to 21.1 ± 0.9 µm. Papillectomy reversed the acetazolamide-induced decrease in diameter, and afferent arteriolar diameter increased significantly to 25.1 ± 1.4 µm (a value 7.4 ± 1.6% greater than the basal diameter). Papillectomy also completely prevented the NS-398-induced decrease in afferent arteriolar diameter during acetazolamide treatment. Afferent arteriolar diameter after papillectomy averaged 25.2 ± 1.4, 24.9 ± 1.4, 25.0 ± 1.4, and 25.6 ± 1.3 µm for 0.01, 0.1, 1, and 10 µM NS-398, respectively. Thus an intact distal nephron volume delivery was required for selective Cox-2 inhibition to elicit afferent arteriolar vasoconstriction.
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Effect of nNOS Inhibition on Enhanced Afferent Arteriolar Responsiveness to Cox-2 Inhibition by Acetazolamide Treatment
The afferent arteriolar responses to NS-398 after acetazolamide treatment and nNOS inhibition are illustrated in Fig. 3. Basal afferent arteriolar diameter averaged 25.8 ± 2.5 µm (n = 6), and acetazolamide treatment significantly decreased afferent arteriolar diameter by 7.4 ± 1.1%. As previously reported (13), treatment with the selective nNOS inhibitor L-SMTC (10 µM) decreased afferent arteriolar diameter by 25.4 ± 1.6% from 24.0 ± 2.5 to 17.9 ± 2.0 µm. Importantly, L-SMTC pretreatment completely prevented the afferent arteriolar constrictor responses to NS-398 during acetazolamide treatment. Afferent arteriolar diameter averaged 18.5 ± 1.8, 18.1 ± 2.0, 17.8 ± 2.0, and 17.7 ± 1.9 µm for 0.01, 0.1, 1, and 10 µM NS-398, respectively. Thus, in the presence of nNOS inhibition during acetazolamide treatment, selective Cox-2 inhibition did not elicit afferent arteriolar vasoconstriction.
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To determine if the loss of vasoconstrictor responses to NS-398 was nonspecific, afferent arteriolar responsiveness to ANG II was also assessed during the L-SMTC plus acetazolamide treatments. Treatment with L-SMTC plus acetazolamide did not influence afferent arteriolar responsiveness to ANG II. As shown in Fig. 4, superfusion with 1 and 10 nM ANG II significantly decreased afferent arteriolar diameters by 17.0 ± 0.8 and 31.5 ± 2.4%, respectively, in kidneys treated with L-SMTC plus acetazolamide (n = 4). The afferent arteriolar constrictor responsiveness to 1 and 10 nM ANG II was similar to that observed in untreated kidneys (17.2 ± 1.1 and 32.7 ± 2.4%, respectively, n = 7).
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Effect of Cox-2 Inhibition on Enhanced Afferent Arteriolar Responsiveness to nNOS Inhibition by Acetazolamide Treatment
Afferent arteriolar responsiveness to L-SMTC was assessed in two unpaired groups consisting of NS-398-treated and acetazolamide plus NS-398-treated kidneys. The responses were compared with those observed previously in untreated kidneys and kidneys treated with acetazolamide (13). We previously reported that acetazolamide led to enhanced afferent arteriolar reactivity to L-SMTC (13). In untreated kidneys, basal afferent arteriolar diameter averaged 21.7 ± 0.6 µm, and 0.1, 1, and 10 µM L-SMTC significantly decreased afferent arteriolar diameter by 5.0 ± 0.2, 8.4 ± 0.5, and 11.3 ± 0.6%, respectively. In acetazolamide-treated kidneys, the afferent arteriolar diameter was reduced from 22.0 ± 0.8 to 20.3 ± 0.7 µm, and L-SMTC decreased afferent arteriolar diameters by 10.3 ± 0.7, 17.5 ± 1.5, and 23.8 ± 1.6% at concentrations of 0.1, 1, and 10 µM, respectively. The maximal response to L-SMTC was significantly greater than that observed in untreated kidneys (13).Figure 5 shows modulation of the L-SMTC response by acetazolamide treatment in afferent arterioles of NS-398-treated kidneys. The baseline diameters utilized in this series of experiments were not significantly different from those utilized in the experiment of our previous study (13). As shown in Fig. 5A, in NS-398-treated kidneys, the basal afferent arteriolar diameter averaged 21.0 ± 0.9 µm (n = 5) and was similar to that observed before NS-398 treatment (21.7 ± 0.2 µm). Exposure to 10 µM L-SMTC significantly decreased afferent arteriolar diameters to 17.8 ± 0.7 µm. In acetazolamide plus NS-398-treated kidneys, the afferent arteriolar diameter was significantly reduced from 20.6 ± 0.8 µm (n = 7) to 19.2 ± 0.7 µm by acetazolamide treatment and further reduced to 16.6 ± 0.6 µm upon exposure to NS-398. Under conditions of Cox-2 inhibition with NS-398 and acetazolamide treatment, afferent arteriolar diameters decreased significantly to 13.9 ± 0.8 µm in response to 10 µM L-SMTC. Figure 5B illustrates these relative responses to L-SMTC, including data previously reported (13). In response to 0.1, 1, and 10 µM L-SMTC, afferent arteriolar diameter decreased significantly by 5.3 ± 0.4, 10.7 ± 1.0, and 15.0 ± 0.5%, respectively, and the reductions in diameter were similar to those observed in untreated kidneys. In acetazolamide plus NS-398-treated kidneys, 0.1, 1, and 10 µM L-SMTC decreased afferent arteriolar diameters by 5.8 ± 1.0, 11.6 ± 1.4, and 16.4 ± 1.9%, respectively. The relative responses to L-SMTC were similar to those observed in the NS-398-treated and untreated kidneys and were significantly less than that observed in acetazolamide-treated kidneys.
