Renin. Renin is the major determinant of the rate of AngII production. It is synthesized, stored, and secreted by exocytosis into the renal arterial circulation by the granular juxtaglomerular cells (Figure 26–2) located in the walls of the afferent arterioles that enter the glomeruli. Renin is an aspartyl protease that cleaves the bond between residues 10 and 11 at the amino terminus of angiotensinogen to generate AngI. The active form of renin is a glycoprotein that contains 340 amino acids. It is synthesized as a preproenzyme of 406 amino acid residues that is processed to prorenin. Prorenin is proteolytically activated by proconvertase 1 or cathepsin B enzymes that remove 43 amino acids (propeptide) from its amino terminus to uncover the active site of renin (Figure 26–3). The active site of renin is located in a cleft between the two homologous lobes of the enzyme. Nonproteolytic activation of prorenin, central to the activation of local (tissue) RAS, occurs when prorenin binds to the prorenin/renin ((pro)renin) receptor, resulting in conformational changes that unfold the propeptide and expose the active catalytic site of the enzyme. (Danser et al., 2005). Both renin and prorenin are stored in the juxtaglomerular cells and, when released, circulate in the blood. The concentration of prorenin in the circulation is ~10-fold greater than that of the active enzyme. The t1/2 of circulating renin is ~15 minutes.
Control of Renin Secretion. The secretion of renin from juxtaglomerular cells is controlled predominantly by three pathways (Figure 26–2):
The first mechanism is the macula densa pathway. The macula densa lies adjacent to the juxtaglomerular cells and is composed of specialized columnar epithelial cells in the wall of that portion of the cortical thick ascending limb that passes between the afferent and efferent arterioles of the glomerulus. A change in NaCl reabsorption by the macula densa results in the transmission to nearby juxtaglomerular cells of chemical signals that modify renin release. Increases in NaCl flux across the macula densa inhibit renin release, whereas decreases in NaCl flux stimulate renin release. ATP, adenosine, and prostaglandins modulate the macula densa pathway. ATP and adenosine are released when NaCl transport increases ATP acts on P2Y receptors to inhibit renin release. Adenosine acts via the A1 adenosine receptor to inhibit renin release. Prostaglandins (PGE2, PGI2) are released when NaCl transport decreases and stimulate renin release through enhancing cyclic AMP formation. Prostaglandin production is stimulated by inducible cyclooxygenase-2 (COX-2). COX-2 and neuronal nitric oxide synthase (nNOS) participate in the mechanism of macula densa–stimulated renin release. The expression of COX-2 and nNOS is upregulated by chronic dietary Na+ restriction; selective inhibition of either COX-2 or nNOS inhibits renin release. The nNOS/NO pathway, in part, may mediate increases in COX-2 expression induced by a low-Na+ diet; however, COX-2 expression in the macula densa is not attenuated in nNOS knockout mice, which suggests that other mechanisms can compensate for nNOS in the regulation of COX-2.
Regulation of the macula densa pathway is more dependent on the luminal concentration of Cl− than Na+. NaCl transport into the macula densa is mediated by the Na+−K+−2Cl− symporter (Figure 26–2B), and the half-maximal concentrations of Na+ and Cl− required for transport via this symporter are 2-3 and 40 mEq/L, respectively. Because the luminal concentration of Na+ at the macula densa usually is much greater than the level required for half-maximal transport, physiological variations in luminal Na+ concentrations at the macula densa have little effect on renin release (i.e., the symporter remains saturated with respect to Na+). On the other hand, physiological changes in Cl− concentrations (20-60 mEq/L) at the macula densa profoundly affect macula densa– mediated renin release.
The second mechanism controlling renin release is the intrarenal baroreceptor pathway. Increases and decreases in blood pressure or renal perfusion pressure in the preglomerular vessels inhibit and stimulate renin release, respectively. The immediate stimulus to secretion is believed to be reduced tension within the wall of the afferent arteriole. The release of renal prostaglandins and biomechanical coupling via stretch-activated ion channels may mediate in part the intrarenal baroreceptor pathway (Wang et al., 1999).
The third mechanism, the β adrenergic receptor pathway, is mediated by the release of norepinephrine from postganglionic sympathetic nerves; activation of β1 receptors on juxtaglomerular cells enhances renin secretion.
