The renin-angiotensin aldosterone system (RAAS) in renal disease
by Dr. A. Titia Lely
University Medical Center Groningen, Groningen, The Netherlands.
Originally published as general introduction to the PhD thesis of Dr. A. Titia Lely which she published in november 2007 under supervision of her promotor Prof. dr. Gerjan Navis and co-promotores Dr. Harry van Goor and Dr. Ron Korstanje at the University Medical Center Groningen in The Netherlands.
An overview is provided on the renin-angiotensin aldosterone system (RAAS) and several components of this system, such as angiotensin 1-7 (ang 1-7) and angiotensin converting enzyme 2 (ACE2). Furthermore both genetic and environmental factors influencing the RAAS and the response to intervention are explained.
Pharmacological blockade of the renin-angiotensin aldosterone system (RAAS) has allowed great advances in the prevention of progressive renal function loss and in cardiovascular prevention and treatment. The RAAS is one of the main regulators of blood pressure, renal hemodynamics and volume homeostasis in normal physiology, and an important mediator of renal and cardiovascular target-organ damage in progressive renal and cardiac failure. Angiotensin-converting enzyme inhibitors (ACEi) were the first class of clinically applicable drugs that specifically block the RAAS. Originally, ACEi were developed as antihypertensives, in particular aimed at the treatment of high renin hypertension. However, 30 years after their introduction they have been shown to be effective in a wide spectrum of renal and cardiovascular diseases, ranging from primary prevention in diabetic nephropathy to the severe end stage of heart failure.
Along with the expanding range of applications, understanding of the (patho-) physiology of the RAAS has increased over the last decades. Novel components have been identified, such as angiotensin 1-7 (ang 1-7) and angiotensin converting enzyme 2 (ACE2). The presence of the RAAS in various organs and tissues is established and better insights in its genetic regulation are gained. Yet, many seemingly simple questions, relevant to clinical practice, remain unanswered. These include the mechanisms underlying the well-established interaction between sodium status and therapeutic efficacy of RAAS-blockade, the role of (genetic) variability of the RAAS in the pathogenesis of renal and cardiovascular disease, and in individual differences in response to RAAS-blockade.
In this thesis we will address those issues from the perspective of the (patho-) physiology of the RAAS, as understanding of the underlying (patho-) physiology may not only help to reconcile seemingly discrepant findings, but also to improve therapeutic application of RAAS-blockade in renal and cardiovascular disease.
 The renin-angiotensin aldosterone system
The role of the RAAS has been studied extensively for more than a century. The history of the discovery of the RAAS began in 1898 with the studies by Tigerstedt and Bergman, who reported the pressor effect of renal extracts; they named the renal substance renin based on its origin . RAAS activation is mediated by a cascade of proteases (figure 1). Its architecture, biochemistry, and functions are more complex than initially assumed. In the classical concept, activation of the RAAS cascade starts with the release of pro-renin from the juxtaglomerular apparatus, in response to specific stimuli such a low perfusion pressure of the afferent arteriole, sympathetic activation, or low sodium/chloride supply to the macula densa. Active renin cleaves the decapeptide angiotensin I (ang I) from its precursor, the liver-derived angiotensinogen. Ang I is a substrate of angiotensin converting enzyme (ACE) or kininase 2, a 170-d zinc metallopeptidase that cleaves two amino acids at the carboxy terminal end, leading to the formation of the octapeptide ang II, the main effector hormone of the system.
Angiotensin II (Ang II) is a potent vasoconstrictor that acts on the systemic as well as the renal vascular bed. Moreover, it stimulates the release of aldosterone from the adrenal gland – leading to distal tubular sodium reabsorption, and directly promotes proximal tubular sodium reabsorption. Together, these effects of ang II play a main role in the homeostatic response to volume depletion. Ang II stimulates cell proliferation and hypertrophy, but has also significant pro-inflammatory and pro-fibrotic properties that appear to be involved in chronic progressive target organ damage in the cardiovascular system and the kidney [2-4]. Ang II exerts these effects mainly through activation of the angiotensin II type 1 receptor (AT-1 receptor). In addition to activation of the RAAS cascade by release of renin into the circulation, activation at tissue level can occur in various pathological conditions [5-8]. However, the regulation of the RAAS in various tissues in health and disease is incompletely characterized.
