Brenner and Rector's The Kidney, 8th ed.

CHAPTER 10. Vasoactive Peptides and the Kidney

Riccardo Candido   Louise M. Burrell   Karin A.M. Jandeleit-Dahm   Mark E. Cooper



Renin-Angiotensin System, 333



Angiotensinogen, 334



Renin, 334



Angiotensin-Converting Enzyme, 336



Angiotensins and Angiotensinases, 338



Angiotensin II Receptors, 338



Renin-Angiotensin System Knockout or Transgenic Models, 340



Physiologic Effects of the Renin-Angiotensin System, 341



Intrarenal Renin-Angiotensin System, 343



Pathophysiologic Effects of the Renin-Angiotensin System in the Kidney, 343



Future Perspectives of the Renin-Angiotensin System, 345



Endothelin, 346



Structure, 346



Synthesis and Secretion, 346



Physiologic Actions of Endothelin on the Kidney, 346



Endothelin and Renal Pathophysiology, 347



Summary, 349



Urotensin II, 349



Role in the Kidney, 350



Summary, 350



Kallikrein-Kinin System, 350



Kininogens, 351



Kallikreins and Kallikrein Inhibitors, 351



Kinin Generation, 351



Kinin Receptors, 352



Kininases, 352



Physiologic Functions of the Kallikrein-Kinin System: Focus on the Kidney, 352



Kallikrein-Kinin System Function in Renal Diseases, 353



Summary, 354



The Natriuretic Peptides, 354



Atrial Natriuretic Peptide—Structure, Processing, and Synthesis, 354



Brain Natriuretic Peptide, 355



Natriuretic Peptide Receptors, 355



Clearance Receptor, 355



Neutral Endopeptidase, 355



Renal Actions of the Natriuretic Peptides, 355



Therapeutic Uses of the Natriuretic Peptides, 355



Summary, 356


The renin-angiotensin system (RAS) cascade has been viewed historically as an integral component of cardiovascular and renal regulation primarily to maintain and modulate blood pressure and water and sodium balance. Recently, the RAS has proved to be an important regulator of cardiovascular and renal structure and function, in addition to salt and water balance.

The RAS has been implicated in the pathophysiology of various diseases including hypertension, cardiac hypertrophy, and myocardial infarction as well as various progressive renal diseases. [1] [2] [3] Of particular interest are newly described roles for the RAS in other situations such as retinal neovascularization[4] and hepatic fibrosis.[5] For this reason, there has been a major effort in the past several years to understand the molecular and cellular mechanisms governing the biosynthesis and the activity of the RAS components, so that novel means might be developed to control their activities. In the classic view of the RAS ( Fig. 10-1 ), the glycoprotein angiotensinogen is secreted into the circulation by the liver, where it is cleaved by renin, an aspartyl protease produced by the juxtaglomerular (JG) cells, to release the decapeptide angiotensin (Ang) I. This peptide is an inactive intermediate, and it is further processed by angiotensin-converting enzyme (ACE), a metalloprotease, into the eight-amino acid peptide Ang II. Ang II binds to high-affinity cell surface receptors, the most well known are the Ang II subtype 1 (AT1) and Ang II subtype 2 (AT2) receptors, which cause a remarkably diverse range of physiologic effects.[6] In addition, Ang II can be formed via non-ACE and nonrenin enzymes including chymase, cathepsin G, cathepsin A, chymostatin-sensitive Ang II-generated enzyme (CAGE), tissue plasminogen activator, and tonin.[7] Ang II induces vascular smooth muscle constriction and raises peripheral vascular resistance. In response to Ang II, renal proximal tubular epithelium increases absorption of salt and water. Within the adrenal gland, zona glomerulosa cells are stimulated to produce aldosterone, which in turn regulates sodium reabsorption from the distal tubule. Ang II elevates the resistance of both efferent and afferent arterioles in the kidney and increases filtration fraction. In the heart, Ang II is associated with positive inotropy and in the brain, it induces a variety of responses including the onset of thirst and increased vasopressin release.[8] These and other actions of Ang II act to increase intracellular volume, peripheral vascular resistance, and blood pressure. In addition to its effects on fluid homeostasis, Ang II has been found to have a myriad of local tissue influences, ranging from growth and repair to a role in ovulation.[9]



FIGURE 10-1  Enzymatic cascade of the RAS: classic and alternative pathways. CAGE, chymostatin-sensitive angiotensin II-generated enzyme; t-PA, tissue plasminogen activator.



Given the physiologic diversity of Ang II, it is not surprising that pharmaceutical companies sought inhibitors of this vasoactive peptide at the level of synthesis and action. Three strategies have already been developed: inhibition of the enzymatic action of renin, inhibition of the enzymatic action of ACE, and competitive inhibition of the binding of Ang II to cell surface receptors. ACE inhibitors and, increasingly, Ang II receptor antagonists are now widely prescribed for the treatment of hypertension, heart failure, myocardial infarction, diabetic nephropathy, and other proteinuric renal diseases.

During the development of angiotensin II receptor antagonists, peptidic and nonpeptidic inhibitors were developed that discriminate between the two major classes of angiotensin II receptors (AT1 and AT2).[10] Whereas the AT2inhibitors are still under experimental investigation, the AT1 receptor blockers have been extensively evaluated in large clinical trials and have been shown to demonstrate significant beneficial end-organ effects not only in hypertension but also in both cardiac[11] and renal diseases.

In addition to the circulating RAS, there are complete RASs within a variety of tissues and organs, the functions of which are quite varied. Local synthesis of all the components of the RAS has been demonstrated within the kidney. Messenger RNA (mRNA) for both angiotensinogen and renin is found in the JG cells and renal tubular cells.[12] AT1 receptors are found on the efferent and afferent arterioles of the glomerulus as well as in mesangial and tubular epithelial cells. Expression of AT2 receptor mRNA is highly localized to interlobular arteries. However, emulsion autoradiography and immunohistochemical staining have recently localized AT2 receptors in the glomeruli and proximal tubules, [13] [14] albeit at much lower levels. Multiple functions have been proposed for the intrarenal RAS.[15] Ang II constriction of the afferent arteriole would have the effect of reducing glomerular flow and glomerular capillary pressure, with a corresponding decrease in glomerular filtration rate (GFR). Conversely, constriction of the efferent arteriole, while also reducing flow, would increase glomerular pressure and GFR. The action of Ang II on mesangial cells results in a morphologic appearance of contraction with a corresponding decrease in the glomerular capillary ultrafiltration coefficient. In the proximal tubule, Ang II regulates sodium and pH balance through modulation of the activity of the sodium/hydrogen (Na+/H+) antiporter. Indeed, there is much compelling evidence that locally produced components of the RAS may play an important role in both the physiology and the pathophysiology of renal function.

This chapter discusses each component of the RAS and its physiologic and pathophysiologic roles in the kidney.


Plasma angiotensinogen is the source of Ang I in all animal species. Physical isolation of this protein has shown that it is heterogeneous in molecular weight (52-60 kDa). The variance in molecular size appears to stem from a difference in glycosylation.

The human angiotensinogen gene is 12 kilobases long, consisting of five exons and four introns, and is present as a single copy in the human genome.[16] Human angiotensinogen has 452 and rat has 453 predicted amino acids.

The liver is the primary site of angiotensinogen mRNA and protein synthesis. Angiotensinogen mRNA expression has also been demonstrated in the central nervous system, kidney, heart, vascular tissues, adrenal glands, fat, and leukocytes.

In the kidney, both in situ studies and reverse transcription-polymerase chain reaction (RT-PCR) reveal that angiotensinogen gene expression is most abundant in the cortex, primarily within the proximal tubule, with smaller amounts in the glomerulus and even less in the outer and inner medulla. [12] [17]

Adrenal angiotensinogen mRNA is most abundant in the zona glomerulosa, consistent with local angiotensin II production being involved in the regulation of aldosterone production.

In the heart, both atria and ventricles have lower levels of angiotensinogen mRNA.[18] Angiotensinogen is found in cerebrospinal fluid, and this is the source of locally produced cerebrospinal fluid Ang II, which appears to be involved in the regulation of thirst and blood pressure through effects on paraventricular structures.

Like α1-antitrypsin, angiotensinogen is an acute-phase reactant and production by the liver is markedly elevated in response to stresses such as bacterial infection and tissue injury. Finally, Ang II, the final active product of the RAS, participates in a positive feedback loop that stimulates the hepatic production of angiotensinogen. This feedback loop would have the effect that during periods of high Ang II production, the peptide stimulates production of its own precursor protein to ensure constant availability of Ang II. Ang II also increases angiotensinogen mRNA production in the kidney and liver,[19] whereas renin appears to inhibit angiotensinogen release.


Renin is produced and stored in granular JG cells, which are modified smooth muscle cells found in the media of afferent arterioles. [2] [20] Renin is synthesized as an inactive precursor form, preprorenin. Cleavage of the signal peptide from the carboxyl terminus of preprorenin results in prorenin, which is also considered to be biologically inactive. Subsequent glycosylation and proteolytic cleavage leads to the formation of renin, a 37- to 40-kDa proteolytic enzyme. Both prorenin and renin are secreted from JG cells. Because prorenin is the major circulating form, it is postulated that significant conversion of prorenin to renin follows secretion. Prorenin-activating enzymes have been localized to neutrophils, endothelial cells, and the kidney.[2] In addition to JG cells, renin production has also been detected in the submandibular gland, liver, brain, prostate, testis, ovary, spleen, pituitary, thymus, and lung.[2] Circulating renin, however, appears to be derived almost entirely from the kidney.

Within the circulation in humans, active renin cleaves a leucine-valine bond within angiotensinogen ( Fig. 10-2 ) to form the decapeptide Ang I. Based on measurements of the enzymatic activity of renin, several investigators have suggested that inhibitors of renin are present in plasma. Indeed, a number of renin-inhibiting substances such as phospholipids, neutral lipids, unsaturated fatty acids, and synthetic analogs of natural renin substrate have been identified.[21] Recently, Nguyen and colleagues have reported, for the first time, the existence of a functional receptor of renin that was localized to the mesangium of glomeruli.[22] The renin receptor is able to trigger intracellular signalling by activating the mitogen-activated protein (MAP) kinase (ERK1 and ERK2) pathway. It also acts as a cofactor by increasing the efficiency of angiotensinogen cleavage by receptor-bound renin, thereby facilitating Ang II generation and action on a cell surface.[22] These findings emphasize the role of the cell surface in Ang II generation and open a new perspective on renin effects, independent of Ang II.



FIGURE 10-2  Comparison of the structure of the RAS components and sites of enzymic cleavage.



Renin Secretion

The majority of renin production occurs in the JG apparatus, where these specialized smooth muscle cells of afferent arterioles contain electron-dense granules that are the major storage sites for renin.[2] Renin-containing cells are found throughout the afferent glomerular arteriole, with greatly increased density near the glomerular hilum, hence the term JG cells.[2] Direct secretion of renin into the afferent arteriolar plasma appears to be facilitated by the fenestrated endothelium overlying JG cells. Such fenestrations are a feature commonly observed in endocrine organs, supporting the concept of the JG apparatus as an endocrine structure. Between the afferent and the efferent glomerular arterioles at the glomerular hilum lies a region containing lacelike (lacis) cells (also termed Goormaghtigh cells or extraglomerular mesangium), the so-called polkisen. The extraglomerular mesangium is in direct contact with both the macula densa region of the ascending limb of the loop of Henle and the intraglomerular mesangium. Lacis cells have numerous cell processes extending from their ends and have connecting gap junctions suggesting electrical coupling to each other and to cells of the glomerular mesangium and glomerular arterioles. The region of the macula densa of the thick ascending limb of Henle (so named because of its appearance of tightly packed nuclei) is in close apposition with the lacis cells as well as the cells of afferent and efferent glomerular arterioles. However, macula densa cells and lacis cells are not joined by gap junctions, despite their close relationship. Rather, the intercellular matrix between macula densa and lacis cells appears continuous, and the basolateral surface of macula densa cells is irregular, with spaces between the cells varying in size, depending on the rate of fluid reabsorption at this site. The renal vasculature and tubules are richly innervated, yet nerve endings do not seem to make direct contact with macula densa or lacis cells. The possible role of innervation to the adjacent vascular or ascending limb cells in the function of the JG apparatus remains to be fully elucidated.

Several mechanisms are involved in the control of renin secretion ( Table 10-1 ).

TABLE 10-1   -- Mechanisms Regulating Renin Secretion

Renal baroreceptors

Mechanisms involving the macula densa

Neural mechanisms

Endocrine and paracrine mechanisms

Intracellular mechanisms




Renal Baroreceptors.

In the kidney, renin secretion is controlled by at least two independent mechanisms: a renal baroreceptor and the macula densa. Renin secretion is inhibited by increased pressure or stretch within the afferent arteriole. By contrast, renin secretion increases in response to decreased stretch. In support of this concept, alterations in renal perfusion pressure were shown to result in changes in renal renin release, even when the confounding influences of the macula densa mechanism and renal innervation were eliminated. It is thought that diminished JG cell stretch hyperpolarizes the cells, resulting in a fall in intracellular calcium concentration and increase renin release.

Mechanisms Involving the Macula Densa.

Renin secretion is also related to the composition of tubule fluid at the macula densa.[23] Renal arterial infusions of sodium chloride inhibit renin secretion. Volume expansion with sodium chloride has a more profound inhibitory effect on renin secretion than comparable expansion with dextran, presumably because of an effect of sodium chloride at the macula densa. Initial studies assumed sodium dependence of renin secretion, but later observations have suggested that suppression of plasma renin activity (PRA) by administration of sodium is dependent upon the concomitant administration of chloride (Cl-). In fact, sodium fails to suppress PRA when administered with other anions. Similarly, PRA is suppressed by potassium chloride and choline chloride but not by potassium bicarbonate or lysine glutamate. Sodium chloride transport, rather than load, appears to be the important signal. Macula densa cells do not have direct contact with the renin-secreting granular cells in the afferent arteriolar wall. Thus, ion transport plus subsequent second-messenger signaling is necessary. In addition, it has been proposed that adenosine released from adenosine triphosphate (ATP) hydrolysis in macula densa cells serves as the chemical signal that inhibits renin release. Another influence may be the fluid resorption into the lacis cells leading to a stretch receptor mechanism controlling renin release. Mesangial cells appear to contain voltage-activated calcium (Ca2+) channels, as well as Ca2+-activated Cl- channels, so that changes in extracellular Cl- concentration might directly affect granular cells. Indeed, whole cell patch-clamp studies have shown a large Ca2+-activated Cl- conductance in plasma membrane JG granular cells. Arachidonic acid metabolites may also be important in renin release, and arachidonic acid infusion increases PRA (see later). It also appears that nitric oxide (NO) may modulate renin release from the macula densa.[23]

Neural Mechanisms.

Renin release is modulated by the central nervous system, primarily via the sympathetic nervous system. Nerve terminals are present on the JG apparatus, and renin secretion is stimulated by electrical stimulation of the renal nerves, by infusion of catecholamines, and by increasing sympathetic nervous system activity. Based primarily on experiments with adrenergic antagonists and agonists, the neural component of renin secretion appears to be mediated by beta-adrenergic receptors, specifically beta1-receptors.[21] Beta-adrenergic stimulation of renin release appears to involve activation of adenylate cyclase and the formation of cyclic adenosine monophosphate (cAMP).

Endocrine and Paracrine Mechanisms.

Several endocrine and paracrine hormones regulate renin secretion by the kidney. Arachidonic acid, prostaglandin E2 (PGE2), 13,14-dihydro-PGE2 (a metabolite of PGE2) and prostacyclin stimulate renin secretion from renal cortical slices in vitro and from both filtering and nonfiltering denervated kidneys in vivo.[21]

Renin release is also stimulated by other agonists that act through cAMP, namely histamine, parathyroid hormone, glucagon, and dopamine.[21] Whether or how these agonists play a role in the day-to-day physiologic control of renin release is still not fully understood.