The three mechanisms regulating renin release are embedded in a feedback regulation (Figure 26–2A). Increased renin secretion enhances the formation of AngII, which stimulates AT1 receptors on juxtaglomerular cells to inhibit renin release, an effect termed short-loop negative feedback. AngII increases arterial blood via AT1 receptors; this effect inhibits renin release by:
activating high-pressure baroreceptors, thereby reducing renal sympathetic tone
increasing pressure in the preglomerular vessels
reducing NaCl reabsorption in the proximal tubule (pressure natriuresis), which increases tubular delivery of NaCl to the macula densa
The inhibition of renin release owing to AngII-induced increases in blood pressure has been termed long-loop negative feedback.
The physiological pathways regulating renin release are influenced by arterial blood pressure, dietary salt intake, and a number of pharmacological agents (Figure 26–2A). Loop diuretics stimulate renin release by decreasing arterial blood pressure and by blocking the reabsorption of NaCl at the macula densa. Nonsteroidal anti-inflammatory drugs (NSAIDs; see Chapter 34) inhibit prostaglandin synthesis and thereby decrease renin release. ACE inhibitors, angiotensin receptor blockers (ARBs), and renin inhibitors interrupt both the short- and long-loop negative feedback mechanisms and therefore increase renin release. Increasing cyclic AMP in the juxtaglomerular cells stimulates renin. Centrally acting sympatholytic drugs (see Chapter 12), as well as β adrenergic receptor antagonists, decrease renin secretion by reducing activation of β adrenergic receptors on juxtaglomerular cells.
Angiotensinogen. The substrate for renin is angiotensinogen, an abundant globular glycoprotein (MW = 55,000-60,000). AngI is cleaved from the amino terminus of angiotensinogen. The human angiotensinogen contains 452 amino acids and is synthesized as preangiotensinogen, which has a 24– or 33–amino acid signal peptide. Angiotensinogen is synthesized and secreted primarily by the liver, although angiotensinogen transcripts also are abundant in fat, certain regions of the central nervous system (CNS), and the kidneys. Angiotensinogen synthesis is stimulated by inflammation, insulin, estrogens, glucocorticoids, thyroid hormone, and AngII. During pregnancy, plasma levels of angiotensinogen increase several-fold owing to increased estrogen.
Circulating levels of angiotensinogen are approximately equal to the Km of renin for its substrate (~1 μM). Consequently, the rate of AngII synthesis, and therefore blood pressure, can be influenced by changes in angiotensinogen levels. For instance, knockout mice lacking angiotensinogen are hypotensive, and there is a progressive relationship among the number of copies of the angiotensinogen gene, plasma levels of angiotensinogen, and arterial blood pressure. Oral contraceptives containing estrogen increase circulating levels of angiotensinogen and can induce hypertension. A missense mutation in the angiotensinogen gene (a methionine to threonine at position 235 of angiotensinogen) that increases plasma levels of angiotensinogen is associated with essential and pregnancy-induced hypertension (Sethi et al., 2003). Angiotensinogen shares sequence homologies that have anti-angiogenic properties with the serpin protein family (Célérier et al., 2002).
Angiotensin-Converting Enzyme (ACE, Kininase II, Dipeptidyl Carboxypeptidase). ACE is an ectoenzyme and glycoprotein with an apparent molecular weight of 170,000. Human ACE contains 1277 amino acid residues and has two homologous domains, each with a catalytic site and a Zn2+-binding region. ACE has a large amino-terminal extracellular domain, a short carboxyl-terminal intracellular domain, and a 17–amino acid hydrophobic region that anchors the ectoenzyme to the cell membrane. ACE is rather nonspecific and cleaves dipeptide units from substrates with diverse amino acid sequences. Preferred substrates have only one free carboxyl group in the carboxyl-terminal amino acid, and proline must not be the penultimate amino acid; thus, the enzyme does not degrade AngII. ACE is identical to kininase II, the enzyme that inactivates bradykinin and other potent vasodilator peptides. Although slow conversion of AngI to AngII occurs in plasma, the very rapid metabolism that occurs in vivo is due largely to the activity of membrane-bound ACE present on the luminal surface of endothelial cells throughout the vascular system.
The ACE gene contains an insertion/deletion polymorphism in intron 16 that explains 47% of the phenotypic variance in serum ACE levels. The deletion allele, associated with higher levels of serum ACE and increased metabolism of bradykinin, may confer an increased risk of hypertension, cardiac hypertrophy, atherosclerosis, and diabetic nephropathy (Hadjadj et al., 2001) but may be protective against Alzheimer's disease (Sayed-Tabatabaei et al., 2006; Castrop et al., 2010).
