Multiple gene approaches to delineate the role of the renin-angiotensin-aldosterone system in nephropathy

Renal function and blood pressure are tightly linked. Physiologically, the kidneys provide a key mechanism of chronic blood pressure control via their infinite gain mechanism [1], whereas elevated blood pressure affects renal function via the pressure natriuresis mechanism [2,3]. Pathophysiologically, long-standing hypertension attenuates pressure natriuresis [4] and can cause, or at least contribute to, renal damage [5], whereas an impaired renal function can lead to, or at least aggravate, arterial hypertension [6]. Hypertension is a well-established risk factor for myocardial infarction, other cardiovascular events and premature death. Recent studies indicate that, independent of the presence of hypertension, even a mild impairment of renal function (i.e. a reduction of glomerular filtration rate by 10 ml/min) is a significant risk factor for myocardial infarction [7], cardiovascular events in general, hospitalization and death from any cause [8]. These complex relationships make it important to better understand the pathogenesis of impaired renal function in hypertensive patients. The renin-angiotensin-aldosterone system (RAAS) is a key regulator of both blood pressure and renal function, and may play an important role in their interaction. Thus, the RAAS at least partly mediates pressure natriuresis [2] and also contributes to blood pressure elevations associated with impaired renal function [9]. Hence, RAAS inhibitors [i.e. both angiotensin-converting enzyme (ACE) inhibitors and angiotensin-receptor blockers] lower blood pressure and can attenuate or even prevent renal damage [9]. However, major inter-individual treatment responses to RAAS inhibition have been noted [10], and it remains difficult to predict responders based on known pathophysiological characteristics [11]. Genetic variability in the genes encoding for one or more components of the RAAS is likely to contribute to the heterogeneous treatment responses to RAAS inhibition. Polymorphisms have been described in the genes encoding several important components of the RAAS, including angiotensinogen [12], ACE [13], angiotensin type 1 (AT1) receptors [14] and aldosterone synthase (also known as CYP11B2) [15]. Most of these studies have focused on the insertion/deletion (I/D) polymorphism in the ACE gene, where the D allele is associated with a dose-dependent increase in plasma ACE activity [13]. Despite numerous studies investigating a possible association between any of these gene polymorphisms on the one hand and the presence of hypertension on the other hand, such relationships have remained elusive [13]. Similarly, a smaller number of studies investigating the relationship between such polymorphisms and blood pressure responses to drugs acting on the RAAS have also yielded inconclusive results [16]. In 1994, associations between diabetic nephropathy and genotype at the locus of the ACE gene were described for the first time [17,18]. Interestingly, one study looked at polymorphisms in the ACE gene using restriction length polymorphisms [18], whereas the other, similar to most later studies in the field, used the I/D polymorphism of the ACE gene [17]. Both studies reported an association of genotype at the ACE locus with progression of diabetic patients to nephropathy, with the D allele of the I/D polymorphism conferring a greater risk. An association of the D allele with the risk of developing diabetic nephropathy was also confirmed in studies of both type 1 [19] and type 2 diabetes [20]. Studies in both type 1 and type 2 diabetes patients also demonstrated an association of this allele with a fast progression of diabetic nephropathy [21,22]. Two studies demonstrated that the D allele was associated with a poorer response to nephroprotective treatment, particularly ACE inhibition, in such patients [23,24]. Studies on graft function in kidney transplant patients also support the view that the D allele of the ACE gene may adversely affect renal function [25-28]. Although the above studies have been very encouraging with regard to a role of ACE gene polymorphisms in the pathophysiology and treatment of diabetic nephropathy and/or renal allograft dysfunction, similar studies in other types of nephropathy have yielded inconsistent results. For example, studies in autosomal dominant polycystic kidney disease have reported adverse effects of the D allele of the ACE gene in some cases [29-32], whereas a greater number of studies did not confirm such associations [33-39]. Similarly, an adverse effect of the D allele of the ACE gene was found in some studies in immunoglobulin (Ig)A nephropathy [40-44] or end-stage renal failure in general [45-48], whereas other studies in IgA nephropathy [49-53] or end-stage renal failure [54-56] did not confirm this. One study in hypertensive nephropathy reported that carriers of the D allele of the ACE gene are at greater risk for developing nephropathy [57]. In the absence of studies demonstrating a beneficial effect of the D allele of the ACE gene with regard to renal function, the cumulative evidence suggests that this allele, which is associated with increased ACE activity [13], conveys a greater susceptibility to develop or progress in nephropathy. However, the large number of studies failing to detect such associations indicates that the