Nitric Oxide Synthesis Inhibition Promotes Renal Production of Carbon Monoxide

Drugs and Solutions

Stannous mesoporphyrin (SnMP; Frontier Scientific), a nonselective HO inhibitor,¹⁵ was dissolved in 50-mmol/L Na₂CO₃. All other drugs were from Sigma Chemical Co. S-nitroso-N-acetylpenicillamine (SNAP), a donor of NO,⁶ was dissolved in 0.1-mol/L potassium phosphate buffer pH 7.4; Ferriprotoporphyrin IX chloride (hemin) was dissolved in 0.1-mol/L NaOH and the pH was adjusted to 7.8 with 0.1-mol/L HCl. The NOS inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) was dissolved in tap water (0.72±0.00 and 0.23±0.02 mg/mL in short- and long-term treatment studies, respectively).

Experimental Protocols

Protocols were approved by the Institutional Animal Care and Use Committee. Studies were conducted on male Sprague-Dawley rats (Charles River, Wilmington, Mass; 300 to 325 g body weight) treated and not treated with l-NAME. l-NAME was provided in the drinking water at a daily dose of 74.3±1.0 and 29.8±0.5 mg/kg in short- and long-term treatment studies lasting 2 days and 6 weeks, respectively.

On the last day of treatment, control and experimental animals anesthetized with thiobutabarbital (50 mg/kg IP) and ketamine (30 mg/kg IM) were prepared for assessment of urinary CO. The rats were instrumented with polyethylene cannulas in the trachea (PE-205) to aid ventilation, the left femoral artery (PE-50) for measurement of blood pressure,⁵ the left femoral vein (PE-50) for administration of drugs and fluids (0.15-mol/L NaCl; 2.7 mL/h throughout the study), and the bladder (PE-60) for urine collection. After a 60-minute equilibration interval, 2 or 3 urine samples (≈100-μL each) for CO analysis were collected consecutively. Animals in the short-term l-NAME treatment protocol were subsequently injected with the HO inhibitor SnMP (40 μmol/kg IV) and, after a 45-minute interval, urine specimens for CO analysis were again collected as described above. In other animals not instrumented for urine collection, the kidneys were excised and frozen in liquid N₂ for later assessment of HO isoform protein expression and CO-generating activity.

Assessment of HO Isoform Expression

Kidneys were homogenized in ice-cold 50-mmol/L Tris-HCl buffer, pH 7.4, containing 1% NP-40, 0.25% sodium deoxycholate, 1-mmol/L EDTA, and 10% protease inhibitor cocktail (Sigma Chemical Co). Homogenates were centrifuged (10 000g for 30 minutes) and the supernatant was saved for protein assay and Western blot analysis in triplicate of HO-1, HO-2, and β-actin according to published procedures¹⁶ utilizing antibodies provided by Stressgen Biotechnologies. Immunocomplexed bands were visualized and quantified by densitometric analysis. Data are expressed as the HO isoform/β-actin ratio.

Assessment of CO-Generating Activity

Kidneys were homogenized in ice-cold 0.1-mol/L potassium phosphate buffer, pH 7.4, containing 1.37-mol/L NaCl, 0.027-mol/L KCl, 0.1-mmol/L butylated hydroxytoluene, and 10% protease inhibitor cocktail (Sigma Chemical Co). Subsequently, the homogenates were centrifuged (10 000 for 15 minutes at 4°C), the protein content of the supernatant was assayed, and an aliquot (10 to 20 μL containing ≈300-μg protein) was reacted with 40-μmol/L heme in the presence and absence of a NADPH-generating system consisting of 10-mmol/L MgCl₂, 6.8-mmol/L glucose-6-phosphate, 3.3-U/mL glucose-6-phosphate dehydrogenase, and 2.6-mmol/L NADPH. When so noted, SNAP (0.01 to 1.0 mmol/L) was included in the incubation mixture to test the effect of NO on the CO-generating activity of homogenates prepared from kidneys of l-NAME-treated rats.

The reaction mixture (1 mL final volume) was incubated for 1 hour at 37°C in 2-mL amber vials capped with rubberized Teflon liners. Subsequently, the vials were placed on ice, an internal standard made of isotopically-labeled CO (¹³C¹⁸O, Sigma Chemical Co.) was injected into the vials, and the CO content of the headspace was determined by gas chromatography/mass spectroscopy, as previously described.³ CO-generating activity is expressed as pmol/mg protein/h. Values obtained in the absence of NADPH (NADPH-independent CO generation) were subtracted from values in the presence of NADPH (total CO generation); the resulting values represent the NADPH-dependent generation of CO, which is an index of HO activity.¹⁷ In contrast, NADPH-independent generation of CO is considered not to involve HO-1 or HO-2, as these enzymes require NADPH for expression of catalytic activity.^1,17 This is consistent with the result of experiments showing that the HO inhibitor chromium mesoporphyrin (50 μmol/L) reduces by 84.8±7.4% (n=4; P<0.05) the NADPH-dependent generation of CO by renal homogenates, without affecting the NADPH-independent generation of the gas.

Analysis of CO in Urine

CO was measured by gas chromatography-mass spectroscopy as previously described,³ in urine specimens collected into amber vials (2 mL), capped with rubberized Teflon liners perforated with one G-23 and one G-30 needle which, respectively, allow the specimens to flow into the vials under isobaric conditions.¹⁸ Immediately after completion of sample collection, the internal standard was added, the needles were removed, the perforations were sealed, and the samples were analyzed.

