‘The road not taken’: role of angiotensin II type 2 receptor in pathophysiology

Gunter Wolf

Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany

Two roads diverged in a wood, and I,

I took the one less traveled by,

And that has made all the difference

From ‘The Road Not Taken’ by Robert Frost (1874–1963).

Introduction

Robert Frost's famous poem from 1915 is usually interpreted as a declaration of individualism reflected in taking the ‘less traveled’ road. However, it has been also argued in a more unphilosophical way that this poem is just a mocking satire on a hesitant hiking friend of Frost's who always speculated what would have happened if he had chosen their path differently.

The two roads of angiotensin II (ANG II) action are AT1 and AT2 receptors [1]. There may be even more types of ANG II receptors, but they are not yet cloned, resembling more small tracks than major highways of ANG II signalling. AT1 receptors are responsible for mediating many of the well-known stimulatory physiological actions of ANG II including secretion of aldosterone, vasoconstriction, and renal sodium reabsorption [2]. Furthermore, the AT1 receptor plays a pivotal role in pathophysiological effects such as ANG II-mediated growth (proliferation or hypertrophy) and profibrogenic actions of the vasopeptide, partly through induction of transforming growth factor-beta [1,2]. Binding of ANG II to AT1 receptors can activate a deluge of G protein-coupled and uncoupled signal transduction pathways such as down regulation of adenylate cyclase, activation of phospholipase A2 and protein kinase C, stimulating the Janus kinase/STAT cascade, and opening of calcium channels. The AT1 receptor is clearly the predominant subtype in the adult kidney and is localized in glomeruli and renal tubules.

Until recently, the role of the AT2 receptor has been less well defined. The AT2 receptor shares only about 32% amino acid sequence identity with the AT1 receptor [3,4]. Its also coupled to G proteins, but employs different signal transduction pathways such as activation of tyrosine and serine/threonine phosphatases [5]. AT2 receptors are abundantly expressed in fetal organs, but with exception of neural tissue and the uterus, only a minority of total ANG II receptors are of the AT2 subtype in adult organs including the kidney [5]. There may be species differences in the localization and abundance of AT2-receptor expression in adult organs [5]. AT2-receptor expression apparently increases in injured tissues and organs undergoing remodelling. Transcription of the AT2 receptor is regulated by various hormones and growth factors [6]. In contrast to the AT1 subtype, ANG II does not down regulate AT2 receptors. Activation of AT2 receptors causes vasodilatation by generation of nitric oxide with a subsequent increase in cGMP [7,8]. Furthermore, binding of ANG II to AT2 receptors inhibits, in certain cells, proliferation, mediates differentiation in neural tissue, and even induces apoptosis [911]. Interestingly, one study suggests that AT2 receptor-mediated apoptosis may be independent of the ligand ANG II and requires only AT2-receptor expression on a specific target cell [12]. As all these effects of AT2-receptor activation are the opposite of those mediated through AT1 receptors, a Ying-Yang hypothesis has been proposed in which AT1 and AT2 receptors have principally contrary functions [5,6].

This intuitively understandable concept has been heavily exploited for pharmaceutical advertisement. Every sales representative of a company that offers an AT1-receptor antagonist carries a little brochure with a cartoon. This cartoon shows binding of ANG II to AT1 receptors in the presence and absence of the company's specific AT1-receptor antagonist. Besides blocking the deleterious effects of ANG II on AT1 receptors, this scheme always explains that the excess ANG II, which could not bind anymore to the occupied AT1 receptor, goes now to the AT2 subtype ‘doing something good’. Although one could certainly argue whether AT2-receptor-induced apoptosis or inhibition of growth is ‘something good’ for a dying kidney in chronic renal disease or the heavily injured heart after myocardial infarction, is everything coming from the AT2 receptor indeed beneficial? Recent evidence suggests that this may not be the case.

AT2 receptors and inflammation

We found that ANG II stimulates the expression of the chemokine RANTES in cultured rat glomerular endothelial cells [13]. Surprisingly, this ANG II-stimulated induction was transduced by AT2 receptors [13]. Furthermore, intraperitoneal infusion of ANG II into normal rats for 4 days significantly stimulated glomerular influx of macrophages/monocytes [13]. This glomerular infiltration was associated with RANTES expression localized to glomerular endothelium (Figure 1Go). Treatment with an AT2-receptor antagonist (PD 123177) attenuated the glomerular macrophage/monocyte influx without normalizing hypertension [13]. This study was the first demonstration that ANG II exerts proinflammatory effects through activation of AT2 receptors. The data were recently confirmed by Ruiz-Ortega and coworkers, who demonstrated that ANG II infusion for 72 h increased glomerular and interstitial inflammatory cells [14]. In agreement with our findings, an AT2-receptor antagonist, but not losartan, significantly reduced this inflammation [14]. The group of Jesus Egido also spearheaded studies demonstrating that ANG II activates nuclear factor {kappa}B (NF-{kappa}B) [15]. NF-{kappa}B is a key proinflammatory factor involved in the transcriptional regulation of many genes including RANTES. Using specific ANG II-receptor antagonists, Ruiz-Ortega et al. convincingly showed that ANG II activates NF-{kappa}B in vascular smooth muscle cells (VSMC) through AT1 and AT2 receptors [15]. Moreover, ANG II still activates NF-{kappa}B in VSMC obtained from AT1 receptor-deficient mice [16]. Treatment with an AT2-receptor antagonist in vivo significantly reduced renal NF-{kappa}B activation in ANG II-infused rats and in a protein-overload model [14,17].



