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Am J Physiol Heart Circ Physiol 291: H2772-H2778, 2006. First published July 14, 2006; doi:10.1152/ajpheart.00528.2006
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Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Fady T. Botros and L. Gabriel Navar

Department of Physiology, Hypertension and Renal Center, Tulane University Health Sciences Center, New Orleans, Louisiana

Submitted 23 May 2006 ; accepted in final form 3 July 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 µmol/l CO solution, or 15 µmol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l N{omega}-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 µmol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 µmol/l CORM-2 or with CO (10 µmol/l) significantly dilated AAs (22.0 ± 0.9 and 22.8 ± 0.9 vs. 18.3 ± 0.9 µm, n = 5, P < 0.05; and 26.0 ± 1.4 vs. 18.8 ± 0.7 µm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 ± 0.7 vs. 17.2 ± 0.6 µm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 ± 1.2 µm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 ± 1.6 µm, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 ± 0.4 vs. 18.8 ± 0.4 µm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 ± 0.7 µm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.

heme oxygenase; renal circulation; soluble guanylate cyclase

CARBON MONOXIDE (CO) is produced biologically via degradation of heme, a reaction that is catalyzed by heme oxygenase (28). Two isoforms of heme oxygenase (HO-1 and HO-2) have been shown to be expressed in the kidney (8, 12). HO-2 is a constitutive isoform that is responsible for basal CO production under normal conditions (1, 18, 19). CO exerts an important role in the regulation of vascular tone and blood pressure (37). CO arising from heme via metabolism by HO exerts a vasodilatory effect (16, 17, 20). Other reports have shown that CO stimulates soluble guanylate cyclase (sGC) (21) and calcium-activated potassium (KCa) channels (31) in vascular smooth muscle, which causes relaxation, and that inhibition of HO causes vascular constriction (37). In addition, Johnson et al. (15) showed that inhibition of HO with chromium mesoporphyrin (CrMP) vasoconstricted isolated first-order gracilis muscle arterioles denuded of endothelium. Inhibition of HO with tin mesoporphyrin significantly magnified the renal vasoconstrictor effect of angiotensin II and enhanced pressure-induced constriction of isolated pressurized renal interlobular arteries (26), and reduced renal blood flow and increased renal vascular resistance, effects that were magnified in the absence of NO (26). Overexpression of HO-1 reduced the pressor responsiveness to angiotensin II (36). In contrast, CO has also been shown to promote endothelium-dependent vasoconstriction by inhibiting endothelial nitric oxide (NO) formation (14). Thorup et al. (29) showed that lower concentrations of CO (0.1 µM) dilate juxtamedullary afferent arterioles via release of NO from the intracellular pool, whereas high levels of CO (10 µM) inhibit NO synthase (NOS) and NO generation. NO has been shown to desensitize KCa channels to CO (35), and inhibition of NOS has been shown to increase renal CO production (25).