Atrial natriuretic peptide (ANP) has been shown to inhibit renin release from isolated JG cells (see later). Other inhibitory hormones include vasopressin, endothelin, and adenosine. [20] [23] It has been postulated that adenosine may serve as the macula densa-derived signal that suppresses renin release in response to enhanced solute transport by ascending limb cells. Inhibition of renin release by endothelin suggests a possible paracrine regulation of renin release.[20]

Regulation of renin secretion by Ang II is probably the most physiologically relevant.[24] Ang II inhibits renin secretion and renin gene expression in a negative feedback loop. Treatment of transgenic mice bearing the human renin gene with an ACE inhibitor increases renin expression in the kidney by 5- to 10-fold.[25] Similarly, ACE inhibition in rats augments renal renin mRNA expression, an effect that is reversed by infusion of Ang II.[26] Indeed, ACE inhibition has been shown to be associated with a marked increase in cells with a JG phenotype expressing renin protein.[27] The effects of Ang II on renin are believed to be direct and not dependent on changes in renal hemodynamics or tubular transport.

Intracellular Mechanisms.

Most investigators have shown that increased extracellular Ca2+ concentrations inhibit renin secretion both in vitro and in vivo and attenuate stimulation of renin release by catecholamines. As previously reported, ANP inhibits renin release. The mechanisms whereby ANP and NO, and thus cyclic guanosine monophosphate (cGMP), inhibit renin release need further clarification. The sum effect of ANP on renin release probably depends on the integration between a variety of concomitant stimuli that either augment or inhibit renin release.

Renin secretion is invariably augmented by agonists that stimulate adenylate cyclase activity in JG cells. The finding of a cAMP-responsive element in the renin gene and the finding that forskolin, a diterpene that directly activates adenylate cyclase activity, markedly enhances renin release further indicate that cAMP is an important second messenger in renin release. Renin release in vitro is increased by direct exposure of renal cortical slices to dibutyryl cAMP, with in vivo infusion of dibutyryl cAMP also augmenting renin release. The phosphodiesterase inhibitor theophylline, which increases cAMP levels, enhances the actions of PGE2 on renin release, providing further evidence that cAMP acts as the second messenger for renin release. cGMP has an important function in the regulation of vascular tone, and an increase in intracellular cGMP induces a vasorelant action. However, no consistent link between glomerular cGMP and renin release has been identified.[28]

Angiotensin-Converting Enzyme

ACE is a zinc-containing dipeptidyl carboxypeptidase that is responsible for the cleavage of the dipeptide His-Leu from the carboxyl end of Ang I to form the octapeptide Ang II (see Fig. 10-2 ). The main site of synthesis of ACE is in the pulmonary vasculature and, it has a molecular weight of approximately 200 kDa. The structure of ACE has now been determined with the recent crystallization of this enzyme.[29] The analysis of the three-dimensional structure of ACE by Natesh and colleagues[29] shows that it bears little similarity to that of carboxypeptidase A. This new finding provides the opportunity to design domain-selective ACE inhibitors that may exhibit new pharmacologic profiles. Whereas renin is extraordinarily precise in its substrate specificity, ACE is enzymatically far more promiscuous. Indeed, many other small peptides (enkephalins, substance P, luteinizing hormone-releasing hormone) can be cleaved by this enzyme. Moreover, ACE cleaves bradykinin into inactive fragments (see later) and thus functionally degrades this potent vasodilator. The same enzyme produces the pressor substance Ang II and inactivates the vasodepressor kinins. It is perhaps because of this wide diversity of substrates that inhibitors of ACE are so effective in the treatment of hypertension and other cardiovascular and renal diseases.

ACE is also located in plasma and in endothelium of pulmonary and other vascular beds, including the kidney. This enzyme is ubiquitous and has an enormous capacity to convert Ang I to Ang II. Thus, the conversion step has not been regarded as rate limiting for Ang II production. Many tissues produce ACE, but it is the production of the enzyme by vascular endothelial cells that is thought to be most important for the regulation of blood pressure. Nascent ACE protein contains a 29–amino acid signal sequence, and its NH2 terminus is extruded from the cell. A COOH-terminal hydrophobic anchor sequence secures the protein to the luminal face of the endothelial cell membrane. Thus, Ang II is formed at the luminal surface of endothelial cells in close proximity to vascular smooth muscle, a critical target organ for this vasoconstrictor. Studies have examined the ACE levels in human sera, and although levels vary by up to fourfold among individuals, no significant association with clinical hypertension has been identified. [30] [31] ACE exists as two isozymes transcribed from a single gene by the differential utilization of two different promoters. In addition, a soluble form of ACE exists that is presumably derived from the vascular endothelium. The larger isozyme, termed somatic ACE, is present as an ectoenzyme in vascular endothelial cells and other somatic tissues including the renal proximal tubule. Analysis of the cDNA shows that somatic ACE contains 1306 amino acids.

A striking feature of the ACE sequence is the presence of two internal homologous domains, each of which in now known to be catalytic. Each domain is composed of 357 amino acids, and overall the two domains are 68% identical in amino acid sequence.

Human ACE in encoded by a single gene located on chromosome 17.[31] It spans 21 kilobases and is made up of 25 exons.[32] Each of the two homologous domains of the enzyme is encoded by a cluster of eight exons (exons 4 to 11 and exons 16 to 23). The similarity of the exon-intron organization of the two clusters strongly suggests that the mammalian ACE gene is the result of an ancestral gene duplication event.

The testis has a distinct form of ACE that is shorter and has only one catalytic site. Whereas the testicular form of ACE seems to be under the control of androgens, DNA elements possibly responsive to glucocorticoids and cAMP have been found in the upstream region of the endothelial promoter.[33] Also, it has been shown that the gene expres-sion of the endothelial ACE is down-regulated by plasma Ang II levels[34] and upregulated by ACE inhibitors[35]and dexamethasone.[36]

Within the kidney, ACE has been localized to glomerular endothelial cells and the proximal tubule brush border.[21] Several potential roles for proximal tubule brush border ACE have been considered. It has been postulated that ACE may play a role in the cleavage of dipeptides from filtered proteins for subsequent uptake and processing by epithelial cells, a role suggested by localization of ACE in intestinal microvilli, a site without Ang II receptors. ACE probably also serves to form Ang II within proximal tubule fluid, thereby affecting reabsorption. In blood vessels, ACE may be located in vascular cells other than the endothelium.[37] In most cells, ACE appears to be located on the external surface of the cell membrane, although there is some evidence that it may also be located intracellularly. It has been demonstrated by Diet and coworkers[38] that ACE is expressed in lipid-laden ma-crophages within the atherosclerotic plaques in humans. Recently, our group has confirmed and extended these ob-servations to diabetes-induced atherosclerotic lesions. We observed that ACE gene expression was significantly increased in diabetic vessels and that ACE protein expression was consistently found to be at the site of macrophage accumulation within the atherosclerotic plaques.[39] Moreover, recently, our group has demonstrated that ACE is highly expressed in areas of active fibrogenesis in bile duct-ligated livers in the rat suggesting a key role for the RAS in the pathogenesis of liver fibrosis.[5]

The role of plasma ACE is still unknown. Interestingly, studies have shown that a deletion polymorphism of the ACE gene is associated with an increase in plasma ACE levels and with target organ damage in hypertension. Specifically, the D allele of the ACE gene has been associated with microalbuminuria, left ventricular hypertrophy, and coronary artery disease, as well as with renal complications in diabetes. [40] [41] In addition, we recently demonstrated that the D allele of the ACE gene was associated with renal failure in patients with essential hypertension.[42] Further observations suggest that the ACE genotype may influence the response to ACE inhibitor therapy. In particular, it has been observed that the beneficial short- and long-term renoprotective effects of ACE inhibition are lower in albuminuric diabetic patients homozygous for the deletion compared with the insertion polymorphism of the ACE gene. [43] [44] By contrast, Parving's group[45] has demonstrated that treatment with the AT1 receptor blocker losartan offers similar short-term renoprotective and blood pressure-lowering effects in albuminuric hypertensive type 1 diabetic patients with the ACE II and DD genotype, indicating that there is no evidence for an interaction between ACE genotype and blockade of the AT1 receptor. However, this finding remains to be confirmed.

Recently, the classical view of the RAS has been challenged by the discovery of the enzyme ACE2. In addition, there is increasing awareness that many agiotensin peptides other than Ang II have biologic activity and physiologic importance.[46] Two separate groups have described the first human homolog of ACE. [47] [48] ACE-related carboxypeptidase (ACE2), like ACE, is a membrane-associated and -secreted enzyme. The ACE2 and ACE catalytic domains are 42% identical in amino acid sequence, and conservation of exon-intron organization further indicates that the two genes evolved from a common ancestor.[47] In contrast to ACE, however, ACE2 is highly tissue specific. Whereas ACE is expressed ubiquitously in the vasculature, human ACE2 is restricted to the heart, kidney, and testis.[47] In addition to endothelial expression, ACE2 is present in smooth muscle in some coronary vessels and focally in tubular epithelium of the kidney. Both ACE and ACE2 cleave Ang I, but their activities are distinct. Whereas ACE is a dipeptidase, ACE2 removes the single C-terminal Leu residue to generate Ang 1-9 ( Fig. 10-3 ). Ang 1-9 has been identified in vivo in rat and human plasma, but its function is unknown.[49] Ang 1-9 is then subjected to further cleavage by ACE to yield Ang 1-7, a vasodilator. [46] [50] In addition, Ang II can be degraded by ACE2 to also yield Ang 1-7 (see Fig. 10-3 ). Thus, ACE2 may function to limit the vasoconstrictor action of Ang II not only by its inactivation but also by the formation of a counteracting vasodilatory angiotensin, Ang 1-7. Although Ang II is considered to be the main effector of the RAS, the reported vasodilatory actions of Ang 1-7, [46] [50] taken together with the discovery of ACE2 and its potential involvement in both Ang II degradation and Ang 1-7 production, adds another level of complexity to the RAS. Thus, the identification and characterization of ACE2 has uncovered an exciting new area of cardiovascular and renal physiology as well as providing possible novel therapeutic targets. In addition, an unexpected function of ACE2 has recently been identified and characterized. Specifically, ACE2 is a functional receptor for coronaviruses, including the coronavirus that cause severe acute respiratory syndrome, and is involved in mediating virus entry and cell fusion.[51] Although not directly relevant to cardiovascular function, this would indicate that the RAS including ACE2 has multiple roles in physiology and various pathophysiologic states.[52]



FIGURE 10-3  A schema showing where ACE and ACE2 cleave angiotensin (Ang) I as well as how the cleavage products are processed by the two peptidases.



Indeed, a recent study[53] has shown that ACE2 mRNA levels are reduced in animal models of hypertension and ACE2 knockout mice exhibit severe defects in cardiac contractility that are restored by concomitant ACE ablation. These findings support the concept that ACE2 acts in a counter-regulatory manner to ACE and may play an important role to modulate the balance between vasoconstrictors and vasodilators in the heart and kidney. Recent immunohistochemical studies have shown that in the kidney, both ACE2 and ACE protein are localized to tubular epithelial cells.[54] Furthermore, we have reported that ACE2 protein expression is reduced in the diabetic kidney and that this reduction is prevented by ACE inhibitor therapy, suggesting that ACE2 might have a renoprotective role in diabetes.[54] Given the findings of altered expression levels of ACE2 in the diseased kidney, it is postulated that there is an important role for ACE2 in angiotensin metabolism, renal physiology, and potentially, a variety of renal diseases.[55]

Angiotensins and Angiotensinases

Ang II has a very short biologic half-life. After intravenous injection, the pressor response in animals lasts only 1 to 3 minutes. This very rapid effect would suggest rapid uptake from the circulation and/or degradation. Seventy percent or more of Ang II is removed in one circulation, through the liver or the femoral or vascular beds. However, Ang II passes undestroyed through the pulmonary circulation.


Ang II, with aspartic acid in position 1, is most susceptible to cleavage by an acid aminopeptidase, known as aminopeptidase A, angiotensinase A, or more precisely, glutamyl aminopeptidase. Glutamyl aminopeptidase is a metalloprotease containing zinc. In vivo, cleavage by glutamyl aminopeptidase is the step that usually begins the degradation of Ang II.[56] Removal of aspartic acid from Ang II forms the heptapeptide Ang 2-8, also called Ang III (see Fig. 10-2 ). This heptapeptide is a less potent vasoconstrictor than Ang II, but it is at least as potent as the octapeptide in the adrenal zona glomerulosa and central nervous system. Indeed, it has been postulated that Ang III is the principal effector of the RAS in the brain. Ang III is even more rapidly hydrolyzed than Ang II in vivo. Like Ang II, the heptapeptide is first cleaved by aminopeptidase action into the hexapeptide Ang 3-8 or Ang IV (see Fig. 10-2 ). The enzyme most active in its further cleavage is arginyl aminopeptidase (angiotensinase B or aminopeptidase B). Another enzyme, aminopeptidase N, can convert Ang III into the hexapeptide Ang IV. Other aminopeptidases that cleave angiotensins include leucyl aminopeptidase, alanyl aminopeptidase, and dipeptidyl peptidase I (also known as cathepsin C).

Ang IV, another breakdown product once thought to be a virtually inactive peptide, has been demonstrated by Kerins and colleagues[57] to be the form of angiotensin that stimulates endothelial expression of plasminogen inactivator inhibitor (PAI)-1. This effect appears to be mediated via the stimulation of an endothelial AT4 receptor because neither AT1 nor AT2 receptor antagonists could inhibit the Ang IV-induced PAI-1 gene expression. [57] [58] There is also some evidence that Ang IV is involved in the pathogenesis of some renal diseases.


Angiotensins are susceptible to hydrolysis by several classical endopeptidases, including trypsin and chymotrypsin, which probably never gain access to these peptides either at their origin in the circulation or at their targets outside of the gut. The endopeptidase most likely to be involved in limiting the duration of action of angiotensin is neutral endopeptidase (NEP), also called enkephalinase. This enzyme has also been throughly studied for its action on ANP (see later). NEP can directly convert Ang I to Ang 1-7.[59] Whereas Ang I is the primary substrate for the formation of Ang 1-7, the heptapeptide may be formed also from Ang II by the cleavage of the Pro[7]-Phe[8] bond by prolyl-endopeptidase and a prolyl carboxypeptidase.[60] Formerly considered physiologically inactive, Ang 1-7 has been recently demonstrated to have effects on the vasculature and on kidney function. In the vasculature, it produces opposite effects to the growth promoting and constrictor actions of Ang II. In the kidney, Ang 1-7 exerts important regulatory effects on the long-term control of arterial pressure, particularly to counterbalance the actions of Ang II. Infusion of Ang 1-7 into the renal artery stimulates marked diuresis and natriuresis. [61] [62] [63] Ang 1-7 is hydrolyzed at the Ile[5]-His[6] bond to form Ang 1-5 and the dipeptide His-Pro by ACE. [64] [65] This observation suggests that chronic treatment with ACE inhibitors is able to prolong the half-life of Ang 1-7.[66] Thus, ACE constitutes a critical convergence point between the pressor and proliferative properties of Ang II and the depressor and antiproliferative actions of Ang 1-7, similarly to that observed for bradykinin (see later).

Angiotensin II Receptors

Ang II is known to interact with at least two distinct Ang II receptor subtypes, designated AT1 and AT2.[67] The characterization of Ang II receptor subtypes was made possible by the discovery and development of selective nonpeptide Ang II receptor antagonists, namely losartan (AT1-selective) and PD123319 (AT2-selective).[68] Virtually all the known biologic actions of Ang II, including vasoconstriction, release of aldosterone, stimulation of sympathetic transmission, and cellular growth, are mediated by the AT1 receptor. [68] [69] The functional role of the AT2 receptor is not fully understood. Recent studies have described a possible role for AT2 receptors in mediating antiproliferation, apoptosis, differentiation, and possibly vasodilation. [70] [71] However, recent evidence indicates that proinflammatory, growth stimulatory, and profibrogenic effects of Ang II may not be solely transduced through AT1 receptors, but also involve activation of AT2 receptors.

