The development of the first drug-like selective angiotensin II type 2 (AT₂) receptor agonist (22) derived from the non-selective angiotensin II type 1 (AT₁) receptor/AT₂ receptor agonist L-162,313 is presented. Compound 22 with a K_i value of 0.4 nM for the AT₂ receptor and a K_i > 10 µM for the AT₁ receptor induces outgrowth of neurite cells, stimulates p42/p44^mapk, enhances in vivo duodenal alkaline secretion in Sprague- Dawley rats and lowers the mean arterial blood pressure in anaesthetised spontaneously hypertensive rats. Thus, the peptidomimetic 22 exerts a similar biological response as the endogenous peptide angiotensin II after selective activation of the AT₂ receptor. In addition, Compound 22 has a bioavailability of 20–30% after oral administration and a half-life estimated to four hours in the rat. Compound 22 will therefore serve as a valuable research tool enabling studies of the function of the AT₂ receptor in more detail.

Introduction. Sodium loading, and subsequent volume expansion, suppresses aldosterone levels in individuals with normal renal function. We hypothesised that loss of renal function impairs this volume-aldosterone relationship.

Materials and methods. With multifrequency bioimpedance spectroscopy, we measured total body water (TBW), extracellular volume (ECV), and intracellular volume in five haemodialysis patients at varied states of hydration and in five healthy volunteers during low-, normal-, and high-salt diets. Serum aldosterone, potassium, and C-reactive protein were measured simultaneously. Scatterplots and general estimating equations were used to examine the relationship among these variables.

Results. In healthy volunteers with salt loading, and in haemodialysis subjects with increased inter-dialytic weight gain, expansion of ECV led to reciprocal declines in serum aldosterone concentrations. The relationship was more profound in healthy volunteers (p<0.001) than in haemodialysis subjects (p=0.1). Notably, haemodialysis subjects posted consistently higher levels of ECV (median 49.6% TBW, IQR 43.9–51.8% compared to 41.1%, 39.9–42.8% in volunteers) and serum aldosterone (median 26.7 ng/dl, IQR 19.8–29.6 compared to 12.4 ng/dl, 8.8–16.0 in volunteers). Serum potassium did not appear to influence aldosterone concentration (p=0.9).

Conclusions. The shift of the volumealdosterone curve in haemodialysis subjects suggests that end-stage kidney disease is a state of high volume and inappropriately high aldosterone. These data have important clinical implications, as dialysis patients may benefit from both volume reduction and mineralocorticoid receptor blockade.

Suppression of angiotensin II formation by angiotensin-converting enzyme inhibitors or blockade of the angiotensin II receptor by angiotensin receptor blockers is a powerful therapeutic strategy to slow the progression of renal disease. However, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers provide only imperfect protection against the progression of chronic kidney disease to end-stage renal failure. Hence, innovative approaches are needed to keep patients with chronic kidney disease off dialysis. Angiotensin II activates at least two receptors, namely the angiotensin II type 1 (AT₁) and angiotensin II type 2 (AT₂) receptors. The majority of the effects of angiotensin II, such as vasoconstriction, inflammation, and matrix deposition, are mediated via the AT₁ receptor. It is thought that the AT₂ receptor counteracts these effects and plays a role in nephroprotection. However, recent data support the notion that the AT₂ receptor transduces pro-inflammatory effects and promotes fibrosis and hypertrophy. Therefore, the question of whether stimulation of the AT₂ receptor could represent a silver bullet for the treatment of chronic kidney disease or may, on the contrary, exert detrimental effects on renal physiology remains unresolved. Recent data from AT₂ receptor-knockout mice demonstrate that the loss of AT₂ receptor signalling is associated with increased renal injury and mortality in chronic kidney disease. This raises the expectation that pharmacological stimulation of the AT₂ receptor may positively influence renal pathologies. However, further research is needed to explore the question whether AT₂ receptor stimulation may represent a new therapeutic strategy for the treatment of chronic kidney disease.

