Role of reactive oxygen species in hyperadrenergic hypertension: biochemical, physiological, and pharmacological evidence from targeted ablation of the chromogranin a (Chga) gene

Abstract

Background: Oxidative stress, an excessive production of reactive oxygen species (ROS) outstripping antioxidant defense mechanisms, occurs in cardiovascular pathologies, including hypertension. In the present study, we used biochemical, physiological, and pharmacological approaches to explore the role of derangements of catecholamines, ROS, and the endothelium-derived relaxing factor nitric oxide (NO(•)) in the development of a hyperadrenergic model of hereditary hypertension: targeted ablation (knockout [KO]) of chromogranin A (Chga) in the mouse.

Methods and results: Homozygous ⁻(/)⁻ Chga gene knockout (KO) mice were compared with wild-type (WT, +/+) control mice. In the KO mouse, elevations of systolic and diastolic blood pressure were accompanied by not only elevated catecholamine (norepinephrine and epinephrine) concentrations but also increased ROS (H₂O₂) and isoprostane (an index of lipid peroxidation), as well as depletion of NO(•). Renal transcript analyses implicated changes in Nox1/2, Xo/Xdh, and Sod1,2 mRNAs in ROS elevation by the KO state. KO alterations in blood pressure, catecholamines, H₂O₂, isoprostane, and NO(•) could be abrogated or even normalized (rescued) by either sympathetic outflow inhibition (with clonidine) or NADPH oxidase inhibition (with apocynin). In cultured renal podocytes, H₂O₂ production was substantially augmented by epinephrine (probably through β₂-adrenergic receptors) and modestly diminished by norepinephrine (probably through α₁-adrenergic receptors).

Conclusions: ROS appear to play a necessary role in the development of hyperadrenergic hypertension in this model, in a process mechanistically linking elevated blood pressure with catecholamine excess, renal transcriptional responses, ROS elevation, lipid peroxidation, and NO(•) depletion. Some of the changes appear to be dependent on transcription, whereas others are immediate. The cycle could be disrupted by inhibition of either sympathetic outflow or NADPH oxidase. Because common genetic variation at the human CHGA locus alters BP, the results have implications for antihypertensive treatment as well as prevention of target-organ consequences of the disease. The results document novel pathophysiological links between the adrenergic system and oxidative stress and suggest new strategies to probe the role and actions of ROS within this setting.

Figure 1

A. Chga targeted ablation on H₂O₂ and lipid peroxidation (isoprostane). Urine isoprostane and H₂O₂ measured and normalized by creatinine. WT vs KO: H₂O₂ (p<0.02) and isoprostane (p<0.002), [n=6 animals/condition]. Results are shown as mean ± one SEM. B. Renal podocytes, catecholamines and H₂O₂. Catecholamines (both epinephrine and norepinephrine; each at 1 μM) were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM. C. Selective adrenergic agonists and renal podocyte H₂O₂. Selective adrenergic agonists (alpha-1: phenylephrine; alpha-2: clonidine; beta: isoproterenol), each at 1 μM, were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM.

Figure 1

A. Chga targeted ablation on H₂O₂ and lipid peroxidation (isoprostane). Urine isoprostane and H₂O₂ measured and normalized by creatinine. WT vs KO: H₂O₂ (p<0.02) and isoprostane (p<0.002), [n=6 animals/condition]. Results are shown as mean ± one SEM. B. Renal podocytes, catecholamines and H₂O₂. Catecholamines (both epinephrine and norepinephrine; each at 1 μM) were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM. C. Selective adrenergic agonists and renal podocyte H₂O₂. Selective adrenergic agonists (alpha-1: phenylephrine; alpha-2: clonidine; beta: isoproterenol), each at 1 μM, were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM.

Figure 1

A. Chga targeted ablation on H₂O₂ and lipid peroxidation (isoprostane). Urine isoprostane and H₂O₂ measured and normalized by creatinine. WT vs KO: H₂O₂ (p<0.02) and isoprostane (p<0.002), [n=6 animals/condition]. Results are shown as mean ± one SEM. B. Renal podocytes, catecholamines and H₂O₂. Catecholamines (both epinephrine and norepinephrine; each at 1 μM) were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM. C. Selective adrenergic agonists and renal podocyte H₂O₂. Selective adrenergic agonists (alpha-1: phenylephrine; alpha-2: clonidine; beta: isoproterenol), each at 1 μM, were used to influence H₂O₂ production in mouse podocytes (n=6 replicate wells per condition). Results are shown as mean ± one SEM.