Exogenous CO constricts isolated pressurized renal interlobular arteries (26) but dilates vessels harvested from rats pretreated with NG-nitro-L-arginine methyl ester (L-NAME) (26). In vivo studies suggest that endogenously produced CO exerts a vasodilatory influence on the renal circulation; inhibition of HO resulted in decreases in renal blood flow and renal function (3, 5, 23, 26, 38). However, most of our knowledge about the effect of the HO-CO system on renal vascular tone is derived from experiments done on isolated perfused vessels but not afferent arterioles, which are the predominant resistance vessels in the kidney. Because of the importance of afferent arterioles in regulating renal blood flow and glomerular function, we examined the role of HO in regulating renal afferent arteriolar tone in intact blood-perfused juxtamedullary nephrons. In this preparation, the afferent arterioles are perfused with blood and have an intrinsic tone without the need for exogenous constrictor agents; also, the dissection procedure preserves the in vivo tubulovascular relationships so that the autoregulation mechanisms are intact (i.e., myogenic tone and tubuloglomerular feedback). Because NO also activates sGC and exerts effects via the same pathways as CO, it was important to determine whether their actions are additive, opposite, or overlapping. Accordingly, the aims of this study were 1) to determine the effect of exogenous CO administration on afferent arteriolar vasoactivity, 2) to determine the role of endogenously produced HO-derived CO on the vasoactivity of renal afferent arterioles, and 3) to determine the interactions between endogenously produced CO and NO in regulating afferent arteriolar function via activation of sGC.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimental protocols were approved by the Tulane Institutional Animal Care and Use Committee. The blood-perfused juxtamedullary nephron technique was utilized as previously described by Casellas et al. (7). Male Sprague-Dawley rats, weighing 350–450 g, were anesthetized with pentobarbital sodium (50 mg/kg ip), and a cannula was placed in the left carotid artery. Captopril was administered via the carotid cannula (10 mg/kg ia). The left kidney pedicle was tied, and the right kidney was perfused through a cannula inserted into the superior mesenteric artery and advanced into the right renal artery. The perfusate was a Tyrode solution containing 5% bovine serum albumin and a mixture of amino acids (pH 7.4). Blood was collected via the carotid cannula in a heparinized syringe (500 U), and the right kidney was then excised. The blood was centrifuged to separate plasma and cellular fractions, erythrocytes were washed twice with saline, and plasma was filtered through a 0.22-µm filter and was then added to the red blood cells to achieve a hematocrit of 33%. This reconstituted blood was filtered through a 5-µm nylon mesh and placed in a closed reservoir pressurized with a 95% O2-5% CO2. The kidney was sectioned longitudinally, and the papilla was lifted to expose the inner cortical surface with the tubules, glomeruli, and related vasculature of the juxtamedullary nephrons while leaving the papilla intact. The arterial supply of the exposed area was isolated by ligating the larger branches of the renal artery with fine suture; the arterial branch supplying the exposed area was ligated at the terminal end. The Tyrode perfusate was replaced with the reconstituted blood, and perfusion pressure was adjusted to 95 ± 5 mmHg by adjusting the rate of gas inflow into the blood reservoir. The inner cortical surface of the kidney was superfused continuously with Tyrode solution containing 1% albumin at 37°C. The tissue was transilluminated under a microscope with a water-immersion objective (40x). Video images of the microvessels were transferred to a video monitor via camera, and the video signal was recorded on DVD for later analysis. Afferent arteriolar inside diameters were measured using a digital image-shearing monitor. A 10-min equilibration period was allowed before the initiation of each experimental protocol. Basal diameters were measured during superfusion with appropriate vehicle for at least 5 min. Afferent arteriolar diameters were averaged for each minute (10).

Chemicals and Drug Preparation

Tricarbonyldichlororuthenium (II) dimer {[Ru(CO)3Cl2]2, Sigma-Aldrich, St. Louis, MO}, known as CORM-2 or CO-releasing molecule, was used as a CO donor and was dissolved in DMSO and added to the superfusing buffer immediately before treating the vessel to achieve a final concentration of 150 or 300 µmol/l. The final concentration of DMSO was 0.5%. Ruthenium chloride (RuCl3, Sigma-Aldrich) was used as a negative control for the CO donor, and DMSO was also added. However, because the CO donor is a dimer, we used 600 µmol/l RuCl3.

CO (Aeriform, Houston, TX), 1 mmol/l CO-saturated stock solution (1 mmol/l), was prepared immediately before an experiment by bubbling the gas in Tyrode solution for 30 min. The CO-saturated solution was used to prepare a 10 µmol/l CO superfusing solution (17).

Chromium mesoporphyrin (CrMP) (Frontier Scientific, Logan, UT), HO inhibitor, 15 mmol/l stock solution in 50 mmol/l Na2CO3, was prepared the day of experiment and added to the superfusing buffer to prepare 15 µmol/l final concentration.