Angiotensin-Converting Enzyme 2. Two groups independently discovered a novel ACE-related carboxypeptidase, now termed ACE2 (Donoghue et al., 2000; Tipnis et al., 2000). Human ACE2 is 805 amino acids in length with a short putative signal sequence. ACE2 contains a single catalytic domain that is 42% identical to the two catalytic domains of ACE. ACE2 cleaves one amino acid from the carboxyl terminal to convert AngI to Ang(1–9) and AngII to Ang(1–7). AngII is the preferred substrate for ACE2 with 400-fold higher affinity than AngI. The physiological significance of ACE2 is still uncertain; it may serve as a counter-regulatory mechanism to oppose the effects of ACE. ACE2 regulates the levels of AngII and limits its effects by converting it to Ang(1–7), which binds to Mas receptors and elicits vasodilator and anti-proliferative responses (Varagic et al., 2008). ACE2 is not inhibited by the standard ACE inhibitors and has no effect on bradykinin. In animals, reduced expression of ACE2 is associated with hypertension, defects in cardiac contractility, and elevated levels of AngII (Crackower et al., 2002). Overexpression of the ACE2 gene prevents AngII-induced cardiac hypertrophy in hypertensive rats (Huentelman et al., 2005). ACE2 also serves as a receptor for the SARS coronavirus (Li et al., 2003).
Angiotensin Peptides. When given intravenously, AngI is rapidly converted to AngII. However, AngI per se is less than 1% as potent as AngII on smooth muscle, the heart, and the adrenal cortex. Angiotensin III (AngIII), also called Ang(2–8), can be formed either by the action of aminopeptidase on AngII or by the action of ACE on Ang(2–10). AngII and AngIII cause qualitatively similar effects. AngII and AngIII stimulate aldosterone secretion with equal potency; however, AngIII is only 25% and 10% as potent as AngII in elevating blood pressure and stimulating the adrenal medulla, respectively.
Ang(1-7) is formed by multiple pathways (Figure 26–1). AngI can be converted to Ang(1–7) by endopeptidases. AngII can be converted to Ang(1–7) by prolylcarboxypeptidase (Ferrario et al., 1997). ACE2 converts AngI to Ang(1–9) and AngII to Ang(1–7); ACE metabolizes Ang(1-9) to Ang(1–7). In animal models, Ang(1–7) opposes many of the effects of AngII: it induces vasodilation, promotes NO production, potentiates the vasodilatory effects of bradykinin, and inhibits AngII-induced activation of ERK1/2; it has anti-angiogenic, anti-proliferative, and anti-thrombotic effects; and is cardioprotective in cardiac ischemia and heart failure. The effects of Ang(1–7) are mediated by a specific Mas receptor. The Mas proto-oncogene encodes an orphan G protein–coupled receptor (Santos et al., 2008; Varagic et al., 2008). Ferrario and colleagues (1997) proposed that Ang(1–7) serves to counterbalance the actions of AngII, which may depend on ACE-AngII/ACE2-Ang(1–7) activity ratio (Santos et al. 2008). ACE inhibitors increase tissue and plasma levels of Ang(1–7), both because AngI levels are increased and diverted away from AngII formation (Figure 26–1) and because ACE contributes to the plasma clearance of Ang(1–7). AT1 receptor blockade boosts the levels of AngII that is converted to Ang(1–7) by ACE2.
Angiotensin IV (AngIV), also called Ang(3–8), is formed from AngIII through the catalytic action of aminopeptidase M and has potent effects on memory and cognition. Central and peripheral actions of AngIV are mediated through specific AT4 receptors identified as insulin-regulated amino peptidases (IRAPs). AngIV binding to AT4 receptors inhibits the catalytic activity of IRAPs and enables accumulation of various neuropeptides linked to memory potentiation. Other actions include renal vasodilation, natriuresis, neuronal differentiation, hypertrophy, inflammation, and extracellular matrix remodeling (Ruiz-Ortega et al., 2007). Analogs of angiotensin IV are being developed for their therapeutic potential in cognition in Alzheimer disease or head injury (Albiston et al., 2007).
Angiotensinases. These include aminopeptidases, endopeptidases, carboxypeptidases, and other peptidases that degrade and inactivate angiotensin peptides; none is specific.
Local (Tissue) Renin–Angiotensin Systems. The traditional view of the RAS as classical endocrine system is oversimplified. The modern view of the RAS also includes the local (tissue) RAS. Local (tissue) RAS is an AngII-producing system that is being recognized for its role in hypertrophy, inflammation, remodeling, and apoptosis. Activation of (tissue) RAS and local AngII production require the binding of renin or prorenin to the specific (pro)renin receptor (PRR), located on cell surfaces. In this regard, it is important to distinguish between extrinsic and intrinsic local RAS:
Extrinsic Local RAS. ACE is present on the luminal face of vascular endothelial cells throughout the circulation, and circulating renin of renal origin can be taken up by the arterial wall and by other tissues (Danser et al., 1994).
