Angiotensin receptor gene variant goes with the flow

Attempts to determine the molecular genetic basis of hypertension started with the renin–angiotensin system (RAS) [1,2]. However, over the years, genetic association findings for RAS gene polymorphisms have generally been disappointing [3–8], with the possible exception of an A1166C variant in the angiotensin type 1 receptor (AT1R) gene (AGT1R) [9,10]. This polymorphism is located in that part of the gene corresponding to the 3′-untranslated region (3′-UTR) of the messenger RNA. The 3′-UTR is important in determination of AGT1R mRNA stability and thus expression and production of AT1R [11]. The 3′-UTR can also change the coupling of the angiotensin receptor to G-proteins from the normal Gαi to Gαq[11]. This involves the binding of proteins to the 3′-UTR of the AGT1R mRNA [12]. Angiotensin II, via phosphorylation of the mRNA binding protein calreticulin, increases AGT1R mRNA decay, and this involves bases 2175–2195 [13]. A role for the 3′-UTR in modulating cell signalling associated with tachyphylaxis in response to AT1R-mediated rises in inositol triphosphate and Ca2+ has also been invoked [14]. Whether the effects of the A1166C polymorphism involve differences in the binding of protein(s) to this portion of the 3′-UTR or whether nearby polymorphisms in the transcript or promoter region are involved is not known. In this regard, essential hypertension has also shown an association with variation in the 5′-untranslated region [15], which is more concerned with the rate of mRNA translation on the ribosome. In this issue of the journal, Spiering et al. [16] report the results of a study of the effects of AT1R blockade with EXP3174, the active metabolite of losartan, on various parameters in hypertensive subjects homozygous for either the A allele (n = 15) or the C allele (n = 14) of this commonly studied polymorphism of AGT1R. They studied subjects both on a high and on a low sodium diet. A high sodium state leads to an elevation in circulating angiotensin II concentration, whereas plasma angiotensin II is reduced in low sodium states. This is because angiotensin II levels reflect the level of secretion of renin, which is high in low sodium states and low in high sodium states. Angiotensin II, the classic biological effector of renin–angiotensin, binds to AT1R in multiple locations in the body, such as vascular wall, proximal tubule, adrenal, brain and heart, leading to increased vascular resistance and sodium/water retention. Thus, when dietary sodium intake is low, occupancy of AT1R is elevated, meaning that fewer vacant sites are available for binding by agonists (such as angiotensin II) or antagonists (such as losartan). The converse applies in a state of high sodium intake. In an earlier study, the present authors examined the effect of exogenously infused angiotensin II [17], whereas their use of EXP3174 in the present study meant that they were examining the role of endogenous angiotensin II [16]. In this new work, Spiering et al. find that, in hypertensive patients on a low salt diet, there were no significant differences in blood pressure responses to EXP3174 between genotypes [16]. By contrast, heart rate increased by 8% in CC subjects, but did not change in those with the AA genotype [16]. They also found that effective renal plasma flow of CC subjects increased only by half as much as it did in AA subjects. Although glomerular filtration rate (GFR) was unchanged, filtration fraction decreased less in CC subjects, and the fall in renal vascular resistance (RVR) in CC was half that in AA. Changes in plasma renin and angiotensin II were similar. When the experiment was performed in subjects on a high salt diet, the fall in systolic/diastolic blood pressure for CC subjects was only 2 mmHg/2 mmHg compared to 12 mmHg/7 mmHg for AA subjects. Once again, heart rate increased more in those who were CC, but changes in the other parameters did not differ between each genotype group. These new findings add to those reported previously by this group for hypertensive subjects on a high sodium diet, in whom the response in heart rate, effective renal plasma flow (ERPF), GFR and RVR to infusion of angiotensin II was more pronounced in CC patients [17]. Similar to the present study, there were no differences in plasma renin or aldosterone, nor in atrial natriuretic peptide. This led the authors to conclude that hypertensive CC subjects possess an elevation in sensitivity, but not reactivity, to angiotensin II, and that this might explain the association of the C allele with hypertension and other cardiovascular abnormalities [17]. Studies of the A1166C variant in relation to physiological response began in 1999 with a report by Hilgers et al. [18], which found no genotypic differences in blood pressure, renal vascular or aldosterone responses to angiotensin II infusion. However, the subjects used in that particular study had normal or only slightly elevated blood pressure and were on a high salt intake. In the same year, Miller et al. [19] noted that C allele carriers (i.e. those with the genotype AC or CC) had lower GFR, ERPF and renal blood flow (RBF) compared to AA in healthy subjects on a 200 mmol/day sodium intake. These sorts of base-line differences were what Spiering et al. [16] observed in their