AT1 Receptors

AT1 receptors selectively bind biphenylimidazoles, including losartan, candesartan, and irbesartan, with high affinity and are rather insensitive to tetrahydroimidazolpyridines, such as PD123319 and PD123177.[68]

The gene for the AT1 receptor was first cloned from rat vascular smooth muscle cells[72] and bovine adrenal gland.[73] The AT1 receptor gene product consists of 359 amino acids and has a molecular mass of 41 kDa. The human genome contains a unique gene coding for the AT1 receptor, which is localized on chromosome 3.[74]

The AT1 receptor belongs to the seven transmembrane class of G-protein-coupled receptors.[75] The transmembrane domain and the extracellular loop play an important role in Ang II binding.[76] The binding site for Ang II is different from the binding site for AT1 receptor antagonists, which interact only with the transmembrane domain of the receptor.[77] Like most G-protein-coupled receptors, the AT1 receptor is also subject to internalization when stimulated by Ang II, a process dependent on specific residues on the cytoplasmic tail.[78]

There are five classical signal transduction mechanisms for the AT1 receptor: activation of phospholipase A2, phospholipase C, phospholipase D, and L-type Ca2+ channels and inhibition of adenylate cyclase.

It has been observed that activation of the AT1 receptor stimulates growth factor pathways, such as tyrosine phosphorylation and phospholipase C-gamma, leading to activation of downstream proteins, including MAP kinases, janus kinases (JAK), and the signal transducers and activators of transcription (STAT) proteins. [79] [80] Ang II-stimulated cellular proliferation and growth has been defined in adrenal medulla and vascular smooth muscle cells. These growth-like effects have been linked to cardiovascular and kidney diseases.

The tissue distribution of AT1 receptors has been studied extensively in humans and animals. AT1 receptors are found primarily in the brain, adrenal glands, heart, vasculature, and kidney, serving to regulate blood pressure and fluid and electrolyte balance. AT1 receptors have been demonstrated in the central nervous system of the rat,[81] rabbit,[82] and human. [83] [84] AT1 receptors are localized to areas of the brain that are exposed to blood-borne Ang II, such as the circumventricular organs, including the subfornical organ, median eminence, vascular organ of the lamina terminalis, anterior pituitary, and the area postrema in the hindbrain.[81] Furthermore, other regions of the hypothalamus, nucleus of the solitary tract, and ventrolateral medulla in the hindbrain also contain a high density of AT1 receptors.[81]

AT1 receptors have also been identified in the adrenal gland of rodents, primates, and humans,[6] where they are localized mainly to the zona glomerulosa of the cortex and chromaffin cells of the medulla. In the heart, the highest density of AT1 receptors is found in the conducting system.[85] Punctate AT1 receptor binding is found in the epicardium surrounding the atria, with low binding seen throughout the atrial and ventricular myocardium.[86] Moreover, AT1 receptors in the vasculature, including the aorta, pulmonary, and mesenteric arteries, are present at high levels on smooth muscle cells and at low levels in the adventitia.[87]

The anatomic distribution of the AT1 receptor in the kidney has been mapped in various species.[87] High levels of AT1 receptor binding occur in glomerular mesangial cells and renal interstitial cells located between the tubules and the vasa recta bundles within the inner stripe of the outer medulla.[88] Moreover, moderate binding is localized to proximal convoluted tubular epithelia.

Ang II stimulation of AT1 receptors in blood vessels causes vasoconstriction, leading to an increase in peripheral vascular tone and systemic blood pressure. AT1 receptors in the heart are known to mediate the positive inotropic and chronotropic effects of Ang II on cardiomyocytes.[89] Ang II is also known to mediate cell growth and proliferation in cardiac myocytes and fibroblasts, as well as in vascular smooth muscle cells. [90] [91] Ang II induces the expression and release of various endogenous growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF). [90] [91] It is now clear that these long-term trophic effects of Ang II occur as a result of activation of AT1 receptors.[92] It is well documented that AT1 receptor activation mediates the Ang II-induced release of catecholamines from the adrenal medulla and aldosterone from the adrenal cortex.[93] Finally, in the kidney Ang II influences sodium and water reabsorption from the proximal tubules and inhibits renin secretion from the macula densa cells via the AT1 receptor.[94]

AT2 Receptors

The AT2 receptor is characterized by its high affinity for PD123319, PD123177, and CGP42112 and its very low affinity for losartan and candesartan.[68] Ang II binds to the AT2 receptor with similar affinity as to the AT1 receptor.[67]

The AT2 receptor has been cloned in a variety of species, including human, [95] [96] rat,[97] and mouse. [98] [99] The AT2 receptor is also a seven transmembrane domain receptor, encoded by a 363–amino acid protein with a molecular mass of 41 kDa, and shares only 34% sequence identity with the AT1 receptor.[100] The AT2 receptor gene has been mapped in humans to chromosome X.[95]

Previously, various second messengers coupled to the AT2 receptor have been described and include indirect negative coupling to guanylate cyclase (inhibition of cGMP production)[100] and activation of potassium channels.[101]There have been new insights into AT2 receptor signalling pathways, including activation of protein phosphatases and protein dephosphorylation, the NO-cGMP system, and phospholipase A2 (release of arachidonic acid). In particular, stimulation of AT2 receptors leads to activation of various phosphatases, such as protein tyrosine phosphatase, MAP kinase phosphatase 1 (MKP-1), [102] [103] SH2-domain-containing phosphatase 1 (SHP-1)[104] and serine/threonine phosphatase 2A,[105] resulting in the inactivation of extracellular signal-regulated kinase (ERK), opening of potassium channels and inhibition of T-type Ca2+ channels.[106] It has been confirmed that the AT2receptor is a G-protein-coupled receptor[13] and that an inhibitory G-protein (Gi) is linked to the AT2 receptor signalling mechanism.[107]

In the human heart, the AT2 receptor is localized mainly to fibroblasts in interstitial regions, with a lower degree of binding seen in the surrounding myocardium. [108] [109] Moreover, AT2 receptors are highly expressed in the adrenal medulla of most species, but expression is much lower in humans. [6] [110] In human kidneys, the AT2 receptor is localized to glomeruli, tubules, and renal blood vessels.[14]

Using emulsion autoradiography, our group demonstrated the presence of AT2 receptors in the glomeruli and proximal tubules of adult rat kidney.[13] These findings are similar to those reported by Ozono and coworkers[14] who demonstrated, using immunohistochemistry and Western blot analysis, that the AT2 receptor is localized mainly to glomeruli, but it is also found at low levels in cortical tubules and interstitial cells.

Because the AT2 receptor is highly abundant in fetal tissues, it is believed to play an important role in fetal development. However, AT2 receptor knockout mice appear to develop and grow normally (see later), suggesting that AT2receptors may not be as crucial as previously thought for fetal development. [111] [112] In mice lacking the AT2 receptor, the drinking response is impaired and locomotion is reduced. In addition, the animals exhibit an increase in the vasopressor response to Ang II.

It has been demonstrated that the AT2 receptor is involved in the production of cGMP,[113] NO,[114] and prostaglandin F2a[115] in the kidney, suggesting an important role in renal function, including vasodilatation and blood pressure regulation. In addition, evidence suggests that, in the kidney, AT2 receptor stimulation induces the bradykinin/NO pathway.[116]

Our group has explored the renal expression of the AT2 receptor in subtotally nephrectomized rats and the effects of AT2 receptor blockade on renal injury.[117] In that study, we observed increased gene expression of the AT2receptor in the kidney, whereas no global differences in AT2 receptor protein expression and binding were found between remnant and control kidneys. However, the AT2 receptor protein was identified specifically in the injured tubules after renal mass reduction. Treatment with the AT2 receptor blocker PD123319 significantly reduced tubulointerstitial injury, tubular cell proliferation, renal inflammatory cell infiltration, and proteinuria.[117] These findings suggest a key role for the AT2 receptor in mediating kidney damage in certain contexts.

Other Angiotensin Receptors

There is mounting evidence for the existence of additional angiotensin receptors, which are pharmacologically distinct from AT1 and AT2 receptors. The angiotensin AT4 receptor is a novel binding site that displays high specificity and affinity for the hexapeptide fragment Ang IV, but with low affinity for Ang II.[118] The binding of Ang IV to the AT4 receptor is insensitive to both losartan and PD123319 but is selectively blocked by the peptide antagonist divalinal-Ang IV.[119] Although it is not clear yet whether the AT4 receptor belongs to the G-protein-coupled receptor superfamily as reported for the AT1 and AT2 receptor subtypes, it has been demonstrated that this transmembrane protein is distributed in many tissues, and in particular, in the brain and kidney. Harding and colleagues[120] have shown that the AT4 receptor is preferentially concentrated in the outer stripe of the medulla. Moreover, using autoradiography Handa and coworkers[121] localized AT4 receptors to the cell body and apical membrane of convoluted and straight proximal tubules not only in the outer medulla but also in the cortex of rat kidney. The same authors demonstrated that human proximal tubular epithelial cells contain functional AT4 receptors that are pharmacologically similar to the AT4 receptor described in more distal segments of the nephron and other renal cells. [122] [123] The functional role of this receptor remains to be fully clarified, but it has been suggested that it may play an important role in mediating cerebral and renal blood flow, memory retention, and neuronal development.[118]

Studies using the selective Ang 1-7 antagonist A-779 provide evidence for an Ang 1-7 receptor distinct from the classical Ang II receptors AT1 and AT2. [124] [125] Recent studies by Santos and colleagues[126] have identified the G protein-coupled receptor Mas as a functional receptor for Ang 1-7 because it binds Ang 1-7 and is involved in mediating biologic actions of this angiotensin peptide. Because Ang 1-7 counteracts Ang II, these findings clearly widen the possibilities for treating cardiovascular diseases using agonists for the Ang 1-7-Mas axis.

Another atypical angiotensin binding site, loosely termed the AT3 receptor, has also been identified in cultured mouse neuroblastoma cells and binds Ang II with high affinity, but has low affinity for Ang III and no affinity for losartan or PD123319.[127]

Renin-Angiotensin System Knockout or Transgenic Models

Targeted gene manipulation has provided significant insights into the physiologic and pathologic roles of the RAS in regulating blood pressure, cardiovascular homeostasis, renal function, and development ( Table 10-2 ). In angiotensinogen-deficient mice, who have complete loss of plasma immunoreactive Ang I,[128] the systolic blood pressure was approximately 20 to 30 mm Hg lower than in wild-type mice.[128] Similarly, in ACE-deficient mice, markedly reduced blood pressure was observed, and interestingly, there was also severe renal disease. [129] [130] The renal papilla in these mice was markedly reduced, and the intrarenal arteries exhibited vascular hyperplasia associated with a perivascular inflammatory infiltrate. Moreover, these animals could not effectively concentrate urine and had an abnormally low urinary sodium-to-potassium ratio despite reduced levels of aldosterone.

TABLE 10-2   -- Differential Phenotypes Among Various Renin-Angiotensin System Knockout Models


Systolic Blood Pressure

Fetal Kidney Development

Postnatal Kidney Development

AGT -/-

Very low (reduction of 20 mm Hg)


Hypertrophy of renal arteries and arterioles, atrophy of the papilla, focal areas of tubular dropout, interstitial inflammation and fibrosis

Renin -/-

Very low (reduction of 20 mm Hg)


Similar to that observed in AGT -/- model

ACE -/-

Very low (reduction of 20 mm Hg)


Similar to that observed in AGT -/- model


Very low (reduction of 20 mm Hg)


Similar to that observed in AGT -/- model

AT1A -/-

Moderately low (reduction of 12 mm Hg)


Normal or slight dilatation of the renal pelvis, mild compression of the papilla, shortening of the renal papilla, and reduction in the area of the inner medulla

AT -/-




AT2 -/-

High (increase of 13 mm Hg)




ACE -/-, ACE knockout model; AGT -/-, angiotensinogen knockout model; AT1A -/-, angiotensin II subtype 1A receptor knockout model; AT -/-, angiotensin II subtype 1B receptor knockout model; AT1A/AT -/-, angiotensin II subtype 1A and 1B receptors knockout model; AT2 -/-, angiotensin II subtype 2 receptor knockout model; Renin -/-, renin knockout model.




Deletion of the gene encoding the AT1A receptor subtype in mice is associated with a significant reduction in blood pressure and an attenuated pressor response to infused Ang II. [131] [132] Conversely, in AT receptor knockout mice, systemic blood pressure is normal, suggesting that the AT1A receptor subtype is the major receptor involved in blood pressure regulation.[133] Deletion of either the AT1A or the AT receptor in mice is not associated with impaired development, survival, or tissue abnormalities. [131] [132] [134] However, deletion of both receptor subtypes results in decreased blood pressure, impaired growth, and renal abnormalities, a phenotype similar to that seen with deletion of angiotensinogen or ACE. [134] [135] Thus, the AT receptor, although considered to be of less importance, may compensate for the effects seen in AT1A knockout mice.

In contrast to AT1 receptor gene deletion, targeted dele-tion of the AT2 receptor gene in mice results in raised blood pressure and enhanced sensitivity to the pressor effects of Ang II. [111] [112] This suggests that the AT2 receptor mediates a vasodepressor effect and may functionally oppose the effects mediated by the AT1 receptor, possibly via bradykinin and NO.[136] Although AT2 receptors are abundant in fetal tissues, such as the heart, kidney, and brain, AT2 receptor knockout mice apparently develop and grow normally. [111] [112] However, these mice have impaired drinking responses to water deprivation and reduced exploratory behavior. [111] [112] More recently, it has been reported that mice lacking the AT2 receptor exhibit anxiety-like behavior[137] and have increased sensitivity to pain.[138] Thus, the AT2 receptor may also play a role in modulating behavioral effects, mood, and the threshold for pain.

The RAS has been the focus of the largest number of transgenic studies reported in the renal literature using animal models overexpressing various components of the RAS.[139] Rats transgenic for either the human renin or the human angiotensin gene have normal plasma Ang II levels despite high circulating levels of renin or angiotensinogen. [140] [141] These negative findings can be explained by the species specificity of the renin-angiotensinogen interaction. Human renin does not act on rat angiotensinogen, and human angiotensinogen does not serve as a substrate for rat renin.[140] However, transgenic mice expressing both human angiotensinogen and human renin genes under the control of the appropriate human promoter[142] develop hypertension and renal fibrosis. Administration of the ACE inhibitor lisinopril to these mice significantly decreased the glomerulosclerosis index without decreasing systolic blood pressure. These results suggest that activation of the renal RAS induces renal sclerosis independently of systemic hypertension. Findings from other animal models support the hypothesis that endogenous Ang II produced locally plays a role in the formation of renal fibrosis, independent of alterations in systemic vascular resistance.[143]

Mullins and coworkers [140] [144] introduced the mouse Ren-2 gene into normotensive rats, thus creating a transgenic strain that expresses high levels of Ren-2 mRNA in many sites including the adrenal gland and to a much lesser extent the kidney. In these rats, fulminant hypertension develops between 5 and 10 weeks of age. Treatment with low-dose ACE inhibitors or Ang II antagonists normalized blood pressure.[145] Despite severe hypertension in Ren-2 transgenic rats, the systemic RAS was not stimulated and plasma levels of active renin, angiotensinogen, Ang I, and Ang II were lower than those seen in control animals.[144] By contrast, the plasma concentration of prorenin was dramatically elevated. This increase in prorenin originated mainly from the adrenal glands with adrenalectomy normalizing blood pressure in these rats.[140] Because the activation of the RAS has been implicated in the progression of chronic renal disease, Ganten and collegues[140] studied progression of glomerular sclerosis after subtotal nephrectomy in Ren-2 transgenic rats. Compared with blood pressure-matched spontaneously hypertensive rats, the transgenic animals had significant acceleration of glomerulosclerosis, consistent with a pathogenetic role for the intrarenal RAS in the progression to renal failure. Similarly, it has been observed that induction of diabetes in Ren-2 transgenic rats was associated with glomerulosclerosis, tubulointerstitial fibrosis, and decline in renal function, features not observed in other rodent models of diabetes.[146] Moreover, blocking the RAS with either an ACE inhibitor or an AT1 receptor blocker preserves renal function and attenuates renal structural damage in this model. These effects on renal disease progression appear to be due to attenuation of expression and activation of the local RAS and not solely the result of reducing systemic blood pressure because an equihypotensive dose of an endothelin antagonist failed to confer a similar degree of renoprotection in this model.

