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Am J Physiol Heart Circ Physiol 293: H333-H342, 2007. First published March 9, 2007; doi:10.1152/ajpheart.00870.2006
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An analysis of the DOCA-salt model of hypertension in HO-1–/– mice and the Gunn rat

Karl A. Nath,1 Livius V. d'Uscio,2,3 Julio P. Juncos,1 Anthony J. Croatt,1 Melissa C. Manriquez,1 Siobhan T. Pittock,4 and Zvonimir S. Katusic2,3

1Division of Nephrology and Hypertension and 2Departments of Anesthesiology, 3Molecular Pharmacology and Experimental Therapeutics, and 4Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 14 August 2006 ; accepted in final form 6 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heme oxygenase-1 (HO-1) is induced in the vasculature in the DOCA-salt model of hypertension in rats. Whereas the HO system and its products may exert vasodilator effects, recent studies have suggested that the HO system may predispose to hypertension. The present study examined the effects of selected components of the HO system, specifically, the HO-1 isozyme and the product bilirubin in the DOCA-salt model of systemic hypertension; the experimental approach employed mutant rodent models, namely, the HO-1–/– mouse and the hyperbilirubinemic Gunn rat. DOCA-salt induced HO-1 protein in the aorta in HO-1+/+ mice and provoked a significant rise in systolic arterial pressure in HO-1–/– mice but not in HO-1+/+ mice; this effect could not be ascribed to impaired urinary sodium excretion or impaired glomerular filtration rate in the DOCA-salt-treated HO-1–/– mice. The administration of DOCA salt to uninephrectomized rats significantly increased systolic arterial pressure in wild-type rats, an effect that was attenuated in the mutant Gunn rat; this reduction in systemic hypertension in the DOCA-salt-treated Gunn rat was not due to a greater induction of HO-1 in the vasculature or to a more avid urinary sodium excretion. DOCA-salt impaired endothelium-dependent and endothelium-independent vasorelaxation in wild-type rats but not in Gunn rats; prior exposure to bilirubin repaired the defect in endothelium-dependent vasorelaxation in aortic rings in DOCA-salt-treated rats. DOCA salt stimulated vascular production of superoxide anion in wild-type but not in Gunn rats. We suggest that HO-1 and the product bilirubin may exert a countervailing effect in the DOCA-salt model of systemic hypertension.

superoxide anion; bilirubin; vascular reactivity; oxidative stress

HEME OXYGENASE (HO) is the rate-limiting enzyme in the degradation of heme, enabling the conversion of the tetrapyrrole ring to biliverdin, with the attendant release of iron and the generation of carbon monoxide (CO); biliverdin is subsequently converted to bilirubin by biliverdin reductase (1–3, 45, 62). HO-1 is the inducible isoform upregulated by diverse forms of stress, including systemic hypertension, whereas HO-2 represents the constitutive isoform. Both HO-1 and HO-2 can contribute to vasodepressor responses through actions of their products (1–3, 45, 49, 73). For example, CO may increase guanylate cyclase activity, stimulate calcium-activated potassium channel activity, and suppress the synthesis of endothelin, all of which may promote vasodilatation (2, 32, 41, 44, 49, 72, 73); additionally, by inhibiting the heme prosthetic group, CO may compromise the ability of the cytochrome P-450 system to generate vasoconstrictors such as 20-HETE (31, 73). Bile pigments scavenge oxidants (22, 37, 63), thereby interrupting this pathway for vasoconstriction. Indeed, in certain models of hypertension, there is evidence attesting to the induction of HO-1 as a countervailing, vasodilator response (1, 2, 7, 29, 49, 70, 71, 73).

Vasodepressor effects of the HO system and its products, however, have not been uniformly observed (23–28, 64). For example, in the Dahl-salt-dependent model of systemic hypertension, induction of HO-1 occurs in the vasculature and is accompanied by endothelial dysfunction, the latter reflected by impaired vascular responses to acetylcholine and N{omega}-nitro- L-arginine methyl ester; such dysfunction is prevented by a metalloporphyrin inhibitor of HO activity (25, 64). In the obese Zucker rat, vascular responses of arterioles are impaired, and whereas HO-1 is not induced in the vasculature, metalloporphyrin inhibitors of HO activity prevent such impairment; additionally, systemic arterial pressure is elevated in the obese Zucker rat, and the administration of metalloporphyrin inhibitors reduces systemic hypertension in this model (28). In the deoxycorticosterone acetate (DOCA)-salt model in the rat, HO-1 is induced in the vasculature, and arterioles in this model of systemic hypertension demonstrate impaired vasodilatory responses to the endothelium-dependent vasodilator, acetylcholine; acute exposure to chromium mesoporphyrin, a compound that effectively inhibits HO activity, enables arterioles to regain a normal vasodilatory response to acetylcholine (26). In studies undertaken in isolated arterioles, CO can directly induce endothelium-dependent vasoconstriction (27). These findings have led to the view that the HO system, possibly via the HO product CO, may contribute to endothelial dysfunction in the DOCA-salt and other models and thereby predispose to systemic hypertension in such conditions.

