Renal Effects of Cyclooxygenase Inhibition When Nitric Oxide Synthesis Is Reduced and Angiotensin II Levels Are Enhanced.

ORIGINAL ARTICLE

Renal Effects of Cyclooxygenase Inhibition When Nitric Oxide Synthesis Is Reduced and Angiotensin II Levels Are Enhanced Ruth López, PhD,*†‡ Maria Teresa Llinás, PhD,*†‡ Elena Salazar, PhD,* and F. Javier Salazar, PhD*†‡

Abstract: The involvement of both cyclooxygenase (COX) isoforms in regulating renal function is well known but their interactions with other regulatory mechanisms, such as angiotensin II (Ang II) and nitric oxide (NO), are not well defined. This study has evaluated the relative contribution of both COX isoforms in regulating renal function when NO synthesis is reduced with and without a simultaneous increment in Ang II levels. The renal responses to a nonselective (meclofenamate) or a selective COX2 (nimesulide) inhibitor were examined in dogs pretreated with L-NAME with or without an intrarenal Ang II infusion. Meclofenamate induced a greater (P , 0.05) renal vasoconstriction than nimesulide in dogs pretreated with L-NAME. This vasoconstriction seems to be Ang II-dependent because it was reduced (P , 0.05) by captopril administration. Meclofenamate also induced a greater (P , 0.05) renal vasoconstriction than that elicited by nimesulide in dogs with reduced NO synthesis and elevated Ang II levels. The renal vasoconstriction induced by nimesulide but not that elicited by meclofenamate in dogs pretreated with L-NAME and Ang II, decreased (P , 0.05) during an extracellular volume expansion. These results demonstrate that the nonselective COX inhibition induces a greater renal vasoconstriction than that elicited by the selective COX2 inhibition when NO synthesis is reduced, and when NO synthesis is reduced and Ang II levels are elevated. Key Words: angiotensin II, nitric oxide, cyclooxygenases, renal blood flow, glomerular filtration rate, extracellular volume expansion (J Cardiovasc Pharmacol Ô 2015;65:465–472)

INTRODUCTION The involvement of cyclooxygenase (COX)-derived prostaglandins (PG) in protecting the renal vasculature from the vasoconstriction secondary to a decrease in nitric oxide (NO) is supported by studies showing that the renal Received for publication July 9, 2014; accepted December 19, 2014. From the *Department of Physiology, School of Medicine, Universidad de Murcia; †Regional Campus of International Excellence “Mare Nostrum”; and ‡Instituto Murciano de Investigación Biomédica. Supported by the Dirección General de Investigación Científica y Técnica of Ministerio de Economía y Competitividad (BFU2013-49098R). The authors report no conflicts of interest. Reprints: F. Javier Salazar, PhD, Department of Physiology, School of Medicine, University of Murcia, 30100 Murcia, Spain (e-mail: salazar@ um.es). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

J Cardiovasc Pharmacol ä Volume 65, Number 5, May 2015

vasoconstrictor effects elicited by a nonselective or a selective COX2 inhibitor are enhanced when NO synthesis is reduced.1–6 It has also been reported that a decrease in NO induces an upregulation of COX2 expression in the kidney.7 However, the relative contribution of COX1- and COX2derived metabolites in regulating renal hemodynamics when NO synthesis is reduced was unknown. The first objective of this study was to examine whether both COX isoforms are involved in producing the PG that protect the renal vasculature from the constriction secondary to a decrease in NO synthesis. The results obtained may have important pathophysiological implications because there are situations such as aging where NO activity seems to be reduced8 and the intake of nonsteroidal anti-inflammatory drugs (NSAIDs) is enhanced.9 The possible role of endogenous angiotensin II (Ang II) levels in mediating the renal vasoconstriction elicited by a selective COX2 inhibitor when NO synthesis is reduced was also unknown. Therefore, the second objective was to evaluate the contribution of endogenous Ang II levels to the renal vasoconstriction induced by the inhibition of COX2 or both COX isoforms when intrarenal NO synthesis is reduced. The results obtained may also have pathophysiological implications because it will be examined to what extent the administration of a converting enzyme inhibitor reduces the renal vasoconstriction induced by NSAIDs when NO synthesis is reduced. The role of NO or both COX isoforms in modulating the renal vasoconstriction elicited by an increase in Ang II has also been examined by several research groups.10–17 However, it remained to be elucidated to what extent both COX isoforms are involved in regulating renal vascular resistance (RVR) when there is an increase in Ang II levels and NO synthesis is reduced. The third objective was to examine whether both COX isoforms are involved in protecting the renal vasculature from the vasoconstriction induced by a slight increment in Ang II levels when NO synthesis is reduced. The results obtained may have pathophysiological and clinical relevance because there are situations such as aging where NO production may be reduced,8 and the intake of NSAIDs9 and renal Ang II content18 are enhanced. It has also been reported that intrarenal Ang II levels are elevated,19 and NO production is reduced8 in hypertensive patients. Therefore, the results obtained may also show to what extent the administration of nonselective or selective COX2 inhibitors induces www.jcvp.org |

