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The rise in blood pressure produced by injection of kidney extracts is due to renin, an acid protease secreted by the kidneys into the bloodstream. This enzyme acts in concert with angiotensin-converting enzyme (ACE) to form angiotensin II (Figure 38–6). It is a glycoprotein with a molecular weight of 37,326 in humans. The molecule is made up of two lobes, or domains, between which the active site of the enzyme is located in a deep cleft. Two aspartic acid residues, one at position 104 and one at position 292 (residue numbers from human preprorenin), are juxtaposed in the cleft and are essential for activity. Thus, renin is an aspartyl protease.
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Like other hormones, renin is synthesized as a large preprohormone. Human preprorenin contains 406 amino acid residues. The prorenin that remains after removal of a leader sequence of 23 amino acid residues from the amino terminal contains 383 amino acid residues, and after removal of the pro sequence from the amino terminal of prorenin, active renin contains 340 amino acid residues. Prorenin has little if any biologic activity.
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Some prorenin is converted to renin in the kidneys, and some is secreted. Prorenin is also secreted by other organs, including the ovaries. After nephrectomy, the prorenin level in the circulation is usually only moderately reduced and may actually rise, but the level of active renin falls to essentially zero. Thus, very little prorenin is converted to renin in the circulation, and active renin is a product primarily, if not exclusively, of the kidneys. Prorenin is secreted constitutively, whereas active renin is formed in the secretory granules of the granular cells in the juxtaglomerular apparatus, the same cells that produce renin (see below). Active renin has a half-life in the circulation of 80 min or less. Its only known function is to cleave the decapeptide angiotensin I from the amino terminal end of angiotensinogen (renin substrate) (Figure 38–7).
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Circulating angiotensinogen is found in the α2-globulin fraction of the plasma (Figure 38–6). It contains about 13% carbohydrate and is made up of 453 amino acid residues. It is synthesized in the liver with a 32-amino-acid signal sequence that is removed in the endoplasmic reticulum. Its circulating level is increased by glucocorticoids, thyroid hormones, estrogens, several cytokines, and angiotensin II.
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ANGIOTENSIN-CONVERTING ENZYME & ANGIOTENSIN II
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ACE is a dipeptidyl carboxypeptidase that splits off histidyl-leucine from the physiologically inactive angiotensin I, forming the octapeptide angiotensin II (Figure 38–7). The same enzyme inactivates bradykinin (Figure 38–6). Increased tissue bradykinin produced when ACE is inhibited acts on B2 receptors to produce the cough that is an annoying side effect in up to 20% of patients treated with ACE inhibitors (see Clinical Box 38–2). Most of the converting enzyme that forms angiotensin II in the circulation is located in endothelial cells. Much of the conversion occurs as the blood passes through the lungs, but conversion also occurs in many other parts of the body.
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ACE is an ectoenzyme that exists in two forms: a somatic form found throughout the body and a germinal form found solely in postmeiotic spermatogenic cells and spermatozoa (see Chapter 23). Both forms have a single transmembrane domain and a short cytoplasmic tail. However, somatic ACE is a 170-kDa protein with two homologous extracellular domains, each containing an active site. Germinal ACE is a 90-kDa protein that has only one extracellular domain and active site. Both enzymes are formed from a single gene. However, the gene has two different promoters, producing two different mRNAs. In male mice in which the ACE gene has been knocked out, blood pressure is lower than normal, but in females it is normal. In addition, fertility is reduced in males but not in females.
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CLINICAL BOX 38–2 Pharmacologic Manipulation of the Renin-Angiotensin System
It is now possible to inhibit the secretion or the effects of renin in a variety of ways. Inhibitors of prostaglandin synthesis such as indomethacin and β-adrenergic blocking drugs such as propranolol reduce renin secretion. The peptide pepstatin and newly developed renin inhibitors such as enalkiren prevent renin from generating angiotensin I. ACE inhibitors such as captopril and enalapril prevent conversion of angiotensin I to angiotensin II. Saralasin and several other analogs of angiotensin II are competitive inhibitors of the action of angiotensin II on both AT1 and AT2 receptors. Losartan (DuP-753) selectively blocks AT1 receptors, and PD-123177 and several other drugs selectively block AT2 receptors.
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METABOLISM OF ANGIOTENSIN II
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Angiotensin II is metabolized rapidly; its half-life in the circulation in humans is 1–2 min. It is metabolized by various peptidases. An aminopeptidase removes the aspartic acid (Asp) residue from the amino terminal of the peptide (Figure 38–7). The resulting heptapeptide has physiologic activity and is sometimes called angiotensin III. Removal of a second amino terminal residue from angiotensin III produces the hexapeptide sometimes called angiotensin IV, which is also said to have some activity. Most, if not all, of the other peptide fragments that are formed are inactive. In addition, aminopeptidase can act on angiotensin I to produce (des-Asp1) angiotensin I, and this compound can be converted directly to angiotensin III by the action of ACE. Angiotensin-metabolizing activity is found in red blood cells and many tissues. In addition, angiotensin II appears to be removed from the circulation by some sort of trapping mechanism in the vascular beds of tissues other than the lungs.