Figure 2. Adrenergic outflow inhibition (clonidine) or NADPH oxidase (Nox) inhibition (apocynin) on BP

Both SBP and DBP were reduced significantly in KO mice by sympathetic inhibition with the α₂-agonist clonidine (125 μg/kg body weight/day for 3 weeks). SBP and DBP were reduced significantly in KO mice by Nox inhibitor apocynin (2 mmol/L) in drinking water for 3 weeks. N for each group (number of mice) is given in the figure inset. Results are shown as mean ± one SEM.

Figure 3. mRNA abundances in kidney

Relative abundances of mRNAs were normalized to β-actin in kidney by real-time PCR (n=12 kidneys were studied from each strain, WT or KO). Differences in C_t (target mRNA versus β-actin mRNA) are expressed on a % scale (see Methods). Results are shown as mean ± one SEM. A. NADPH oxidase (Nox) isoforms. B. RedOx targets. C. Nos (nitric oxide synthase) isoforms.

Figure 3. mRNA abundances in kidney

Relative abundances of mRNAs were normalized to β-actin in kidney by real-time PCR (n=12 kidneys were studied from each strain, WT or KO). Differences in C_t (target mRNA versus β-actin mRNA) are expressed on a % scale (see Methods). Results are shown as mean ± one SEM. A. NADPH oxidase (Nox) isoforms. B. RedOx targets. C. Nos (nitric oxide synthase) isoforms.

Figure 3. mRNA abundances in kidney

Relative abundances of mRNAs were normalized to β-actin in kidney by real-time PCR (n=12 kidneys were studied from each strain, WT or KO). Differences in C_t (target mRNA versus β-actin mRNA) are expressed on a % scale (see Methods). Results are shown as mean ± one SEM. A. NADPH oxidase (Nox) isoforms. B. RedOx targets. C. Nos (nitric oxide synthase) isoforms.

Figure 4A. Oxygen radicals (H₂O₂): Response to treatment by sympathetic inhibition or NADPH oxidase blockade

Urine H₂O₂ level (amplex red fluorescence/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. B. Lipid peroxidation (isoprostane): Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urine isoprostane level (ng/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. C. Catecholamine secretion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma catecholamine levels (ng/ml) of WT, KO, KO+clonidine and KO+apocynin mice [n=8 per condition] were measured by HPLC, in plasma obtained from anesthetized mice. Results are shown as mean ± one SEM. D. Nitric oxide (NO•) depletion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urinary excretion of nitrate+nitrite was taken as an index of NO• production. Urine levels of NO• (μmol/mg creatinine) were measured in WT (n=5), KO (n=8), KO+clonidine (n=7) and KO+apocynin (n=7) mice. Results are shown as mean ± one SEM. E. Nitric oxide (NO•) depletion in the circulation: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma levels of NO• (nmol/ml) were measured in WT (n=10), KO (n=10), KO+clonidine (n=8). and KO+apocynin (n=8) mice. Results are shown as mean ± one SEM.

Figure 4A. Oxygen radicals (H₂O₂): Response to treatment by sympathetic inhibition or NADPH oxidase blockade

Urine H₂O₂ level (amplex red fluorescence/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. B. Lipid peroxidation (isoprostane): Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urine isoprostane level (ng/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. C. Catecholamine secretion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma catecholamine levels (ng/ml) of WT, KO, KO+clonidine and KO+apocynin mice [n=8 per condition] were measured by HPLC, in plasma obtained from anesthetized mice. Results are shown as mean ± one SEM. D. Nitric oxide (NO•) depletion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urinary excretion of nitrate+nitrite was taken as an index of NO• production. Urine levels of NO• (μmol/mg creatinine) were measured in WT (n=5), KO (n=8), KO+clonidine (n=7) and KO+apocynin (n=7) mice. Results are shown as mean ± one SEM. E. Nitric oxide (NO•) depletion in the circulation: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma levels of NO• (nmol/ml) were measured in WT (n=10), KO (n=10), KO+clonidine (n=8). and KO+apocynin (n=8) mice. Results are shown as mean ± one SEM.