N{omega}-nitro-L-arginine (L-NNA, Sigma-Aldrich) was prepared in 2 N HCl stock solution (1 M) and added to the superfusing buffer (1% albumin in Tyrode’s salt buffer, pH = 7.4), final concentration was 1 mmol/l.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (Sigma-Aldrich), 20 mmol/l stock solution, was prepared in DMSO and stored at –20°C. ODQ was diluted to a final concentration of 10 µmol/l into the superfusing buffer.

Experimental Protocols

Protocol 1: effect of exogenous CO on afferent arteriolar diameter in control kidneys. Afferent arterioles were superfused with vehicle buffer, followed by 150 and 300 µmol/l CORM-2 (CO donor) and by a final recovery period. As a negative control, a separate group was superfused with 600 µmol/l RuCl3 for the same period. An additional protocol was performed in which afferent arterioles were superfused with 10 µmol/l CO solution for 10 min, followed by a final recovery period.

Protocol 2: effect of HO inhibition on afferent arteriolar diameter in control kidneys. Afferent arteriolar diameters were measured before, during, and after exposure to 15 µmol/l CrMP (HO inhibitor) for 10 min. Applying 15 µmol/l CrMP for 10 min has been shown to be an effective dose and time for HO inhibition (4, 17). This protocol was performed to determine the physiological role of HO in regulating afferent arterioles in normal conditions. Separate vehicle experiments were also performed where afferent arterioles were superfused with 50 µmol/l Na2CO3 for 5 min.

Protocol 3: effect of HO inhibition on afferent arteriolar diameter during NOS inhibition with L-NNA. Afferent arterioles were superfused with vehicle buffer followed by 1 mmol/l L-NNA (NOS inhibitor) to block NO synthesis. CrMP (15 µmol/l) was superfused during NOS inhibition. Each treatment period was 5 min. This protocol was performed to determine the effects of HO inhibition during NOS inhibition. Time-control experiments for L-NNA were also performed, where afferent arterioles were superfused only with L-NNA for 10 min.

Protocol 4: afferent arteriolar response to sGC inhibition alone or concurrently with HO inhibition. Afferent arterioles were superfused with vehicle buffer, followed by 10 µmol/l ODQ (sGC inhibitor) for 15 min. CrMP (15 µmol/l) was then superfused for 10 min concurrently with ODQ. This protocol was performed to determine the effect of sGC inhibition on afferent arteriolar diameter and to determine the effect of HO inhibition during sGC blockade on afferent arteriolar diameter.

Protocol 5: effect of exogenous CO on afferent arteriolar diameter during NOS inhibition with L-NNA. Afferent arterioles were superfused with vehicle buffer, followed by 1 mmol/l L-NNA (NOS inhibitor); 300 µmol/l CORM-2 was then superfused during NOS inhibition. Each treatment period was ~9 min. This protocol was designed to determine the effect of exogenous CO administration on the afferent arteriole during inhibition of NO production.

Statistical Analysis

Afferent arteriolar internal diameters were measured using a calibrated shear monitor connected to Biopac system. One measurement was collected every second, and measurements were averaged every minute and used for time-course figures. Average of diameters during the final 2 to 3 min of each treatment period was used to determine the final response. Results are presented as means ± SE for a number (n) of different vessels. Repeated-measures one-way ANOVA followed by Bonferroni's multiple comparison test was used to analyze changes in afferent arteriolar diameter in response to different treatments. Repeated-measures two-way ANOVA followed by unpaired t-test was used to analyze the difference between different groups. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Effect of Exogenous CO on Afferent Arteriolar Diameter