Metalloporphyrin inhibitors of HO activity may exert effects that are not restricted to the inhibition of HO activity. These compounds may influence other vasoactive systems, such as nitric oxide, guanylate cyclase, and atrial natriuretic peptide (19, 60); metalloporphyrins can also undergo photosensitizing reactions that generate oxidants (12, 66), and, in this regard, the oxidant hydrogen peroxide, under certain conditions, may be a potent vasodilator (61). Additionally, metalloporphyrin inhibitors can inhibit proteins, such as caspases, which influence tissue survival (6). Finally, some members of the metalloporphyrin class of compounds, such as tin protoporphyrin, although clearly effective inhibitors of HO activity, can promote reciprocal induction of the HO-1 gene and protein (57); it is quite possible that the HO-1 gene and protein, independent of HO activity, may exert cellular effects. Thus, although selected metalloporphyrins are very effective inhibitors of HO activity, it is possible that these compounds may exert vascular and other effects not necessarily reflecting the inhibition of HO activity.

The present studies undertook an examination of the vasoactive effects of selected and relevant components of the HO system by using mutant rodent models and thereby obviating the need for the administration of metalloporphyrins; these specific components included the HO-1 isozyme and a product of the HO system, namely, bilirubin. We selected the DOCA-salt model since this model is a well-established, clinically relevant model of systemic hypertension and is associated with the upregulation of HO-1 (26). This model can be readily applied to mutant rodent models, and, finally, the mechanisms that contribute to systemic hypertension in this model are relatively well defined. The present studies employed the homozygous null mutant HO-1 mouse as a way of examining the effect of genetic deficiency of HO-1 (33, 46, 48, 54, 55, 70), hypothesizing that if HO-1 exerts vasorelaxant effects, a deficiency of HO-1 would predispose to an increase in systolic blood pressure. The present studies also examined the effect of a product of the HO system, bilirubin, by utilizing the hyperbilirubinemic Gunn rat (53), hypothesizing that if bilirubin promotes vasorelaxant effects, this mutant rat strain would evince less systemic hypertension in this model.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All studies were approved by our Institution Animal Care and Use Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Studies in HO-1+/+ and HO-1–/– Mice

Homozygous HO-1 null mutant mice, employed in this and in prior studies by this laboratory (46, 48, 54), were generated by targeted disruption of the HO-1 gene (55). Colonies of mice were maintained by breeding HO-1–/– males with HO-1+/– females. Offspring were genotyped at the time of weaning by using polymerase chain reaction to amplify the wild-type and mutant alleles of genomic DNA from tail samples. HO-1+/+ mice were used as controls. HO-1+/+ and HO-1–/– mice were age matched with the mean ages of both groups (~12 wk), and groups comprised equal numbers of male and female mice. The DOCA-salt model was based on prior studies of this model in mice (56). HO-1+/+ and HO-1–/– mice were injected weekly with DOCA in olive oil [6 mg subcutaneous (sc)] and received 0.9% saline in the drinking water; additional groups of HO-1+/+ and HO-1–/– mice were injected with vehicle (olive oil, sc) and received tap water. In applying the DOCA-salt model to HO-1+/+ and HO-1–/– mice, uninephrectomy was not performed. Mean body weights of HO-1+/+ mice subjected to vehicle or DOCA salt and HO-1–/– mice subjected to vehicle or DOCA salt were not significantly different among these four groups when compared at the start or at the conclusion of the study. Systolic arterial pressures were obtained by tail-cuff plethysmography, as described in our prior studies using transgenic sickle mice (47). The determination of water intake and urine output over 24 h in awake mice was undertaken in metabolic banks; urine sodium concentration in this and in other protocols was determined by flame photometry.

Clearance studies for the assessment of renal function in DOCA-salt-treated HO-1+/+ and HO-1–/– mice. These studies were performed as previously described (30). Mice were anesthetized with pentobarbital sodium (60 mg/kg body wt ip) and placed on a temperature-regulated table to maintain body temperature at 37°C. A tracheostomy was performed using polyethylene (PE)-160 tubing, and PE-10 tubing was used to cannulate the carotid artery for measurements of mean arterial pressure, the jugular vein for infusion of fluids, and the urinary bladder for the collection of urinary samples. Mean arterial pressure was recorded throughout the experiment. To maintain hydration during the clearance studies, a solution of 0.9% saline containing 2.25% bovine serum albumin was infused at a rate of 0.25 µl·min–1·g–1 body wt throughout the experiment. After the surgical preparation, mice were allowed to recover for 40 min, after which urine was collected for 30 min for the determination of urinary sodium excretion. Following the collection of urine, the infusion fluid was switched to a solution of 0.9% saline containing 2.25% bovine serum albumin with the addition of 0.75% FITC-inulin (FITC-I, Sigma-Aldrich, St. Louis, MO) (42). One hour after beginning the infusion of FITC-I, urine was collected for 30 min, at the end of which a blood sample was taken. Glomerular filtration rate (GFR) was determined by the clearance of FITC-I. FITC-I concentrations in urine and blood were measured using a fluorescence reader (FL 600, BIO-TEK, Winooski, VT).

Studies in Wild-Type and Gunn Rats

Male Gunn and control wild-type Wistar rats were obtained from Harlan (Indianapolis, IN), and all experiments were conducted on rats ~8 wk old. Rats were fed standard rat chow and given tap water ad libitum until the induction of the DOCA-salt model. As shown in our previous study, hyperbilirubinemia was confirmed in the Gunn rat from spectrophotometric measurements of plasma bilirubin, with approximate values of 5 and 118 µM in wild-type and Gunn rats, respectively (53).