Data Analysis

Results are expressed as means±SEM. Data on urinary CO concentration and excretion are the average of 2 or 3 consecutive collection periods. Results were analyzed as appropriate by Student t test for unpaired observations or by a 2-way ANOVA followed by the Newman-Keuls post hoc test. The null hypothesis was rejected at P<0.05.

Effects of Short-Term l-NAME Treatment on the Renal Heme-HO System

The mean arterial pressure of rats treated with l-NAME for 2 days exceeded that of untreated rats (140±3 versus 106±3 mm Hg, P<0.05). Shown in Figure 1, untreated (n=3) and l-NAME-treated (n=3) rats did not differ significantly from each other in terms of renal expression of HO-1 and HO-2 protein, relative to β-actin expression. Also shown in Figure 1, untreated (n=5) and l-NAME-treated (n=5) rats did not differ in terms of CO-generating activity of renal homogenates incubated with heme in the absence of NADPH. However, the CO-generating activity of renal homogenates incubated with heme in the presence of NADPH was 2.5-fold higher (P<0.05) in samples derived from l-NAME-treated rats than from the untreated controls. Consequently, the NADPH-dependent component of CO-generating activity, a measure of HO activity, was increased (P<0.05) 2.7-fold in kidneys from rats treated with the NOS inhibitor relative to levels in control kidneys. Inclusion of the NO donor SNAP into incubation mixtures of heme and homogenates of kidney from l-NAME-treated animals (n=4) decreased (P<0.05) NADPH-dependent, but not NADPH-independent, generation of CO over the concentration range 100 to 1000 μmol/L (Figure 2). Thus, NO inhibits HO-catalyzed generation of CO by renal homogenates.

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Figure 1. A, Assessment of HO-1 and -2 expression by immunoblotting, and B, of CO-generating activity, in kidneys of untreated rats and rats treated with l-NAME for 2 days. In A, each blot is representative of determinations conducted in triplicate in different kidneys; numbers under the blots indicate the ratio between the density of HO isoforms and β-actin bands. In B, ▵ indicates the difference between CO-generating activity in the absence and presence of NADPH; this value is a measure of HO activity. Results are mean±SEM. *P<0.05 relative to values of CO generation in the absence of NADPH. †P<0.05 relative to corresponding data in untreated rats.

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Figure 2. Effect of SNAP on CO-generating activity of renal homogenates derived from rats treated with l-NAME for 2 days. CO-generating activity was assayed in the absence and presence of NADPH. ▵ indicates the difference between CO-generating activity in the absence and presence of NADPH. Results are mean±SEM. *P<0.05 relative to values of CO generation in the absence of NADPH. †P<0.05 relative to control data in the absence of SNAP.

Figure 3 displays data on urinary CO concentration and excretion in untreated (n=5) and l-NAME treated (n=7) rats before and after administration of SnMP. Before treatment with SnMP, values of urinary CO concentration and excretion rate in untreated rats were exceeded (P<0.05) by corresponding values in rats treated with l-NAME for 2 days. The administration of the HO inhibitor SnMP acutely decreased (P<0.05) urinary CO concentration and excretion rate in rats with and without l-NAME treatment. Mean arterial pressure was not affected significantly by SnMP administration to untreated (106±3 versus 100±2 mm Hg) or l-NAME-treated rats (140±3 versus 133±3 mm Hg). Urine volume was similarly unaffected consistently by SnMP in untreated (8.8±4.0 versus 28.4±14.2 μL/min, P=0.2795) or l-NAME treated rats (7.7±1.8 versus 13.5±4.4 μL/min, P=0.2568).

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Figure 3. Urinary CO concentration and excretion rate, before and after treatment with stannous mesoporphyrin (SnMP, 40 μmol/kg IV), in untreated rats and rats treated with l-NAME for 2 days. Results are mean±SEM. *P<0.05 relative to data obtained before SnMP treatment. †P<0.05 relative to corresponding data in untreated rats.

Effects of Long-Term l-NAME Treatment on the Renal Heme-HO System

The mean arterial pressure of rats treated with l-NAME for 6 weeks (n=5) exceeded that of untreated rats (n=5, 144±5 versus 111±4 mm Hg, P<0.05). As shown in Figure 4, HO-1 and HO-2 proteins were similarly expressed in kidneys of untreated and l-NAME treated animals. In contrast, the values of urinary CO concentration and excretion in rats treated with the NOS inhibitor were clearly elevated (P<0.05) relative to corresponding values in untreated rats. Hence, renal HO isoform expression and urinary CO were similarly affected by short- and long-term treatment with l-NAME.

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Figure 4. A, Assessment of HO-1 and -2 expression by immunobloting, and B, of urinary CO concentration and excretion, in untreated rats and rats treated with l-NAME for 6 weeks. In A, numbers under the blots indicate the ratio between the density of HO isoforms and β-actin bands. Data are mean±SEM. †P<0.05 relative to corresponding data in untreated rats.

Perspectives

Previous reports established that NOS inhibition enhances the functional relevance of the heme-HO system, facilitating CO-induced vasodilation and intensifying the vasoconstriction that accompanies inhibition of HO.^2,5,13,14 Such observations, in conjunction with the present finding that NO inhibition upregulates renal CO generation, are compelling reasons to consider the heme-HO system a potential player in the homeostatic response to disorders that feature deficient synthesis or bioavailability of NO. Indeed, there is evidence that the renal heme-HO system subserves a renoprotective role in angiotensin-dependent models of hypertension in which NO bioavailability is believed to be diminished.^22,23