Physiologic Effects of the Renin-Angiotensin System

The predominant function of the RAS is regulation of vascular tone and renal salt excretion in response to changes in extracellular fluid volume or blood pressure. Ang II represents the effector limb of this hormonal system, acting on several organs, including the vascular system, heart, adrenal glands, central nervous system, and kidney. We focus on the renal effects of Ang II.

Effects of Angiotensin II on Renal Hemodynamics

In the kidney, the primary action of Ang II is on the small-diameter resistance arterioles supplying the glomeruli. It is well established that both endogenous and exogenous Ang II affect preglomerular (afferent) as well as efferent arteriolar tone. The tendency of the filtration fraction to rise is due to smaller reductions in GFR relative to a larger decrease in renal blood flow. This is interpreted as consistent with a predominant action of Ang II on the postglomerular (efferent) arterioles. Micropuncture studies indicate that Ang II usually decreases the filtration coefficient and increases permeability to macromolecules.[147] The precise in vivo role of the contractile mesangial cells in producing changes in capillary hydraulic conductivity and/or capillary surface area is not fully known.[147]

Segmental Vascular Resistance.

Early studies on single nephron function demonstrated that Ang II increases total resistance as a result of contraction of preglomerular arteries and afferent and efferent arterioles. [147] [148] The most convincing in vivo evidence for direct actions of Ang II on the afferent arteriole was obtained during infusion of Ang I or Ang II into the renal artery. Several studies using direct microscopic visualization and calcium-sensitive dye fluorescence provide convincing evidence that Ang II constricts both afferent and efferent arterioles, with similar potency or slightly greater effects on the efferent arteriole. [149] [150] There are high concentrations of Ang II in the vicinity of the JG apparatus and glomerular arterioles with at least a hundredfold increase in intrarenal versus systemic Ang II concentrations (nM versus pM).[151]

Paracrine/Autocrine Agents.

In addition to direct effects on vascular smooth muscle cells, Ang II can stimulate release of other vasoactive factors from endothelial cells. Accordingly, the vascular actions of Ang II can be modulated by paracrine and autocrine agents produced in response to Ang II, with either buffering or amplifying effects. The most common integrated response to Ang II is net vasoconstriction. Within the kidney, these secondary vasoactive agents may originate from endothelial, vascular smooth muscle, or mesangial cells (and perhaps reno medullary interstitial cells). Most widely known are the protective or opposing effects provided by NO and vasodilator products of arachidonic acid metabolism, notably PGE2, PGI2, and possibly epoxyeicosatrienoic acid.

Endothelin-1 may be another endothelial factor that regulates Ang II-induced vasoconstriction.[152] Ang II can increase gene expression and synthesis of endothelin-1 in vascular smooth muscle cells.[153] Blockade of both endothelin A and endothelin B receptors with bosentan attenuates the vasoconstrictor and proteinuric effects of chronic Ang II infusion.[154] Other modulatory factors include adenosine and ATP, dopamine, and lipoxygenase and cytochrome P-450 metabolites derived from arachidonic acid.[148]

Effects of Angiotensin II on Renal Autoregulatory Mechanisms

The renal vasculature plays an important role in protecting the glomerulus and tubules from large changes in arterial pressure that occur during normal daily activity as well as during stress. These autoregulatory mechanisms continually adjust renal vasomotor tone to counterbalance fluctuations in arterial pressure to maintain renal blood flow and GFR constant, thereby blunting the natriuretic effects of increases in arterial pressure.[147] Whole kidney blood flow studies show that renal blood flow is regulated near constancy during acute changes in systolic arterial pressure between 90 and 180 mm Hg. Such renal autoregulation is thought to be mediated by two basic mechanisms, both of which involve the afferent arteriole. One is a pressure-induced myogenic response of vascular smooth muscle cells in the interlobular arteries and afferent arterioles. The other is a tubuloglomerular feedback (TGF) loop involving the JG apparatus. This TGF system functions as a negative feedback system, regulating afferent arteriolar tone as a function of solute delivery and transport by macula densa cells at the start of the distal tubule.

Effects of Angiotensin II on Tubular Transport

Direct actions of Ang II on tubular transport function have been suggested for more than 2 decades. In sodium-depleted dogs, chronic blockade of Ang II formation decreased blood pressure and increased urinary sodium excretion, independent of any changes in circulating aldosterone levels. Similarly, it has been observed that in sodium-depleted dogs with activation of the RAS, increases in sodium excretion in response to increases in arterial pressure were attenuated, compared with dogs maintained on normal sodium diets. Furthermore, with administration of the ACE inhibitor captopril, absolute and fractional sodium excretion increased at all levels of arterial pressure, an effect that could not be explained by changes in the filtered load of sodium. These studies and others provide compelling evidence that increases in fractional sodium excretion in response to RAS blockade are greater than can be accounted for by associated changes in GFR or renal blood flow. Accordingly, it is suggested that Ang II exerts direct stimulatory effects on tubular transport.

In the proximal tubule, Ang II plays a central role in promoting the reabsorption of sodium, fluid, and bicarbonate (HCO3-). A luminal Na+/H+ exchanger is activated by Ang II, resulting in sodium uptake from the lumen into the cells. [155] [156] The increased activity of the Na+/H+ exchanger promotes HCO3- transport by the basolateral Na+/HCO3- cotransporter, which may be directly affected by Ang II. [157] [158] Ang II also increases basolateral Na+-K+-ATPase activity in the proximal tubule, thereby contributing to sodium transport.[155] In addition, Ang II can modify sodium-independent H+ secretion by insertion of H+-ATPase-containing vesicles into the brush border membrane.[159] Finally, Garvin[160] has investigated the effect of Ang II on glucose and fluid absorption in isolated, perfused rat proximal tubules and determined that Ang II stimulates Na+/glucose cotransport in this nephron segment.

There is a well-characterized biphasic effect of Ang II on transport activities in the proximal tubule. Low concentrations of Ang II (<10-9 M) appear to stimulate fluid reabsorption whereas high concentrations of Ang II (>10-9 M) inhibit transport.

Most studies suggest that both inhibitory and stimulatory effects of Ang II are mediated by the AT1 receptor subtype. [68] [69] This notion is supported by the use of specific antagonists against AT1 and AT2 receptors in a number of studies. [161] [162] For example, Quan and Baum[163] demonstrated that luminally applied AT1 or AT2 receptor antagonists decreased endogenous Ang II-stimulated proximal tubule volume reabsorption. Clearly, studies assessing transport in tubules isolated from both AT1 and AT2 receptor-deficient mice may clarify the role of these receptor subtypes on proximal tubule transport.

Some studies suggest that the inhibitory effects of high-dose Ang II on transport may be mediated via AT2 receptors in the proximal tubule. Jacobs and Douglas[164] have localized AT2 receptor function to apical membranes of rabbit proximal tubule cells, associated with stimulation of arachidonic acid release through phospholipase A2. It has been suggested that the effect described previously appears to be mediated by a novel mechanism involving the AT2receptor, with coupling to the G protein β-γ subunit and stimulation of phospholipase A2 activity, arachidonate release, p21ras, and MAP kinase.[165] Effects of Ang II on other sites within the nephron have also been reported, including stimulation of bicarbonate transport in the superficial loop of Henle and stimulation of apical Na+/H+ exchange in the early distal tubule. In the late distal tubule, Ang II activates apical amiloride-sensitive sodium channels on principal cells and also potently stimulates bicarbonate reabsorption at this site in an AT1 receptor-dependent manner. The effects of Ang II on the collecting duct have been less well studied, with lower concentrations of Ang II having no effect on bicarbonate transport.[166] Ang II appears to directly modulate water transport in the inner medulla, with AT1 receptors having been identified in these inner medullary collecting ducts.[167] Finally, there is indirect evidence for interactions of Ang II and AT1 receptors in renomedullary interstitial cells, which may be important in the regulation of the renal medullary microcirculation. These cells are situated in the medullary interstititum between and anchored closely into the basement membranes of the loop of Henle and vasa recta blood vessels.[168] These cells are distinct from two other cell types, macrophages and dendritic cells, which are also present in the renal medullary interstitium.[169] A number of studies have been performed exploring the effects of Ang II in inducing vasoconstriction of medullary blood vessels. These effects are considered partly to occur via paracrine actions on renomedullary interstitial cells in addition to direct actions on these vessels.

Intrarenal Renin-Angiotensin System

Although traditionally the RAS has been thought primarily as an endocrine system that delivers circulating Ang II to target tissues, significant insights have been generated during the past 20 years regarding the capacity of kidney tissue to directly synthesize Ang II. It has now been convincingly shown that the major components of the RAS (angiotensinogen, renin, ACE, and AT1 and AT2 receptors) are synthesized within the kidney, in both glomeruli and tubules. [24] [170] [171]

Renin production and secretion from the JG apparatus are controlled by intrarenal as well as by systemic factors. Ang II is clearly produced within the kidney, and ACE, which is found on peritubular and capillary endothelial cells, plays a role in its production. It has been demonstrated that intrarenal Ang II production occurs within the kidney in the interstitium, within JG cells, and within tubular cells. [24] [170] [171]

The interstitium has long been considered to be an important site for the RAS within the kidney. Renin, angiotensinogen, Ang I, and Ang II are present not only in the intravascular compartment of the kidney but also in renal lymph with the interstitium containing high levels of Ang II.[172]

The colocalization of renin and Ang II in JG cell granules originally led to the concept that Ang II may also be synthesized in JG cells. Indeed, it has been shown that Ang II peptides are generated within JG cells, presumably by a mechanism which involves the action of endogenous renin on internalized, exogenous angiotensinogen.[173]

mRNAs for angiotensinogen, renin, ACE, and AT1 and AT2 receptors have been localized to proximal tubule cells, and the corresponding proteins have also been identified in this segment by immunohistochemistry. [24] [170] [171] In studies performed by Seikaly and coworkers[174] and Braam and colleagues,[175] the lumen of the proximal tubule was shown to contain concentrations of Ang II ranging from 100- to 1000-fold higher than the concentrations normally present in plasma. It appears that Ang II may be formed within proximal tubule cells, and secreted into the tubular lumen, or converted from intraluminal Ang I by the presence of apical membrane-bound ACE.[176]Interestingly, in rats maintained on salt-restricted diets, Tank and coworkers[177] reported a significant increase in proximal tubule renin mRNA expression, suggesting an enhanced capacity for local Ang II generation. Several studies by our group reported similar findings of proximal tubular renin expression in association with immunoreactive Ang II in two different models of progressive renal injury, subtotal nephrectomy,[178] and diabetes in transgenic Ren-2 rats.[146] Together, these data indicate that the proximal tubule has an endogenous RAS and that locally generated Ang II could exert autocrine and/or paracrine effects in this segment.

Advances have also been made in our understanding of the distribution of Ang II receptors along the nephron. Radioligand binding studies demonstrated that Ang II binding sites were present in discrete nephron segments in the rat, including the proximal convoluted tubule (where the density of receptors is highest), pars recta, loop of Henle, distal convoluted tubule, and cortical and medullary collecting ducts. It appears that the majority of tubular Ang II receptors are of the AT1 subtype. Immunohistochemical studies have revealed the presence of AT1 receptors along the entire nephron, on apical and basolateral membranes of proximal tubule cells, but also in other segments including the macula densa, distal tubule, and inner medullary collecting ducts.[167] There is evidence that AT1 receptors are also expressed on renomedullary interstitial cells and that these receptors are regulated in a manner similar to those in glomerular mesangial cells during alterations in the activity of the RAS.

In contrast, it is estimated that up to 10% to 15% of intrarenal Ang II receptors in the adult may be of the AT2 subtype. AT2 receptors are abundant in the fetal kidney, with diminished expression immediately after birth. In the adult rat kidney, these receptors have been localized to cortical and medullary tubular segments, with up-regulation following sodium depletion.[14]

Pathophysiologic Effects of the Renin-Angiotensin System in the Kidney

Most forms of progressive renal disease lead to a common histologic end point: the end-stage kidney, which is usually fibrotic and reduced in mass. In both humans and animals with chronic renal disease, the evolution of glomerulosclerosis is characterized by progressive involvement of segments within individual glomeruli, eventual global sclerosis and a decrease in the number of glomeruli, and obliteration of tubular structures.[21] Impairment of glomerular and tubular function correlates to a certain extent with histologic changes.[21] Tubular atrophy is manifested by a progressive impairment of the kidney's capacity to concentrate the urine or excrete acid.[21] A number of kidney diseases, and their progression to end-stage renal failure, are driven by the autocrine, paracrine, and endocrine effects of Ang II. Moreover, despite the beneficial effects of ACE inhibitors, [179] [180] the findings that the systemic RAS is not activated in most types of chronic renal disease has led to the suggestion that it is the local intrarenal RAS that may be a particularly important determinant in the progression of renal disease ( Fig. 10-4 ).



FIGURE 10-4  Pathophysiologic mechanisms for the intrarenal RAS in mediating renal injury. bFGF, basic fibroblast growth factor; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF-kB, nuclear factor-kB; PDGF, platelet-derived growth factor; PKC, protein kinase C; TGF-b, transforming growth factor-β; TNF, tumor necrosis factor.



Effects of Angiotensin II on the Kidney

In Vitro Studies

Diabetes mellitus, systemic hypertension, and inflammatory disease are important causes of chronic progressive renal disease. [181] [182] [183] The glomerular findings in these diseases include mesangial expansion and excessive accumulation of extracellular matrix proteins. [181] [182] [183] This leads to glomerular capillary obliteration and decline in the GFR, ultimately leading to renal failure.[184] The three major cell types in the glomerulus, mesangial, endothelial, and epithelial are all implicated in progressive renal diseases and respond to Ang II.

Besides its hemodynamic effects in the kidney, Ang II has been shown to have various important direct actions on mesangial cells. Indeed, these nonhemodynamic effects of Ang II may play a crucial role in Ang II-mediated glomerular injury. In cultured murine mesangial cells, Ang II stimulates cellular hypertrophy, and this effect is blocked by AT1 receptor antagonism.[185] On the other hand, other investigators [186] [187] have shown that Ang II causes not only cellular hypertrophy but also proliferation in mesangial cells.

Accumulating evidence supports the notion that TGF-β1 plays a key role in progression of glomerulosclerosis by directly enhancing mesangial hypertrophy and extracellular matrix production. [188] [189] The treatment of rat mesangial cells with Ang II increased mRNA and protein levels for TGF-β1 and extracellular matrix components including biglycan, fibronectin, and collagen type I. Furthermore, a neutralizing antibody to TGF-β1 blocked Ang II-induced mesangial cell hypertrophy. A wide range of other in vitro findings, including assessment of PAI-1 and fibronectin expression, supports the notion that TGF-β1 is a key mediator in Ang II-induced glomerulosclerosis. A range of inflammatory pathways is also activated by Ang II in cultured mesangial cells. This includes activation of nuclear factor-kB (NF-kB) and increased expression of the NF-kB-dependent chemokine, monocyte chemoattractant protein (MCP)-1.[190] An inhibitor of NF-kB activation, the antioxidant, pyrrolidine dithiocarbamate, inhibited not only Ang II-induced NF-kB activation but also MCP-1 gene expression.[190]

Cultured rat glomerular endothelial cells have been found to possess not only AT1 receptors but also AT2 receptors. Ang II treatment stimulated mRNA and protein synthesis of RANTES, a chemokine with chemoattractant properties for macrophages/monocytes.[191] This effect was blocked by the AT2 receptor ligands PD123177 and CGP-42112, but not by the AT1 receptor antagonist losartan, suggesting a possible role for the endothelial AT2 receptor in Ang II-induced RANTES expression and the subsequent development of glomerular inflammation.[191] Glomerular epithelial cells, which play an important role in the glomerular filtration barrier, have both AT1 and AT2 receptors. Ang II has been shown to increase cAMP accumulation in these cells via the AT1 but not the AT2 receptor.[192] However, the significance of this observation is as yet unknown.