All components of the RAAS are highly expressed in the developing kidney in a pattern that suggests a role for ang II in renal development. In support of this notion, pharmacological interruption of AT-1 receptor-mediated effects in animals by ACEi or AT-1 receptor blockade with an ongoing nephrogenesis produces specific renal abnormalities characterized by papillary atrophy, abnormal wall thickening of intrarenal arterioles, tubular atrophy associated with expansion of the interstitium, and a marked impairment in urinary concentrating ability .
ACEi are widely used for the treatment of cardiovascular and renal diseases. However, their mechanisms of action is not completely understood . In general, it is assumed that blockade of ang II generation at tissue level rather than ang II generation in the circulation is relevant to their therapeutic effects [11-13]. Moreover, the interference of ACE with the formation of other substances (e.g. bradykinin, angiotensin 1-7, N-acetyl-Ser-Asp-Lys-Pro) may contribute to the beneficial effect of ACEi [14-16]. It is a consistent observation that correction of volume overload, or induction of mild volume depletion by diuretics and/or dietary sodium restriction (that is, actions that induce activation of the RAAS), increases the therapeutic efficacy of ACEi in hypertension and renal disease [17-21].
 Angiotensin-converting enzyme
ACE plays a central role in the RAAS through generation of the peptide ang II from its inactive precursor ang I. ACE is an ecto-enzyme that is primarily attached to cell membranes, but it can also be shed by alpha-secretasis and as a result be found in the circulation. It contains two catalytic domains, which differ in their function. Whereas the C-terminal accounts for approximately 75% of ang II-forming capacity, the N-terminal domain has major functions in the inactivation of bradykinin, neurotensin, and substance-P. In addition, a germinal form (90kd, with only one catalytic domain) has been identified, which has an important function in sperm differentiation and fertilization . Moreover, ACE is thought to act in the intestine as a hydrolyser of dietary peptides . As ACE catalyzes a multitude of enzymatic processes, it has even been referred to as the most promiscuous enzyme in the human body.
Human ACE is strongly expressed in vascular endothelial cells throughout the body, with the highest expression in the pulmonary capillaries. In the kidney it is also abundantly expressed on the brush border of the proximal tubular epithelial cells. The extra-cellular localization of ACE on endothelial cells creates an ideal position for the interaction with its substrates ang I and bradykinin. ACE is shed from the endothelium at a high rate by proteolytic cleavage, which accounts for the circulating form of ACE . An unidentified membrane-bound secretase catalyzes the cleavage/secretion process . Interestingly, tissue ACE can be increased by several disease-associated triggers and thus might be involved in a vicious circle of tissue damage . Elevation of ACE levels and enhanced generation of ang II could further promote tissue damage by its pro-inflammatory and pro-fibrotic actions. The relevance of tissue ACE activity has been emphasized in several studies . ACE mRNA is expressed in virtually all tissues, and biochemical measurements of ACE activity illustrate that >90% of ACE is located in tissue . Tissue ACE is now recognized as a key factor in cardiovascular and renal diseases . Moreover, during ACE inhibition, the blood pressure response to exogenous ang I much more closely parallels the effects of the drug on tissue rather than on plasma ACE . However, in human, it is difficult to obtain a valid assessment of tissue ACE, which hampers unraveling its role. It would be relevant in this respect to know whether the level of ACE in the circulation provides a reflection of ACE in particular tissues.
Studies with genetically manipulated mice show in more detail the complex role of tissue ACE. Complete ACE knockout mice have low blood pressure, the inability to concentrate urine, and a mal development of the kidney. In contrast, mice who have plasma ACE but no tissue ACE have low blood pressure and cannot concentrate urine, but they have normal indices of renal function. Cole et al. investigated the fine control of body physiology by the RAAS and developed a novel promoter swapping approach and generated a mice with selectively ACE expression in the liver, without vascular expression [28;29]. These mice shows that endothelial expression of ACE is not required for blood pressure control or normal renal function.
