Physiology and Regulation of the Renin–Angiotensin–Aldosterone System

1

The Classical Circulating Renin–Angiotensin System

The classical renin–angiotensin system (RAS) ( Fig. 1.1 ) begins with the biosynthesis of the glycoprotein hormone, renin, by the juxtaglomerular (JG) cells of the renal afferent arteriole ( Fig. 1.2 ). Renin is encoded by a single gene, and renin mRNA is translated into preprorenin, containing 401 amino acids. In the JG cell endoplasmic reticulum, a 20-amino-acid signal peptide is cleaved from preprorenin, leaving prorenin, which is packaged into secretory granules in the Golgi apparatus, where it is further processed into “active” renin by severance of a 46-amino-acid peptide from the N-terminal region of the molecule. Mature, “active” renin is a glycosylated carboxypeptidase with a molecular weight of ∼44 kDa. “Active” renin is released from the JG cell by a process of exocytosis involving stimulus-secretion coupling. In contrast, “inactive” prorenin is released constitutively across the cell membrane. Prorenin is converted to “active” renin by a trypsin-like proteolytic activation step.

In the past, renin has been considered to have no intrinsic biological activity, serving solely as an enzyme catalytically cleaving angiotensinogen (Agt), the only known precursor of Ang peptides, to form the decapeptide Ang I ( Fig. 1.1 ). Liver-derived Agt provides the majority of systemic circulating angiotensin (Ang) peptides, but Agt is also synthesized and constitutively released in other tissues, including the heart, vasculature, kidney, and adipose tissue. Angiotensin-converting enzyme (ACE), a glycoprotein (molecular weight 180 kDa) with two active carboxy-terminal enzymatic sites, hydrolyzes the inactive Ang I into biologically active Ang II ( Fig. 1.1 ). ACE exists in two molecular forms: soluble and particulate. ACE is localized on the plasma membranes of various cell types, including vascular endothelial cells, the apical brush border (microvilli) of epithelial cells (e.g., renal proximal tubule cells), and neuroepithelial cells. In addition to cleaving Ang I to Ang II, ACE metabolizes bradykinin (BK), an active vasodilator and natriuretic autacoid, to BK (1-7), an inactive metabolite ( Fig. 1.1 ). ACE, therefore, increases the production of a potent vasoconstrictor, Ang II, while simultaneously degrading a vasodilator, BK. ACE also metabolizes substance P into inactive fragments.

Unlike renin and Agt, which have relatively long plasma half-lives, Ang II and other Ang peptides are degraded within seconds by peptidases, collectively termed angiotensinases, at different amino acid sites, to form fragments, mainly des-aspartyl ¹ -Ang II (Ang III), Ang (1-7), and Ang (3-8) (Ang IV). Ang II is converted to Ang III by aminopeptidase A, and Ang III is converted to Ang IV by aminopeptidase N (APN). However, the expression levels and functional significance of these two critical enzymes, especially at the tissue level, are not completely understood, and the functional role of the peptide fragments produced is largely unknown.

The vast majority of cardiovascular, renal, and adrenal actions of Ang II are mediated by the Ang type-1 (AT ₁ ) receptor, a seven-transmembrane G protein–coupled receptor that is widely distributed in these tissues, which is coupled positively to protein kinase C and negatively coupled to adenylyl cyclase. As shown in Fig. 1.3 , AT ₁ receptors mediate vascular smooth muscle cell contraction, aldosterone secretion, thirst, sympathetic nervous system stimulation, renal tubular Na ⁺ reabsorption, and cardiac ionotropic and chronotropic responses, among many other actions. Ang II also binds to another cloned receptor, the Ang type-2 (AT ₂ ) receptor, but until recently the cell signaling mechanisms and functions of the AT ₂ receptor were unknown.

2

Renin Biosynthesis and Secretion

Renin catalytic cleavage of Agt is the rate-limiting biochemical step in the RAS. The renal JG cell is thought to be the only source of circulating renin because following bilateral nephrectomy renin quantitatively disappears from the circulation. However, nephrectomy does not alter circulating levels of prorenin, indicating that nonrenal tissues (e.g., adrenal glands, testes, ovaries, placenta, and eyes) both produce and secrete prorenin into the circulation. In addition, many organs, such as the heart, can take up renin from the circulation by unclear mechanisms (see Chapter 14 ).