Studies on cultured renomedullary interstitial cells have suggested that interactions between Ang II and these cells may exert several important influences in the kidney in a manner similar to that seen with respect to Ang II and mesangial cells.[193] Like mesangial cells, in which AT1A receptors are expressed and Ang II stimulates protein synthesis,[194] Ang II also acts in these cells on AT1A receptors to increase [3H]-thymidine incorporation and induces extracellular matrix accumulation.[195] The proliferative effects of Ang II on renomedullary interstitial cells in vitro may be physiologically important both in maintaining normal structural arrangements in the renal medulla and in the pathogenesis of progressive renal disease.[196]

In Vivo Studies

Ang II infusion in vivo in rats leads to glomerulosclerosis.[197] A range of cellular and molecular mechanisms linking glomerular injury to Ang II have been identified in these studies.[198] Continuous Ang II infusion in rats led to dramatic up-regulation of alpha-smooth muscle actin in glomerular mesangial cells and desmin in epithelial cells.[199] Continuous administration of Ang II in rats for 7 days caused increases in glomerular expression of TGF-β1 and collagen type I.[200] Moreover, infusion of Ang II in rats for 4 days significantly stimulated glomerular expression of chemokine RANTES and increased glomerular macrophage/monocyte influx. Notably, oral treatment with the AT2 receptor antagonist PD123177 did not affect blood pressure but did attenuate glomerular RANTES expression and glomerular macrophage/monocyte influx.[191]

Our group[13] has demonstrated that subcutaneous infusion of Ang II for 14 days in normotensive rats induced proliferation and apoptosis of proximal tubular epithelial cells. The administration of the AT2 receptor antagonist PD123319 or the AT1 receptor antagonist valsartan was associated with a reduction in renal injury and attenuation of cell proliferation and apoptosis following Ang II infusion.[13] Furthermore, in that study, Ang II infusion was associated with increased osteopontin gene and protein expression, which could be reduced by treatment with either AT1 or AT2 receptor blockers. These findings on the matrix protein osteopontin should be interpreted in the light of recent findings from microarray studies in vascular smooth muscle cells in which osteopontin was shown to be a gene that is highly responsive to exogenous Ang II.[201] Interestingly, our findings indicated that not only the AT1 but also the AT2 receptor have a role in mediating Ang II-induced proliferation and apoptosis in proximal tubular cells and expression of osteopontin. These observations are in agreement with other studies previously mentioned, which suggest a role for the AT2 receptor in mediat-ing cellular processes, including cell recruitment in the kidney.[191]

Role of the Renin-Angiotensin System in the Pathophysiology of Kidney Diseases

Studies by Hostetter and Brenner[21] first suggested that increases in capillary pressure and/or flow cause glomerular injury. Furthermore, Anderson and colleagues[21] showed in the subtotally nephrectomized rat model that chronic ACE inhibition normalized both systemic and glomerular capillary pressure. These striking hemodynamic effects were associated with prevention of glomerular injury in the remnant kidney. More recent studies, in the same experimental model (remnant kidney model), demonstrated that the Ang II antagonist candesartan significantly reduced the expression of glomerular alpha-smooth muscle actin and desmin, while decreasing urinary albumin excretion and attenuating glomerulosclerosis.[202]

Although the glomerulus is often the primary site of injury in renal disease, it is the extent of tubulointerstitial rather than glomerular injury that correlates most closely with and predicts future loss of renal function in patients with primary glomerular disease. Following subtotal nephrectomy, our group demonstrated that renin synthesis is suppressed at the JG apparatus but appears de novo in the tubular epithelium. Furthemore, we observed in the same study that the JG apparatus and tubule responded in a divergent manner to ACE inhibition with regard to renin synthesis. As seen in an intact kidney, ACE inhibition led to increased JG apparatus renin expression with proximal extension into the afferent arteriole, whereas in the tubule, this intervention led to suppression of renin production.[203] These findings provide evidence that within the kidney, the regulation of the RAS differs between the JG apparatus and the tubules. The relevance of this altered pattern of renin synthesis is confirmed by concomitant de novo appearance of the effector molecule of the RAS, Ang II in the renal tubule in response to subtotal nephrectomy. The expression of renin by tubular epithelial cells described in this study may reflect a phenotypic change that occurs as a nonspecific response to injury.[178] Activation in this model of the tubular RAS was associated with an increased expression of TGF-β1 within the tubular epithelium and an increase in collagen IV expression. Interruption of the RAS by ACE inhibition was associated with disappearance of aberrant tubular expression of renin and Ang II in association with the restoration of high levels of renin expression in the JG apparatus. Furthermore, ACE inhibition significantly reduced expression of the Ang II-induced mediator of renal fibrosis, TGF-β1 in association with amelioration of the functional and structural manifestations of renal injury.[178]

It has been recently demonstrated that the integrity of the podocyte and specifically the slit diaphragm is crucial to the process of glomerular filtration.[204] The recently discovered protein nephrin is a major constituent of the molecular structure of the slit pore, and its absence has been implicated in the pathogenesis of the congenital nephrotic syndrome of the Finnish type.[205] Down-regulation of nephrin has been implicated in various models of proteinuria including puromycin aminonucleoside nephrosis,[206] mercuric-chloride-treated rat,[207] and more recently, in the diabetic rat and subtotal nephrectomy model.[117] In the subtotal nephrectomy model, it was observed that increased proteinuria was associated with reduced gene and protein expression of this slit diaphragm protein. This alteration in nephrin expression was prevented by both the AT1 receptor blocker valsartan and the AT2 receptor antagonist PD123319, suggesting a role for the RAS in influencing expression of nephrin. In addition, both antagonists were antiproteinuric in association with reduced cellular proliferation. Moreover, it seems that the combination of the AT1 and the AT2 receptor antagonists may confer additive renoprotective effects.

Although not extensively reviewed here, a role for Ang II per se in a range of other renal diseases has been described including cyclosporine nephrotoxicity, deoxycortocosterone acetate (DOCA)-salt hypertension, and ureteral obstruction.

Unlike the role of Ang II in the progression of chronic renal failure, there is less direct evidence that the RAS is involved in the mediation of acute kidney injury (AKI). However, PRA and renal renin content increase in experimental ischemic renal failure.[208] An important pathogenic role for the RAS in the development of AKI during hypertensive crises in scleroderma has long been recognized, and this was one of the first conditions in which ACE inhibitors were considered appropriate therapy.[209]

Role of the Renin-Angiotensin System in the Pathophysiology of Diabetic Nephropathy

Diabetic nephropathy is the leading cause of end-stage renal disease in the Western world. Measurements of circulating components of the RAS in experimental or human diabetes mellitus do not appear to accurately predict the state of activation of the RAS or its response to blockade at the kidney level.[210] Although measurements of components of the RAS in plasma have, in general, suggested suppression of this system in diabetes, there is increasing evidence for activation of the local intrarenal RAS in the diabetic kidney. Indeed, in the proximal tubule, there is evidence for up-regulation of renin and angiotensinogen expression. [211] [212] An increase in ACE levels within the glomerulus has been reported in diabetes, as have direct effects of high extracellular glucose levels on mesangial cell expression of RAS components. Despite the possibility of increased local production of Ang II, several studies have described suppression of AT1 and AT2 receptor mRNA and protein expression in the diabetic kidney.[213] It remains to be determined whether the balance of intrarenal AT1 and AT2 receptors is important in determining the cellular responses to Ang II in diabetic nephropathy.

The importance of the RAS in mediating the kidney damage in diabetes has been demostrated by a large number of clinical and experimental studies showing that ACE inhibitors as well as AT1 receptor antagonists decrease proteinuria and slow the progression of diabetic nephropathy in both type 1 and type 2 diabetes. [214] [215] [216] [217] Studies by Hostetter and Brenner[21] first showed that increases in glomerular capillary pressure and flow were responsible for the above-normal elevation of GFR in diabetic rats. The hemodynamic abnormalities in rats with diabetes were different from those observed in rats with renal insufficiency, as a result of subtotal nephrectomy. Specifically, diabetic rats exhibited an increase in glomerular capillary pressure without any increase in systemic blood pressure. Chronic Ang II blockade, however, was found to reduce glomerular pressure in diabetes as well as in experimental renal insufficiency. These hemodynamic changes were associated with protection of the glomerulus from accelerated renal sclerotic injury that was seen in experimental diabetes.

Increased TGF-β and type IV collagen expression have been demonstrated in experimental diabetic nephropathy [218] [219] as well as in human diabetic nephropathy.[220] Although most studies of diabetic nephropathy have addressed the glomerular changes, there is increasing interest in the tubulointerstitial abnormalities in this disease. Indeed, tubulointerstitial changes in diabetic nephopathy are closely related to declining renal function.

In a series of studies in diabetic rodents, investigators have explored the effects of agents that interrupt the RAS on growth factors and extracellular matrix protein expression. These studies have demonstrated renoprotective effects of these agents in both hypertensive and normotensive models of diabetic nephropathy. In particular, the tubulointerstitial lesions observed in experimental diabetes were attenuated by ACE inhibition in association with reduced proximal tubular TGF-β expression.[221] A range of other Ang II-mediated effects that are relevant to diabetic kidney disease include stimulation of proliferative cytokines such as PDGF, induction of oxidative stress, activation of NF-kB, and enhancement of expression of chemokines and cytokines that are proinflammatory such as RANTES and MCP-1.[221]

Future Perspectives of the Renin-Angiotensin System

Twenty years ago, it was assumed that Ang II, the main effector of the RAS, was a systemic circulating hormone that was considered primarily to be a peripheral vasoconstrictor involved in blood pressure regulation, a regulator of glomerular filtration, and a secretagog for aldosterone. Major scientific advances in this area have changed this simple view of Ang II, and it is increasingly recognized that specific organs exhibit their own local RASs, which act independently from their systemic counterparts interacting with specific Ang II receptors. In parallel with these findings, it became clear that Ang II has many additional properties above and beyond being a simple vasoconstrictor. In fact, it has been clearly demonstrated that Ang II is directly involved in the control of tubular transport and cell growth and that it has profibrogenic and proinflammatory effects. In addition, it has gradually become clear that not only Ang II but also related peptides such as Ang III, Ang IV, and Ang 1-7 have specific effects independent of the parent peptide. Among the local RASs, the renal RAS has been particularly characterized. It is now known that specific cell populations such as renal proximal tubular cells exhibit all components of the RAS. The RAS plays a major role in the pathogenesis of hypertension, cardiovascular, and renal diseases. Moreover, the RAS has been demonstrated to mediate the progression of glomerular and tubulointerstitial injury in numerous experimental and clinical conditions. These observations provide a clear explanation for the beneficial effects observed for the ACE inhibitors and AT1 receptor blockers in renal diseases. The future elucidation of the increasing complexity of this system will greatly assist in the rational use of agents that interrupt the RAS in chronic progressive renal injury.



Endothelins (ETs) are potent endothelium-derived vasoconstrictor peptides first described by Yanagisawa and coworkers in 1988. Three structurally and pharmacologically distinct ET isoforms (endothelin-1, -2, and -3 [ET-1, -2, and -3]) have been described. All three isoforms consist of 21 amino acids, are highly homologous, and share a common structure. ET-1 is considered to be the most dominant isoform in the cardiovascular system.

Synthesis and Secretion

ETs are synthesized via posttranslational proteolytic cleavage of specific prohormones. Dibasic pair specific processing endopeptidases, which recognize Arg-Arg or Lys-Arg paired amino acids, cleave prepro ETs and reduce their size from approximately 203 to 39 amino acids. These proETs are subsequently proteolytically cleaved by ET-converting enzymes, yielding mature ETs. These endothelin-converting enzymes (ECEs) are the key enzymes in the endothelin biosynthetic pathways that catalyze the conversion of big ET, the biologically inactive precursor of mature ET. ECEs are type II membrane bound metalloproteases and share significant amino acid sequence identity with neutral endopeptidase 24.11. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity.

Polarized endothelial cells secrete the majority of the ET-1 into the basolateral compartment.[222] Secretion occurs at a constant level, suggesting constitutive pathways. However, a variety of triggers stimulate ET synthesis via transcriptional regulation ( Table 10-3 ).

TABLE 10-3   -- Endothelin Gene and Protein Expression


Vasoactive Peptides

Growth Factors

Angiotensin II

Epidermal growth factor


Insulin-like growth factor


Transforming growth factor-β (TGF-β)








Thromboxane A2


Tissue plasminogen-activating factor

Inflammatory Mediators






Tumor necrosis factor-α (TNF-α)

Shear stress

Phorbol esters


Oxidized low-density lipoproteins








   Protein kinase A activators

   Nitric oxide

   ACE inhibitors




ET stimulation is endothelium dependent and requires de novo protein synthesis because protein synthesis inhibitors such as cycloheximide prevent the release of the mature peptide. However, ET production is not exclusively released by the endothelium but also by nonvascular tissues, albeit at much lower levels than by endothelial cells.

Numerous cells in the kidney produce ETs, including glomerular endothelial cells,[223] glomerular epithelial cells,[224] mesangial cells,[225] and tubular epithelial cells.[226] The kidney synthesizes ET-3 as well as ET-1.[226] In microdissected rat kidney nephron segments, ET-1 mRNA was reported to be in glomeruli and innermedullary collecting ducts but was undetectable in other nephron segments. [227] [228]

ECE mRNA has been found to be more abundant in the renal medulla than in the cortex. However, in disease states such as chronic heart failure, there is up-regulation of ECE mRNA expression, predominantly in the renal cortex.[229] In human kidney, ECE-1 was localized to endothelial and tubular epithelial cells in the cortex and medulla of kidneys.[230]

ETs bind to two G-protein-coupled receptors, the ET(A) and ET(B) receptors. [231] [232] The specific distribution of the two distinct ET receptors has been determined using RT-PCR in microdissected rat nephrons. ET(A) receptors are found in the proximal straight tubule, and both ET receptor subtypes are found in glomeruli and in the afferent and efferent arterioles. The ET(B) receptor has been demonstrated in the proximal convoluted tubule, cortical inner and outer medullary collecting duct, and medullary thick ascending limb. ET(B) receptors have also been identified on podocytes.[233]

Physiologic Actions of Endothelin on the Kidney

The kidney is both the source and an important target for ETs. The effects of ETs include regulation of vascular and mesangial tone, regulation of sodium and water excretion, and cell proliferation and matrix formation. The highest concentrations of ET are found in the renal medulla, where it mediates natriuretic and diuretic effects through the ET(B) receptor and is regulated by sodium intake.[234] The ET(B) receptor is considered to exert predominantly renoprotec-tive effects such as natriuresis and vasodilation via NO and prostaglandins.

ET exerts its hemodynamic effects in almost all vessels, but the sensitivity of the different vascular beds to this peptide varies considerably. The renal and the mesenteric vasculatures have the greatest susceptibility to the actions of endothelins. ET-1 increases renal vascular resistance via contraction of the glomerular arterioles and arcuate and interlobular arteries and decreases blood flow.[235] Long-lasting vasoconstriction that is mediated by the ET(A) receptor is temporarily preceded by transient vasodilation. Vasodilation results from ET(B) receptor mediated release of NO but possibly also involves PGE2 synthesis and cAMP release from mesangial cells.[235] ET(B) receptors may also be involved in the clearance of ET-1 from the plasma.[236] Micropuncture techniques have demonstrated that ET-1 results in a decline in net filtration pressure and a reduction in the glomerular ultrafiltration coefficient, as a result of constriction of pre- and postglomerular arterioles, reduction of blood flow, and mesangial contraction. ET also influences tubular reabsorption and secretion.

In the glomerular tuft, mesangial cells are important targets for ETs. ET-1 induces mesangial cell contraction and mitogenesis. Contraction of the mesangium by ET-1 may reduce glomerular ultrafiltration in vivo, as is the case in post-ischemic renal failure (see later).