465

Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

López et al


significant changes of renal function in these hypertensive patients. The studies to accomplish these objectives have been performed before and during an acute extracellular volume expansion (ECVE) because NO and Ang II are involved in modulating the renal response to an ECVE.6,20–23 The hypothesis was that the greater renal vasoconstriction induced by a nonselective than a selective COX2 inhibitor, when NO synthesis is reduced and Ang II levels are enhanced, would be more evident when ECV is elevated. The results obtained may also have important implications because they may help to better understand the mechanisms involved in the development of a sodium-sensitive hypertension during aging and in some hypertensive patients.

used does not induce changes in renal hemodynamic when infused alone and is effective in reducing NO synthesis4,6,22 but the inhibition is not complete because greater doses of 2,27 L-NAME induce a renal and systemic vasoconstriction.

METHODS Experiments were performed using dogs of either gender (16–29 kg) maintained on a standard laboratory diet and free access to water. All experimental procedures were designed in accordance with the rules of European Union and approved by the University of Murcia Institutional Animal Care and Use Committee. Surgical preparation was performed in anesthetized dogs (30 mg/kg intravenous, sodium pentobarbital) as described.4,6,21–26 Catheters were placed for measuring mean arterial pressure (MAP) and for intravenous infusion of inulin, additional anesthesia, and nimesulide or meclofenamate. Left kidney was exposed through flank incision and the ureter cannulated. Left renal artery was fitted with an electromagnetic flowprobe that was connected to a flowmeter. Distal to the flowprobe, a curved 23-gauge needle attached to polyethylene tubing was inserted into the renal artery and connected to a peristaltic pump for infusion of saline and different solutions (L-NAME, captopril, and Ang II) depending of the protocol. Finally, a 45-minute stabilization period was allowed before experimental maneuvers begun.

Group 2 (n = 5) The experimental protocol was similar to that described for group 1 with the exception that meclofenamate (10 mg$kg21$min21), instead nimesulide was intravenously administered throughout the experiment. Meclofenamate reduces by .75% the urinary excretion of PGE2 and 6ketoPGF1a.6,23,24 The renal effects of meclofenamate at the dose used in this study were reported in previous studies of our group.6,23,24,26 Experiments in groups 3 and 4 were performed to accomplish the second objective of this study.

Group 3 (n = 6) After determination of two 15-minute clearances, an infusion of nimesulide and L-NAME started, at doses and durations similar to those described in group 1. Fifteen minutes later, captopril (0.8 mg$kg21$min21) was infused into the renal artery throughout the experiment. Thirty minutes after initiating captopril infusion, 2 more 15-minute clearances were obtained, and a 5% ECVE during 45 minutes was performed. Two clearances were obtained during the last 10 minutes of saline infusion and 10 minutes after cessation of the expansion. The dose of captopril employed is effective in blocking 70% of the fall in renal blood flow (RBF) induced by an intrarenal Ang I bolus of 0.8 mg.21–23

Group 4 (n = 5) The experimental protocol was similar to that described for group 3 with the exception that meclofenamate (10 mg$kg21$min21), instead nimesulide was intravenously administered throughout the experiment.

Experimental Groups

The first objective was accomplished by comparing the renal effects elicited by a selective COX2 (group 1) and a nonselective COX (group 2) inhibitor in dogs treated with a subpressor dose of L-NAME when given alone.4,6,22

The importance of COX2 or both COX isoforms in modulating the renal vasoconstriction induced by an increment of Ang II when NO synthesis is reduced (third objective) was examined in the anesthetized dogs included in group 5 or 6, respectively.

Group 1 (n = 8)

Group 5 (n = 7)

After two 15-minute control clearances, L-NAME (1 mg$kg21$min21, intrarenal) and nimesulide (bolus, 0.75 mg/kg; continuous intravenous infusion, 5 mg$kg21$min21) were infused throughout the experiment. Thirty minutes after the initiation of nimesulide and L-NAME infusions, 2 more 15-minute clearances were obtained, and a 5% ECVE during 45 minutes with isotonic saline infusion were performed. Two clearances were also obtained during the last 10 minutes of saline infusion and 10 minutes after cessation of this infusion. The effectiveness of nimesulide to inhibit COX2 activity was confirmed in studies showing that the dose used reduces PGE2 and 6ketoPGF1a by 40% but does not induce changes in renal hemodynamics when administered alone to dogs with normal sodium intake.4,25,26 The dose of L-NAME

After two 15-minute control clearances, an infusion of captopril (0.8 mg$kg21$min21) and Ang II (1 ng$kg21$min21) was started into the left renal artery. Fifteen minutes after captopril and Ang II infusions were inititated, L-NAME infusion started at the dose used in group 1. Fifteen minutes later, nimesulide was also administered as in groups 1 and 3. Thirty minutes after nimesulide infusion started, two 15-minute clearances were obtained. A 5% ECVE during 45 minutes then begun with clearances determined during the last 10 minutes of saline infusion and 10 minutes after the cessation of the expansion. Captopril was administered to avoid significant changes in the intrarenal Ang II levels throughout the experiment. The dose of Ang II infused does not induce significant changes in renal hemodynamics when administered alone.22,23

466

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

| www.jcvp.org



Group 6 (n = 5) The experimental protocol was similar to that described for group 5 with the exception that meclofenamate (10 mg$kg21$min21), instead nimesulide was intravenously administered throughout the experiment.