The primary means by which the RAAS contributes to acute changes in extracellular fluid volume and BP homeostasis is by varying the level of renin in the circulation. This process is mediated by active renin release from secretory granules of JG cells. A primary mechanism of renin release is the afferent arteriolar baroreceptor, which increases renin release when arterial (and renal) perfusion pressure decreases and vice versa. In addition, JG cells are innervated by sympathetic neurons, the activation of which stimulates norepinephrine release and subsequent stimulation of β ₁ -adrenergic receptors triggering renin release. Therefore, as shown in Fig. 1.4 , β ₁ -adrenergic receptor blockade suppresses renin release by direct action at JG cells. JG cells also express both AT ₁ and AT ₂ receptors, and circulating Ang II participates in a short-loop negative feedback mechanism to inhibit renin release by binding to both of these receptors. Conversely, blockade of the RAS increases renin release and circulating renin levels (see Fig. 1.5 for ACE inhibitors and Fig. 1.6 for AT ₁ receptor blockers). Indeed, chronic RAS blockade by AT ₁ receptor antagonists or ACE inhibitors induces recruitment of new renin-secreting cells beyond the JG apparatus in the renal microvasculature, further augmenting renin secretion. Another renin secretory control mechanism is the macula densa segment of the early distal tubule, which relays a signal to the JG cell to increase renin release when a reduction in Na ⁺ and/or Cl ⁻ in the distal tubule is detected.

3

The (Pro)Renin Receptor

Although renin has been considered as the enzyme responsible for cleaving the decapeptide Ang I from substrate Agt and has been thought to have no direct biological actions, recent studies demonstrate that renin can bind to human glomerular mesangial cell membranes in culture and that binding causes cell hypertrophy and increased levels of plasminogen activator inhibitor. The bound renin is not internalized or degraded. A (pro)renin receptor (PRR) has now been cloned from mesangial cells, and its functional significance remains in the process of being clarified. The receptor is a 350-amino-acid protein with a single transmembrane domain that specifically binds both renin and prorenin ( Fig. 1.7 ). Binding induces the activation of the extracellular signal-related mitogen-activated protein (MAP) kinases (ERK 1 and ERK 2) associated with serine and tyrosine phosphorylation and a fourfold increase in the catalytic conversion of Agt to Ang I ( Fig. 1.8 ). Activation of the PRR directly by prorenin or renin increases vacuolar H ⁺ -ATPase activity and ATP-dependent proton pumps that acidify intracellular compartments, including lysosomes, endosomes, and synaptic vesicles. The receptor is localized on renal mesangial cell membranes, the apical membranes of cortical collecting duct intercalated cells, and in the subendothelial layer of both coronary and renal arteries associated with vascular smooth muscle cells and colocalizes with renin. The receptor is also expressed in visceral adipocytes. In renal mesangial cells, the PRR mediates transforming growth factor-β production via MAP kinase phosphorylation ( Fig. 1.9 ). In addition, MAP kinase activation occurs in collecting duct cells, vascular smooth muscle cells, monocytes, and neurons resulting in increased cell proliferation, production of transforming growth factor β1, upregulation of profibrotic factors plasminogen activator inhibitor-1, fibronectin, and collagen. Importantly, prorenin- and renin-PRR interaction requires pharmacological prorenin and renin concentrations; prorenin transgenic animals display an Ang II-dependent phenotype and PRR deletion is lethal, casting doubt as to whether prorenin- and renin-PRR interaction occurs in normal physiology. Although the possibility of a direct biological role of renin and prorenin via the PRR exists, the functional importance of this receptor other than catalytic conversion of Agt to Ang I awaits further investigation.

4

Angiotensin-Converting Enzyme

ACE inactivates two vasodilator peptides, BK and kallidin. BK is both a direct and an indirect vasodilator via stimulation of NO and cGMP and also by the release of vasodilator prostaglandins, PGE ₂ and prostacyclin. Thus, when an ACE inhibitor is employed ( Fig. 1.5 ), not only the synthesis of Ang II is inhibited but also the formation of BK, NO, and prostaglandins is facilitated. ACE inhibition induces cross talk between the BK B ₂ receptor and ACE on the plasma membrane, abrogating B ₂ receptor desensitization and potentiating both the levels of BK and the vasodilator action of BK at its B ₂ receptor. Also, in the presence of ACE inhibition an alternative pathway of Ang II production via chymase may be activated ( Fig. 1.5 ). The chymase pathway may serve as a major route of Ang II formation in the heart, especially in the presence of ACE inhibition.