To induce the DOCA-salt model in rats, uninephrectomized wild-type or Gunn rats were injected twice weekly with DOCA in olive oil (25 mg/kg body wt sc) and received 0.9% saline in the drinking water; additional groups of wild-type or Gunn rats with two intact kidneys were injected with vehicle (olive oil, sc) and received tap water (21, 26). DOCA salt was thus administered to wild-type and Gunn rats that underwent uninephrectomy, whereas vehicle (olive oil) was administered to wild-type and Gunn rats that underwent sham-operated nephrectomy. Uninephrectomy or sham-operated nephrectomy was performed under anesthesia induced by ketamine (90 mg/kg body wt) and xylazine (10 mg/kg body wt). Systolic arterial pressures were obtained by tail-cuff plethysmography, as described in our prior studies of angiotensin II-induced (21, 53) and DOCA-salt-induced (21) systemic hypertension in rats. Urinary protein concentration was determined by the Coomassie method (21).

Studies of vasomotor function in rats. This was performed by methods described in detail in our prior publications (14, 15, 47, 53). Briefly, isolated aortic rings (4 mm length) were connected to a force transducer for recording of isometric force and placed in an organ bath filled with 25 ml modified Krebs-Ringer solution containing (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 calcium EDTA, and 11.1 glucose (pH 7.4), maintained at 37°C and bubbled with a gaseous mixture of 94% O2-6% CO2. Aortic rings were stretched progressively to their optimal tension (2.5 g) in response to 80 mM KCl. Endothelium-dependent relaxations in response to acetylcholine (10–9–10–5 M) and endothelium-independent relaxations in response to the nitric oxide donor diethylammonium(z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate; 10–9–10–5 M) were cumulatively obtained during submaximal contractions to phenylephrine.

Quantitative analysis of vascular production of superoxide anion. Intracellular superoxide anion production in intact arteries was quantified using the recently described HPLC/fluorescence assay that employs dihydroethidium as a probe (74). A stable fluorescent product 2-hydroxyethidium is formed from the reaction between dihydroethidium and superoxide anion. We also employed the superoxide dismutase mimetic manganese (III) tetra(4-benzoic acid)porphyrin chloride (MnTBAP) and defined and determined the production of superoxide anion as the tissue content of 2-hydroxyethidium in vascular tissue that was MnTBAP inhibitable. Briefly, aortic rings were isolated from rats and were dissected free from connective tissue in cold (4°C) modified Krebs-HEPES buffer (pH 7.4) containing (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA, 11.1 glucose, and 20 HEPES. Segments of arteries (15 mm) were incubated in Krebs-HEPES buffer in the absence or presence of MnTBAP (10–5 M) for 15 min, after which dihydroethidium (50 µM, Molecular Probes) was added, and all samples were incubated for an additional 15 min at 37°C. The arteries were washed of dihydroethidium and incubated in Krebs-HEPES buffer for an additional 1 h. The arteries were then homogenized in 4°C cold methanol and centrifuged at 10,000 rpm to remove debris. The supernatant was analyzed by HPLC/fluorescence (Beckman Coulter) in 37% acetonitrile in 0.1% trifluoroacetic acid aqueous solution. Data were quantified using as a standard, the product, 2-hydroxyethidium, generated from the reaction between dihydroethidium and Fremy's salt as described (75), and data were normalized for tissue protein content.

Western Blot Analysis

Western analysis was performed on soluble protein extracts from mouse aorta and kidney microsomal fractions for the expression of HO-1 protein and is described in detail in our prior studies (35, 54). Antibodies to HO-1 (catalog no. SPA-895, StressGen, Ann Arbor, MI) and beta-actin (catalog no. 612657, BD Transduction, San Diego, CA) were employed as primary antibodies followed by goat anti-mouse or anti-rabbit IgG secondary antibodies as appropriate. Visualization was achieved using a chemiluminescence method (Amersham Pharmacia, Piscataway, NJ). Equal protein loading was confirmed by immunoblotting for beta-actin for analysis of vascular extracts, and Ponceau staining was used for kidney microsomal preparations.

Statistical Analysis

All values are expressed as means ± SE. ANOVA with Bonferroni's test was used for analyses involving more than two groups, whereas the Student's t-test was used for analyses involving two groups. In studies of vascular reactivity, concentration-response curves of the different groups were compared by ANOVA for repeated measurements followed by Bonferroni's correction. Single values were compared with one-way ANOVA with Bonferroni's correction for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Studies in HO-1+/+ and HO-1–/– Mice

Before the administration of DOCA salt, HO-1+/+ and HO-1–/– mice exhibited quite comparable systolic arterial pressure [117 ± 2 vs. 118 ± 3 mmHg, P = not significant (NS), n = 13 in each group]. Over the course of study, systolic arterial pressure did not increase either in the vehicle-treated and DOCA-salt-treated HO-1+/+ mice or in the vehicle-treated HO-1–/– mice and was not significantly different among these groups. However, by 3 wk after the initiation of DOCA salt, systolic arterial pressure rose in DOCA-salt-treated HO-1–/– mice, which, by 4 wk, was significantly greater than that observed in the DOCA-salt-treated HO-1+/+ mice and, by 5 and 6 wk, was significantly greater than in all other groups (Fig. 1). Thus HO-1+/+ mice accommodate DOCA salt, as administered in the currently employed protocol, without any significant alteration in systolic arterial pressure, whereas HO-1–/– mice exhibit a sustained and significant rise in systolic arterial pressure, which approximates some 20 mmHg. In these studies, we demonstrated by Western blot analysis that the administration of DOCA salt to HO-1+/+ mice induced HO-1 protein in the aorta, whereas neither the vehicle-treated nor the DOCA-salt-treated HO-1–/– mice expressed HO-1 in the aorta (Fig. 2).