Afferent arterioles were treated for 9 min with two concentrations (150 and 300 µmol/l) of the CO donor [Ru(CO)3Cl2]2 (CORM-2). As shown in Fig. 1A, afferent arterioles responded to treatment with 150 µmol/l CORM-2 with vasodilatation, which achieved significance during the 6th min of treatment (21.4 ± 0.6 vs. 18.3 ± 0.9 µm, n = 5, P < 0.05). The afferent arterioles continued to dilate during treatment with 300 µmol/l CORM-2, with maximal vasodilatation reached 6 min after treatment with 300 µmol/l CORM-2 (23.6 ± 0.9 µm, n = 5). As reported previously (22), the vasodilation was sustained and arteriolar diameters were not restored to control values during the 5-min recovery period. When afferent arterioles were treated with 600 µmol/l RuCl3 as a negative control for CORM-2, there was not a significant increase in arteriolar diameter (Fig. 1A).


Figure 1
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  		Fig. 1. Effect of exogenous CO on afferent arteriole (AA) diameter. A: effects of superfusing with the CO donor CO releasing molecule (CORM-2) or ruthenium chloride (RuCl3) as a negative control on internal AA diameter. A,a: basal AA diameter (vehicle treated). A,b: AA response to treatment with 150 µmol/l CORM-2 for 5 min. A,c: AA response to 300 µmol/l CORM-2. A,d: averaged internal diameters of different AAs in response to treatment with CORM-2 (n = 5) or in response to 600 µmol/l RuCl3 (n = 6). Results are expressed as means ± SE for each group. *P < 0.05 vs. basal diameter. B: effects of superfusing with 10 µmol/l CO solution for 10 min on AA diameter. Results are expressed as means ± SE for each group. *P < 0.05 vs. basal diameter; #P < 0.05 vs. diameter at final minute of CO treatment (n = 5).

 
Superfusing with 10 µmol/l CO solution for 10 min significantly dilated afferent arterioles, which reached an average of 26.0 ± 1.4 µm compared with 18.8 ± 0.7 µm of basal diameter, n = 5, P < 0.05 (Fig. 1B). When the superfusion solution was returned to a CO-free solution, afferent arteriolar diameters rapidly returned back to control values (18.3 ± 1.8 µm, n = 5) (Fig. 1B).

Effect of HO Inhibition on Afferent Arteriolar Diameter

Afferent arterioles were treated 5 to 10 min with 15 µmol/l CrMP to inhibit HO; diameters were averaged every minute. As illustrated in Fig. 2A, inhibition of HO did not alter afferent arteriolar diameter over a period of 10 min. Afferent arteriolar diameters averaged 17.5 ± 0.7 (n = 7) and 17.4 ± 0.9 µm (n = 5) after 5 and 10 min of treatment with CrMP versus 17.2 ± 0.6 (n = 7) µm of basal diameter (Fig. 2A). Another set of experiments showed that afferent arteriolar diameters did not change when superfused with vehicle solution containing 50 µmol/l Na2CO3 for 5 min (18.3 ± 0.4 vs. 18.5 ± 0.4 µm, n = 3, P > 0.05).


Figure 2
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  		Fig. 2. Effect of heme oxygenase (HO) inhibition alone by chromium mesoporphyrin (CrMP) or HO inhibition after nitric oxide synthase inhibition by N{omega}-nitro-L-arginine (L-NNA). A: effects of superfusing with 15 µmol/l CrMP, the HO inhibitor, for 10 min on internal AA diameter. Results are expressed as means ± SE for each group. B: effects of superfusing with 1 mmol/l L-NNA for 5 min, followed by 5 min of concurrent superfusion with 15 µmol/l CrMP, the HO inhibitor on the internal AA diameter (n = 6). Comparison of effects of superfusing with 1 mmol/l L-NNA alone for 10 min are shown (n = 6). Results are expressed as means ± SE for each group. *P < 0.05 vs. basal diameter; #P < 0.05 vs. L-NNA.