Intrinsic Local RAS. Many tissues—including the brain, pituitary, blood vessels, heart, kidney, and adrenal gland—express mRNAs for renin, angiotensinogen, and/or ACE, and various cells cultured from these tissues produce renin, angiotensinogen, ACE, and angiotensins I, II, and III. Thus, it appears that local RASs exist independently of the renal/hepatic-based system and may influence vascular, cardiac, and renal function and structure (Paul et al., 2006).
The (Pro)Renin Receptor. The PRR is the functional receptor, located on the cell surface, that binds prorenin and renin with high affinity (KD ~6 and 20 nM, respectively) and specificity (Batenburg and Danser, 2008). Human PRR is a 350–amino acid protein that shares no homology with any membrane protein. The PRR gene is located in the locus p11.4 of the X chromosome and is named ATP6ap2 (ATPase-associated protein). Knockout of the PRR gene is lethal. In humans, mutations in the PRR gene are associated with mental retardation and epilepsy, suggesting an important role in cognition, brain development, and survival (Nguyen and Danser, 2008; Ramser et al., 2005). Prorenin and renin also bind to mannose-6-phosphate receptor (M6P), an insulin-like growth factor II receptor that functions as a clearance receptor.
Binding of (pro)renin to PRR enhances the catalytic activity of renin by 4-5 fold and induces non-proteolytic activation of prorenin (Figure 26-3). Bound, activated (pro)renin catalyzes the conversion of angiotensinogen to AngI, which is subsequently converted to AngII by ACE located on the cell surface. Locally produced AngII binds to AT1 receptors and activates intracellular signaling events that regulate cell growth, collagen deposition, fibrosis, inflammation, and apoptosis (Nguyen and Danser, 2008).
The binding of (pro)renin to PRR also induces AngII-independent signaling events that include activation of ERK1/2, p38, tyrosine kinases, TGF-β gene expression, and plasminogen activator inhibitor type 1 (PAI-1). These signaling pathways are not blocked by ACE inhibitors or AT1 receptor antagonists and are reported to contribute to fibrosis, nephrosis, and organ damage (Kaneshiro et al., 2007; Nguyen and Danser, 2008; see Figure 26-4). PRR is abundant in the heart, brain, eye, adrenals, placenta, adipose tissue, liver, and kidneys. Overexpression of the human PRR in transgenic animals increases plasma aldosterone levels in the absence of changes in plasma renin levels and induces hypertension and nephropathy. Rats overexpressing PRR exhibit increased expression of COX-2 in the macula densa and develop proteinuria and glomerulosclerosis that increase with aging (Kaneshiro et al., 2006; Kaneshiro et al., 2007).
Prorenin is no longer considered the inactive precursor of renin. Prorenin is capable of activating local (tissue) RAS and AngII-dependent and independent events that may contribute to organ damage. Circulating plasma concentrations of prorenin are 10-fold higher than renin in healthy subjects but are elevated to 100-fold in diabetic patients and are associated with increased risk of nephropathy, renal fibrosis, and retinopathy (Danser and Deinum, 2005; Nguyen and Danser, 2008). The interaction of prorenin with PRR has become a target for therapeutic interventions.
Alternative Pathways for Angiotensin Biosynthesis. Angiotensinogen may be converted to AngI or directly to AngII by cathepsin G and tonin. Other enzymes that convert AngI to AngII include cathepsin G, chymostatin-sensitive AngII-generating enzyme, and heart chymase. Chymase contributes to the local tissue conversion of AngI to AngII, particularly in the heart and kidneys (Paul et al., 2006).
Angiotensin Receptors. AngII and AngIII couple to specific GPCRs designated AT1 and AT2 (de Gasparo et al., 2000). The AT1 receptor has a 10,000-fold higher affinity for losartan (and related biphenyl tetrazole derivatives) than the AT2 receptor. The AT1 receptor (359 amino acids) and the AT2 receptor (363 amino acids) share 34% sequence homology.
Most of the known biological effects of AngII are mediated by the AT1 receptor. The AT1 receptor gene contains a polymorphism (A-to-C transversion in position 1166) associated with hypertension, hypertrophic cardiomyopathy, and coronary artery vasoconstriction. Moreover, the C allele synergizes with the ACE deletion allele with regard to increased risk of coronary artery disease (Álvarez et al., 1998). Preeclampsia is associated with the development of agonistic auto-antibodies against the AT1 receptor (Wallukat et al., 1999).