Figure 4A. Oxygen radicals (H₂O₂): Response to treatment by sympathetic inhibition or NADPH oxidase blockade

Urine H₂O₂ level (amplex red fluorescence/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. B. Lipid peroxidation (isoprostane): Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urine isoprostane level (ng/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. C. Catecholamine secretion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma catecholamine levels (ng/ml) of WT, KO, KO+clonidine and KO+apocynin mice [n=8 per condition] were measured by HPLC, in plasma obtained from anesthetized mice. Results are shown as mean ± one SEM. D. Nitric oxide (NO•) depletion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urinary excretion of nitrate+nitrite was taken as an index of NO• production. Urine levels of NO• (μmol/mg creatinine) were measured in WT (n=5), KO (n=8), KO+clonidine (n=7) and KO+apocynin (n=7) mice. Results are shown as mean ± one SEM. E. Nitric oxide (NO•) depletion in the circulation: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma levels of NO• (nmol/ml) were measured in WT (n=10), KO (n=10), KO+clonidine (n=8). and KO+apocynin (n=8) mice. Results are shown as mean ± one SEM.

Figure 4A. Oxygen radicals (H₂O₂): Response to treatment by sympathetic inhibition or NADPH oxidase blockade

Urine H₂O₂ level (amplex red fluorescence/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. B. Lipid peroxidation (isoprostane): Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urine isoprostane level (ng/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. C. Catecholamine secretion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma catecholamine levels (ng/ml) of WT, KO, KO+clonidine and KO+apocynin mice [n=8 per condition] were measured by HPLC, in plasma obtained from anesthetized mice. Results are shown as mean ± one SEM. D. Nitric oxide (NO•) depletion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urinary excretion of nitrate+nitrite was taken as an index of NO• production. Urine levels of NO• (μmol/mg creatinine) were measured in WT (n=5), KO (n=8), KO+clonidine (n=7) and KO+apocynin (n=7) mice. Results are shown as mean ± one SEM. E. Nitric oxide (NO•) depletion in the circulation: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma levels of NO• (nmol/ml) were measured in WT (n=10), KO (n=10), KO+clonidine (n=8). and KO+apocynin (n=8) mice. Results are shown as mean ± one SEM.

Figure 4A. Oxygen radicals (H₂O₂): Response to treatment by sympathetic inhibition or NADPH oxidase blockade

Urine H₂O₂ level (amplex red fluorescence/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. B. Lipid peroxidation (isoprostane): Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urine isoprostane level (ng/mg creatinine) is presented in WT (n=6), KO (n=6), KO+clonidine (n=8), or KO+apocynin (n=8) mice. Results are shown as mean ± one SEM. C. Catecholamine secretion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma catecholamine levels (ng/ml) of WT, KO, KO+clonidine and KO+apocynin mice [n=8 per condition] were measured by HPLC, in plasma obtained from anesthetized mice. Results are shown as mean ± one SEM. D. Nitric oxide (NO•) depletion: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Urinary excretion of nitrate+nitrite was taken as an index of NO• production. Urine levels of NO• (μmol/mg creatinine) were measured in WT (n=5), KO (n=8), KO+clonidine (n=7) and KO+apocynin (n=7) mice. Results are shown as mean ± one SEM. E. Nitric oxide (NO•) depletion in the circulation: Response to treatment by sympathetic inhibition or NADPH oxidase (Nox) blockade. Plasma levels of NO• (nmol/ml) were measured in WT (n=10), KO (n=10), KO+clonidine (n=8). and KO+apocynin (n=8) mice. Results are shown as mean ± one SEM.

Figure 5. Oxygen radicals and hyper-adrenergic hypertension: Hypothesis integrating experimental results from this study in targeted ablation of the mouse Chga locus

Directional arrows indicate proposed cause-and-effect relationships.