ET synthesis in endothelial and mesangial cells is increased after exposure to proinflammatory agents and shear stress (see Table 10-3 ), supporting the view that ET-1 serves as a biologic signal in glomerular injury and inflammation. A large number of proinflammatory stimuli induce ET-1 synthesis, including Ang II, TGF-b, thromboxane A2, thrombin, hypoxia, and shear stress. In glomerular injury, infiltrating inflammatory cells such as macrophages, neutrophils, and mast cells may also become important sources of ET-1. Receptor interactions with ET trigger cell contraction, proliferation, and matrix synthesis. In vitro, ET-1 stimulates proliferation of human renal interstitial fibroblasts and gene expression of collagen I, TGF-b, matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2. All these effects are blocked by ET(A) receptor blockade.[237] In response to injury, stimulation of ET isopeptide synthesis may cause complex rearrangement of actin microfilament bundles and transform mesangial cells from a quiescent to an activated status. The resulting long-term changes in glomerular cell phenotype would then contribute to progressive renal disease and, ultimately, glomerulosclerosis and tubulointerstitial injury.

Endothelin and Renal Pathophysiology

The kidney is an important source and target for ETs. ET has been implicated in several disorders associated with the renal endothelium including cyclosporine toxicity, vascular rejection of kidney transplants, various forms of AKI and chronic renal failure, and hepatorenal syndrome ( Table 10-4 ). Renal vasoconstriction and reductions in GFR are characteristic of these disorders. In chronic progressive renal injury of diverse etiologies, the promitogenic and proinflammatory actions of ET may be even more important.

TABLE 10-4   -- Endothelin and Renal Pathophysiology


ET Antagonists

Renal Effect

BP Effect


Renal ablation


Proteinuria ↓




Renal injury ↓




Proteinuria ↓


Renal injury ↓, survival ↑



Bosentan, ET(A) blocker BMS 193884

No beneficial effect on fibrosis, renal injury, proteinuria


Bosentan +AT1 blocker

Combination no additional effect to AT1 blocker



ACEi vs ET(A) and combination

GSI all treatments ↓



Better on GSI and TI, not on albumin excretion



ET(A) 127722

Response to exogenous big ET ↓

No effect


ET(A) PD 155080

No adverse effect on creatinine clearance or proteinuria


ET(A) BMS 182874 or ET(A/B) Ro 46-2005


No effect


Only ET(A) TI ↓



No effect on glomerular hypertrophy



ET(A) LU 135252

Serum creatinine and proteinuria, plasma and urinary ET-1 ↓


Transgenic ET-1 mice


Renal fibrosis ↓

No effect


SHR + salt

ET(A) blocker LU 135252

Albuminuria ↓ vascular hypertrophy ↓

No effect


PA nephrosis

NEP/ECE inhibitor CGS 26302

Renal injury ↓



PHN nephritis

ET(A) LU 135252 and trandolapril

Both proteinuria ↓



GSI and TI ↓

Combination superior


ET(A) blocker

Matrix proteins↓, chemokines and cytokines ↓



FR 139317




ET(B) blocker

No effect on ECM or growth factors



ET(A/B) PD 142893 and ET(A) blocker

Proteinuria ↓, glomerular damage ↓


ET(A) 135252 or ET(A/B) 224332

Both: ECM ↓



Fibronectin ↓

Collgen IV ↓

Proteinuria ↓ 50%

6 months diabetes


Thickening of glomerular basement membrane ↓ matrix deposition ↓ fibronectin and collagen ↓



LU135252 or trandolapril

Diabetic Ren2

Bosentan versus valsartan

No effect on glomerulosclerosis index, tubulointerstitial injury TGF-β and collagen IV


Diabetic SHR

Bosentan + amlodipine

Similar to cilazapril TGF-β, collagen and fibrosis ↓



Renal injury ↓



Renal injury ↓


Galactose feeding


Renal injury ↓



DOCA salt renal fibrosis

ET(A) A 127722

Improved hemodynamics but no effect on renal injury in all treatment groups



AT1 (candesartan) and combination

Radiocontrast nephropathy

ET(A) 127722

Plasma creatinine ↓



Proteinuria ↓

Stroke prone SHR

ET(A) BMS 182874

Survival ↑, renal injury ↓

No effect


ET(A) BMS 182874

TGF-β, bFGF, MMP-2, procollagen I ↓


Chronic renal allograft rejection


No prevention of rejection, no improvement of survival




Prevention of rejection


Proliferative nephritis


Proteinuria and injury ↓, renal function↑, urinary ET-1 excretion ↓

BP normal



ACEi, ACE inhibitor; BP, blood pressure; ECE, endothelin-converting enzyme; ECM, extracellular matrix; ET, endothelin; GSI, glomerulosclerotic index; MMP-2, matrix metalloproteinase-2; NEP, neutral endopeptidase; TI, tubulointerstitial injury.




Acute Renal Ischemia

The role of ET has been extensively investigated in a range of renal disorders including acute renal ischemia, cyclosporine-induced nephrotoxicity, and renal allograft rejection. In these experimental models, ET(A) and dual ET(A)/ET(B) antagonists have, in general, demonstrated a degree of renoprotection, although this has not been a universal finding. [238] [239] [240] [241]

Chronic Renal Disease/Fibrosis

The renal ET system appears to be involved in the pathogenesis of kidney fibrosis as well as blood pressure regulation by regulating tubular sodium excretion. It has been demonstrated that renal tubular cells synthesize ETs and that protein overload of these cells induces a dose-dependent increase in the synthesis and release of ET-1. [242] [243] This peptide accumulates in the interstitium and participates in the activation of a sequence of events that leads to interstitial inflammation and, ultimately, renal scarring. In several animal models of proteinuric progressive nephropathies, the enhanced renal ET-1 expression as well as the excretion of the peptide in the urine correlated with urinary protein excretion. Similarly, in patients with chronic renal disease an association has been found between increased urinary ET-1 excretion and renal damage. In nephrotic patients, not only is ET-1 localized to endothelial cells but de novo expression of ET-1 also occurs in tubular cells, suggesting a possible relationship between proteinuria and renal ET-1 production.[244] In patients with remission of proteinuria, urinary ET-1 levels decreased, whereas in patients with persistent proteinuria, ET-1 levels remained elevated.

Transgenic mice models selectively overexpressing the ET gene represent an opportunity to investigate directly the mechanisms of ET-mediated vascular and renal changes. Human ET-1 transgenic mice demonstrate heightened expression of ET-1 in the kidney.[245] Although blood pressure was similar to that in control animals, the kidneys of these animals demonstrated interstitial fibrosis and glomerulosclerosis in association with increased extracellular matrix protein expression both in the glomeruli and in the interstitium. From studies in mice with overexpression of ET-1, it was observed that ET did not directly cause hypertension but triggered renal injury that led to increased susceptibility to salt-induced hypertension.[246] Indeed, the ET antagonist bosentan was effective in reducing renal fibrosis in this model independent of effects on blood pressure. [247] [248]

A strong argument in favor of ET-1 as a mediator of renal injury derives from preclinical studies with selective and nonselective ET receptor antagonists that have become available over the last decade. These studies, performed in Ren-2 transgenic animals and in the subtotal nephrectomy model, have demonstrated variable findings on renal protection and suggest that ET antagonists are not as effective as agents that interrupt the RAS.

Diabetic Nephropathy

ETs may contribute to both the pathogenesis and the progression of diabetic nephropathy by at least two separate mechanisms. First, ET acts as a vasoconstrictor with subsequent cortical and inner medullary hypoperfusion. Second, as a trophic agent, ET causes extracellular matrix deposition, a prominent pathologic feature of diabetic nephropathy. Nevertheless, compared with the role of the RAS in the development and progression of diabetic nephropathy, the effects of the ET system remain less clear.

In diabetic population studies, measuring plasma and urine ET levels have been conflicting. In general, in uncomplicated type 1 or 2 diabetes, plasma ET-1 levels are usually not elevated. If albuminuria is present, plasma ET-1 levels may be elevated and could reflect generalized endothelial dysfunction and damage. Furthermore, correlations between plasma ET-1 levels and the degree of albuminuria have been demonstrated. In the presence of diabetic macrovascular complications, plasma ET-1 levels are consistently elevated. There is now accumulating evidence that smoking aggravates and accelerates diabetic and nondiabetic nephropathies by increasing the renal ET-system and impairing endothelial vasodilation.[249]

The effect of glucose per se on ET production remains controversial. [250] [251] [252] [253] In high glucose conditions, mesangial cells lose their contractile response to ET-1 in association with filamentous F-actin disassembly and a reduction in cell size. Loss of the contractile response of mesangial cells to ET-1 occurs in the presence of normal Ca2+ signalling and normal myosin light chain phosphorylation. Recently, it has been shown that these changes are mediated by protein kinase C-ζ. [254] [255] [256] In contrast, if mesangial cells are exposed to high glucose, mesangial cell p38 responsiveness to ET-1, Ang II, and PDGF, and consequent CREB (cAMP responsive element binding) phosphorylation are enhanced through a PKC-independent pathway.[256]

Insulin itself has been shown to stimulate ET-1 release and ET- receptor gene expression.[250] Rats on a high-fructose diet develop hyperinsulinemia, hypertriglyceridemia, and hypertension and subsequently develop renal and cardiac injury. A novel dual ET(A/B) inhibitor, enrasentan, has been reported to prevent the rise in blood pressure as well as renal and cardiac injury in this model.[257]

In several different animal models of type 1 and 2 diabetes, plasma ET-1 levels were increased. However, most of these studies were unable to detect any change at the receptor level. [258] [259] Studies performed by our group using in vitro autoradiographic techniques did not show a significant difference in renal ET receptor distribution between control and streptozotocin-diabetic rats.[260]

In various animal models of diabetes, there have been reports showing renoprotection by treatment with ET(A) blockade. For example, the renoprotective effect of ET(A) blockade with FR 139317 was associated with a reduction in the mRNA levels of various extracellular matrix proteins including type IV collagen and laminin as well as a reduction in cytokines and growth factors including TNF-α, PDGF-B, TGF-b, and bFGF. [261] [262]

Blockade of the ET(B) receptor alone by selective ET(B) blockers has not proven to be beneficial in diabetic nephropathy and had no effect on extracelluar matrix deposition or growth factor expression.[261] The ET(B) receptor is now thought to confer renoprotection by increasing natriuresis and diuresis as well as by promoting vasodilation. Indeed, diabetic ET(B) receptor-deficient rats develop severe low-renin hypertension and progressive renal failure. These mice have high ET-1 plasma levels, suggesting a clearance role for the ET(B) receptor. This study supports a protective role for the ET(B) receptor in the progression of diabetic nephropathy.[263]

Further studies exploring nonselective ET(A/B) receptor blockers or ET(A) antagonists in diabetes have not led to consistent findings. These studies performed in animal models of type 1 and type 2 diabetes have demonstrated variable findings on the development and progression of diabetic nephropathy and have confirmed that the RAS plays a more critical role in the physiopathology of experimental diabetic nephropathy than does the ET pathway. [264] [265] [266] [267] [268]

Rather than considering approaches blocking ET-dependent events as monotherapy, it may be worth considering these agents as part of a combination regimen. For example, the combination of bosentan and amlodipine conferred similar renoprotection to a treatment with the ACE inhibitor cilazapril as monotherapy in diabetic SHR. These effects occurred in association with reduced urinary excretion of TGF-b, renal fibrosis, and collagen accumulation. However, that study did not include control groups treated with bosentan or amlodipine alone.[269] In another study, a novel treatment strategy was employed including a dual inhibitor CGS 26303 that blocked both NEP and ECE (NEP/ECE). This treatment was shown to be effective in reducing renal injury and blood pressure in diabetic SHR and was similar in efficacy to a dual NEP/ACE inhibitor.[270]

Human Studies

ET antagonism in experimental hypertension may result in regression of vascular damage, prevention of stroke and renal failure, and improvement of heart failure. Whether the same is true in human hypertension remains to be established. [271] [272] In humans, moderate to severe hypertension was associated with enhanced expression of pre-pro ET1 mRNA in the endothelium of subcutaneous resistance arteries.[273] Severity of blood pressure, salt-sensitivity, and insulin resistance may be common denominators of involvement of the ET system in hypertension. In essential hypertension, bosentan reduced blood pressure to a similar extent to enalapril without reflex neurohumoral activation.[274] In 47 patients with essential hypertension, salt-depleted-salt-sensitive hypertensive patients exhibited enhanced catecholamine-stimulated ET-1 release. This response pattern was associated with a better response to ET blocker treatment than in nonselected patients.[275]


ETs are important at several stages in embryonic development, in normal postnatal growth, and in cardiovascular and renal homeostasis under healthy conditions. In addition, there is now overwhelming evidence that ET-1 plays an important pathophysiologic role in conditions of decompensated vascular homeostasis.

ET receptor antagonists hold the potential to improve the outcome in patients with various cardiovascular disorders. Most of the progress has been achieved in heart failure and pulmonary hypertension with some exciting preliminary data in the field of atherosclerosis.

With respect to renal disease, inhibitors of the RAS appear to be superior to inhibition of the ET system. Thus, if there is a role for ET antagonists in renal disease, it is likely to be in the context of concomitant RAS blockade.


Urotensin II (U-II) was initially isolated from the goby urophysis, a neurosecretory system in the caudal portion of the spinal cord of fish that is functionally similar to the human hypothalamic-pituitary system.[276] Named urotensin for its smooth muscle-stimulating activity, it has notably hemodynamic, gastrointestinal, reproductive, osmoregulatory, and metabolic functions in fish. Subsequntly, homologs of U-II were identified in tissues of many other animals such as rat and ultimately man. [277] [278]

Human U-II, cloned in 1998, is a cyclic dodecapeptide that is derived from post-translational processing of two distinct precursors ( Fig. 10-5 ), which are alternate splice variants. [279] [280]



FIGURE 10-5  Comparison of the structure of urotensin II and its precursors and sites of enzymic cleavage.



In 1999, Ames and colleagues demonstrated that U-II was the ligand for the rat orphan receptor known as GPR14/SENR, which had been cloned by two independent groups.[279] The U-II receptor, known as UT, is a seven transmembrane, G-protein-coupled receptor encoded on chromosome 17q25.3.[281] It shares significant structural similarity with somastatin receptor subtype 4 and the opioid receptors. It has been demonstrated, ex vivo, that vessels taken from UT receptor knockout mice fail to vasoconstrict in the presence of U-II, demonstrating that this receptor is required for U-II-mediated vasoconstriction.[282]

Binding of U-II to UT leads to activation of the G protein, leading to activation of protein kinase C, calmodulin, and phospholipase C, as evidenced by inhibition of vasoconstriction by specific inhibitors to these enzymes. [281] [283]

U-II has also been demonstrated to be a vascular smooth muscle mitogen. Further studies have linked U-II to the ERK/MAP and RhoA/Rho kinase pathways. [284] [285]

Role in the Kidney

In fish, U-II affects sodium transport, lipid, and glucose metabolism.[276] The urinary human U-II (hU-II) concentration is about three orders of magnitude greater than the plasma concentration.[286] U-II may play a role in the regulation of GFR via tubuloglomerular feedback and reflex control of GFR ( Fig. 10-6 ).[287] In the kidney, U-II has vasodilator and natriuretic effects (see Fig. 10-6 ). Increases in renal blood flow and GFR were observed after the infusion of synthetic human U-II into the renal artery of anesthetized rats, and this can be completely inhibited by an NO synthase inhibitor.[288]



FIGURE 10-6  Biologic actions of urotensin II in several major organ systems in humans. ACTH, adrenocorticotropic hormone; VSMC, vascular smooth muscle cells.