 
Effect of HO Inhibition on Afferent Arteriolar Diameter During NOS Inhibition with L-NNA

As illustrated in Fig. 2B, and consistent with previous reports (24), L-NNA significantly reduced afferent arteriolar diameter from 17.3 ± 0.9 to 14.6 ± 1.2 µm, n = 6, P < 0.05. As shown in Fig. 2B, subsequent inhibition of HO with CrMP during NOS inhibition further reduced afferent arteriolar diameter to 11.7 ± 1.6 µm (n = 6), indicating that HO-derived product(s) contribute to the maintenance of afferent arteriolar diameter during NOS inhibition. In arterioles not treated with CrMP, the L-NNA reduction in diameter remained stable.

Afferent Arteriolar Response to sGC Inhibition and Concurrent HO Inhibition

As shown in Fig. 3, inhibition of sGC, by superfusing with 10 µmol/l ODQ, gradually constricted afferent arterioles, and a maximal response was achieved by the 13th min. Afferent arteriolar diameter did not change for the additional 2 min of treatment. After 15 min, afferent arteriolar diameters averaged 15.7 ± 0.4 versus 18.8 ± 0.4 µm basal diameter, n = 6, P < 0.05, demonstrating that basal cGMP production regulates afferent arteriolar tone (Fig. 3). To determine whether HO inhibition during sGC inhibition would cause further constriction, we added 15 µmol/l CrMP to the superfusing buffer for an additional 10 min. HO inhibition for 10 min did not cause any further decreases in diameter (15.5 ± 0.7 µm), demonstrating that inhibition of HO does not alter afferent arteriolar diameter when cGMP production is inhibited (Fig. 3).


Figure 3
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  		Fig. 3. Effects of superfusing with 10 µmol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the soluble guanylate cyclase inhibitor, for 15 min, followed by 10 min of concurrent superfusion with 15 µmol/l CrMP, the HO inhibitor on the internal AA diameter. Results are expressed as means ± SE for each group. *P < 0.05 vs. basal diameter (n = 6).

 
Effect of Exogenous CO on Afferent Arteriolar Diameter During NOS Inhibition with L-NNA

Inhibition of NOS by L-NNA significantly constricted afferent arterioles from an average of 18.8 ± 0.7 to 16.1 ± 0.6 µm, n = 7, P < 0.05 (Fig. 4). When 300 µmol/l CORM-2 was superfused in the presence of 1 mmol/l L-NNA, a dilatory response ensued that reversed the L-NNA-induced vasoconstriction (18.6 ± 0.7 µm, n = 7) (Fig. 4). The dilatory response to CORM-2 in L-NNA-treated arterioles was not significantly different from that in untreated arterioles.


Figure 4
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  		Fig. 4. A: final AA diameter measurements in response to L-NNA alone, L-NNA followed by CrMP or CORM-2, or ODQ followed by CrMP compared with responses to L-NNA alone as a time control. B: results are expressed as percentage of basal diameters and as means ± SE for each group. *P < 0.05 vs. basal; #P < 0.05 vs. treatment 1.

 
As shown in Fig. 4, inhibition of sGC resulted in afferent arteriolar constriction (17.7 ± 2.5%) compared with that achieved by NOS inhibition (14.2 ± 4.3%). In contrast, HO inhibition during NOS inhibition resulted in further vasoconstriction, uncovering a CO-mediated vasodilatory influence when NO production is inhibited. In the presence of an intact NO system, HO inhibition did not alter afferent arteriolar diameter (4.4 ± 2.1%) and HO inhibition during sGC inhibition did not cause further constriction (16.1 ± 2.5%) (Fig. 4), indicating that the effects of HO may be partially mediated by activating sGC but are masked in the presence of NO.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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GRANTS
 REFERENCES
 