 Genetic control of ACE levels – ACE I/D genotype
Plasma ACE levels are remarkably stable when measured repeatedly in the same individual, whereas inter individual differences are large . This suggests strong long-term control of plasma levels, possibly with genetic origins. The human ACE gene is located on chromosome 17q23 and includes 26 exons. The coding sequence codes for a 1306 amino-acid protein, including a signal peptide. In 1990, Rigat et al. found a polymorphism involving the presence (insertion I) or absence (deletion D) of a 287-bp sequence of DNA in intron 16 of the ACE gene (figure 2) . Mean ACE activity levels in DD carriers were approximately twice as high as in II genotype individuals. Subjects with the ID genotype had intermediate levels, indicating co-dominancy. The ACE I/D genotype accounted for approximately half (47%) of the observed variance in ACE level. Subsequent studies showed that the I/D polymorphism is also associated with differences in ACE levels in various tissues and organs, i.e the kidney, the heart, lymphocytes, and testis [32-34].
 ACE I/D genotype and response to ACE inhibition
Since the discovery of the ACE I/D genotype it has been speculated that the differences in plasma ACE activity associated with the ACE genotype might affect the therapeutic response to ACEi, explaining inter individual variability in cardiovascular or renal response to equivalent doses of ACEi . Two opposite hypotheses were elicited in this respect. First, subjects with the DD genotype might respond less well to ACEi, as a consequence of their higher ACE activity. The normal dose of ACEi would not effectively block their ACE activity. Alternatively, the DD subjects could have a better response because their pathophysiology is more “ACE-driven” . Many association studies have investigated the possible effect modification on response to ACEi for different indications such as hypertension , diabetic and non-diabetic renal disease [35;42;44;45] and coronary artery disease [46;47]. Table 1 shows an overview of the articles published on non-diabetic renal patients. However, there are very few randomized controlled clinical trials on this issue. Moreover, there are inconsistencies in trial findings. In a very well performed systematic review Scharplatz et al. stated that there is a lack of sufficient data . As a result the extent of effect modification of the genotype on therapy response remains unclear. Unfortunately, after 17 years of ACE genotyping involving thousands of subjects, we still do not know whether ACE genotyping helps in predicting the therapy response to ACEi.
 Gene environment interaction
It is difficult to attribute the cause, or progression, of a complex disease to a single genetic factor. In complex disorders usually multiple genes are involved with potentially important genetic interactions. Furthermore, many environmental factors can exert influences on the natural course of the diseases, as well as on therapy response, resulting in multiple gene-environment interactions. Very large studies are needed to account for environmental factors when studying the influence of genes on the cause or progression of renal disease by association studies, e.g. “the epidemiological approach”. Increasing study size, or turning to meta-analysis, usually increases heterogeneity in the patient populations. This heterogeneity can result in bias by introducing undetected interacting factors. A great number of association studies on the ACE I/D genotype and the progression of cardiovascular and renal diseases have been performed, with conflicting results [35;36;49-53]. Conclusive evidence for a role of the ACE I/D genotype in the progression of disease would require prospective studies with patients selected by genotype. Previous work at our department showed a clear gene-environment interaction between sodium status and the ACE I/D genotype. First, cross sectional data in renal patients showed a correlation between sodium intake and therapy response to ACEi in DD subjects, which was absent in the other genotypes . Furthermore, in healthy subjects with the DD genotype an enhanced response to ang I infusion was shown during high sodium intake only . These results support presence of gene-environment interaction between the ACE I/D genotype and sodium status. Future prospective studies on the genotype should account for this interaction.