Analytical Methods Renal clearances were taken during each experimental period to determine glomerular filtration rate (GFR), urinary sodium excretion (UNaV) and urine flow rate (UV). Blood samples for hematocrit, plasma sodium, and inulin concentrations were also obtained. GFR was measured by the clearance of inulin. Inulin concentrations were analyzed by the anthrone method. Sodium concentration was measured by flame photometry.

Statistical Analysis

Data are expressed as means 6 SEM. Data for the 2 clearances obtained during the control period and infusions of the different substances, before and during the ECVE, were averaged for statistical comparisons because the fluid and solute excretions were in steady-state conditions. The significance of differences between values of each period in the same group was evaluated by using a one-way analysis of variance and the Fisher’s exact test. Significance of differences between the values obtained in different groups was calculated by using a 2-way analysis of variance and the Fisher’s exact test. A P , 0.05 was considered statistically significant.

RESULTS Group 1 Table 1 shows that MAP remained elevated throughout the experiment during L-NAME and nimesulide administration. The administration of both inhibitors did not modify GFR but induced a decrease of RBF (22% 6 3%, P , 0.05) and an increase in RVR (35% 6 4%, P , 0.05) before ECVE (Table 1, Figs. 1, 2 and 3). Both, RBF and RVR returned to control levels during ECVE (Figs. 2, 3). The simultaneous reduction in COX2 and NO synthases activity

TABLE 1. Changes in MAP and Renal Function Induced by the Simultaneous Administration of L-NAME and Nimesulide (Nime), Before and During an ECVE L-NAME

Control MAP, mm Hg GFR, mL/min RBF, mL/min RVR, mm Hg$mL21$min21 UNaV, mEq/min UV, mL/min

122 37 188 0.65

6 6 6 6

5 5 21 0.09

50 6 12 0.26 6 0.06

Pre-ECVE 129 38 147 0.88

6 6 6 6

5* 5 17* 0.11*

19 6 5* 0.13 6 0.02*

+ Nime ECVE 132 35 176 0.74

6 6 6 6

6* 4 16 0.07

463 6 56* 5.53 6 0.45*

*P , 0.05 versus control.


COX1–COX2 Inhibition During Changes in NO and Ang II

induced a fall (P , 0.05) in UNaV and UV before ECVE. As expected, UNaV and UV increased (P , 0.05) during ECVE (Table 1).

Group 2 The administration of L-NAME and meclofenamate elicited an increase (P , 0.05) in MAP that was similar before and during ECVE (Table 2). The reduction of NO synthesis and the activity of both COX isoforms before ECVE led to a fall in GFR (42% 6 9%, P , 0.05) (Fig. 1), a fall in RBF (39% 6 5%, P , 0.05) (Fig. 2), and to an elevation in RVR (77% 6 4%, P , 0.05) (Fig. 3) that were greater than that found in group 1. During ECVE, GFR returned to control levels (Fig. 1) but RVR remained elevated (Fig. 3) compared with the values found during control period. Both UNaV and UV decreased before ECVE and increased during ECVE (P , 0.05) (Table 2). However, the increments in UNaV and UV during ECVE were of a lower magnitude than those found in group 1.

Group 3 The MAP and renal responses to COX-2 and NO synthesis inhibition, when the intrarenal Ang II synthesis was inhibited, are shown in Table 3 and Figures 1, 2 and 3. No significant changes in GFR and RVR were found (Figs. 1, 3). Regarding RBF, it decreased (P , 0.05) before ECVE during COX-2 and NO inhibition, but the fall (6% 6 1%) was lower (P , 0.05) than that found in group 1 (Fig. 2), in which the converting enzyme activity was not modified by the intrarenal captopril infusion. During ECVE, RBF increased to levels not significantly different to those found during the control period. The responses of UNaV and UV in this group (Table 3) were similar to those found in group 1 (Table 1).

Group 4 The simultaneous reduction in NO and Ang II levels in association with the inhibition of both COX isoforms did not induce significant changes in MAP, GFR, and RVR before or during ECVE (Table 4, Figs. 1, 3) and led to a small decrease in RBF before (6% 6 1%, P , 0.05) but not during ECVE (Table 4, Fig. 2). The fall in UNaV and UV before ECVE and the elevation in both excretory parameters during ECVE in this group were similar to those induced by COX2 inhibition in group 3 (Table 3).