Figure 1
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  		Fig. 1. Serial changes in systolic arterial pressure in vehicle (Veh)-treated heme oxygenase (HO)-1+/+ (n = 5) and DOCA-salt-treated HO-1+/+ (n = 8) mice and vehicle-treated HO-1–/– (n = 5) and DOCA-salt-treated HO-1–/– (n = 8) mice after 4, 5, 6 wk of administration of DOCA salt or vehicle. *P < 0.05 vs. DOCA-salt-treated HO-1+/+ mice; {dagger}P < 0.05 vs. all other groups.

 

Figure 2
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  		Fig. 2. Western blot analysis for the expression of HO-1 in the aorta in vehicle-treated HO-1+/+ and DOCA-salt-treated HO-1+/+ mice and vehicle-treated HO-1–/– and DOCA-salt-treated HO-1–/– mice. Equivalency of protein loading was verified by immunoblotting for beta-actin.

 
To determine whether the higher systolic arterial pressure in HO-1–/– mice compared with HO-1+/+ mice in response to DOCA salt was due to an inability to excrete salt or impaired renal function, two additional protocols were conducted in DOCA-salt-treated HO-1+/+ and HO-1–/– mice; collectively, these protocols evaluated water consumption, urine output, urinary sodium excretion, GFR, and proteinuria. These studies were undertaken 2 to 3 wk after the initiation of DOCA salt, a time point selected and based on the premise that if increased salt retention was the cause for the increase in systolic blood pressure in HO-1–/– mice, such differences would be more likely evinced during the phase just preceding the onset of differences in systolic blood pressure as observed by tail-cuff measurement. In studies undertaken over 24 h in awake mice in metabolic banks, there were no significant differences between DOCA-salt-treated HO-1+/+ mice and DOCA-salt-treated HO-1–/– mice either for water consumption (6.0 ± 1.7 vs. 10.3 ± 1.7 ml/24 h, P = NS, n = 4 in each group), urinary flow rate (3.0 ± 0.9 vs. 3.9 ± 1.1 ml/24 h, P = NS, n = 4 in each group), or urinary sodium excretion (0.8 ± 0.3 vs. 1.4 ± 0.4 meq/24 h, P = NS, n = 4 in each group). Renal clearance studies under general anesthesia were also performed in separate groups of HO-1+/+ mice and HO-1–/– mice treated with DOCA salt and in which mean arterial pressure, GFR, proteinuria, and urinary sodium excretion were determined. Clearance studies afford the determination of the best overall index of renal function, namely, GFR, as measured by the clearance of inulin. These studies are summarized in Table 1. As demonstrated, 2 to 3 wk after the initiation of DOCA salt, a time point at which differences in blood pressure by tail-cuff measurement are not discernible in HO-1+/+ and HO-1–/– mice, there is already a significant increase in mean arterial pressure in HO-1–/– mice subjected to DOCA salt. Importantly, GFR, as determined by clearance of inulin, was not different between these two groups. With the use of an additional marker of renal function, namely, urinary protein excretion determined either by an absolute urinary protein excretory rate or urinary protein excretion indexed for urinary creatinine excretion, renal function was again comparably matched in HO-1+/+ and HO-1–/– mice subjected to DOCA salt. Additionally, in these clearance studies under general anesthesia, the urinary sodium excretory rate was significantly increased in the HO-1–/– mice compared with the HO-1+/+ mice subjected to DOCA salt (0.6 ± 0.2 vs. 2.3 ± 0.7 meq/24 h, P < 0.05). From these studies, it is concluded that the elevation in mean arterial pressure cannot be simply ascribed to an impairment in kidney function or an impairment in urinary excretion of sodium in HO-1–/– mice.


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  		Table 1. Kidney function as assessed by clearance studies performed under anesthesia in DOCA-salt-treated HO-1+/+ and HO-1–/– mice

 
The expression of HO-1 in the kidney in HO-1+/+ and HO-1–/– mice subjected to DOCA salt or vehicle was also evaluated; these studies were undertaken in the kidneys obtained from HO-1+/+ and HO-1–/– mice subjected to DOCA salt or vehicle and in which weekly measurements of blood pressure were performed. As shown in Fig. 3, HO-1 is not induced in the kidney in HO-1+/+ mice (unlike the induction seen in the aorta as shown in Fig. 2), and neither the vehicle-treated HO-1–/– mice nor the DOCA-salt-treated HO-1–/– mice evinced expression of HO-1.


Figure 3
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  		Fig. 3. Western blot analysis for the expression of HO-1 in microsomes prepared from the kidney in vehicle-treated HO-1+/+ and DOCA-salt-treated HO-1+/+ mice and vehicle-treated HO-1–/– and DOCA-salt-treated HO-1–/– mice. Equivalency of protein loading was verified by Ponceau staining (data not shown).