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To test if the preconstriction observed during the acetazolamide plus NS-398 treatment suppressed the vasoconstrictor responses to L-SMTC, the afferent arteriolar responses to L-SMTC were also assessed in a group of kidneys treated with acetazolamide and norepinephrine (NE; Levophed bitartrate; Sanofi Winthrop Pharmaceuticals, New York, NY). In this group, the basal afferent arteriolar diameter averaged 21.7 ± 1.9 µm (n = 5) and was reduced to 20.0 ± 1.4 µm by acetazolamide treatment and further decreased to 17.3 ± 1.1 µm upon continuous exposure to 200 nM NE. The percent decrease in afferent arteriolar diameter averaged 19.8 ± 2.1% and was similar to that observed during acetazolamide plus NS-398 treatments (19.6 ± 1.2%). In acetazolamide plus NE-treated kidneys, 0.1, 1, and 10 µM L-SMTC significantly decreased afferent arteriolar diameters by 12.9 ± 1.2, 20.0 ± 1.9, and 28.0 ± 0.5%, respectively. The maximal response to L-SMTC was similar to that obtained in acetazolamide-treated kidneys and significantly greater than that observed in untreated kidneys, NS-398-treated kidneys, and acetazolamide plus NS-398-treated kidneys.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Recent studies have demonstrated that Cox-2 is constitutively present in the macula densa segment of rat kidneys (11, 35) and in glomerular podocytes of human kidneys (15). Its unique localization has suggested a potential role of constitutive Cox-2 in regulating the sensitivity of TGF responses. The present study has demonstrated that the selective Cox-2 inhibitor NS-398 significantly decreased afferent but not efferent arteriolar diameters during increased distal nephron volume delivery elicited by treatment with acetazolamide. In addition, interruption of distal tubular flow by papillectomy reversed the acetazolamide-induced decrease in afferent arteriolar diameter, demonstrating that acetazolamide was exerting its effect via the TGF mechanism. These results indicate that acetazolamide-induced increases in distal volume delivery stimulate production of vasodilatory Cox-2 metabolites. Because these vasodilatory Cox-2 metabolites only influence afferent arterioles, which are the effectors of the TGF mechanism, it is suggested that Cox-2 in the macula densa segment participates in modulating the sensitivity of the TGF mechanism via a vasodilatory metabolite.
Cox-2 inhibition with NS-398 did not influence afferent or efferent arteriolar diameters in untreated kidneys, indicating a minimal influence of Cox-2 metabolites at normal distal nephron volume delivery. However, the contribution of Cox-2 was manifested during increased distal nephron volume delivery elicited by acetazolamide treatment. Although it is possible that Cox-2 could be induced by 5-10 min of increases in distal tubular flow, the more likely explanation is that constitutive Cox-2 protein was activated or stimulated by some factor or factors associated with increased distal tubular flow.
The unique localization of nNOS (2, 36) and constitutive Cox-2 (11, 35) in and around the macula densa suggests that these predominantly vasodilatory pathways play an important role in the tubular flow-dependent responses and may interact with each other (13, 33). Because NO has been demonstrated to stimulate constitutive Cox enzymes in the kidney (27), nNOS-derived NO may stimulate constitutive Cox-2 in and around the macula densa. Experiments were also performed to delineate the interactive influence of nNOS and Cox-2 on afferent arteriolar tone during increased distal nephron volume delivery. Pretreatment with the selective nNOS inhibitor L-SMTC completely prevented the NS-398-induced decrease in afferent arteriolar diameter during acetazolamide treatment. Because acetazolamide treatment combined with L-SMTC elicited a greater vasoconstriction than acetazolamide treatment alone, it is possible that such a large preexisting vasoconstriction may have suppressed further vasoconstriction. However, acetazolamide plus L-SMTC treatment did not suppress the afferent arteriolar vasoconstriction to ANG II. These results indicate that nNOS-derived NO contributes to the Cox-2-dependent afferent arteriolar vasodilation during increased distal volume delivery.