Group 5 Table 5 shows the MAP and renal responses to the administration of L-NAME and nimesulide when intrarenal Ang II levels were maintained constant by the administration of captopril and Ang II. It can be observed that MAP increased (P , 0.01) to similar levels before and during ECVE in response to Ang II infusion. The renal vasoconstriction elicited by L-NAME and nimesulide in this group was more pronounced (P , 0.05) than that found in groups 1 and 3. Figures 1, 2 and 3 show that GFR and RBF decreased (28% 6 4% and 37% 6 3%, respectively) and RVR increased (77% 6 6%, P , 0.05) before ECVE. GFR returned to control levels, RBF remained reduced (24% 6 5%, P , 0.05), and RVR remained elevated (49% 6 5%, www.jcvp.org |

467


López et al


FIGURE 1. GFR changes before (Pre-ECVE) and during an ECVE in response to a selective COX2 inhibitor or a nonselective COX inhibitor in dogs pretreated with L-NAME (left), L-NAME + captopril (Capt) (middle), or L-NAME + Capt + Ang II (right). *P , 0.05 versus control. #P , 0.05 versus nimesulide.

P , 0.05) during ECVE when L-NAME and nimesulide were administered to dogs in which intrarenal Ang II levels were maintained elevated. Table 5 shows that UNaV and UV decreased (P , 0.01) before ECVE and increased (P , 0.01) in response to the acute ECVE during the simultaneous reduction in NO production and COX2 inhibition. However, the increases in both excretory parameters during ECVE were of a lower magnitude (P , 0.05) than those found in groups 1 and 3.

Group 6 Tables 5 and 6 show that the increment in MAP elicited by the inhibition of both COX isoforms is similar to that induced by COX2 inhibition alone when NO production is reduced and Ang II levels are elevated. However, the renal vasoconstriction secondary to the inhibition of both COX isoforms (Table 6) is more pronounced (P , 0.05) than that elicited by COX2 inhibition (Table 5) under this experimental situation. The administration of L-NAME and meclofenamate before ECVE led to a decrease GFR (61% 6 9%, P , 0.05) (Fig. 1), a fall in RBF (61% 6 7%, P , 0.05) (Fig. 2) and an elevation in RVR (186% 6 8%, P , 0.05) (Fig. 3) when intrarenal Ang II levels were enhanced. Contrary to what was

found in the other experimental groups, the renal vasoconstriction remained unaltered during ECVE because GFR (59% 6 8%, P , 0.05) (Fig. 1) and RBF (59% 6 6%, P , 0.05) (Fig. 2) were decreased and RVR was enhanced (173% 6 6%, P , 0.05) (Fig. 3). The increase in UNaV and UV induced by ECVE was lower in dogs in which Ang II was infused with meclofenamate and L-NAME (Table 6) than in those treated with nimesulide and L-NAME (Table 5).

DISCUSSION The main objective of this study was to examine the relative contribution of COX1 and COX2-derived metabolites in modulating renal hemodynamic when NO synthesis is reduced with or without a simultaneous increment in intrarenal Ang II levels. This study reports novel findings showing that there are important interactions between Ang II, NO, and COX1- and COX2-derived metabolites in regulating renal function when ECV is normal or enhanced. It is worthy to note that the contribution of both COX isoforms in regulating renal hemodynamics was examined during Ang II or L-NAME administration at doses that do not induce changes in renal hemodynamics when administered alone.4,6,20,22,23 Therefore, the observed renal effects could simulate those

FIGURE 2. Renal blood flow changes before (Pre-ECVE) and during an ECVE in response to a selective COX2 inhibitor or a nonselective COX inhibitor in dogs pretreated with L-NAME (left), L-NAME + captopril (Capt) (middle), or L-NAME + Capt + Ang II (right). *P , 0.05 versus control. #P , 0.05 versus nimesulide.

468

| www.jcvp.org





FIGURE 3. RVR changes before (Pre-ECVE) and during an ECVE in response to a selective COX2 inhibitor or a nonselective COX inhibitor in dogs pretreated with L-NAME (left), L-NAME + captopril (Capt) (middle), or L-NAME + Capt + Ang II (right). *P , 0.05 versus control. #P , 0.05 versus nimesulide.

elicited by the administration of NSAIDs to patients with moderated increments in intrarenal Ang II levels or a slight reduction in NO synthesis. The results reported in this study do not allow us to define the mechanistic insight in signal transduction involved in the interactions between NO, COX1, COX2, and Ang II in regulating renal function. Additional studies are obviously needed to examine the intrinsic mechanisms involved in these interactions.