The characterization of the metal carbonyl compounds as pharmacologically relevant CO donors (22) enabled us to elicit local release of exogenous CO to the afferent arterioles. [Ru(CO)3Cl2]2, CORM-2, has been shown previously to be an effective CO donor (22) and caused dilation in isolated aortic rings. When used as a pretreatment, CORM-2 significantly suppressed L-NAME-induced pressor responses in rats (22). In addition, pretreatment with CORM-2 1 h before the onset of renal ischemia significantly decreased the levels of plasma creatinine 24 h after reperfusion, suggesting a protective effect (30). Release of CO from [Ru(CO)3Cl2]2 has also been shown to activate the calcium-sensitive potassium (BK) channels expressed in human embryonic kidney cells (HEK293) (34). The present study demonstrates that treating afferent arterioles with CORM-2 resulted in significant vasodilation. We also superfused afferent arterioles with 10 µmol/l CO solution to compare the effects with those produced by CORM-2. CO also elicited marked dilation of afferent arterioles. These results support the conclusion that CO is an afferent arteriolar dilator and explain previous results showing that acute renal administration of Mn2(CO)10, a CO donor known as CORM-1, increased renal blood flow and glomerular filtration rate (5). The observation that afferent arterioles recovered to basal diameters when superfusion with CO was discontinued, although they did not when superfusion with CORM-2 was discontinued, indicates that CORM-2 infiltrated the tissues and may have entered the cells and continued to release CO for a prolonged period. This sustained dilation was also observed in the study by Motterlini et al. (22) when aortic rings previously treated with CORM-2 failed to regain contraction in response to phenylephrine.

Although HO inhibition did not affect afferent arteriolar diameter during control conditions, inhibition of HO during NOS inhibition led to further and significant constriction of afferent arterioles. These data indicate that HO-derived product(s) partially counteract the vasoconstriction resulting from NOS inhibition. These results also indicate the presence of a vasodilatory influence of HO-derived CO, but the endogenously produced CO may not exert that influence as long as NO is plentiful. The influence of CO only becomes unmasked after blockade of NOS. Both NO and CO can bind to the heme center of sGC to increase its activity, resulting in higher levels of cGMP. However, the conformational change in sGC induced by NO results in 100- to 400-fold increase in activity, whereas CO induces a 4- to 6-fold activation of this enzyme (27, 32). Because NO is much more potent than CO in activating sGC, the effects of endogenous CO on sGC may be negligible compared with the effects of NO on sGC. Furthermore, both CO and NO can activate KCa; this stimulatory effect has been shown to rely on the specific interactions of these gases with {alpha}- and beta-subunits of KCa, respectively (35). Interestingly, pretreatment of smooth muscle cells with NO abolished the effects of subsequently applied CO on KCa channels (35), indicating that NO desensitizes KCa channels to CO. In our study, when NOS was inhibited to reduce endogenous NO levels, the influence of endogenous CO to prevent excess vasoconstriction was unmasked, indicating that both NO and CO may work through common pathways that are fully activated in the presence of NO, while only partially activated by CO in the absence of NO. As shown in Fig. 4, inhibition of sGC resulted in afferent arteriolar constriction compared with that achieved by NOS inhibition. HO inhibition during NOS inhibition resulted in a greater overall vasoconstriction, suggesting the presence of an additional sGC-independent mechanism. Thus endogenous CO may provide a reserve renoprotective role to maintain the glomerular circulation under conditions of limited NO production such as occurs during endothelial dysfunction. This explanation is further supported by the finding that inhibition of sGC significantly constricted afferent arterioles and concurrent HO inhibition failed to cause any additional constriction, indicating that during sGC inhibition, the effects of HO are still masked in the presence of NO. The alternative explanation is that treatment with L-NNA stimulates basal HO activity which then leads to augmented CO levels, allowing a greater vasodilatory influence to partially offset the vasoconstriction caused by reduced NO levels. This possibility is supported by the observation that L-NAME-treated rats displayed increased urinary CO concentrations, and renal homogenates from these rats showed higher HO activity compared with that of untreated rats (25).