We observed further that pretreatment with NS-398 inhibited the acetazolamide-induced enhancement of the afferent arteriolar constrictor response to L-SMTC. The prevention of enhanced L-SMTC response did not depend on the preexisting large vasoconstriction caused by NS-398 plus acetazolamide treatment, because NE-induced preconstriction did not influence the acetazolamide-induced enhancement of the afferent arteriolar constrictor response to L-SMTC. Therefore, Cox-2 is involved in the nNOS-derived NO-mediated counteraction of TGF response. In the present study, nNOS inhibition still constricted afferent arterioles in the presence of Cox-2 inhibition, but if nNOS was first inhibited, Cox-2 inhibition no longer influenced afferent arterioles. On the basis of these data, it is suggested that Cox-2 is stimulated directly or indirectly as a result of nNOS activation. The data also suggest that Cox-2 activity substantially depends on nNOS activity to provoke its vasodilatory effects on afferent arterioles but that the net effect of nNOS on the TGF mechanism is not completely dependent on Cox-2 activity.
Although it is also possible that increased distal nephron volume delivery might directly stimulate Cox-2 activity, leading to the production of vasodilatory Cox-2 metabolites which, in turn, would stimulate nNOS-derived NO production and/or release, currently available data are less supportive of this explanation. The administration of a stable analog of prostacyclin or exogenous prostaglandin E2 during Cox inhibition with indomethacin did not influence nitrite production in untreated rat mesangial cells (32). In bovine aortic endothelial cells, the stable analog of prostacyclin directly suppressed NO production (37). These studies indicate that vasodilatory Cox metabolites inhibit or do not influence NO synthesis. It is therefore less likely that Cox-2 metabolites stimulate nNOS activity in the macula densa segment.
In anesthetized dogs, acute inhibition of overall NOS activities enhanced the decrease in renal blood flow caused by the nonselective Cox inhibitor indomethacin (24). In the presence of a higher pressor dose of the nonselective NOS inhibitor, nitro-L-arginine methyl ester, addition of indomethacin resulted in renal vasoconstriction (3). Additionally, studies in vascular tissues (4, 12, 22) and in kidneys (3) have reported that chronic NOS inhibition stimulates Cox-1- or Cox-2-mediated production of vasodilatory prostaglandins. These studies suggest that Cox activity may compensate for the renal hemodynamic effects of the NO system. However, in the presence of a subpressor dose of nitro-L-arginine methyl ester, indomethacin did not alter renal vascular tone (3), and a study examining isolated renal arterioles did not find any interactions between NOS and Cox activity (1). Thus evaluation of the interaction between the overall kidney NOS and Cox activities in renal hemodynamics remains unclear. The present study indicates that, in and around the macula densa, nNOS-derived NO acts indirectly on the afferent arterioles by stimulating production of vasodilatory Cox-2 metabolites in addition to its direct effects. Because the magnitude of nNOS and Cox-2 activity in the macula densa segment may be small compared with the overall kidney NOS and Cox activities, studies of overall NOS and Cox activity may mask important interactions between nNOS and Cox-2 in modulating the TGF response.
Significant contributions of both prostaglandins and nNOS to the TGF mechanism have been observed in microperfusion studies using superficial cortical nephrons (29). Therefore, the present findings in juxtamedullary nephrons may also be applicable to midcortical and superficial cortical nephrons. However, Cox inhibition has been shown to decrease renal medullary blood flow without altering cortical blood flow (23). Because afferent and efferent arterioles of juxtamedullary nephrons supply renal medullary blood flow, the interactive influences of Cox-2 and nNOS may be more important in the regulation of juxtamedullary microcirculation compared with midcortical and superficial cortical microcirculation.
The TGF mechanism responds to increases in distal tubular flow past the macula densa segment with an increase in afferent arteriolar tone. Our previous study demonstrated that the TGF-mediated afferent arteriolar constriction is counteracted by increased activity of macula densa nNOS (13). From the present data, we conclude that, during increased activity of the TGF mechanism, the increased level of nNOS-derived NO not only dilates afferent arterioles directly but also stimulates Cox-2 activity to generate vasodilatory metabolites, which contribute further to buffering the magnitude of TGF-mediated afferent arteriolar constriction.
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ACKNOWLEDGEMENTS |
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We thank Anthony K. Cook for technical assistance.
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FOOTNOTES |
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This work was supported by J. Walter Libby Fellowship Grant from the American Heart Association, Lousiana Affiliate (A. Ichihara), Grant-in-Aid no. 95009790 from the American Heart Association (J. D. Imig), and National Heart, Lung, and Blood Institute Grant HL-18426. E. W. Inscho is an Established Investigator of the American Heart Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. Ichihara, Dept. of Physiology, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112.
Received 6 May 1998; accepted in final form 9 July 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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, n = 6) and kidneys treated with
10 mM acetazolamide in the blood perfusate (
,
n = 9).
* P < 0.05 vs. control
diameter.
) and ACZ-treated kidneys (
) reproduced
from Ref. 
P < 0.05 for the
response in ACZ-treated kidneys vs. the response in untreated kidneys
or NS-398-treated kidneys.
§ P < 0.05 for the
response in ACZ-treated kidneys vs. the response in ACZ + NS-398-treated kidneys. ns, No significant difference between the
responses in the 3 groups.