Since the discovery of a renin-angiotensin system (RAS) in the brain, several studies have linked this central RAS to neurological disorders such as ischaemia, Alzheimer’s disease and depression. In the last decade, evidence has accumulated that the central RAS might also play a role in Parkinson’s disease. Although the exact cause of this progressive neurodegenerative disorder of the basal ganglia remains unidentified, inflammation and oxidative stress have been suggested to be key factors in the pathogenesis and the progression of the disease. Since angiotensin II is a pro-inflammatory compound that can induce the production of reactive oxygen species due to activation of the NADPHdependent oxidase complex, this peptide might contribute to dopaminergic cell death. In this review, three different strategies to interfere with the pathogenesis or the progression of Parkinson’s disease are discussed. They include inhibition of the angiotensin-converting enzyme, blockade of the angiotensin II type 1 receptor and stimulation of the angiotensin II type 2 receptor.

Recent knowledge demonstrated that the reninangiotensin system (RAS) functions as a local renal paracrine system. All components of the RAS are present within the kidney and include angiotensinogen, renin, angiotensin I, angiotensinconverting enzymes, angiotensin II, the angiotensin II type 1 (AT₁) receptor and the angiotensin II type 2 (AT₂) receptor. Angiotensin II is the major effector hormone of the RAS and contributes to a variety of renal and cardiovascular physiologic and pathologic mechanisms through stimulation of AT₁ and AT₂ receptors. Angiotensin receptor blockers were developed based on the advanced knowledge of the AT₁ receptor contribution to development of a variety of kidney, vascular and cardiac diseases including but not limited to hypertension, diabetic nephropathy, heart failure, myocardial infarction and atherosclerosis. In contrast, knowledge concerning the role of the AT₂ receptor in health and disease is still emerging. The AT₂ receptor is believed to counterbalance the effects of the AT₁ receptor through influencing cellular differentiation, vasodilation, inhibition of cellular proliferation and hypertrophy, nitric oxide production and natriuresis. Thus, the pursuit of a specific AT₂ receptor agonist is a potentially fruitful area for combating renal and cardiovascular diseases. This review focuses on the role of the AT₂ receptor in the kidney.

Studying the angiotensin type 2 receptor (AT₂) has been problematic in the past because a pharmacological tool for direct, specific in vitro and in vivo stimulation of the receptor has been lacking. Consequently, current knowledge about AT₂ receptor signalling and function had to be obtained by indirect approaches, like studying animals or cells with genetically altered AT₂ receptor expression levels, inhibitory experiments using specific AT₂ receptor antagonists, stimulation with angiotensin II under concomitant angiotensin II type 1 receptor blockade or stimulation with the peptide agonist CGP42112A, which has additional AT₂ receptor antagonistic properties. The recently developed non-peptide AT₂ receptor agonist Compound 21 now, for the first time, allows direct, selective and specific AT₂ receptor stimulation in vitro and in vivo. This new tool will certainly revolutionise AT₂ receptor research, enable many new insights into AT₂ receptor function and may also have the potential to become a future medical drug. This article reviews milestone findings about AT₂ receptor functional properties obtained by ‘conventional’ experimental approaches within the last 20 years. Moreover, it provides an overview of the first results obtained by direct AT₂ receptor stimulation with Compound 21, comprising effects on alkaline secretion, neurite outgrowth, blood pressure and post-infarct cardiac function.