Functional roles for the AT2 receptors are less well defined, but they may counterbalance many of the effects of the AT1 receptors by having antiproliferative, proapoptotic, vasodilatory, natriuretic, and antihypertensive effects (Jones et al., 2008; Carey et al., 2001). The AT2 receptor is distributed widely in fetal tissues, but its distribution is more restricted in adults. Although the AT2 receptor generally is conceptualized as a cardiovascular protective receptor, its overexpression and activation may contribute to myocyte hypertrophy and cardiac fibrosis (D'Amore et al., 2005; Ichihara et al., 2001). Expression of AT2 receptors is upregulated in cardiovascular diseases, including heart failure, cardiac fibrosis, and ischemic heart disease; however, the significance of increased AT2 receptor expression is unclear (Jones et al., 2008).
The Mas receptor mediates the effects of Ang(1–7), which include vasodilation and anti-proliferation. Deletion of the Mas gene in transgenic mice reveals cardiac dysfunction (Santos et al., 2008).
The AT4 receptor (IRAP; see Figure 26-2) mediates the effects of AngIV. This receptor is a single transmembrane protein (1025 amino acids) that co-localizes with the glucose transporter GLUT4. AT4 receptors are detectable in a number of tissues, such as heart, vasculature, adrenal cortex, and brain regions processing sensory and motor functions (Chai et al., 2004).
Angiotensin Receptor–Effector Coupling. AT1 receptors activate a large array of signal-transduction systems to produce effects that vary with cell type and that are a combination of primary and secondary responses. AT1 receptors couple to several heterotrimeric G proteins, including Gq, G12/13, and Gi. In most cell types, AT1 receptors couple to Gq to activate the PLCβ–IP3–Ca2+ pathway. Secondary to Gq activation, activation of PKC, PLA2, and PLD and eicosanoid production, as well as activation of Ca2+-dependent and MAP kinases and the Ca2+–calmodulin–dependent activation of NOS may occur. Activation of Gi may occur and will reduce the activity of adenylyl cyclase, lowering cellular cyclic AMP content; however, there also is evidence for Gq → Gs cross-talk such that activation of the AT1–Gq–PLC pathway enhances cyclic AMP production (Meszaros et al., 2000; Epperson et al., 2004). The βγ subunits of Gi and activation of G12/13 lead to activation of tyrosine kinases and small G proteins such as Rho. Ultimately, the JAK/STAT pathway may be activated and a variety of transcriptional regulatory factors induced. By these mechanisms, angiotensin influences the expression of a host of gene products relating to cell growth and the production of components of the extracellular matrix. AT1 receptors also stimulate the activity of a membrane-bound NADH/NADPH oxidase that generates reactive oxygen species (ROS). ROS may contribute to biochemical effects (activation of MAP kinase, tyrosine kinase, and phosphatases; inactivation of NO; and expression of monocyte chemoattractant protein-1) and physiological effects (acute effects on renal function, chronic effects on blood pressure, and vascular hypertrophy and inflammation) (Mehta and Griendling, 2007; Higuchi et al., 2007). The relative importance of these myriad signal-transduction pathways in mediating biological responses to AngII is tissue specific. The presence of other receptors may alter the response to AT1 receptor activation. For example, AT1 receptors heterodimerize with bradykinin B2 receptors, a process that enhances AngII sensitivity in preeclampsia (AbdAlla et al., 2002).
Less is known about AT2 receptor–effector coupling. Signaling from AT2 receptors is mediated by G protein-dependent and independent pathways. Consequences of AT2 receptor activation include activation of phosphoprotein phosphatases, K+ channels, synthesis of NO and cyclic GMP, bradykinin production, and inhibition of Ca2+ channel functions (Jones et al., 2008). AT2 receptors may possess constitutive activity. Overexpression of AT2 receptors has been reported to induce NO production in vascular smooth muscle cells and hypertrophy in cardiac myocytes through an intrinsic activity of the AT2 receptor independent of angiotensin binding (D'Amore et al., 2005; Jones et al., 2008). Homo-oligomerization of AT2 receptors also has been reported to induce apoptosis (Miura et al., 2005). The AT2 receptor may bind directly to and antagonize the AT1 receptor (AbdAlla et al., 2001) and can form heterodimers with the bradykinin B2 receptor to enhance NO production (Abadir, 2006).