5

The ACE-2/Angiotensin (1-7)/ Mas Receptor Pathway

A second ACE, ACE-2, has recently been discovered ( Fig. 1.10 ). ACE-2 is a zinc metalloproteinase consisting of 805 amino acids with significant sequence homology to ACE. Unlike ACE, however, ACE-2 functions as a carboxypeptidase rather than a dipeptidyl-carboxypeptidase. In contrast to ACE, ACE-2 hydrolyzes Ang I to Ang (1-9), but the major pathway is the conversion of Ang II to Ang (1-7) ( Fig. 1.10 ). ACE-2 also degrades BK to [des-Arg ⁹ ]-BK, an inactive metabolite. In marked contrast to ACE, ACE-2 does not convert Ang I to Ang II and its enzyme activity is not blocked with ACE inhibitors. Thus, ACE-2 is effectively an inhibitor of Ang II formation by stimulating alternate pathways for Ang I and, particularly, Ang II degradation. ACE-2 has been localized to the cell membranes of cardiac myocytes, renal endothelial and tubule cells, and the testis. ACE-2 gene ablation does not alter BP but impairs cardiac contractility and induces increased Ang II levels, suggesting that ACE-2 may at least partially nullify the physiological actions of ACE.

The heptapeptide fragment of Ang II and Ang (1-7) ( Fig. 1.10 ) has been discovered to have biological activity (see Chapter 14 for additional details). Ang (1-7) can be formed directly from Ang I by a two-step process involving conversion to Ang (1-9) by ACE-2 followed by conversion to Ang (1-7) by endopeptidases. However, as stated above, the major pathway for Ang (1-7) formation is directly from Ang II by the action of ACE-2 ( Fig. 1.10 ). Interestingly, the major catabolic pathway for inactivation of Ang (1-7) is by ACE ( Fig. 1.10 ). Thus, ACE inhibitor administration markedly increases the level of Ang (1-7). The kidney is a major target organ for Ang (1-7). Although a specific Ang (1-7) receptor has not been cloned, the peptide is an endogenous agonist for the Mas oncogene, which mediates the majority of its actions ( Fig. 1.10 ). Ang (1-7) binds to and activates the mas receptor, inducing phosphatidylinositol 3-kinase/Akt pathway leading to activation of endothelial NO synthase, the consequent NO release–inducing vasodilation. The peptide is formed in the kidney, where it has specific actions via a non-AT ₁ or non-AT ₂ receptor. These actions include increased GFR, inhibition of Na/K/ATPase, vasorelaxation, natriuresis, diuresis, and downregulation of AT ₁ receptors, all of which are blocked by the specific Ang (1-7) antagonist (D-Ala ⁷ )-Ang (1-7) and are mediated at least in part by NO and prostacyclin. Most of these renal effects of Ang (1-7) oppose those of Ang II via the AT ₁ receptor.

6

AT ₁ Receptors

Ang II, the major effector peptide of the RAS, binds to two major receptors AT ₁ and AT ₂ , which generally oppose each other. The AT ₁ receptor is widely distributed in the vasculature, heart, and kidney. Actions of Ang II mediated by the AT ₁ receptor include vasoconstriction; SNS activation; aldosterone, vasopressin, and endothelin secretion; plasminogen activator inhibitor biosynthesis; platelet aggregation; thrombosis; cardiac contractility; superoxide formation; VSM growth; and collagen formation ( Fig. 1.3 ). These actions are conducted by both G protein–coupled and G protein–independent pathways and involve phospholipases C, A ₂ , and D activation; increased intracellular Ca ⁺⁺ and inositol 1,4,5-trisphosphate; activation of MAP kinases; ERKs and the JAK/STAT pathway; enhanced protein phosphorylation; and stimulation of early growth response genes. Tyrosine phosphorylation and stimulation of MAP kinase phosphorylation are the major intracellular signaling pathways for the AT ₁ receptor. Ang II, via AT ₁ receptors, activates c-SRC generating reactive oxygen species (ROS) via NADPH oxidase (NOX1). Many of the detrimental tissue effects of Ang II, including vascular smooth muscle contraction, hyperplasia/hypertrophy, fibrosis, and inflammation, involve the actions of ROS on these intracellular signaling pathways.