Interactions Between Both COX Isoforms and NO in the Regulation of Renal Function The cross talk between COX and NOS pathways in the regulation of renal function has been suggested in numerous studies.1–5,7,25,28,29 In support of an important interaction between both pathways, it has been shown that the renal effects elicited by a selective COX2 inhibitor are much more evident when endogenous NO synthesis is reduced.1–5,25 The possibility that COX2 and NOS expressions are reciprocally regulated in the kidney is supported by studies showing that a reduction in NO induces an upregulation of renal COX2 expression7 and that a decrease in COX2 leads to an elevation in neural NOS expression and NOS activity in the kidney.30 However, no previous study has compared the renal effects

TABLE 2. Changes in MAP and Renal Function Induced by the Simultaneous Administration of L-NAME and Meclofenamate (Meclof), Before and During an ECVE L-NAME


123 35 190 0.65

6 6 6 6

3 3 12 0.06

49 6 15 0.31 6 0.06

Pre-ECVE 135 21 117 1.15

6 6 6 6

4* 4* 11* 0.09*

9 6 3* 0.07 6 0.01*

elicited by the inhibition of both COX isoforms with those induced by inhibiting only COX2 when NO synthesis is reduced. The results obtained confirm that there is an interaction between NO and COX2-derived metabolites in regulating renal hemodynamics2,4,5,7,25,26 because the administration of nimesulide and L-NAME leads to a significant fall in RBF, but the administration of each of these inhibitors alone do not modify renal hemodynamics.4,21,22,25 This study reports novel findings suggesting that COX1-derived metabolites also play an important role in modulating the renal hemodynamic response to a decrease in NO. In support of this hypothesis, Figures 1, 2 and 3 show that the nonselective COX inhibition induces a renal vasoconstriction that is significantly greater than that induced by the selective COX2 inhibitor when endogenous NO synthesis is reduced.

Role of Endogenous Ang II in the Renal Vasoconstriction Elicited by COX Inhibition and NO Synthesis Inhibition Previous studies have examined whether the effects elicited by NO or COX inhibition are secondary to those induced by Ang II levels.21,23,24,30 However, it was unknown whether endogenous Ang II levels play an important role in

TABLE 3. Changes in MAP and Renal Function Induced by the Simultaneous Administration of L-NAME, Nimesulide (Nime), and Captopril (Capt), Before and During an ECVE

+ Meclof

L-NAME

ECVE 131 36 165 0.79

6 6 6 6

2* 3 19* 0.08*

243 6 69* 3.09 6 1.12*




129 33 178 0.72

6 6 6 6

6 1 3 0.04

56 6 23 0.35 6 0.13

+ Nime + Capt

Pre-ECVE 132 32 167 0.79

6 6 6 6

5 1 4* 0.05

23 6 10* 0.19 6 0.04*

ECVE 131 33 174 0.75

6 6 6 6

5 2 2 0.03

355 6 43* 4.10 6 0.56*


www.jcvp.org |

469


López et al


TABLE 4. Changes in MAP and Renal function Induced by the Simultaneous Administration of L-NAME, Meclofenamate (Meclof), and Captopril (Capt), Before and During an ECVE

TABLE 6. Changes in MAP and Renal Function Induced by the Simultaneous Administration of L-NAME, Meclofename (Meclof), Captopril (Capt), and Angiotensin II (Ang II), Before and During an ECVE

L-NAME


126 31 182 0.69

6 6 6 6

+ Meclof + Capt

Pre-ECVE

4 3 12 0.06

48 6 11 0.32 6 0.06

126 30 172 0.73

6 6 6 6

5 4 14* 0.05

27 6 9* 0.21 6 0.05*

L-NAME

ECVE 127 31 176 0.72

6 6 6 6

4 3 11 0.06

401 6 53* 4.03 6 0.42*



123 34 194 0.63

6 6 6 6

4 4 16 0.07

52 6 7 0.34 6 0.06

+ Meclof + Capt + Ang II

Pre-ECVE 135 12 75 1.88

6 6 6 6

5* 2* 11* 0.03*

6 6 2* 0.06 6 0.01*

ECVE 136 13 79 1.72

6 6 6 6

5* 2* 14* 0.04*

83 6 25* 0.88 6 0.22*


mediating the renal vasoconstriction elicited by the simultaneous reduction in COX2 and NOS activities. The results of this study suggest that a decrease in COX1 and/or COX2 activity, when NO synthesis is reduced, increases RVR presumably by accentuating Ang II-induced constriction of the renal vasculature. These results may have clinical implications because they suggest that the renal vasoconstriction elicited by a NSAID in patients with a reduced NO production can be prevented or treated with a converting enzyme inhibitor or an AT1 receptor antagonist.