Previous studies (5, 26) have shown that acute inhibition of HO decreases renal blood flow in normal kidneys, whereas others (13, 23, 38) have demonstrated lack of hemodynamic effects. These contradictory findings may be due to differences in protocols used or in the specificity of HO inhibitors utilized. Our study provides evidence that HO inhibition in normal kidneys does not cause afferent arteriolar constriction. Our results are also in agreement with several other reports (15, 26) showing that the effects of HO inhibition are significantly augmented under conditions where NOS is inhibited and also in agreement with previous reports (3, 5, 23, 26, 38) that have suggested a renal vasodilatory role of endogenous CO. Under normal conditions, renal HO activity is mainly derived from HO-2, which is expressed in various renal segments, whereas HO-1 is barely expressed (6, 8) and renal HO-1 is only induced in response to pathological stimuli (12). Because we used normal kidneys in our experiments, it seems likely that renal HO-2 exerts a major role in regulating afferent arterioles. HO-2–/– mice showed higher plasma creatinine levels that were exacerbated in streptozotocin-treated diabetic mice, suggesting an impairment of renal function in HO-2-deficient mice (11). The present study further supports and explains the beneficial protective effects conferred by HO upregulation in different kidney diseases (12). Renal HO-1 has been shown to be upregulated in angiotensin II-dependent hypertension, and inhibition of HO decreased creatinine clearance and exacerbated the hypertension (3). HO-1 has been shown to be induced in kidneys from rats exposed to chronic hypoxia, and inhibition of HO decreased renal blood flow, suggesting a vasodilatory influence of HO (23). In addition, one-kidney, one-clip (1K1C) HO-1–/– mice displayed an exacerbated renovascular hypertension and acute renal failure compared with 1K1C wild-type mice, indicating that HO-1 may serve to counteract the reduced renal function and sodium retention that occur in this form of renovascular hypertension by promoting renal vasodilation and improving renal function (33). Biliverdin and bilirubin have been shown to be potent antioxidants, and kidneys pretreated with bilirubin have been shown to be protected during ischemia reperfusion injury (2). Although bilirubin may sustain vascular homeostasis by protecting vascular cells from oxidative stress and by inhibiting the adhesion and infiltration of leukocytes into the vessel wall, there is scarce evidence that bilirubin regulates vascular function under normal conditions (9). We cannot exclude that bilirubin may modulate the response to CO. In fact, our preparation is not bilirubin-free because afferent arterioles were perfused with blood collected from normal animals. Thus the effects of HO inhibition cannot be attributed to absence of bilirubin. Our results provide direct evidence of a vasodilatory influence mediated by CO production.

In summary, these results indicate that exogenous CO exerts a substantial vasodilatory effect on renal afferent arterioles. However, in control conditions, inhibition of HO does not alter afferent arteriolar diameters, suggesting minimal influence of endogenous CO in normal kidneys. When NO production is inhibited, HO inhibition causes vasoconstriction, suggesting activation or unmasking of a residual CO vasodilatory renoprotective influence.

These results provide direct evidence supporting a role for HO and CO in the regulation of the renal microcirculation. The presence of an active HO system in the kidney suggests that the HO-CO system may provide a reserve renoprotective role to maintain the integrity of the renal microcirculation under conditions where NO production is compromised by endothelial dysfunction.


   GRANTS
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 ABSTRACT
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 GRANTS
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-18426, Center of Biomedical Research Excellence grant P20-RR-017659 from the Institutional Award Program of the National Center for Research Resources, and Health Excellence Fund grant from the Louisiana Board of Regents. F. T. Botros was supported by a fellowship from the Consortium for Southeastern Hypertension Centers and is currently a fellow of the National Kidney Foundation.


   ACKNOWLEDGMENTS
 
We thank Drs. MingGuo Feng and Lisa Harrison-Bernard for assistance in learning the juxtamedullary nephron preparation and Debbie Olavarrieta for assistance in manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. T. Botros, Dept. of Physiology, Hypertension and Renal Center, Tulane Univ. Health Sciences Center, New Orleans, LA 70112 (e-mail: fbotros{at}tulane.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
 GRANTS
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