 
Studies in the Mutant Hyperbilirubinemic Gunn Rat

We utilized the hyperbilirubinemic Gunn rat to determine whether a product of the HO system, specifically, bilirubin, can confer vasodepressor effects. Systolic arterial pressure determined in wild-type and Gunn rats before the administration of DOCA salt revealed quite comparable and not significantly different values (136 ± 3 vs. 135 ± 4 mmHg, P = NS, n = 15 and 13, respectively). One week after the initiation of DOCA salt, systolic arterial pressure was significantly increased in the DOCA-salt-treated wild-type rat, and there was a tendency for a lower systolic arterial pressure in the DOCA-salt-treated Gunn rats (Fig. 4). By the second and third week after the imposition of DOCA salt, systolic arterial pressure was significantly lower in DOCA-salt-treated Gunn rats compared with DOCA-salt-treated wild-type rats (Fig. 4). Western blot analysis of the aorta demonstrated that DOCA salt induced HO-1 protein in the aorta in wild-type and Gunn rats, the latter exhibiting more variable induction (Fig. 5); thus, the milder elevation in systolic arterial pressure in the Gunn rat following the imposition of DOCA salt cannot be ascribed to a greater induction of HO-1 in the vasculature in the Gunn rat.


Figure 4
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  		Fig. 4. Serial changes in systolic arterial pressure in vehicle-treated wild-type (WT Veh, n = 6) and DOCA-salt-treated WT (WT DOCA, n = 9) rats and vehicle-treated Gunn (Gunn Veh, n = 5) and DOCA-salt-treated Gunn (Gunn DOCA, n = 8) rats after 1, 2, and 3 wk of administration of DOCA salt or vehicle. #P < 0.05 vs. vehicle-treated WT and vehicle-treated Gunn rats; *P < 0.05 vs. DOCA-salt-treated WT rats.

 

Figure 5
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  		Fig. 5. Western blot analysis for the expression of HO-1 in the aorta in vehicle-treated WT and DOCA-salt-treated WT rats and vehicle-treated Gunn and DOCA-salt-treated Gunn rats. Equivalency of protein loading was verified by immunoblotting for beta-actin.

 
Prior studies demonstrate that the Gunn rat can exhibit a urinary concentrating defect (43), and when subjected to fasting and water deprivation, the Gunn rat may also exhibit increased urinary excretion of sodium (50). Studies were thus undertaken to determine whether the reduced severity of systemic hypertension in the Gunn rat upon the imposition of DOCA salt reflected more avid salt excretion; urinary sodium excretion was determined at 1 and 2 wk after initiation of DOCA salt in wild-type and Gunn rats. As demonstrated in Fig. 6, urinary sodium excretion was markedly increased in both groups, and at each time point, there were no significant differences in urinary sodium excretion in the DOCA-salt-treated wild-type and Gunn rats.


Figure 6
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  		Fig. 6. Urinary sodium excretion in vehicle-treated WT (n = 6) and DOCA-salt-treated WT (n = 9) rats and vehicle-treated Gunn (n = 5) and DOCA-salt-treated Gunn (n = 8) rats after 1 and 2 wk of administration of DOCA salt or vehicle. *P < 0.05 vs. respective vehicle-treated group at that time point. There were no significant differences between DOCA-salt-treated WT and DOCA-salt-treated Gunn rats at either time point.

 
Since the DOCA-salt model increases urinary protein excretion and since the latter is an index of renal injury that reflects, at least in part, the severity of systemic hypertension in this model, urinary protein excretory rates were measured. Urinary protein excretory rates determined in wild-type and Gunn rats before the administration of DOCA salt revealed quite comparable and not significantly different values (5 ± 1 vs. 4 ± 1 mg/24 h, P = NS, n = 15 and 13 in each group, respectively); thus, under basal, unstressed conditions, and before the imposition of DOCA salt or a surgical procedure, wild-type and Gunn rats, and at this age, exhibit comparable rates for urinary protein excretion. As shown in Fig. 7, DOCA salt induced a significant rise in urinary protein excretion at 2 wk, and this was blunted in the DOCA-salt-treated Gunn rat compared with the DOCA-salt-treated wild-type rat. Assessment of renal expression of HO-1 demonstrated that DOCA salt induced, modestly and variably, HO-1 in the kidney in wild-type and Gunn rats (Fig. 8); thus, the reduced urinary protein excretion in Gunn rats compared with wild-type rats following the imposition of DOCA salt cannot be ascribed to a greater induction of HO-1 in the kidney.


Figure 7
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  		Fig. 7. Serial changes in urinary protein excretory rates (in mg/24 h) in vehicle-treated WT (n = 6) and DOCA-salt-treated WT (n = 9) rats and vehicle-treated Gunn (n = 5) and DOCA-salt-treated Gunn (n = 8) rats after 1 and 2 wk of administration of DOCA salt or vehicle. #P < 0.05 vs. vehicle-treated WT rats; *P < 0.05 vs. DOCA-salt-treated WT rats; {dagger}P < 0.05 vs. vehicle-treated WT and vehicle-treated Gunn rats.

 

Figure 8
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  		Fig. 8. Western blot analysis for the expression of HO-1 in microsomes prepared from the kidney in vehicle-treated WT and DOCA-salt-treated WT rats and vehicle-treated Gunn and DOCA-salt-treated Gunn rats. Equivalency of protein loading was verified by Ponceau staining (data not shown).