Angiotensin II (Ang II) is considered the major final mediator of the renin-angiotensin system. The actions of Ang II have been implicated in many cardiovascular conditions, such as hypertension, atherosclerosis, coronary heart disease, restenosis, and heart failure. Ang II can act through two different receptors: Ang II type 1 (AT₁) receptor and Ang II type 2 (AT₂) receptor. The AT₁ receptor is ubiquitously expressed in the cardiovascular system and mediates most of the physiological and pathophysiological actions of Ang II. The AT₂ receptor is highly expressed in the developing foetus, but its expression is very low in the cardiovascular system of the normal adult. Expression of the AT₂ receptor can be modulated by pathological states associated with tissue remodelling or inflammation such as hypertension, atherosclerosis, and myocardial infarction. The precise role of the AT₂ receptor remains under debate. However, it appears that the AT₂ receptor plays a vasodilatory role, and may be enhanced as a countervailing mechanism in cardiac hypertrophy, and in presence of vascular injury in hypertension and atherosclerosis. Signalling pathways induced by the stimulation of the AT₂ receptor are poorly understood, but three main mechanisms have been described: (a) activation of protein phosphatases causing protein dephosphorylation; (b) activation of bradykinin/nitric oxide/cyclic guanosine 3’,5’-monophosphate pathway; and (c) stimulation of phospholipase A₂ and release of arachidonic acid. Vasodilatory effects of the AT₂ receptor, probably the only well-established role of the AT₂ receptor, have been attributed to the second of these mechanisms. The participation of the AT₂ receptor in cardiovascular remodelling and inflammation is more controversial. In vitro, AT₂ receptor stimulation clearly inhibits cardiac and vascular smooth muscle growth and proliferation, and stimulates apoptosis. In vivo, the situation is less clear, and depending on the studies, the AT₂ receptor appears to be required for cardiac hypertrophic growth or contrariwise, the AT₂ receptor has demonstrated no effects on cardiac hypertrophy. Similar controversial findings have been reported in atherosclerosis. Here we discuss the role of the AT₂ receptor on cardiovascular structure and disease, and the signalling pathways induced by its activation.

Considerable progress in our understanding of the role of the angiotensin II type 2 (AT₂) receptor in the development of cardiac hypertrophy and coronary artery disease has been achieved using in vitro and in vivo animal models. Our understanding in humans, however, has been hindered by the lack of availability of specific AT₂ receptor agonists and antagonists suitable for human study. Nevertheless, an alternative approach involving genotyping humans for a functional polymorphism within the AT₂ receptor gene (–1332G/A) has been used in several association studies to elucidate the pathogenic role of the AT₂ receptor in cardiovascular disease. Both the A allele and the G allele have independently been associated with left ventricular remodelling. However, the methods of measuring left ventricular mass, sodium balance, age and degree of remodelling appear to influence the outcome. An association of carriers of the G allele and premature coronary artery disease has also been established, particularly in males presenting with stenotic atherosclerosis requiring revascularisation. At the molecular level, it remains unclear as to whether carriers of the G allele express more or fewer AT₂ receptors when compared to carriers of the A allele. Consequently, it is presently not possible to definitively interpret the role of the AT₂ receptor in human cardiovascular disease from these association studies.

The renin-angiotensin system (RAS) is well known for its vital involvement in body fluid homeostasis and circulation. However, very little research has been devoted to the impact of this regulatory system on the gastrointestinal (GI) system. This is surprising because the GI tract is fundamental for the intake and excretion of fluid and electrolytes (and nutrients), and it accommodates a large proportion of bodily haemodynamics and host defence systems. The RAS is well expressed and active in the GI tract, although the exact roles for the key mediator angiotensin II (Ang II) and its receptors in general, and the type 2 (AT₂) receptor in particular, are not completely settled. There are several reports showing Ang II regulation of intestinal fluid and electrolyte transport. For example, mucosaprotective duodenal bicarbonate-rich secretion is inhibited by Ang II via type 1 (AT₁) receptor-mediated facilitation of sympathoadrenergic activity, but this secretory process can also be stimulated by Ang II via AT₂ receptors. Novel data from human oesophagus and jejunum suggest that the AT₁ receptor mediates muscular contractions and that the AT₂ receptor regulates epithelial functions. Data are accumulating suggesting involvement of AT₁ and AT₂ receptors in GI inflammation and carcinogenesis. The picture of the RAS and AT₂ receptor in the GI tract is, however, far from complete. Much more basic research is needed with regard to GI pathophysiology before concluding clinical significance and potential applicability of pharmacological interferences with the RAS.