The plasma U-II concentration is twofold higher in patients with renal dysfunction not on hemodialysis and threefold higher in patients on hemodialysis compared with healthy individuals.[289] Although there is no correlation between blood pressure and urinary U-II levels, a higher urinary U-II level was observed in patients with essential hypertension, patients with both glomerular disease and hypertension, and patients with renal tubular disorders but not in normotensive patients with glomerular disease.[286] Abundant U-II-like immunoactivity is observed in tubular epithelial cells and collecting ducts with lower expression in capillaries and glomerular endothelium in the normal kidney as well as renal clear-cell carcinoma. [278] [287]

In type 2 diabetic patients, plasma and urinary U-II levels are higher in those with renal dysfunction than in those with normal renal function.[290] This may be due to increased production of U-II by various organs as well as by renal tubular cells as a result of renal damage.[286] In diabetic nephropathy, there are dramatic increases in the expression of U-II and the UT receptor in tubular epithelial cells.[291]

U-II and its receptor have been extensively investigated in various nonrenal contexts including cardiovascular disease, the nervous system, and diabetes and the metabolic syndrome. U-II appears to have a powerful vasoconstrictor action, promotes fatty acid release, and appears to be highly expressed in certain sites within the peripheral and central nervous systems (see Fig. 10-6 ). [280] [281] [292] [293] There are only very limited renal data including two studies using the specific nonpeptide U-II receptor antagonist palosuran. Intravenous administration of palosuran protected against renal ischemia in a rat model,[294] perhaps by inhibiting U-II-mediated renal vasoconstriction. Furthermore, palosuran has been reported to decrease albuminuria in a diabetic rat model.[295] Clinical studies of palosuran are now in progress to examine its effect on diabetic nephropathy.


U-II is the most potent vasoconstrictor known, causing endothelium-independent vasoconstriction and endothelium-dependent vasodilation. There is increasing evidence that U-II is associated with renal dysfunction, various cardiovascular diseases, atherosclerosis, diabetes, and hypertension, although the results of some studies are ambiguous. More research is needed to elucidate the physiology and pathophysiology of U-II and its receptor. Plasma and urinary concentrations of U-II are elevated in several cardiorenal and metabolic disease states in humans, including hypertension, heart failure, renal disease, and diabetes. The rapid development of research tools, such as knockout mice and novel UT receptor antagonists, will advance our understanding of the physiology and pharmacology of U-II and the UT receptor and may provide a novel treatment for cardiorenal diseases.


The kallikrein-kinin system (KKS) is a complex multienzymatic system, the main components of which are the enzyme kallikrein, the substrate kininogen, effector hormones or kinins (lysyl-bradykinin, bradykinin), and metabolizing enzymes (several kininases, the most relevant being kininase I and II and NEP) ( Fig. 10-7 ). The kinins were discovered in 1909 when Abelous reported an acute fall in blood pressure induced by experimental injection of urine. Kinins are formed from partial hydrolysis of kininogens by a family of kininogenases called kallikreins. Kinins produce their effects by the binding and activation of specific cell surface receptors. At least two types of kinin receptors have been described, B1 and B2. The B1 receptor is activated predominantly by desArg[9]-bradykinin, a natural degradation product of bradykinin produced by the enzyme kininase I. Although it is generally agreed that B1 receptors are inducible by tissue injury,[296] it has been suggested that B1 receptors may also be functionally expressed under normal conditions in the vasculature and the kidney. [297] [298] The B2 receptor is activated by lys-bradykinin and bradykinin and mediates all the known physiologic actions of kinins, including the regulation of organ blood flow, systemic blood pressure, transepithelial water and electrolyte transport, cellular growth, capillary permeability, and the inflammatory response.[299]



FIGURE 10-7  Enzymatic cascade of the KKS. ACE, angiotensin-converting enzyme; HMW, high molecular weight; LMW, low molecular weight; NEP, neutral endopeptidase.



Kinins have a very short half-life (<30 sec) and are degraded by a number of peptidases. Kininase II, which is the same as ACE, is the predominant kinin-degrading enzyme.[300] Other important enzymes in the degradation pathway include NEP and kininase.[300] Because of their short half-life, kinins are paracrine rather than true endocrine hormones.

The discovery of specific and potent kinin receptor antagonists, such as Hoe 140, [301] [302] and the availability of animal models with defined genetic modifications of the KKS have facilitated the efforts to uncover the functional relevance of endogenous kinins. In the kidney, kinins play a significant role in the modulation of renal hemodynamics and sodium excretion and therefore participate in the regulation of blood volume. [303] [304] Kinins have bifunctional effects on cell growth. Bradykinin increases IP3 and intracellular calcium and stimulates the proliferation of a variety of mesenchymal cells, including fibroblasts, vascular smooth muscle cells, and glomerular mesangial cells. [305] [306] [307] [308] Conversely, in the injured vascular wall, kinins may be responsible for the antiproliferative action of ACE inhibitors by stimulating the production of NO.[309]


The single human kininogen gene is localized to chromosome 3q26-qter and is close to two closely related genes, the α-2-HS-glycoprotein and the histidine-rich glycoprotein. [310] [311] It codes for the production of both high-molecular-weight (HMW) kininogen (626 amino acids and 88-120 kDa) and low-molecular-weight (LMW) kininogen (409 amino acids and 50-68 kDa) via alternative splicing from 11 exons spread over a 27-kilobase pair span.

In the human kidney, kallikrein has been demonstrated in connecting tubule cells (see later) and kininogen in the principal cells of the same tubule just preceding the collecting duct. The site is juxtaposed to the afferent arteriole, where synthesized kallikrein (known to traffic to basolateral membranes of these epithelial cells) might affect interstitial cell or vascular wall function via local kinin formation. Alternatively, the known kallikrein trafficking to the apical membranes of these epithelial cells, with subsequent tubular secretion, might result in kininogen being present in the adjacent principal cells to generate kinin locally, thereby modulating tubular ion and water transport.

Kallikreins and Kallikrein Inhibitors

Kallikrein exists in two major forms, plasma and tissue or glandular kallikrein. Renal kallikrein is the so-called true tissue kallikrein, a serine protease. The human genes are clustered on chromosome 19 at q13.2-13.2 and the enzyme is expressed in the epithelial or secretory cells of various ducts, including salivary, sweat, pancreatic, prostatic, intestinal, and distal nephron.[312] Some studies suggest that renal kallikrein mRNA is also detectable by in situ hybridization at the glomerular vascular pole.

The tissue kallikreins are acid glycoproteins, variably and extensively glycosylated.[312] The purified human renal enzyme is synthesized as a zymogen (prokallikrein) with an attached 17–amino acid signal peptide preceding a 7–amino acid activation sequence, which must be cleaved to activate the enzyme.

In the human kidney, kallikrein is localized to tubular segments that, according to cytologic criteria, correspond to the connecting tubule. Close anatomic contact between the kallikrein-containing tubules and the afferent arteriole of the JG apparatus is consistently observed. The close anatomic association of the kallikrein-containing cells with the renin-containing cells at the afferent arteriole close to the JG apparatus suggest a physiologic function and is consistent with a paracrine function for the KKS in the regulation of renal blood flow, GFR, and renin release.

Kallikrein, which is not filtered under normal conditions, crosses the glomerular basement membrane in pathologic conditions such as chronic renal failure. In the rat remnant kidney model, kallikrein is consistently observed in reabsorption droplets of the proximal tubule and is almost certainly secondary to an alteration in glomerular permeability, grossly manifested as proteinuria.[313]

Once activated, renal kallikrein cleaves both HMW and LMW kininogens to release Lys-bradykinin (kallidin). In most mammals including humans, tissue kallikrein cleaves Lys-bradykinin from kininogens, whereas plasma kallikrein releases bradykinin.

Renal kallikrein gene expression is modulated by physiologic factors such as development, thyroid hormones, glucocorticoids, and salt intake. [314] [315] [316] [317] Interestingly, high dietary salt intake down-regulates renal kallikrein synthesis and enzymatic activity in both newborn and adult rats.[316]

Kinin Generation

Two independent KKSs can be distinguished in humans that involve specific subtypes of both kallikreins and kininogens.[299] The circulating plasma KKS consists of the HMW kininogen and plasma prekallikrein, both of which are synthesized in the liver and secreted as plasma proteins. Plasma kallikrein is proteolytically cleaved by the activity of an endothelial cell-borne prekallikrein activator. Previous investigation has identified Ang II and bradykinin as substrates of the same processing enzyme. As reported previously in the RAS section of this chapter, prolylcarboxypeptidase can activate the biologically inert Ang I or the vasoconstrictor Ang II to form Ang 1-7, a biologically active peptide that induces vasodilation by stimulating NO formation. [50] [318] Interestingly, the finding that prolylcarboxypeptidase also activates prekallikrein indicates that it can produce two biologically active peptides, bradykinin and Ang 1-7, each of which could potentially reduce blood pressure, counterbalancing the vasoconstrictor effects of Ang II.

Apart from the plasma KKS, there are also tissue-specific systems consisting of locally synthesized or liver-derived LMW kininogen and tissue kallikrein, a serine protease that has been demonstrated in various glands, as previously described.[299] Unlike the plasma KKS, a continuous synthesis and secretion of kallikrein can occur in these organ-specific tissue systems so that kallidin can be produced physiologically from local and plasma-derived LMW kininogen. Some of these tissue KKSs have been shown to express LMW-kininogen. This applies expecially to the kidney, where large quantities of kininogen and kallikrein are synthesized by the tubular epithelium and are secreted in the urine. Kinin formed within the kidney is detectable in urine, renal interstitial fluid, and even in renal venous blood. However, both the local and the systemic half-life of kinins is widely considered to be very short, in the order of 10 to 30 seconds. Owing to the fact that kinins are produced continuously and that there is a clear evidence for powerful effects of kinins on renal vasculature resistance, electrolyte and water excretion, and renal function modulators (such as renin and angiotensin, eicosanoids, cathecholamines, NO, vasopressin, and ET), it is presumed that the renal KKS contributes to physiologic regulatory processes within the mammalian kidney.[299]

The activity of the renal KKS is usually inferred from measurements of urinary kallikrein. However, the activity of this system could be regulated not only by the amount of kallikrein secreted in the tubular fluid but also by the substrate concentration, the presence of kallikrein inhibitors, the pH and ionic composition of the tubular fluid, the presence of kininases, and the sensitivity of the target organs. In most studies, urinary kallikrein excretion has been the only parameter measured. The activity of the renal KKS can also be assessed by measuring kinins formed in the renal vascular compartment or by determining urinary kinin excretion, because kinins are the biologically active component of this system. However, kinins are rapidly catabolized in the kidney and blood, and in urine while in the bladder. Thus, renal vein kinins and urinary excretion of kinins may not reflect the intrarenal activity of the system. Campbell and colleagues[319] have developed high-performance liquid chromatography-based radioimmunoassays for the specific measurement of hydroxylated and nonhydroxylated bradykinin and kallidin peptides and their metabolites. Using this technique, it was observed that the levels of kinin peptides in urine were several orders of magnitude higher than in plasma or tissue, and kallidin peptides were more abundant than bradykinin peptides in urine.[320]

Kinin Receptors

The most prominent effects of kinins are mediated via the B2 kinin-receptor subtype. This receptor is a constitutively expressed G-protein-coupled receptor that is present in a large number of organs and tissues (e.g., endothelium, fibroblasts, glandular epithelium, kidney, heart, skeletal muscle, central nervous system, as well as in the smooth muscle of blood vessels, vas deferens, trachea, intestine, uterus, and bladder). The B2 receptor is stimulated by both bradykinin and kallidin. By contrast, bradykinin has hardly any effect on the B1 receptor subtype.

The signal transduction mechanisms of the kinin receptors are well characterized only for the B2 subtype. This receptor is responsible for the vasodilatatory activity of kinins at its location on endothelial cells. B2 receptors stimulate phospholipase C and intracellular production of both IP3 and diacylglycerol via activation of certain G-proteins such as Gaq and Gai.

Substantially less information exists about the functionality and signal transduction mechanisms of the B1 receptor subtype. The amino acid sequence is 36% identical with that of the B2 receptor. The B1 receptors, which are physiologically present in only a few tissues, are also associated with vasoactive and inflammatory effects. This receptor subtype does not appear to play a major role in the kidney.


Kinins are cleaved by a number of peptidases that have been described as kininases ( Fig. 10-8 ). Apart from the metabolites desArg[9]-bradykinin and desArg[10]-kallidin, all kinin cleavage products are biologically inactive, and thus, kininase activities can significantly influence kinin effectiveness. These include various carboxypeptidases, ACE, and NEP. ACE, which has been described in detail in the RAS section of this chapter, truncates its own reaction product, 1-7 bradykinin, further to 1-5 bradykinin.



FIGURE 10-8  Comparison of the structure of tissue KKS components and sites of enzymic cleavage. ACE, angiotensin-converting enzyme; LMW, low molecular weight; NEP, neutral endopeptidase.



NEP is a membrane enzyme that, like ACE, cleaves bradykinin at the 7-8 position, but without breaking down the resulting 1-7 bradykinin peptide any farther. NEP has been demonstrated in renal tubules, intestinal epithelium, the central nervous system, the prostate, the heart, and the epididymis. It has also been localized to fibroblasts, endothelial cells, and granulocytes. [321] [322] Most of the kininase activity present in urine and seminal fluid is derived from NEP. Like ACE, NEP also has a broad substrate specificity for a variety of the other substances, including substance P, Ang II, ANP, brain natriuretic peptide (BNP), ET-11, big-ET, enkephalins, oxytocin, and gastrin.[321]

At the NH2-terminal end of bradykinin, only a proline-specific exopeptidase, aminopeptidase P, is able to attack the molecule owing to the presence of two proline residues. After bradykinin breakdown by aminopeptidase P, the resulting peptide 2-9-bradykinin is susceptible to other proteases including the endothelial enzyme dipeptidyl-aminopeptidase IV that reduces this metabolite to 4-9-bradykinin. A variety of other peptidases are also known to be capable of breaking down kinins, including a range of rarely discussed enzymes such as aminopeptidase M.

Physiologic Functions of the Kallikrein-Kinin System: Focus on the Kidney

Kinins can provoke a variety of biologic effects including effects on inflammation, coagulation, and vascular permeability.

Concerning the importance of kinins in the control of blood pressure and organ perfusion, the local KKSs in the myocardium, vascular system, and kidney are the most important. The vasodilatatory effects of kinins are related to an increased release of the mediators NO, PGI2, and endothelium-derived hyperpolarizing factor (EDHF) from the endothelium.[323] Whereas the increase in kidney perfusion is primarily mediated by prostaglandins, NO appears to be essential for the anti-ischemic effect of kinins on the isolated heart.[324] Many of the beneficial effects of kinins can be explained by their ability to stimulate endothelial mediators. Regarding NO and prostacyclin, in addition to their vasodilatory activity, they can also inhibit platelet aggregation and granulocyte adhesion and reduce the release of cardiac catecholamines. The ability of NO to act as a free radical scavenger can also be considered as a further beneficial effect.[325]

Recent studies using animal models with defined genetic modifications have helped to demonstrate the role of the KKS in normal physiology and in disease states. The kininogen-deficient Brown-Norway Katholiek strain of rat shows increased sensitivity to the pressor effects of increased dietary salt, mineralocorticoid administration and Ang II infusion, and an impairment of the cardioprotective effects of preconditioning.[326]

The B2 receptor gene knockout mouse is reported to have elevated blood pressure, increased heart weight-to-body weight ratio, and an exaggerated pressor response to Ang II infusion and chronic dietary salt loading.[327] In addition to various cardiac defects, bradykinin B2 receptor gene knockout mice also demonstrate increased urinary concentration in response to vasopressin, indicating that endogenous kinins acting through the B2 receptor oppose the antidiuretic effect of vasopressin.[328]

Wang and coworkers[329] have observed that two transgenic mouse lines expressing the human B2 receptor showed a significant reduction in blood pressure. Furthermore, administration of Hoe 140, a bradykinin B2 receptor antagonist, restored the blood pressure of the transgenic mice to normal levels within 1 hour, the effect diminishing within 4 hours. The transgenic mice displayed an enhanced blood pressure-lowering effect induced by a bolus intra-aortic injection of kinin and showed an increased response in kinin-induced uterine smooth muscle contractility compared with control littermates. These observations suggest that overexpression of the human bradykinin B2 receptor causes a sustained reduction in blood pressure in transgenic mice, confirming an important physiologic role for the KKS in the control of systemic blood pressure. Moreover, it has recently been demonstrated that transgenic mice overexpressing human tissue kallikrein showed a sustained reduction in blood pressure throughout their life span, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein.[330]

In the kidney, kinins have been reported to increase renal blood flow and papillary blood flow and mediate the hyperfiltration induced by high-protein diet. Furthermore, kinins inhibit in cortical collecting ducts the osmotic response to antidiuretic hormone and reduce net sodium reabsorption. Kinins also inhibit conductive sodium entry in inner medullary collecting duct cells and induce the release of renin in isolated glomeruli.[331]

There are observations that bradykinin can reset tubuloglomerular feedback.[332] In particular, it has been suggested that endogenously produced kinins in the normal rat may, via effects upon prostaglandin production, lower tubuloglomerular feedback sensitivity.

