Contribution of different Nox homologues to cardiac remodeling in two-kidney two-clip renovascular hypertensive rats: Effect of valsartan

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Abstract

Growing evidences have shown that hypertension, cardiac hypertrophy and fibrosis were associated with an overactivity of NAD(P)H oxidase. It is unknown, however, which isoform of NAD(P)H oxidase yields O2radical dot formation in heart and aorta in two-kidney, two-clip (2K2C) hypertensive rats in vivo and thus is responsible for the development of cardiac remodeling. We examined the pathological change of NAD(P)H oxidase homologues and tested the effect of valsartan on the cardiac remodeling in 2K2C renovascular hypertensive rats. Four weeks after male Sprague–Dawley rats accepted 2K2C or sham operation, 2K2C hypertensive (>160 mmHg) rats were divided into vehicle-treated (2K2C) and valsartan (30 mg kg−1 per day, for 6 weeks)-treated (2K2C + Val) groups, which were compared with sham-operated controls (Sham). At week 10, 2K2C hypertensive rats showed increased serum level of angiotensin II (Ang II), MDA and blood pressure (BP), obvious cardiac hypertrophy and fibrosis, increased O2radical dot production and NAD(P)H oxidase activity and expression in aorta and heart. The heart in 2K2C hypertensive rats preferred to use NADH as substrate while the aorta used both NADH and NADPH. Valsartan treatment decreased BP, ameliorated cardiac hypertrophy and fibrosis, decreased O2radical dot production and NAD(P)H oxidase activity in aorta and heart. Nox2 and Nox4 protein expression increased in heart, while Nox1 and Nox4 increased in aorta in 2K2C hypertensive rats, which were all normalized after valsartan treatment. In conclusion, these data indicate that different Nox expression might account for substrate preference and the formation of O2radical dot by NAD(P)H oxidase resulting from elevated Ang II in the 2K2C model contributes to the development of renovascular hypertension and subsequent cardiac remodeling.

Introduction

Human renovascular disease constitutes a relatively rare form of secondary hypertension, frequently related to the activation of the renin-angiotensin system (RAS), as a result of the fall in the renal blood flow and pressure perfusion produced after renal artery stenosis [1]. In situation such as hypertension, the heart responds to increased afterload by initiating adaptive remodeling processes. These include cardiomyocyte hypertrophy, fibrosis, etc., which are deleterious over time and ultimately result in progressive heart failure.

An increase in oxidative stress resulting from excessive generation of reactive oxygen species (ROS) is implicated in the development of renovascular hypertension. Recent work suggests a role for vascular NAD(P)H oxidases in the development of hypertension, cardiac hypertrophy and interstitial fibrosis [2]. Vascular NAD(P)H oxidases (also named nonphagocytic NAD(P)H oxidases), similar to the phagocyte NADPH oxidase, are the major enzymatic sources of ROS in the cardiovascular system and the only enzyme discussed so far whose primary function appears to be ROS production [3]. In contrast to the phagocyte oxidase, which uses NADPH exclusively as an electron donor, there have been many reports of possible NADH consumption by the vascular oxidase, which was thus called a NAD(P)H oxidase [4]. Each member of the NAD(P)H oxidase family contains a catalytic subunit termed Nox that forms a heterodimer with a lower molecular weight subunit called p22phox; this heterodimeric cytochrome is the site of electron transfer from NADPH or NADH to molecular O2, resulting in the formation of O2radical dot. Five Nox homologues (Nox1–5) have been identified to date and the biochemical regulation of different Nox homologue varies significantly. All vascular cell types as well as cardiomyocytes and cardiac fibroblasts express components of the prototypical NADPH oxidase (phagocyte NADPH oxidase), including its cell membrane-associated subunits, p22phox and gp91phox (Nox2), and cytosolic subunits, p47phox, p67phox, and p40phox, but they also exhibit specific patterns of Nox expression, with several cell types expressing more than one homologue (including Nox2 and other homologues Nox1 or Nox4) [5]. Nox1 is highly expressed in cultured vascular smooth muscle cells (VSMCs) but is not significantly expressed in cardiomyocytes or endothelial cells. Nox2 is abundantly expressed in cardiomyocytes, endothelial cells and fibroblasts. Nox4 appears to be the most widely expressed homologue, being found in endothelial cells, cardiomyocytes and fibroblasts [4], [6].

It is still unknown, however, which homologue of NAD(P)H oxidase yields O2radical dot formation in heart and aorta in renovascular hypertensive rats in vivo and thus is responsible for the subsequent development of cardiac remodeling. Therefore, we used a rat model with partial constriction of the bilateral renal arteries, in which obvious cardiac remodeling occurred by the sustained elevation of blood pressure which was introduced with two-kidney, two-clip (2K2C) operation. We investigated the pathological change of NAD(P)H oxidase homologues to elucidate which one play a role in the development of cardiac remodeling and tested the effect of valsartan, a selective angiotensin II type 1 receptor (AT1R) antagonist, in this 2K2C renovascular hypertension model.

Section snippets

Materials

Dihydroethidine (DHE), diphenylene iodonium (DPI), diethyldithiocarbamate (DDC), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), monoclonal anti-α-tubulin were from Sigma Aldrich, bis-N-methylacridinium nitrate (lucigenin) was from Alexis Biochemicals, polyclonal Nox2 (gp91phox) antibody was from BD transduction, polyclonal Nox1 (Mox1), Nox4 and p47phox antibodies and horseradish peroxidase (HRP)-conjugated rabbit anti-goat, goat anti-rabbit, and goat anti-mouse immunoglobulin G (IgG)

Characterization of the 2K2C animals

We originally had 15 rats in every group and the numbers were reduced to 14 rats in the Sham group, and 13 in 2K2C and 13 in 2K2C + Val in the end because of the operation stress.

As shown in Table 1, the BP of Sham group was stable at about 125 mmHg, while those of 2K2C-operated rats increased to about 180 mmHg at week 4. After 2K2C-operated rats were treated with valsartan for 6 weeks, the BP decreased to about 140 mmHg. Cardiac hypertrophy in 2K2C hypertensive rats, measured by the ratio of left

Discussion

The major novel findings of this study are that Nox2 and Nox4 expression increased in LV, and Nox1 and Nox4 increased in aorta in 2K2C hypertensive rats, which may contribute to the different substrate preference of NAD(P)H oxidase in LV and aorta. The present study demonstrate for the first time down-regulation of NAD(P)H oxidase by valsartan might have made an important contribution to the control of BP and inhibition of cardiac remodeling in 2K2C hypertensive rats.

Human renovascular disease

Acknowledgments

This work was supported by Major Program in Key Field of People's Government of Guangdong Province, PR of China (No: 2003A30904); Key Program of Ministry of Education, PR of China (No: 104146); Key Natural Science Fund of Guangdong province, PR of China (No: 04105349); Key Program of Guangdong Province, PR of China (No: 2003B31713) and Key Program of Guangzhou city, PR of China (No: 2003Z1-E5011).

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