Kinin B1 receptor enhances the oxidative stress in a rat model of insulin resistance: outcome in hypertension, allodynia and metabolic complications

Abstract

Background: Kinin B(1) receptor (B(1)R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B(1)R activation could perpetuate the oxidative stress which leads to diabetic complications.

Methods and findings: Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8-12 weeks. A selective B(1)R antagonist (SSR240612) was administered acutely (3-30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B(1)R expression, aortic superoxide anion (O(2)(*-)) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3-30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O(2)(*-), NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B(1)R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O(2)(*-) in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10-100 microM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe(8)]des-Arg(9)-BK (20 microM; B(1)R agonist). Data show that the greater aortic O(2)(*-) production induced by the B(1)R agonist was blocked only by apocynin.

Conclusions: Activation of kinin B(1)R increased O(2)(*-) through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B(1)R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B(1)R gene expression in this model.

Figure 1. Acute effect of orally administered SSR240612 on systolic blood pressure in 12-week glucose-fed rats.

Doses of 10 mg/kg and 30 mg/kg of SSR240612 were given to control rats and doses of 3, 10, 30 mg/kg of SSR240612 or vehicle were given to glucose-fed rats. Data are mean ± s.e.m of values obtained from (n) rats and represent changes of systolic blood pressure in mmHg (A) or Area Under the Curve (B). Statistical comparison with vehicle (V) in glucose-fed rats (+) is indicated by + P< 0.05.

Figure 2. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on systolic blood pressure in glucose-fed rats.

From day (D) 1 to 7, measurements were taken prior to the morning treatment with SSR240612. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by ***†††P< 0.001.

Figure 3. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on (A) tactile allodynia and (B) cold allodynia in glucose-fed rats.

From day (D) 1 to 7, measurements were taken prior to the morning treatment with SSR240612. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by †P< 0.05, ††P< 0.01, ***†††P< 0.001.

Figure 4. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on B₁R expression in the thoracic spinal cord.

A: Quantification of specific density of [¹²⁵I]-HPP-desArg¹⁰-Hoe 140 to B₁R in various spinal cord laminae (Lam). B: Autoradiograms of thoracic spinal cord for B₁R. The non-specific binding (NS) was obtained by the co-addition of 1 µM R-715 (B₁R antagonist) with [¹²⁵I]-HPP-desArg¹⁰-Hoe 140. C: Bars represent fold change in gene expression for B₁R. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by * P< 0.05, **††P< 0.01, ***P< 0.001.

Figure 5. B₁R mRNA levels in aorta, liver and skeletal muscle after orally administered SSR240612 (10 mg/kg/day for 7 days).

Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by † P< 0.05, ††P< 0.01, ***†††P< 0.001.

Figure 6. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on oxidative stress.

(A) Basal superoxide anion production, (B) NADPH oxidase activity in the aorta of glucose-fed rats. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by **††P< 0.01, ***†††P< 0.001.

Figure 7. Superoxide anion production on histological sections of the aorta marked with dihydroethidine.

Control (A, B) and glucose-treated rats (C, D) after 7-day treatment with Vehicle (A, C) or 10 mg/kg/day SSR240612 (B, D). Asterisk (*) indicates the lumen side of the section. The arrow (in A) represents the elastic lamina of the smooth muscle cells while the two arrows (in C) show the increase of fluorescent ethidium bromide in the nucleus of smooth muscle cells in the aorta of glucose-treated rat. The fluorescent marker is reduced after treatment with SSR240612 (in D). Scale bar is 20 µm in each panel. Images are representative of 4 aortas in each group.

Figure 8. Fluorescence intensities of superoxide (dihydroethidium) staining in aortic sections from control and 8-week glucose-treated rats shown in Figure 7 .

Data are expressed as fluorescence intensity in arbitrary unit (a.u.) and represent the mean ± s.e.m of 4 rats in each group. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by ***†††P< 0.001.

Figure 9. Effects of in vitro B₁R activation on superoxide anion production in the aorta of glucose-fed rats pre-treated or not with oxidative enzyme inhibitors.

Basal and stimulated production of superoxide anion in the presence of the B₁R agonist Sar[D-Phe⁸]des-Arg⁹-BK (20 µM) were measured in the presence of (A) Allopurinol (xanthine oxidase inhibitor), (B) L-NAME (inhibitor of all NOS isoforms) and (C) Apocynin (NADPH oxidase inhibitor) at the indicated concentrations (see methods). Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with control (*), glucose (+) or Sar[D-Phe⁸]des-Arg⁹-BK (†) is indicated by * ⁺ P< 0.05, ** ^{++ ††}P< 0.01.

Figure 10. Effects of in vivo B₁R activation on superoxide anion production in the aorta of glucose-fed rats pre-treated or not with the NADPH oxidase inhibitor.

Basal and stimulated production of superoxide anion with the B₁R agonist Sar[D-Phe⁸]des-Arg⁹-BK (1 mg/kg, i.p.). Apocynin (50 mg/kg, i.p.) was administered 30 min prior to the B₁R agonist (see methods). Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with glucose (+) or Sar[D-Phe⁸]des-Arg⁹-BK (†) is indicated by ⁺ P< 0.05, ^{++ ††}P< 0.01, ⁺⁺⁺P< 0.001.

Figure 11. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on vascular expression of superoxide dismutase.

The expression of MnSOD was measured at (A) mRNA level by qRT-PCR, and (B) at protein level by Western blot in the aorta of glucose-fed rats. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by *^†P< 0.05, ^††P< 0.01.

Figure 12. Effect of orally administered SSR240612 (10 mg/kg/day for 7 days) on vascular expression of catalase.

The expression of catalase was measured at protein level by Western blot in the aorta of glucose-fed rats. Data are mean ± s.e.m of values obtained from (n) rats. Statistical comparison with controls (*) or glucose-fed rats + vehicle (†) is indicated by ^†P< 0.05, ^**P< 0.01.