 
We next determined whether the Gunn rat was also resistant to the derangements in vascular reactivity that are known to occur in the DOCA-salt model. Endothelium-dependent relaxation was impaired in the DOCA-salt-treated wild-type rat compared with the vehicle-treated wild-type rat (Fig. 9). This impairment in endothelium-dependent relaxation was completely prevented in the Gunn rat treated with DOCA salt (Fig. 9); as shown by the calculated EC50 values (Table 2), the sensitivity to acetylcholine was decreased in the DOCA-salt-treated wild-type rat, and this decrease was prevented in the DOCA-salt-treated Gunn rat. Additionally, endothelium-independent relaxation was also impaired in the DOCA-salt-treated wild-type rat compared with the vehicle-treated wild-type rat, and such impairment was attenuated in the DOCA-salt-treated Gunn rat (Fig. 10); as shown by the calculated EC50 values (Table 2), the sensitivity to DEA-NONOate was decreased in the DOCA-salt-treated wild-type rat, and this decrease was prevented in the DOCA-salt-treated Gunn rat.


Figure 9
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  		Fig. 9. Concentration-response curves to acetylcholine in rat aorta. Data are shown as means ± SE (n = 6–10 rats) and expressed as percent relaxation of the contraction to phenylephrine. *Endothelium-dependent relaxation in the DOCA-salt-treated WT rat is significantly impaired compared with that in all other groups. See Table 2 for comparisons of EC50 values.

 

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  		Table 2. Sensitivity to acetylcholine and DEA-NONOate in rat aorta in studies depicted in Figs. 9 and 10

 

Figure 10
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  		Fig. 10. Concentration-response curves to the nitric oxide donor diethylammonium(z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in rat aorta. Data are shown as means ± SE (n = 7–10 rats) and expressed as percent relaxation of the contraction to phenylephrine. The endothelium-independent relaxation in the DOCA-salt-treated WT rat is significantly impaired compared with all other groups. See Table 2 for comparisons of EC50 values.

 
Since production of superoxide anion is increased and incriminated in the pathogenesis of systemic hypertension in this model, we measured the generation of superoxide anion by the vasculature. We employed a newly introduced and highly specific HPLC method for evaluating the production of superoxide anion, which is based on the detection of 2-hydroxyethidium; net generation of superoxide anion was defined and determined as MnTBAP-inhibitable production of 2-hydroxyethidium. With the use of this method, DOCA-salt-treated wild-type rats exhibited a greater generation of superoxide anion, whereas the DOCA-salt-treated Gunn rats failed to exhibit increased production of superoxide anion (Fig. 11).


Figure 11
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  		Fig. 11. Quantitative analysis of production of superoxide anion in rat aorta as determined by MnTBAP-inhibitable tissue content of 2-hydroxyethidium. Results are expressed (in µmol/mg protein) as means ± SE (n = 4–9 rats). *P < 0.05 vs. all other groups.

 
In our prior studies, we demonstrated that bilirubin at a concentration of 10–5 M can completely scavenge superoxide anion generated by either angiotensin II-treated smooth muscle cells or aortic segments harvested from angiotensin II-infused rats (53). Because of such oxidant-scavenging capability, we thus examined whether bilirubin can repair the defect in endothelium-dependent vasorelaxation observed in aortic segments from DOCA-salt-treated wild-type rats. As demonstrated in Fig. 12, preincubation with bilirubin at a concentration of 10–5 M completely reversed the impairment in vasorelaxation responses observed in DOCA-salt-treated wild-type rats; as shown by the calculated EC50 values (Table 3), the sensitivity to acetylcholine was decreased in the DOCA-salt-treated wild-type rat, and this decrease was prevented by prior exposure to bilirubin. From these data, we speculate that in the Gunn rat, hyperbilirubinemia per se may prevent the impairment in endothelium-dependent vasorelaxation as is otherwise induced when DOCA salt is imposed in the wild-type rat.


Figure 12
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  		Fig. 12. Effect of bilirubin on concentration-response curves to acetylcholine in rat aorta. These studies were performed in aortic rings harvested from vehicle-treated and DOCA-salt-treated WT rats and in aortic rings from either group that were incubated in the presence or absence of bilirubin (10–5 M) for 20 min. Data are shown as means ± SE (n = 5 rats) and expressed as percent relaxation to phenylephrine. The concentration-response curve in the DOCA-salt-treated group is significantly impaired compared with all other groups; prior incubation of aortic rings from DOCA-salt-treated rats with bilirubin corrected this defect in vasorelaxation response to acetylcholine. See Table 3 for comparison of EC50 values.

 

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  		Table 3. Sensitivity to acetylcholine in rat aorta in studies depicted in Fig. 12: effect of bilirubin (10–5 mol/l)

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of the present study was to employ genetic models, independent of pharmacological approaches, to evaluate the functional significance of relevant and selected components of the HO system in the development of DOCA-salt-induced systemic hypertension; these selected components of the HO system included the HO-1 isozyme and the product bilirubin. The functional significance of HO-1 was first assessed by utilizing a mutant mouse unable to express HO-1; subsequently, the effect of bilirubin was evaluated using an approach based on the Gunn rat.

HO-1+/+ mice responded to the stress of DOCA salt, as administered in the current study, with increased expression of HO-1 in the vasculature and without any elevation in systemic arterial pressure, whereas HO-1–/– mice exhibited a sustained and significant elevation in systemic arterial pressure when subjected to DOCA salt. The finding that DOCA salt provokes an increase in systolic arterial pressure when HO-1 is absent, but not when HO-1 is induced, implies that HO-1 is required by vascular responses that maintain normotension when mice are stressed by DOCA salt. Additional studies revealed that at a time point at which DOCA salt had induced higher systemic arterial pressures in HO-1–/– mice compared with HO-1+/+ mice, HO-1–/– mice exhibited comparable renal function and no impairment in urinary excretion of sodium; these findings would indicate that the higher systemic arterial pressure in the DOCA-salt-treated HO-1–/– mice cannot be simply ascribed to impaired renal function or impaired ability to excrete salt. From these findings, we suggest that HO-1 contributes to a countervailing vasodepressor response following the administration of DOCA salt.