Effects of Ang II on Renal Hemodynamics When NOS and COX Activities Are Reduced

This is also the first study that has examined the relative contribution of both COX isoforms in regulating renal hemodynamics when there is an increment in intrarenal Ang II levels and NO synthesis is reduced. The interactions between Ang II, COX2, and NO has been examined in rats with low sodium intake but renal hemodynamic changes were not evaluated.31,32 Our hypothesis was that both COX isoforms play an important role in protecting the renal vasculature in response to a small increment of intrarenal Ang II levels when NO synthesis is reduced. The interaction between Ang II and NO has been demonstrated in studies showing that

TABLE 5. Changes in MAP and Renal Function Induced by the Simultaneous Administration of L-NAME, Nimesulide (Nime), Captopril (Capt), and Angiotensin II (Ang II), Before and During an ECVE L-NAME


124 35 218 0.57

2 2 16 0.07

45 6 13 0.30 6 0.05


470

6 6 6 6

| www.jcvp.org

+ Nime + Capt + Ang II

Pre-ECVE 136 25 135 1.01

6 6 6 6

2* 2* 6* 0.03*

12 6 3* 0.10 6 0.02*

ECVE 138 31 163 0.85

6 6 6 6

2* 2 11* 0.04*

217 6 63* 2.96 6 0.68*

an elevation in Ang II leads to an increase in cortical NOS activity and neural NOS protein abundance12,16,33 and suggesting that the Ang II-induced renal vasoconstriction is partly attenuated by the vasodilator effects elicited by endogenous NO.10,11,22 The increase of NO in response to Ang II seems to occur through the activation of AT2 receptors in microarteries27 and by activating AT1 receptors in the macula densa.34 The importance of COX-derived PG in protecting the renal vasculature from the Ang II effects has been examined in several studies13,14,23,35,36 with contradictory results that may be explained by the different protocols performed. The hypothesis that COX2-derived PG antagonize the renal vasoconstrictor Ang II effects is supported by studies showing that an increase in Ang II elicits an upregulation of renal COX2 expression15 and leads to a COX2-dependent increment in the renal PG levels.17 Taken together with those already reported,21–23 our results suggest that there is an important interaction between NO and both COX isoforms in modulating the renal vasoconstrictor effects of Ang II. Our previous studies show that Ang II and meclofenamate administration leads to an 11% decrease in RBF with no changes in GFR,23 and that Ang II and L-NAME administration induces a 23% fall in RBF without affecting GFR.22 However, the Ang II infusion when NO synthesis and both COX isoforms are inhibited induces a fall (61%) in RBF and GFR. Apparently, when the activity of one or both COX isoforms is reduced, there is a greater increment in NO to compensate the renal vasoconstrictor effect elicited by a small increment in Ang II levels. It could also be proposed that when NO production is diminished, there is a greater effect of COX-derived metabolites in modulating the Ang II effects. Both COX isoforms seem to play a similar role in the production of vasodilatory PG that modulate the vasoconstriction induced by Ang II before ECVE when NO synthesis is reduced. This hypothesis is supported by the fact that COX2 inhibition induced a 37% fall in RBF and a 28% fall in GFR when Ang II was infused to dogs with a reduced NO synthesis. However, COX1 seems to play a more important role than COX2 when ECV is enhanced in modulating the renal vasoconstriction elicited by Ang II if NO synthesis is reduced. In support of this notion, our results show that Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.




during ECVE, there was only a reduction in RBF (24%, P , 0.05) in dogs treated with nimesulide, L-NAME, and Ang II, and a 59% fall (P , 0.05) in both RBF and GFR in dogs treated with meclofenamate, L-NAME, and Ang II. Although COX1 is involved in the production of several prostanoids with different effects on renal function,37 our results suggest that in the dog kidney, COX1 is mainly involved in the production of vasodilatory PGs when NO synthesis is reduced with and without an elevation in Ang II levels. The interpretation of the renal excretory responses found is not easy because they can be secondary not only to the tubular effects but also to the renal vasoconstriction elicited by changes in Ang II, NO, and COX activity. Previous studies have shown that Ang II and NO levels are involved in regulating the excretory response to an ECVE.20,21,23 It is also possible that the tubular effects of Ang II during ECVE are influenced by endogenous NOand COX-derived metabolites because these tubular effects are modulated by an increase in NO through the activation of AT2 receptors,38 and it has been proposed that a COX2dependent increment of PGE2 in the renal medulla counteracts the antinatriuretic effects of Ang II.17