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Fig. 1. Immunohistochemistry ffor RANTES. (a) Infusion with solvent for 4 days. No glomerular RANTES expression is detectable. (b) Infusion with ANG II for 4 days induces glomerular expression of RANTES protein. This ANG II-mediated stimulation of the chemokine was transdcued through AT2 receptors [12].

 
We used an alternative approach to study a potential role of AT2 receptors in NF-{kappa}B activation and selectively overexpressed AT1 and AT2 receptors in COS7 cells that do normally not bear ANG II-receptors [18]. As shown in Figure 2Go, COS7 cells transfected with an AT2-receptor expression construct demonstrated NF-{kappa}B activation after treatment with 10-7 M ANG II for 30 min. This NF-{kappa}B activation was completely blocked by 10-6 M PD123319. We additionally studied PC12 cells, a rat pheochromocytoma cell line only expressing AT2 receptors, and also found ANG II-mediated NF-{kappa}B activation [18]. As gel shift experiments only address NF-{kappa}B binding to consensus sites in vitro, we also performed experiments with a NF-{kappa}B reporter construct transiently transfected into the cells [18]. These experiments also revealed that ANG II is able to transactivate NF-{kappa}B through AT2 receptor signalling [18]. Thus, all these data clearly demonstrate that ANG II can activate NF-{kappa}B in vitro and in vivo through AT2 receptors. Whether AT2 receptor-mediated proinflammatory events play a role in human renal pathophysiology is not known.



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Fig. 2. NF-{kappa}B binding in vitro as detected with electrophoretic mobility assay. COS7 cells that normally do not express ANG II-receptors were transiently transfected with an AT-receptor expression construct in which the rat AT2 receptor is under control of the CMV promoter [18]. A strong increase in NF-{kappa}B binding was seen after 30 minutes stimulation with ANG II (10-7 mol/l). Co-incubation with the AT2-receptor antagonist PD 123319 significantly reduced ANG II-mediated NF-{kappa}B activation. The specificity of the reactions was established using nuclear proteins from either ANG II-treated AT2 receptor transfected COS7 cells with a 100-fold excess of unlabeled NF-{kappa}B (competitor) or unrelated OCT1 oligonucleotides (non-competitor).

 

AT2 receptors, growth stimulation, and fibrosis

There is also evidence that the growth stimulatory and profibrogenic effects of ANG II may not be solely transduced through AT1 receptors. Levy et al. reported that rats subcutaneously infused for 3 weeks with ANG II exhibited significant aortic hypertrophy and fibrosis [19]. This was not influenced by AT1-receptor antagonist treatment despite normalization of hypertension [19]. However, the AT2-receptor antagonist PD123310 significantly reduced media thickness and collagen content of the aorta [19]. Retroviral gene transfer was used to overexpress AT2 receptors in cultured VSCM [20]. These cells increased cell-associated and secretory collagen synthesis after stimulation with the AT2-receptor agonist CGP42212A [20]. The profibrogenic effect was completely blocked by an AT2-receptor antagonist but not by losartan [20]. In another study, performed in a rat fetal VSMC cell line that expressed AT2 receptors, ANG II causes cellular hypertrophy as defined by an increase in RNA and protein content with concomitant DNA replication [21]. This ANG II-induced hypertrophy was mediated through AT2 receptors [21]. Targeted deletion of mouse AT2 receptors prevents cardiac hypertrophy resulting from pressure overload [22]. ANG II-infusion for 3 weeks induced cardiac hypertrophy and fibrosis in wild-type mice, but not in AT2 receptor ‘knockout’ animals [23]. Interestingly, ANG II-mediated stimulation of transforming growth factor-beta, collagen type III, and fibronectin mRNA expression was attenuated in AT2 receptor-deficient mice [23]. Although this study has been criticized for its methodology [24], it nevertheless provides evidence that ANG II-induced growth stimulation in vivo could be transduced through AT2 receptors under certain circumstances. On the other hand, overexpression of AT2 receptors in a cardiac specific manner failed to increase apoptosis of cardiomyocytes even after prolonged ANG II infusion [25].

A recent study by Cao and coworkers revealed that infusion of ANG II into rats for 14 days induced in proximal tubular cells an increase in proliferation as well as apoptosis [26]. Interestingly, these effects were attenuated by the AT2-receptor blocker PD123319, albeit an AT1-receptor antagonist was also effective in attenuating the ANG II-induced proliferation and apoptosis [26].

Do the diverging roads make a difference?

Recent preliminary results from the RENAAL, IDNT, and IRMA studies provide compelling evidence that AT1-receptor antagonists slow the progression of chronic renal disease in patients with type 2 diabetes [27]. Yet, no data are available showing that this therapy is equal, superior, or even inferior to treatment with ACE inhibitors. Maybe this is an academic discussion because the future will probably lie in the combination therapy with ACE inhibitors and AT1-receptor antagonists [28]. Nevertheless, cell culture and animal studies have clearly demonstrated that activation of the AT2 receptor could have important pathophysiological consequences. The message is that activation of AT2 receptors may not always be beneficial by antagonizing AT1 receptor-mediated effects. And that may make all the difference.

Notes

Correspondence and offprint requests to: Gunter Wolf, MD, Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, University Hospital Eppendorf, Pavilion 61, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: Wolf{at}uke.uni\|[hyphen]\|hamburg.de Back

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