Studies in the hyperbilirubinemic Gunn rat demonstrated that resistance to the pressor effect of DOCA salt occurred in this mutant strain. By 2 and 3 wk after the administration of DOCA salt, systolic arterial pressure was markedly lower in the Gunn rat, and this was associated with reduced urinary excretion of protein, the latter representing a biomarker that reflects, at least in part, the extent of elevation in systolic arterial pressure in this model. This attenuation in systemic arterial pressure in the Gunn rat subjected to DOCA salt could not be ascribed to increased urinary excretion of sodium or more vigorous induction of HO-1 in the vasculature. Such reduction in systolic arterial pressure was accompanied by normalization of endothelium-dependent and endothelium-independent relaxation. Thus hyperbilirubinemic states, as exhibited by the mutant Gunn rat, conferred resistance to the pressor effect of DOCA salt as demonstrated by measurements of systolic arterial pressure in vivo and studies of vascular reactivity in vitro.

To explore the basis for the vasodepressor responses observed in the Gunn rat, we drew on the substantial body of literature attesting to the role of oxidative stress in the pathogenesis of systemic hypertension in the DOCA-salt model. Generation of superoxide anion by the vasculature in this model is significantly increased, and assorted antioxidant strategies inhibit such generation of superoxide anion while concomitantly reducing systemic arterial pressure (4, 5, 38, 40). The pressor effect of increased generation of superoxide anion is derived from, at least in part, the scavenging of nitric oxide by superoxide anion, and thus the deprivation of nitric oxide in the vasculature. Increased generation of superoxide anion in the DOCA-salt model may originate from any one of the following sources: 1) increased production by the superoxide anion-generating enzyme NADPH oxidase (4, 38, 40); 2) impairment in enzymes, such as manganese superoxide dismutase, which scavenge superoxide anion (39); 3) increased generation of superoxide anion by mitochondria (9); and 4) the uncoupling of endothelial nitric oxide synthase (eNOS) as a consequence of limited and suboptimal availability of the essential cofactor for eNOS, tetrahydrobiopterin (36).

To quantitate superoxide anion generated by the vasculature, we utilized a newly described and highly specific HPLC-based method (74, 75), since concerns have been expressed regarding the specificity of methods currently and widely employed for such assays. Our studies demonstrate that aortic segments in DOCA-salt-treated wild-type rats exhibit production of superoxide anion significantly greater than that observed in vehicle-treated wild-type rats and DOCA-salt-treated Gunn rats. From these findings, we conclude that the amelioration of systemic hypertension observed in the Gunn rat subjected to DOCA-salt arises from marked attenuation in vascular generation of superoxide anion. A possible mechanism for such reduced vascular generation of superoxide anion resides in the scavenging of superoxide anion by bilirubin. Bilirubin is a highly efficient scavenger of oxidants, including superoxide anion, as shown by a large number of studies (22, 37, 53, 63); in our prior studies, for example, pathophysiologically relevant amounts of bilirubin (10–5 M) completely quenched the generation of superoxide anion by angiotensin II-stimulated smooth muscle cells in vitro and by aortic rings taken from angiotensin II-infused rats (53).

Scavenging of superoxide anion by bilirubin may occur not only in endothelial and smooth muscle cells of the vasculature in the DOCA-salt model but also in the nervous system. Quite recently, increased generation of superoxide anion by sympathetic neurons has been demonstrated in DOCA-salt hypertension and is incriminated in the evolution of hypertension in this model (11, 51). The pathogenesis of systemic hypertension in the DOCA-salt model may also arise from mechanisms in the central nervous system (8), and abnormalities in central noradrenergic innervation are described in the Gunn rat (52). Thus the resistance to the pressor effect of DOCA salt, as observed in the Gunn rat, may also reflect effects occurring in the central and peripheral nervous systems.

The lack of detection of increased amounts of superoxide anion from the vasculature in the hyperbilirubinemic Gunn rat may arise not only from the scavenging of superoxide anion but also from diminished production. In this regard, a critical stimulus for NADPH oxidase and other sources of oxidants resides in the vasoconstricting peptide endothelin-1. Endothelin-1 is induced in the vessel wall in the DOCA-salt model, and such induction localizes to the endothelium (40, 58, 59). Maneuvers that inhibit the effects of endothelin-1 reduce systemic arterial pressure in this model, decrease superoxide anion production, and limit the extent of vascular remodeling and hypertrophy that evolve in this model (10, 38, 40, 59). Evidence is available that HO-1 and its products can inhibit production of endothelin-1; for example, expression of endothelin-1 is markedly exaggerated in HO-1–/– mice compared with HO-1+/+ mice subjected to renovascular hypertension (70), and CO, a product of HO activity, suppresses expression of endothelin-1 (44). Endothelin-1 is inducible by oxidative stress (58), and thus antioxidants (such as bilirubin) may suppress such induction of endothelin-1. We thus speculate that the pressor response in HO-1–/– mice subjected to DOCA salt may also reflect heightened expression of endothelin-1, whereas the reduced pressor response in Gunn rats subjected to DOCA salt may involve, at least in part, suppressed expression of endothelin-1. Analysis of endothelin-1 in these mutant models is beyond the scope of the present study.