9. Harley C, Wagner S. The prevalence of cardiorenal risk factors in patients prescribed nonsteroidal anti-inflammatory drugs: data from managed care. Clin Ther. 2003;25:139–149. 10. Alberola AM, Salazar FJ, Nakamura T, et al. Interactions between angiotensin II and nitric oxide in control of renal hemodynamics in conscious dogs. Am J Physiol Regul Integr Comp Physiol. 1994;267:R1472–R1478. 11. Cervenka L, Kramer HJ, Malý J, et al. Role of nNOS in regulation of renal function in angiotensin II-induced hypertension. Hypertension. 2001;38:280–285. 12. Chin SY, Pandey KN, Shi SJ, et al. Increased activity and expression of Ca(2+)-dependent NOS in renal cortex of ANG II-infused hypertensive rats. Am J Physiol. 1999;277:F797–F804. 13. González JD, Llinás MT, Moreno C, et al. Renal effects of prolonged cyclooxygenase inhibition when angiotensin II levels are elevated. J Cardiovasc Pharmacol. 2002;36:236–241. 14. Green T, Gonzalez A, Mitchell K, et al. The complex interplay between COX-2 and Ang II in regulating kidney function. Curr Opin Nephrol Hypertens. 2012;21:7–14. 15. Jaimes EA, Tian RX, Pearse D, et al. Up-regulation of glomerular COX-2 by angiotensin II: role of reactive oxygen species. Kidney Int. 2005;68:2143–2153. 16. Murakami K, Tsuchiya K, Naruse K, et al. Nitric oxide synthase I immunoreactivity in the macula densa of the kidney is angiotensin II dependent. Kidney Int Suppl. 1997;63:S208–S210. 17. Qi Z, Hao C, Langenbach RL, et al. Opposite effects of cyclooxygenase1 and –2 activity on the pressor response to angiotensin II. J Clin Invest. 2002;110:61–69. 18. Thompson MM, Oyama TT, Kelly FJ, et al. Activity and responsiveness of the rennin-angiotensin system in the aging rat. Am J Physiol. 2000; 279:R1787–R1794. 19. Hollenberg NK, Willimas GH. Angiotensin and the renal circulation in hypertension. Circulation. 1988;77(suppl 1):59–63. 20. Alberola AM, Pinilla JM, Quesada T, et al. Role of nitric oxide in mediating renal response to volume expansion. Hypertension. 1992;19:780–784. 21. Llinás MT, González JD, Nava E, et al. Role of angiotensin II in the renal effects induced by nitric oxide and prostaglandin synthesis inhibition. J Am Soc Nephrol. 1997;8:543–550. 22. Llinás MT, González JD, Salazar FJ. Interactions between angiotensin II and nitric oxide in the renal response to volume expansion. Am J Physiol Regul Integr Comp Physiol. 1995;269:R504–R510. 23. Pinilla JM, Alberola A, González JD, et al. Role of prostaglandins on the renal effects of angiotensin and interstitial pressure during volume expansion. Am J Physiol Regul Integr Comp Physiol. 1993;265:R1469–R1474. 24. Llinás MT, González JD, Rodriguez F, et al. Renal effects induced by nitric oxide and prostaglandin synthesis reduction. Effects of trandolapril and verapamil. Hypertension. 1998;31:657–664. 25. López R, Llinás MT, Roig F, et al. Role of nitric oxide and cycloxygenase-2 in regulating the renal hemodynamic response to norepinephrine. Am J Physiol Regul Integr Comp Physiol. 2002;284:R488– R493. 26. Rodríguez F, Llinás MT, Moreno C, et al. Role of cyclooxygenase-2derived metabolites and NO in renal response to bradykinin. Hypertension. 2001;37:129–134. 27. Batenburg WW, Garrelds IM, Bernasconi CC, et al. Angiotensin II type 2 receptor-mediated vasodilation in human coronary microarteries. Circulation. 2004;109:2296–2301. 28. López R, Roig F, Llinás MT, et al. Role of cyclooxygenase-2 in the control of renal haemodynamics and excretory function. Acta Physiol Scand. 2003;177:1–7. 29. Paliege A, Mizel D, Medina C, et al. Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E2. Am J Physiol Ren Physiol. 2004;287:F152–F159. 30. Perinotto P, Biggi A, Carra N, et al. Angiotensin II and prostaglandin interactions on systemic and renal effects of L-NAME in humans. J Am Soc Nephrol. 2001;12:1706–1712. 31. Harris RC, Cheng HF, Wang JL, et al. Interactions of the reninangiotensin system and neuronal nitric oxide synthase in regulation of cyclooxygenase-2 in the macula densa. Acta Physiol Scand. 2000;168: 47–51. 32. Kammerl MC, Richthammer V, Kurtz A, et al. Angiotensin II feedback is a regulator of renocortical renin, COX-2, and nNOS expression. Am J Physiol Regul Integr Comp Physiol. 2002;282:R1613–R1617.

