Serelaxin reduces oxidative stress and asymmetric dimethylarginine in angiotensin II-induced hypertension

Abstract

Recent findings suggest the therapeutic action of relaxin during hypertension is dependent on nitric oxide synthase (NOS) activation; however, the mechanisms underlying the beneficial effects of relaxin on the NOS system have not been fully elucidated. We hypothesized that the protective effects of relaxin include reducing both oxidative stress and the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). We examined the effect of Serelaxin [human recombinant relaxin-2 (RLX)] in male Sprague-Dawley rats given high-dose angiotensin (ANG) II (400 ng·kg(-1)·min(-1) sc) for 6 wk or shams. RLX was administered (4 μg/h sc) to half of the rats in each group after 2 wk of ANG II for the remaining 4 wk. ANG II induced hypertension and proteinuria, reduced NO oxidation products (NOx), and increased oxidative stress (NADPH oxidase activity, thiobarbituric acid-reactive substances, and 8-isoprostane excretion) and plasma ADMA. While RLX had no effect on sham rats, RLX attenuated the ANG II-dependent hypertension (165 ± 5 vs. 135 ± 13 mmHg, P < 0.05) and proteinuria at 6 wk (62 ± 6 vs. 41 ± 4 mg·day(-1)·100 g(-1), P < 0.05) and normalized oxidative stress and circulating ADMA, in association with restored NOx excretion and kidney cortex NOx. We found that RLX had no impact on the ADMA-regulatory enzymes protein arginine methyltransferase and dimethylarginine-dimethylaminohydrolase (DDAH). Furthermore, RLX treatment did not increase DDAH activity in kidney cortex or liver. These data suggest that benefits of RLX treatment include reduced ADMA levels and increased NO bioavailability, possibly due to its antioxidant effects.

Keywords: NADPH oxidase; dimethylarginine dimethylaminohydrolase; nitric oxide; protein arginine methyltransferase-1.

Fig. 1.

Relaxin reduces blood pressure and proteinuria and preserves nitric oxide (NO) production during high-dose ANG II hypertension. Mean arterial pressure (MAP), urinary protein excretion (UProtV), kidney cortex NO metabolite (NO_x) content, and urinary NO_x excretion rate in control (CON), relaxin-treated (RLX), ANG II hypertensive (ANG), and ANG II hypertensive with relaxin treatment (ANG+RLX) rats; n = 6–9. *P < 0.05 vs. control. +P < 0.05 vs. ANG.

Fig. 2.

Relaxin treatment does not affect renal cortex abundance of NO synthase (NOS) 1 (A and B) or NOS3 (C). Western blot analysis of expression of NOS isoforms in the kidney cortex from control (CON), relaxin-treated (RLX), ANG II hypertensive (ANG), and ANG II hypertensive with relaxin treatment (ANG+RLX) rats; n = 6–9. *P < 0.05 vs. control. D: representative Western blots for the expression of NOS1 and NOS3. MW, molecular weight marker; +, positive control; C, control; R, relaxin-treated; A, ANG II hypertensive; AR, ANG II hypertensive with relaxin treatment.

Fig. 3.

Relaxin reduces oxidative stress during high-dose ANG II infusion. NADPH oxidase activity levels (A) in the kidney cortex and urinary excretion of thiobarbituric acid-reactive substances (TBARS; B) and 8-isoprostane (C) in CON, RLX, ANG, and ANG+RLX rats; n = 6–8. *P < 0.05 vs. control. +P < 0.05 vs. ANG.

Fig. 4.

Relaxin reduces plasma asymmetric dimethylarginine (ADMA) levels during high-dose ANG II hypertension. Plasma concentrations of ADMA (A) and l-arginine (B) as measured by HPLC and the ratio of plasma l-arginine-to-plasma ADMA (C) in CON, RLX, ANG, and ANG+RLX rats; n = 6–9. *P < 0.05 vs. control. +P < 0.05 vs. ANG.

Fig. 5.

Relaxin has no effect on protein arginine methyltransferase (PRMT) abundance in the kidney cortex, liver, or lung. Western blot analysis of expression of PRMT isoforms in the renal cortex (A-B), liver (C-D), and lung (E-F) of CON, RLX, ANG, and ANG+RLX rats; n = 6–8. G: representative Western blots for the expression of PRMT-1 and PRMT-3.

Fig. 6.

Relaxin does not increase dimethylarginine dimethylaminohydrolase (DDAH) abundance or activity in the kidney cortex or liver. Western blot analysis of expression of DDAH isoforms (A-D) and total DDAH enzymatic activity (E-F) in the kidney cortex and liver from CON, RLX, ANG, and ANG+RLX rats. DDAH activity is expressed as μmol citrulline produced per g of protein per min; n = 6–8. *P < 0.05 vs. control. G: representative Western blots for the expression of DDAH-1 and DDAH-2.