In addition to an increased expression of endothelin-1, other mechanisms that may be considered and explored as possible contributors to the elevated systolic arterial pressure in DOCA-salt-treated HO-1–/– mice include the following: diminished generation of the vasorelaxant gaseous product CO in the vasculature because of the deficiency of HO-1; diminished scavenging of superoxide anion by bilirubin in the vasculature as lower amounts of bilirubin are produced when HO-1 is deficient; diminished catabolism of superoxide anion in the vasculature of HO-1–/– mice since, as recently shown, the superoxide-scavenging enzyme superoxide dismutase can be induced by HO (65); and finally, the proinflammatory state occasioned by the deficiency of HO-1, and which promotes, in turn, oxidant and pressor effects (45).

At least two considerations may be relevant to the absence of elevation in systolic arterial pressure in wild-type mice subjected to DOCA salt. The first consideration pertains to the fact that in applying the DOCA-salt model, as previously utilized in mice (56), to HO-1–/– mice in the current study, we elected not to perform uninephrectomy since the latter would impose anesthetic and surgical stress on HO-1–/– mice in addition to the effect of reduced renal mass. HO-1–/– mice are remarkably sensitive to imposed stress (33, 46, 48, 54, 55), and such anesthetic and surgical stress may confer confounding variables in interpreting the hemodynamic response of HO-1–/– mice to DOCA salt. Moreover, it is important to underscore that the rationale for uninephrectomy in the DOCA-salt model resides, simply and only, in accentuating the relative excess of salt by reducing, relative to dietary salt intake, the capacity to excrete salt, the latter provided essentially by functional renal mass. The presence of two intact kidneys in mice subjected to DOCA salt thus ameliorates the pressor effect of the conventionally applied DOCA-salt model and may account for the lack of elevation in systolic arterial pressure observed in HO-1+/+ mice; nonetheless, HO-1–/– mice, also with two intact kidneys, were unable to withstand the imposition of DOCA salt without an elevation in systolic arterial pressure. The second consideration may relate to the clear and accumulating evidence that models of systemic hypertension and renal injury established in rats, when applied to mice, are often characterized by less severe changes (34, 69). For example, the remnant kidney model imposed in mice often fails to recapitulate the extent of renal injury as observed in the remnant kidney model in rats (34); the chronic administration of relatively large doses of angiotensin II by osmotic minipumps in mice imposes a rather modest elevation in systemic arterial pressure and may not induce oxidative stress or renal injury (69).

We suggest that the present findings regarding the salutary effects of HO-1 and bilirubin in the DOCA-salt model are broadly relevant to cardiovascular disease. Cardiovascular diseases may be exacerbated by increased dietary salt, can be amenable to therapies that block mineralocorticoid receptors, and often involve oxidative stress. As shown by several lines of evidence, HO-1 and its products may reduce the severity of cardiovascular disease. Coronary artery disease in nondiabetic and diabetic subjects and restenosis after vascular stenting or angioplasty are all less likely to occur in individuals expressing polymorphisms in the HO-1 gene that are associated with higher HO activity (18). This protective effect of HO-1 may be accounted for, at least in part, by bilirubin. In humans, higher systemic levels of bilirubin are associated with a decreased risk for cardiovascular disease including atherosclerosis (13), and cardiovascular disease is less likely to occur in patients with Gilbert's syndrome, the latter characterized by unconjugated hyperbilirubinemia (67). Quite recent evidence indicates that, in healthy subjects, higher levels of bilirubin are associated with a greater preservation of coronary flow reserve (20), enhanced flow mediated-dilatation in the brachial artery (17), and reduced intima-media thickness in the carotid artery (68). In this regard, it is notable that, in our studies, pathophysiologically relevant amounts of bilirubin completely reversed the impairment in endothelium-dependent vasorelaxation in aortic rings harvested from DOCA-salt-treated wild-type rats. Relevant to this finding is the fact that endothelial dysfunction is quite commonly a precursor to and an accompaniment of systemic hypertension and assorted cardiovascular diseases (16).

In summary, the present findings demonstrate that the HO-1 system and its product bilirubin confer vasodepressor effects in the DOCA-salt model. The hyperbilirubinemic Gunn rat is markedly resistant to the pressor effect of the DOCA-salt model, a resistance that is attended by the attenuation in the vascular generation of superoxide anion. This attenuation in DOCA-salt hypertension exhibited by the Gunn rat demonstrates that the resistance to pressor responses by this mutant strain is not restricted to states characterized by high circulating levels of angiotensin II, as shown by our prior studies (53), but also extends to states exhibiting suppressed systemic levels of renin and angiotensin II, as occurs in the DOCA-salt model. Finally, we suggest that our present findings may offer insights relevant to the current clinical interest in HO-1 and hyperbilirubinemia as prognostic indexes for cardiovascular disease.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were funded by National Institutes of Health Grants DK-47060 (to K. A. Nath) and HL-53524 (to Z. S. Katusic).


   ACKNOWLEDGMENTS
 
We thank Dr. John C. Burnett, Jr., for the use of the flame photometer for the measurement of urinary sodium. We thank Sharon Heppelmann for secretarial expertise and Allan Ackerman for technical expertise in the preparation of this work.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Nath, Mayo Clinic College of Medicine, 200 First St. SW, Guggenheim 542, Rochester, MN 55905 (e-mail: nath.karl{at}mayo.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.


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