SUMMARY

This study reports novel findings suggesting that: (1) both COX isoforms play an important role in the regulation of renal hemodynamics when there is a decrease of NO synthesis, (2) the renal vasoconstriction elicited by the inhibition of one or both COX isoforms when NO synthesis is reduced is secondary mainly to the effects induced by endogenous Ang II levels, and (3) the renal vasoconstrictor effects of Ang II are countered by the production of COX1and COX2-derived vasodilators when NO synthesis is reduced being more important for COX1 than COX2 when ECV is enhanced. REFERENCES 1. Baylis C, Slangen B, Hussain S, et al. Relationship between basal NO release and cyclooxygenase products in the normal rat kidney. Am J Physiol. 1996;271:R1327–R1334. 2. Beierwaltes WH. Cyclooxygenase-2 products compensate for inhibition of nitric oxide regulation of renal perfusion. Am J Physiol Ren Physiol 2002;283:F68–F72. 3. González JD, Llinás MT, Nava E, et al. Role of nitric oxide and prostaglandins in the long-term control of renal function. Hypertension. 1998; 32:33–38. 4. Llinás MT, Rodríguez F, Moreno C, et al. Role of cyclooxygenase-2derived metabolites and nitric oxide in regulating renal function. Am J Physiol Regul Integr Comp Physiol. 2000;279:R1641–R1646. 5. Roig F, Llinás MT, Lopez R, et al. Role of cyclooxygenase-2 in the prolonged regulation of renal function. Hypertension. 2002;40:721–728. 6. Salazar FJ, Llinás MT, González JD, et al. Role of prostaglandins and nitric oxide in mediating renal response to volume expansion. Am J Physiol Regul Integr Comp Physiol. 1995;268:R1442–R1448. 7. Kommareddy M, McAllister RM, Ganjam VK, et al. Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition. Vet Pathol. 2011;48:1125–1133. 8. Lüscher TF, Dohi Y, Tschdi M. Endothelium-dependent regulation of resistance arteries: alterations with aging and hypertension. J Cardiovasc Pharmacol. 1992;19(suppl 5):S34–S42.


www.jcvp.org |

471


López et al


33. Moreno C, López A, Llinás MT, et al. Changes in NOS activity and protein expression during acute and prolonged ANG II administration. Am J Physiol Regul Integr Comp Physiol. 2002;282:R31–R37. 34. Liu R, Persson AE. Angiotensin II stimulates calcium and nitric oxide release from macula densa cells through AT1 receptors. Hypertension. 2004;43:649–653. 35. Green T, Rodríguez J, Navar LG. Augmented COX-2 effects on renal function during varying states of angiotensin II. Am J Physiol Ren Physiol. 2010;299:R954–R962.

36. Salazar FJ, Llinás MT. Renal hemodynamic effects elicited by acute cyclooxygenase-2 inhibition are not related to angiotensin II levels. Am J Physiol Ren Physiol 2010;299:F952–F953. 37. Navar LG, Inscho EW, Majid DSA, et al. Paracrine regulation of the renal microcirculation. Physiol Rev. 1996;76:425–536. 38. Herrera M, Garvin J. Angiotensin II stimulates thick ascending limb NO production via AT2 receptors and Akt1-dependent nitricoxide synthase 3 (NOS3) activation. J Biol Chem. 2010;285:14932– 14940.

472


| www.jcvp.org


Inhibition of synthesis of endothelium-derived nitric oxide in conscious dogs. Hemodynamic, renal, and hormonal effects.

Combined angiotensin receptor blockade and neprilysin inhibition attenuates angiotensin-II mediated renal cellular collagen synthesis.

Renal angiotensin-converting enzyme upregulation: a prerequisite for nitric oxide synthase inhibition-induced hypertension?

Inhibition of renal nitric oxide synthesis with NG-monomethyl-L-arginine and NG-nitro-L-arginine.

Angiotensin receptors modulate the renal hemodynamic effects of nitric oxide in conscious newborn lambs.

Renal vasoconstriction is modulated by nitric oxide.

Renal effects of angiotensin II inhibition during increases in renal venous pressure.

Renal dysfunction is associated with a reduced contribution of nitric oxide and enhanced vasoconstriction after a congenital renal mass reduction in sheep.

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease.

Intranasal angiotensin II in humans reduces blood pressure when angiotensin II type 1 receptors are blocked.

Cardiorespiratory effects induced by 2-nitrate-1,3-dibuthoxypropan are reduced by nitric oxide scavenger in rats.

Renal prostaglandin synthesis and angiotensin-converting enzyme inhibition.

Role of Angiotensin II type 1 receptor on renal NAD(P)H oxidase, oxidative stress and inflammation in nitric oxide inhibition induced-hypertension.

Inhibition of nitric oxide synthesis in septic shock: how much is beneficial?

Exercise vasodilation is greater in women: contributions of nitric oxide synthase and cyclooxygenase.

Nitric oxide is a mediator of hypoxic coronary vasodilatation. Relation to adenosine and cyclooxygenase-derived metabolites.

Cyclooxygenase-2 Inhibition Limits Angiotensin II-Induced DNA Oxidation and Protein Nitration in Humans.

Renal medullary cyclooxygenase-2 and (pro)renin receptor expression during angiotensin II-dependent hypertension.

Effects of intravitreal injection of bevacizumab on nitric oxide levels.

HDL levels and oxidizability during myocardial infarction are associated with reduced endothelial-mediated vasodilation and nitric oxide bioavailability.

Nitric oxide and angiotensin II. Glomerular and tubular interaction in the rat.

Modulators of nitric oxide synthesis.

guanosine 3',5'-cyclic monophosphate pathway.

Nitric oxide generation by the organic nitrate NDBP attenuates oxidative stress and angiotensin II-mediated hypertension.