relationship may not be that simple. Three main factors are most likely to explain the inconsistencies in the above studies. First, an altered RAAS activity is likely not to be the only factor controlling the development or progression of nephropathy. Second, given the likely involvement of additional factors, many of the above studies may have been underpowered to detect the contribution of the RAAS. Unfortunately, power calculations are largely absent in most reported studies. Third, ACE is not the only player in the RAAS. Therefore, it is possible that polymorphisms other than that in the ACE gene also contribute to the development of renal failure. These include polymorphisms in angiotensinogen, the AT1 angiotensin receptor gene and the aldosterone synthase gene. However, studies investigating a possible role of polymorphisms of the angiotensinogen gene [27-29,32,35,38,43,44,46,47,49,55,58], the AT1 receptor gene [27-29,32,44,55] and the aldosterone synthase gene [47,55] have also yielded inconsistent results. In the present issue of the journal, Fabris et al. [59] report on the impact of such polymorphisms on hypertension-associated renal insufficiency. Their study is based upon a comparison between 86 hypertensive subjects with renal insufficiency and 172 hypertensive patients without renal damage who were matched for age and duration of hypertension. In these groups, they assessed the presence of the M235T polymorphism of the angiotensinogen gene, the I/D polymorphism of the ACE gene, the A1166C polymorphism of the AT1 receptor gene and the -344C/T polymorphism of aldosterone synthase. They report that patients with nephropathy are significantly more likely to carry the D allele of the ACE gene, which is in agreement with a previous report in hypertensive nephropathy [57]. The T allele of the angiotensinogen gene and the C allele of the AT1 receptor gene were found more frequently in subjects with nephropathy, but this barely missed statistical significance with the given sample size (P = 0.054 and 0.065, respectively), and the genotype at the aldosterone synthase gene was not in Hardy-Weinberg equilibrium in the control group. Given that the data on each of these polymorphisms for several nephropathy forms have remained somewhat inconclusive, these data in their own right are interesting but cannot yet be taken as definitive evidence. However, it should be considered that these genes act in concert to elicit angiotensin II and aldosterone-mediated responses in the organism. Therefore, it is possible that specific combinations of polymorphisms in multiple genes encoding for components of the RAAS are a better predictor for the development of renal failure than either of them alone, particularly if any given polymorphism alone has only a moderate effect. Indeed, a study in IgA nephropathy patients detected a significant adverse effect of the D allele of the ACE gene only in a subgroup of subjects who were homozygous for the M allele of the angiotensinogen gene [49]. Fabris et al. [59] now report for the first time on combinations of up to four loci as possible predictors of nephropathy. Although the individual polymorphisms had only a minor effect on the overall risk of developing renal insufficiency, some of their combinations were associated with an odds ratio in excess of 4. These adverse genotype combinations mostly included the D allele of the ACE gene, the T allele of the angiotensinogen gene, the A allele of the AT1 receptor gene and the C allele of the aldosterone synthase gene. Another recent study in the journal has reported an association of polymorphism of the angiotensinogen, AT1 receptor and aldosterone synthase (but not ACE) genes with hypertension, where the A allele of the AT1 receptor and the T allele of aldosterone synthase are associated with risk; the three variants together appeared to have an additive effect in this regard [60]. A further recent study used a similar approach with regard to hypertension and proximal tubular sodium handling in the kidney [61]. The authors report that carriers of the D allele of the ACE gene in combination with homozygosity for the M allele of the angiotensinogen gene, the A allele of the AT1 receptor gene and the C allele of the aldosterone synthase gene, had an odds ratio of 3.4 for being hypertensive, which was associated with significant alterations of proximal tubular sodium handling [61]. Interestingly, the three studies disagree with regard to which alleles of the various genes convey adverse effects. This highlights an important problem in such studies. By definition, specific allelic combinations occur much more rarely than each allele alone. For example, in the study by Fabris et al. [59], most combinations associated with marked odds ratio increases for renal insufficiency were found in less than 10 patients. This results in major reductions of statistical power to reliably detect existing associations between genotype and phenotype. Therefore, the present data should not be over-interpreted as proving a hypothesis, but rather as exploratory. Nevertheless, they illustrate a promising approach for a better understanding of the genetic risk factors for complex diseases. Future investigators in this interesting field would be well advised to strive for large multinational collaborations to enable collection of sample sizes that are sufficient to analyse multiple gene loci.