subjects, with the differences being significant on the low salt diet, but becoming non-significant trends on the high salt diet. Miller et al. [19] also examined the effect of angiotensin II infusion and, on the basis of unaltered GFR in AC/CC subjects and despite RBF subsiding similarly in each genotype group, concluded that the efferent arteriole of C allele carriers had a greater constrictor response than in AA subjects. This same group also tested losartan and observed a decrease in mean arterial pressure, together with an increase in GFR, in the AC/CC group, but no changes for those with the AA genotype. Spiering et al. [16] have rightly criticized this study because of the mixed alleles in the genotype group that were used to test the effect of the recessive C allele, meaning that the domineering effect of the A allele in heterozygous subjects has the potential to confuse the results, which were different from those in the present study. Furthermore, in these kinds of acute experiments, intravenous infusion of the active metabolite, rather than oral administration of the prodrug as in the study by Miller et al. [19] implies that the present findings of Spiering et al. [16] are more meaningful. It would thus appear that the AT1R is more responsive to angiotensin II in subjects with the C allele of the A1166C gene polymorphism. Such enhanced responsiveness is apparent both systemically and within the renal vasculature. What is the reason for this? Is it that CC subjects have higher angiotensin II? It is more likely that there are more AT1Rs or that a more sensitive/higher affinity form of AT1R is present in subjects who possess the CC genotype. Another possibility is that the receptor antagonist EXP3174 is not as effective in CC patients. However, in apparent contrast to earlier expectations [17], Spiering et al. [16] found no evidence for a genotypic difference in receptor number or affinity, as had been noted previously by others [20]. Nevertheless, this and the earlier study only tested receptor properties for platelets, which have very few AT1Rs [20], meaning that the possibility of a difference in number or affinity of receptors on the blood vessel wall is possible, and this requires investigation. In an effort to shed some light on the mechanisms involved, experiments have been performed in vitro using isolated segments of the internal mammary artery of coronary artery bypass patients [21]. Those with the CC genotype showed greater responsiveness to angiotensin II than did A allele carriers [21]. The difference was apparent for vessels of placebo-treated and angiotensin-converting enzyme inhibitor-treated patients, even though the magnitude of the response was greater in the latter. There are a number of effects of angiotensin II within the kidney. Thus, it would be interesting to examine whether proximal tubule reabsorption of sodium also differs between AA and CC subjects. Apart from contributing to any effect of the C allele on blood pressure, such a genotypic difference could lead to differences in tubular interstitial pressure. In this regard, one study failed to find an association between the A1166C variant and indices of renal sodium handling by itself, but an association was seen in CC subjects who were also homozygous for commonly studied variants of other genes of the renin–angiotensin–aldosterone system [22]. Also worthy of investigation would be effects on the ultrafiltration coefficient (Kf) for each genotype. What also remains to be resolved is the molecular basis for any biological effect associated with the A1166C variant of AGT1R. For any polymorphism, a particular genetic association finding means either that the polymorphism is itself responsible for the association observed, or is in linkage disequilibrium (i.e. is co-inherited) with a polymorphism nearby in the gene or adjacent DNA. However, linkage disequilibrium of the A1166C variant with any other variant of AGT1R has not been observed. This includes none of the seven variants in the 5′-flanking DNA 1.5 kb upstream of the transcription start site, nor one in the 5′-untranslated region (exons 1–4) or a T/C variant in the protein encoding portion (exon 5) [23]. There is also a A44221G polymorphism in exon 5 that is associated with hypertension in African-Americans, but such an association did not apply in Whites [24]. Thus, the possibility remains of an effect of the A1166C variant itself. Given its presence in the 3′-UTR, could there be a differential effect of the A1166C variant on the binding of a protein that affects mRNA stability? The protein AUF1 affects AGT1R mRNA stability and this involves binding to an AU-rich region in the distal portion of the 3′-UTR [25]. However, such binding was similar for each genotype [25]. Nevertheless, the number of proteins that bind to the 3′-UTR of a mRNA to affect stability can be considerable [26], meaning that there is much scope for further research in the elucidation of a mechanism possibly involving A1166C and one or more proteins that control the stability of AGT1R mRNA. In conclusion, the A1166C variant remains of continued interest in the aetiology of hypertension, as well as cardiovascular risk generally, irrespective of blood pressure [27]. The new findings of Spiering et al. [16] encourage such ongoing investigations.