Recent clinical studies indicate that blockade of the renin-angiotensin system is important to prevent stroke, and accumulating results of basic research also indicate the possible involvement of the central renin-angiotensin system in ischaemic brain damage and cognition. When the angiotensin II type 1 receptor is blocked by an angiotensin type 1 receptor blocker, unbound angiotensin II acts preferentially on the angiotensin II type 2 (AT₂) receptor. These results suggest the pathophysiological importance of the AT₂ receptor in the clinical use of angiotensin type 1 receptor blockers, which are widely used in patients with hypertension with the expectation of a decrease in the onset of cardiovascular and cerebrovascular disease. We review here the possible roles of AT₂ receptor activation in the brain, focusing on ischaemic stroke, cognitive function and neurogenesis, and potential effects of specific AT₂ receptor agonists.

The angiotensin AT2 receptor (AT2R) represents an important component of the renin-angiotensin system since it is involved in the (patho) physiology of different cardiovascular and neuronal diseases. Furthermore, AT2 receptors can partly mediate beneficial effects of angiotensin AT1 receptor (AT1R) blockers, and direct pharmacological AT2 receptor agonism emerges as a novel therapeutic strategy. This review discusses the constitutive and ligand-mediated activity as well as the signal transduction of the AT2 receptor, focusing on adapter proteins which directly bind to this receptor. Direct protein-protein interaction partners of the AT2 receptor described so far include the transcription factor promyelocytic zinc finger protein, AT2 receptor binding protein and the AT1 receptor. In addition, the putative crosstalk of the AT2 receptor with the renin/prorenin receptor (RER) via the promyelocytic zinc finger protein (PLZF) and the role of oestrogens on the regulation of the AT2 receptor are presented.Conceiving the coupling of the AT2 receptor to different adapter proteins with distinct and partly opposing cellular effects and the implications of its constitutive activity might help to overcome the current controversies on the (patho)physiological role of the AT2 receptor.

Introduction. The angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) polymorphism is associated with ACE activity and has been discussed as a risk factor for pre-eclampsia. Disturbances of uteroplacental circulation are involved in the pathogenesis of pre-eclampsia. In this study, we tested whether the ACE I/D genotype is associated with history of foetal loss (FL) or uteroplacental dysfunction (UPD).

Patients and methods. ACE I/D genotype was determined in 312 women presenting with a history of FL and 112 women admitted because of UPD. The association of the ACE I/D genotype with FL or UPD was assessed in a case-control study using 527 patients with diagnoses other than FL or UPD. To exclude potential biases due to associations of this genotype with other diagnoses, we additionally performed a case-control study using 553 healthy controls.

Results. ACE I/D genotype was significantly associated with history of FL in both case-control studies (patient controls: odds ratio 1.52, p<0.02; healthy controls: odds ratio 1.48, p=0.02). There was no evidence for allele-dose dependency. No association of the ACE I/D genotype with UPD could be detected.

Conclusions. The ACE I/D genotype exhibits a statistically significant association with a history of FL. These results corroborate an involvement of the renin-angiotensin system in pregnancy complications.

Introduction. We applied pressure stress to human aortic endothelial cells (HAEC) and investigated whether mechanical pressure stress and/or angiotensin II (Ang II) affected angiotensin-converting enzyme (ACE) 2. We then tested whether the administration of nifedipine had a demonstrable and possibly beneficial effect.

Methods. A pulsatile atmospheric pressure with or without Ang II was loaded on HAECs. The expression of ACE2 was studied by immunoblots and reverse transcription/real-time polymerase chain reaction.

Results. The pulsatile mechanical pressure increased the expression of ACE2 mRNA by approximately 80%. Supplementation of Ang II (1 µM) with pulsatile mechanical pressure decreased the expression of ACE2 mRNA by approximately 54%. Pulsatile atmospheric pressure increased ACE2 protein, but supplementation of Ang II (1 µM) also increased ACE2 protein, and the latter failed to show significant change compared to pressurized control without Ang II. Ang II administration reduced ACE2 protein in the membranous fraction under pressurized condition. Administration of nifedipine (1 µM) protected cells from this ACE2 protein reduction at the HAEC membrane.