 Novel directions in the RAAS
All components of the RAAS are present in various organs and tissues including the heart, kidneys, fat tissue and vasculature . Recently novel players in the RAAS have been recognised, including new metabolising enzymes (e.g. ACE2) and smaller angiotensin peptides (e.g. ang 1-7) that are biologically relevant and can modulate and antagonize the effect of ang II (figure 1). First identified in 2000 [56;57], ACE2 is an ACE homologue and carboxypeptidase that cleaves a single residue from ang I to generate ang 1–9, and degrades ang II to ang 1–7 . High levels of ACE2 were found in the kidney, heart and vasculature [59-61]. Initially, it was hypothesized that a disruption between the balance between ACE and ACE2, resulting in increased ang II levels, would lead to hypertension and organ damage . Evidence in experimental hypertension and diabetes support this hypothesis and shows decreased levels of ACE2 during these conditions [63;64]. These reports confirm the hypothesis that ACE2 is relevant to the pathogenesis of cardiovascular and renal damage. Ang 1-7 can be formed from ang I via subsequent cleavage by ACE and ACE2, or directly from ang I by endopeptidases. Ang 1-7 has vasodilator and antiproliferative properties, and has been found to antagonize the effects of ang II at various levels . Ang 1-7 has been proposed to contribute to the beneficial effect of RAAS blockade, as both systemic and tissue ang 1-7 are considerably elevated during treatment with ACEi or AT1-receptor antagonist [66;67]. Antagonizing ang 1-7 ameliorates the antihypertensive actions of ACEi and AT1-receptor antagonists [68;69]. To summarize, it has become clear that the RAAS has two main arms, with opposing effects namely the ACE - ang II - AT1-receptor axis, and ACE2 - ang 1-7 - mas-receptor axis. The balance between these arms will determine the net effects on (patho-) physiology.
In recent years, it has become increasingly clear that adipose tissue has endocrine functions. Interestingly, all RAAS components are present in the adipocyte. The RAAS is known to be activated remarkably in obesity [70-72]. Expression of the AT1-receptor in human adipocytes is two-fold higher in adipocytes obtained from obese, hypertensive women . Furthermore, adipocytes secrete a variety of hormones, cytokines, growth factors and other bioactive compounds – collectively known as adipokines. Adiponectin is a novel adipokine that is produced exclusively in adipocytes . Levels are inversely related to the amount of adipose tissue present in the body . Decreased concentrations of adiponectin are associated with an increased risk for the development of diabetes [75;76], essential hypertension , and myocardial infarction . These associations are attributed to anti-atherogenic actions of adiponectin, as well as its favourable effects on insulin sensitivity . The mechanisms involved in the regulation of plasma adiponectin are of considerable interest, but are largely unknown. Interestingly, in diabetic  and hypertensive patients , inhibition of the RAAS, by AT-1 receptor blockade or ACEi increases plasma adiponectin. These studies suggest an interaction between the RAAS and adiponectin.
 Aim of the thesis
The complex role of the RAAS and specifically ACE and its genotype in the susceptibility to renal damage and therapy response to ACEi has been studied extensively. Yet, as noted above, several issues relevant to clinical practice remain unanswered. The role of (genetic) variability in the RAAS, in particular ACE activity, in the pathogenesis of renal damage, and in individual differences in response to RAAS blockade is still unresolved. Moreover, the old question of the mechanisms underlying the interaction between sodium status and therapeutic efficacy of RAAS blockade has not been answered either. In this thesis we will address these issues from a perspective of integrative (patho-) physiology, combining insights from new discoveries with the older concepts of modulation of sodium status.
Starting from observations on the impact of ACE I/D genotype on rate of renal function loss before and during intervention, we will address the role of ACE and its genotype as a determinant of natural course of renal damage, and therapy response, respectively. In addition, we will investigate whether new players in the RAAS, ang 1-7 and ACE2, i.e. RAAS components counterbalancing the effects of activation of the classical RAAS pathway leading to ang II generation, might allow novel insights into the interaction of volume status and the effect of ACEi, and the role of the intrarenal RAAS in renal damage, respectively. Moreover, combining the above two angles, modulation of sodium status will be used to unravel the association between ACE I/D genotype and response to ACEi, thus analyzing this association in terms of gene-environment interaction.
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