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Renin is usually measured by incubating the sample to be assayed and measuring by immunoassay the amount of angiotensin I generated. This measures the plasma renin activity (PRA) of the sample. Deficiency of angiotensinogen as well as renin can cause low PRA values, and to avoid this problem, exogenous angiotensinogen is often added, so that plasma renin concentration (PRC) rather than PRA is measured. The normal PRA in supine subjects eating a normal amount of sodium is approximately 1 ng of angiotensin I generated per milliliter per hour. The plasma angiotensin II concentration in such subjects is about 25 pg/mL (approximately 25 pmol/L).
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ACTIONS OF ANGIOTENSINS
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Angiotensin I appears to function solely as the precursor of angiotensin II and does not have any other established action.
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Angiotensin II produces arteriolar constriction and a rise in systolic and diastolic blood pressure. It is one of the most potent vasoconstrictors known, being four to eight times as active as norepinephrine on a weight basis in normal individuals. However, its pressor activity is decreased in Na+-depleted individuals and in patients with cirrhosis and some other diseases. In these conditions, circulating angiotensin II is increased, and this down-regulates the angiotensin receptors in vascular smooth muscle. Consequently, there is less response to injected angiotensin II.
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Angiotensin II also acts directly on the adrenal cortex to increase the secretion of aldosterone, and the renin-angiotensin system is a major regulator of aldosterone secretion. Additional actions of angiotensin II include facilitation of the release of norepinephrine by a direct action on postganglionic sympathetic neurons, contraction of mesangial cells with a resultant decrease in GFR (see Chapter 37), and a direct effect on the renal tubules to increase Na+ reabsorption.
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Angiotensin II also acts on the brain to decrease the sensitivity of the baroreflex, and this potentiates the pressor effect of angiotensin II. In addition, it acts on the brain to increase water intake and increase the secretion of vasopressin and ACTH. It does not penetrate the blood-brain barrier, but it triggers these responses by acting on the circumventricular organs, four small structures in the brain that are outside the blood-brain barrier (see Chapter 33). One of these structures, the area postrema, is primarily responsible for the pressor potentiation, whereas two of the others, the subfornical organ (SFO) and the OVLT, are responsible for the increase in water intake (dipsogenic effect). It is not certain which of the circumventricular organs are responsible for the increases in vasopressin and ACTH secretion.
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Angiotensin III [(des-Asp1) angiotensin II] has about 40% of the pressor activity of angiotensin II, but 100% of the aldosterone-stimulating activity. It has been suggested that angiotensin III is the natural aldosterone-stimulating peptide, whereas angiotensin II is the blood pressure–regulating peptide. However, this appears not to be the case, and instead angiotensin III is simply a breakdown product with some biologic activity. The same is probably true of angiotensin IV, though some researchers have argued that it has unique effects in the brain.
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TISSUE RENIN-ANGIOTENSIN SYSTEMS
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In addition to the system that generates circulating angiotensin II, many different tissues contain independent renin-angiotensin systems that generate angiotensin II, apparently for local use. Components of the renin-angiotensin system are found in the walls of blood vessels and in the uterus, the placenta, and the fetal membranes. Amniotic fluid has a high concentration of prorenin. In addition, tissue renin-angiotensin systems, or at least several components of the renin-angiotensin system, are present in the eyes, exocrine portion of the pancreas, heart, fat, adrenal cortex, testis, ovary, anterior and intermediate lobes of the pituitary, pineal, and brain. Tissue renin contributes very little to the circulating renin pool, because PRA falls to undetectable levels after the kidneys are removed. The functions of these tissue renin-angiotensin systems are unsettled, though evidence is accumulating that angiotensin II is a significant growth factor in the heart and blood vessels. ACE inhibitors or AT1 receptor blockers are now the treatment of choice for heart failure, and part of their value may be due to inhibition of the growth effects of angiotensin II.
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ANGIOTENSIN II RECEPTORS
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There are at least two classes of angiotensin II receptors. AT1 receptors are serpentine receptors coupled by a G-protein (Gq) to phospholipase C, and angiotensin II increases the cytosolic free Ca2+ level. It also activates numerous tyrosine kinases. In vascular smooth muscle, AT1 receptors are associated with caveolae (see Chapter 2), and AII increases production of caveolin-1, one of the three isoforms of the protein that is characteristic of caveolae. In rodents, two different but closely related AT1 subtypes, AT1A and AT1B, are coded by two separate genes. The AT1A subtype is found in blood vessel walls, the brain, and many other organs. It mediates most of the known effects of angiotensin II. The AT1B subtype is found in the anterior pituitary and the adrenal cortex. In humans, an AT1 receptor gene is present on chromosome 3. There may be a second AT1 type, but it is still unsettled whether distinct AT1A and AT1B subtypes occur.