Conclusions. These results indicate that pulsatile mechanical pressure and Ang II affect ACE2 in HAECs. Our findings suggest that blood pressure reduction with a calcium channel blocker is beneficial for the conservation of ACE2, and may provide a potential therapeutic target beyond blood pressure lowering in hypertensive patients.

Introduction. High basal renin-angiotensin system (RAS) activity is associated with increased risk of severe hypoglycaemia in type 1 diabetes. We tested whether this might be explained by more pronounced cognitive dysfunction during hypoglycaemia in patients with high RAS activity than in patients with low RAS activity.

Materials and methods. Nine patients with type 1 diabetes and high and nine with low RAS activity were subjected to hypoglycaemia and euglycaemia in a cross-over study using an intravenous insulin infusion protocol. Cognitive function, electroencephalography, auditory evoked potentials and hypoglycaemic symptoms were recorded.

Results. At a hypoglycaemic nadir of 2.2 (SD 0.3) mmol/L the high RAS group displayed significant deterioration in cognitive performance during hypoglycaemia in the three most complex reaction time tasks. In the low RAS group, hypoglycaemia led to cognitive dysfunction in only one reaction time task. The high RAS group reported lower symptom scores during hypoglycaemia than the low RAS group, suggesting poorer hypoglycaemia awareness.

Conclusion. High RAS activity is associated with increased cognitive dysfunction and blunted symptoms during mild hypoglycaemia compared to low RAS activity. This may explain why high RAS activity is a risk factor for severe hypoglycaemia in type 1 diabetes.

Combining an angiotensin-converting enzyme inhibitor (ACE-I) and angiotensin II receptor blocker (ARB) lowers blood pressure (BP) by 4/3 mmHg compared to either agent alone, although this additive effect may be abolished with maximal monotherapy dosing. The recent ONTARGET study showed no reduction in primary outcomes when an ACE-I-ARB combination was compared to an ACE-I alone, despite 2.4/1.4 mmHg lower BP in the former group. In proteinuric chronic kidney disease, an ACE-I-ARB combination reduces proteinuria and disease progression more than monotherapy, but the ONTARGET study showed an increase in renal endpoints in the combined group. Aliskiren offers a novel approach to renin-angiotensin system (RAS) inhibition. As monotherapy in hypertension, aliskiren is of similar efficacy to thiazides, calcium channel blockers and ARBs. In combination with other RAS inhibitors at maximal dosage aliskiren has a small synergistic effect on BP (additional 4/2 mmHg reduction). Early data suggest a role for aliskiren in preventing end-organ damage but, considering the ONTARGET results with an ACE-I-ARB combination, outcome studies are needed before the use of aliskiren can be recommended in combination with other RAS inhibitors. As monotherapy, aliskiren should probably be reserved for use as an alternative to ACE-Is or ARBs, where these are ineffective or poorly tolerated.

Introduction. Primary aldosteronism (PA) is caused by autonomous hypersecretion of aldosterone from the adrenal cortex, classically from an adenoma, resulting in sodium and water retention, hypokalaemia and raised blood pressure. The sodium and water retention causes suppression of renin release. The possible cardiac sequelae of aldosterone excess are encountered primarily in patients with secondary hyperaldosteronism due to heart failure, where plasma renin, angiotensin and aldosterone levels are all raised. However, there is also evidence that primary aldosterone excess, in the presence of low renin levels, may also be cardiotoxic.

Patients. In this report, we describe five patients with PA, who developed atrial fibrillation (AF) in the absence of structural cardiac lesions and in one case despite good control of blood pressure and electrolytes.

Conclusion. In patients with hypertension and AF, who have no evidence of coronary disease or any other underlying cause of AF with preserved systolic function, a diagnosis of PA should be considered.