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There are also AT2 receptors, which are coded in humans by a gene on the X chromosome. Like the AT1 receptors, they have seven transmembrane domains, but their actions are different. They act via a G-protein to activate various phosphatases which in turn antagonize growth effects and open K+ channels. In addition, AT2 receptor activation increases the production of NO and therefore increases intracellular cyclic 3,5-guanosine monophosphate (cGMP). The overall physiologic consequences of these second-messenger effects are unsettled. AT2 receptors are more plentiful in fetal and neonatal life, but they persist in the brain and other organs in adults.
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The AT1 receptors in the arterioles and the AT1 receptors in the adrenal cortex are regulated in opposite ways: an excess of angiotensin II down-regulates the vascular receptors, but it up-regulates the adrenocortical receptors, making the gland more sensitive to the aldosterone-stimulating effect of the peptide.
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THE JUXTAGLOMERULAR APPARATUS
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The renin in kidney extracts and the bloodstream is produced by the juxtaglomerular cells (JG cells). These epitheloid cells are located in the media of the afferent arterioles as they enter the glomeruli (Figure 38–8). The membrane-lined secretory granules in them have been shown to contain renin. Renin is also found in agranular lacis cells that are located in the junction between the afferent and efferent arterioles, but its significance in this location is unknown.
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At the point where the afferent arteriole enters the glomerulus and the efferent arteriole leaves it, the tubule of the nephron touches the arterioles of the glomerulus from which it arose. At this location, which marks the start of the distal convolution, there is a modified region of tubular epithelium called the macula densa (Figure 38–8). The macula densa is in close proximity to the JG cells. The lacis cells, the JG cells, and the macula densa constitute the juxtaglomerular apparatus.
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REGULATION OF RENIN SECRETION
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Several different factors regulate renin secretion (Table 38–2), and the rate of renin secretion at any given time is determined by the summed activity of these factors. One factor is an intra-renal baroreceptor mechanism that causes renin secretion to decrease when arteriolar pressure at the level of the JG cells increases and to increase when arteriolar pressure at this level falls. Another renin-regulating sensor is in the macula densa. Renin secretion is inversely proportional to the amount of Na+ and Cl− entering the distal renal tubules from the loop of Henle. Presumably, these electrolytes enter the macula densa cells via the Na–K–2Cl− transporters in their apical membranes, and the increase in some fashion triggers a signal that decreases renin secretion in the juxtaglomerular cells in the adjacent afferent arterioles. A possible mediator is NO, but the identity of the signal remains unsettled. Renin secretion also varies inversely with the plasma K+ level, but the effect of K+ appears to be mediated by the changes it produces in Na+ and Cl− delivery to the macula densa.
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Angiotensin II feeds back to inhibit renin secretion by a direct action on the JG cells. Vasopressin also inhibits renin secretion in vitro and in vivo, although there is some debate about whether its in vivo effect is direct or indirect.
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Finally, increased activity of the sympathetic nervous system increases renin secretion. The increase is mediated both by increased circulating catecholamines and by norepinephrine secreted by postganglionic renal sympathetic nerves. The catecholamines act mainly on β1-adrenergic receptors on the JG cells and renin release is mediated by an increase in intracellular cAMP.
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The principal conditions that increase renin secretion in humans are listed in Table 38–3. Most of the listed conditions decrease central venous pressure, which triggers an increase in sympathetic activity, and some also decrease renal arteriolar pressure (see Clinical Box 38–3). Renal artery constriction and constriction of the aorta proximal to the renal arteries produces a decrease in renal arteriolar pressure. Psychological stimuli increase the activity of the renal nerves.
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CLINICAL BOX 38–3 Role of Renin in Clinical Hypertension
Constriction of one renal artery causes a prompt increase in renin secretion and the development of sustained hypertension (renal or Goldblatt hypertension). Removal of the ischemic kidney or the arterial constriction cures the hypertension if it has not persisted too long. In general, the hypertension produced by constricting one renal artery with the other kidney intact (one-clip, two-kidney Goldblatt hypertension) is associated with increased circulating renin. The clinical counterpart of this condition is renal hypertension due to atheromatous narrowing of one renal artery or other abnormalities of the renal circulation. However, plasma-renin activity is usually normal in one-clip one-kidney Goldblatt hypertension. The explanation of the hypertension in this situation is unsettled. However, many patients with hypertension respond to treatment with ACE inhibitors or losartan even when their renal circulation appears to be normal and they have normal or even low plasma-renin activity.