The natriuretic effect of kinins is either due to inhibition of sodium reabsorption in the distal part of the nephron or to changes in deep nephron reabsorption. Kinins may affect sodium reabsorption as a result of a direct effect on the transport of sodium along the nephron, a vasodilator effect, changes in the osmotic gradient of the renal medulla, or a combination of all three effects. These findings imply that the renal KKS is involved in the physiologic regulation of blood pressure, body fluid, and sodium balance. Further experimental studies, both in vitro and in vivo, predominantly using Hoe 140, suggest that bradykinin is antihypertrophic and antiproliferative. This has been reported in mesangial cells, fibroblasts, and renomedullary interstitial cells.

Kallikrein-Kinin System Function in Renal Diseases

Role of the Kallikrein-Kinin System in the Pathogenesis of Hypertension

Based on findings from gene knockout animals, it is hypothesised that impaired kinin levels/action may be involved in the pathogenesis of primary or secondary hypertension. Considering the genetically determined causes of hypertension, the kidney in particular may play an important role. A reduced activity of kallikrein has been observed in the urine of hypertensive patients and rats. Furthermore, epidemiologic studies have shown that a genetically determined impairment of renal kallikrein excretion is associated with the development of high blood pressure and is apparent even prior to the manifestation of clinical hypertension. However, the interpretation of such results is limited by the fact that any preexisting or hypertension-induced kidney disease might also be the underlying cause for a reduction in renal kallikrein excretion. Therefore, the epidemiologic association cannot conclusively be considered to indicate a causal role of reduced renal kallikrein in the pathogenesis of hypertension. Other associations between renal kallikrein excretion and hypertension-related factors, such as age, race, and dietary sodium and potassium intake of the patients, have also been reported.[333] Hence, the fact that reduced renal kallikrein activity is associated with hypertension remains to be explained fully at a mechanistic level.

Role of the Kallikrein-Kinin System in the Pathogenesis of Renal Diseases: Focus on Diabetes

Rats with streptozotocin-induced diabetes mellitus have markedly altered KKS function.[334] Early in the course of the diabetic state, these animals, if treated with insulin, show glomerular hyperfiltration along with increased renal kallikrein synthesis levels and urinary excretion.[334] Treatment of such animals with aprotinin or a B2 receptor antagonist reduces renal blood flow and GFR.[334] These studies have been confirmed in patients with type 1 diabetes.[335] Diabetic subjects with glomerular hyperfiltration showed greater active kallikrein and PGE2 excretion than diabetic patients with normal GFR or control nondiabetic subjects. Kallikrein levels correlated directly with GFR and distal tubular sodium reabsorption. These findings in diabetic rat models and patients provide evidence that the renal KKS is a contributor to the renal adaptation to diabetes and may play a role in diabetic nephropathy. Our own group[336] has explored the possible role of the bradykinin in mediating the renoprotective effects of ACE inhibitors in experimental diabetic nephropathy. In that study, the administration of the bradykinin B2 receptor antagonist Hoe 140 to diabetic rats did not attenuate the antialbuminuric effects of the ACE inhibitor, nor did it have any effect itself. Moreover, treatment with Hoe 140 did not attenuate the beneficial effect of ACE inhibition on glomerular morphology.[336] These findings demonstrate that blockade of Ang II is the major pathway responsible for renoprotection afforded by ACE inhibition and implies that there is no or only a minor involvement of the KKS to the long-term changes of the kidney in diabetes. However, the importance of the KKS in diabetic renal disease has recently been reconsidered with a study in Akita diabetic B2 receptor knockout mice demonstrating accelerated renal injury.[337]

In patients with chronic renal failure, kallikrein excretion has been reported to be greatly increased, and it has been postulated that an increase in kallikrein activity per nephron may play a role in the maintenance of high sodium excretion and blood flow in the surviving nephrons.

In patients with both renal parenchymal disease and hypertension, kallikrein excretion is conspicuously decreased, and the severity of hypertension inversely correlates with kallikrein excretion. Kallikrein excretion in these patients was lower than in patients with essential hypertension of comparable severity without renal failure.[21]


In the last decade, our knowledge of the KKS, in particular the renal KKS, has been significantly advanced. There are two main classes of KKSs: the plasma KKS and the tissue KKS. Studies involving receptors antagonists, kininogen-deficient animals, B2 receptor knockout mice, and genetic activation of the KKS have suggested a physiologic role for this system in the regulation of blood pressure, blood flow distribution, coagulation, and sodium and water excretion. Moreover, recent observations suggest a crucial role for the KKS and, in particular, for the B2 receptor in mediating renal growth and development. Data are available that indicate that the KKS interacts with other renal hormonal systems such as the prostaglandins and the RAS. Indeed, the KKS may participate in mediating some of the effects of treatment with ACE inhibitors. Abnormalities of the KKS have been found in hypertension, diabetes mellitus, and chronic renal failure, and it has been postulated that this system may participate in the pathogenesis and pathophysiology of these diseases. Future studies are needed to assess whether new therapeutic strategies based on stimulation or interruption of KKS actions will be important to the treatment or perhaps even the prevention of some of these common disorders.


The natriuretic peptides are a well characterized family of hormones that play a major role in salt and water homeostasis.[338] The first member of the family to be described was ANP, but at least five structurally similar but genetically distinct peptides including BNP, C-type natriuretic peptide (CNP),[339] dendroaspis natriuretic peptide,[340] and urodilantin [341] [342] also exist. There are other peptides involved in salt and water balance including adrenomedullin, guanylin, uroguanylin,[343] and oubain-like factor, but these are not discussed further in this review.

ANP and BNP are similar in their ability to act as endogenous antagonists of the RAS to cause natriuresis and diuresis, vasodilation, and suppression of the sympathetic nervous system, as well as inhibiting cell growth and reducing secretion of aldosterone and renin. [338] [344] [345]

The natriuretic peptides (NPs) play an important role in the regulation of cardiovascular, renal, and endocrine function,[344] but their therapeutic potential is limited by their peptide nature and the need for intravenous administration. Despite this, several studies have used intravenous administration of the NPs to treat heart failure and AKI in humans, with limited success. An alternative approach is to increase endogenous NP levels by inhibition of the enzymatic degradation by NEP EC (NEP). [321] [346] [347] [348] Selective orally active NEP inhibitors have been demonstrated to protect the NPs from inactivation in vivo and to potentiate their biologic actions. Novel compounds that simultaneously inhibit both NEP and ACE have been developed and are known as vasopepeptidase inhibitors (VPIs). [349] [350] Although these agents have been successful in various animal models of disease, the first of these compounds, omapatrilat, that was used in large clinical trials in hypertension (OCTAVE) and in heart failure (OVERTURE), showed adverse effects that slowed the further development of these VPIs. [351] [352]

Atrial Natriuretic Peptide—Structure, Processing, and Synthesis

The main site of synthesis of ANP is the cardiac atria, and the main stimulus to release is wall tension secondary to increased intravascular volume. In the adult heart, ANP mRNA levels are approximately 30- to 50-fold higher in the atria than observed in the ventricle. However, ventricular expression is dramatically increased in the developing heart[338] and during hemodynamic overload such as heart failure and hypertension.[353] There are also extracardiac sites of ANP gene expression including the kidney, lung, brain, adrenal gland, and liver. [21] [354] [355] In the kidney, alternate processing of proANP adds four amino acids to the N-terminus of ANP to generate a 32–amino acid peptide, proANP 95-126 or urodilatin.[356]

Brain Natriuretic Peptide

BNP is synthesised predominantly from the heart.[338] BNP is found in the highest concentrations in the cardiac ventricles, where it is constitutively expressed. Like ANP, the expression of BNP is regulated by changes in intracardiac pressure and/or stretch. [357] [358] Cardiac BNP gene expression and plasma levels increase in heart failure,[359] hypertension, [360] [361] and renal failure. [362] [363] [364] The physiologic actions of BNP are qualitatively similar to those of ANP and include effects on the kidney (natriuresis and diuresis), the vasculature (decrease in blood pressure and intravascular fluid volume), endocrine systems (inhibition of plasma renin and aldosterone secretion), and the brain (central vasodepressor activity).[338] In pathophysiologic states such as heart failure and renal failure, the levels of BNP often exceed those of ANP, which may reflect slower clearance of BNP by the degradation system and suggests differences in the regulation of BNP compared with that of ANP.

Natriuretic Peptide Receptors

NPs mediate their effects through binding to high-affinity receptors on the cell surface with subsequent intracellular generation of cGMP.[338] Only two of these receptors, NPR-A and NPR-B, exhibit the intracellular guanylyl cyclase (GC) catalytic domain.[365] Upon ligand binding, a change in receptor conformation allows cytosolic factors to interact with the kinase-like domain, leading to activation of GC and the consequent generation of cGMP, the second messenger of the NPs.

NPR-A mRNA is expressed mainly in the kidney, in the glomeruli, in the renal vasculature, and in the proximal tubules and is highest in the innermedullary collecting duct (IMCD), [366] [367] consistent with the known physiologic actions of ANP on these structures.[368]

Clearance Receptor

The inactivation of the NPs occurs via two pathways, binding to the clearance receptor NPR-C and enzymatic degradation. This results in a half-life of the NPs in the range of minutes. The NPR-C clears the NPs through receptor-mediated uptake, internalization, and lysosomal hydrolysis of the NPs, with rapid and efficient recycling of internalized receptors to the cell surface. NPR-C is the most abundant of the receptors, accounting for more than 95% of the total receptor population, and is expressed at high density in kidney, vascular endothelium, smooth muscle cell, and the heart.[369] The NPR-C binds all members of the NP family with high affinity.

Neutral Endopeptidase

Enzymatic degradation of the NPs takes place in the lung, liver, and kidney, and the main enzyme responsible for this degradation is NEP. [321] [346] [348] NEP, originally referred to as enkephalinase because of its ability to degrade opioid peptides within the brain, was subsequently shown to be identical to an already well-characterized zinc metallopeptidase present in the kidney. NEP has a ubiquitous tissue distribution and multiple functions, sharing structural similarities with various metallopeptidases including aminopeptidase (APN), ACE, ECE, and carboxypeptidases A, B, and E.[321] NEP is most abundant in the brush borders of the proximal tubules of the kidney, where it rapidly degrades filtered ANP, thus preventing the peptide from reaching more distal luminal receptors. Despite its lack of substrate specificity in vitro, the primary function of NEP in vivo is to metabolize the NPs.

Renal Actions of the Natriuretic Peptides

The NPs are endogenous antagonists of the RAS. Levels of ANP and BNP rise in response to volume expansion and pressure overload and have actions to antagonize the effects of Ang II on blood pressure, renal tubular reabsorption, vascular tone and growth, and aldosterone secretion.[370]

The glomerulus and the IMCD are the two regions of the nephron at which most NP receptors have been identified. Not surprisingly, the natriuretic and diuretic actions of the NPs result from both hemodynamic effects and direct tubular actions.[371] The most notable hemodynamic action is to increase GFR by enhancing glomerular hydrostatic pressure as a result of afferent arteriolar dilation and efferent arteriolar constriction. The contrasting effects of the NPs on the afferent and efferent arterioles differ from the actions of classical vasodilators such as BK. The NPs also increase accumulation of cGMP in mesangial cells to cause relaxation of these cells and increase the effective surface area for filtration.[372] The NPs are considered to have direct tubular actions. ANP inhibits Ang II-induced sodium and water transport in the proximal tubule, tubular water transport through vasopressin antagonism in the cortical collecting duct, and the distal tubular actions of aldosterone.[21] In addition, NPs induce increases in sodium delivery to the macula densa, thereby indirectly inhibiting renin secretion and subsequent secretion of Ang II and aldosterone.

The NPs decrease blood pressure by a number of pathways. ANP reduces cardiac preload by shifting intravascular fluid into the extravascular compartment through increases in capillary hydrostatic permeability. ANP also increases venous capacitance and reduces extracellular fluid volume,[370] as well as reduces peripheral vascular resistance. ANP also reduces sympathetic tone, suppresses the release of catecholamines, and reduces central sympathetic outflow. BNP has cardiovascular effects similar to those of ANP, whereas CNP is a more potent dilator than either ANP or BNP.

Therapeutic Uses of the Natriuretic Peptides

Given the properties of the NPs, their efficacy in the treatment of diseases such as hypertension, heart failure, and renal insufficiency has been examined in both experimental models and humans. The following section focuses on the therapeutic uses of the NPs in humans. In essential hypertension, ANP infusion lowers blood pressure and increases urinary sodium excretion.[338] In heart failure, ANP decreases systemic vascular resistance, increases natriuresis and diuresis,[338] and is registered for the treatment of pulmonary edema in Japan, under the name Carperitide (Fujisawa, Japan).[373] Although experimental studies in acute renal dysfunction had encouraging results in terms of improvement in renal function, studies in humans have been disappointing.[374] Anartide is a 25–amino acid synthetic form of ANP (102-106) and was used in a multicenter, randomized, double-blind, placebo-controlled clinical trial in 504 critically ill patients with acute tubular necrosis.[375] Anaritide did not improve the overall rate of dialysis-free survival, and although it may have improved dialysis-free survival in patients with oliguria, it actually worsened survival in patients without oliguria with acute tubular necrosis. In a larger study of 1222 patients with oliguric AKI, a 24-hour infusion of anaritide (0.2 mg/kg/min) had similar effects on the primary end-point of dialysis-free survival (21%) to placebo (15%, p=.22).[376] Thus, ANP is not considered to be of use in the treatment of AKI.[377]

With regard to BNP, an infusion increases sodium excretion and lowers blood pressure in mild to moderate hypertension.[378] As the natriuretic and blood pressure-lowering effects of BNP are two- to threefold greater than ANP,[379] BNP may represents a more beneficial therapeutic modality than ANP. Indeed, BNP or nesiritide (Scios, Sunnyvale, CA, USA), gained U.S. Food and Drug Administration approval as the first new parenteral agent approved for heart failure therapy in more than a decade.[380] Nesiritide is identical to endogenous BNP and has been evaluated in clinical trials involving more than 700 subjects. The rapid and sustained beneficial hemodynamic effects of nesiritide supports its use as a first-line intravenous therapy for patients with symptomatic decompensated CHF.[381] However, a recent meta-analysis of the clinical trials of BNP in acutely decompensated heart failure and data from the company suggest that the risk of worsening renal dysfunction increases with its use in heart failure.[382] To date, there are no data as to the efficacy of BNP in renal failure per se in humans.

Neural Endopeptidase Inhibition

Although there has been some success with ANP and BNP infusions, such a mode of administration is not suitable for the long-term treatment of chronic diseases in humans. As there has been little success with orally active analogs or alternative routes of peptide administration, efforts to use the biologic actions of the NPs as a therapy have centered on NEP inhibitors. Although NEP inhibitors do elevate plasma levels of the NPs and under certain experimental conditions cause the expected responses such as diuresis and natriuresis and peripheral vasodilatation, clinical trials in hypertension and heart failure have, in general, had disappointing results. This may relate to the biologic activity of the compounds themselves, but is probably also due to the fact that a fall in blood pressure activates the RAS. Therefore, any increase in the NPs, be it from infusions or inhibition of breakdown, is unable to overcome an activated RAS. The biologic actions of the NPs are restored in the presence of an inhibited RAS, which has led to the development of compounds that simultaneously inhibit NEP and ACE, known as VPIs. These compounds may offer advantages in the treatment of hypertension, heart failure, and renal disease.

Dual Inhibition of Neural Endopeptidase and Angiotensin-Converting Enzyme

The VPIs have been rationally designed, taking into account the similar structural characteristics of the catalytic site of both peptidases, NEP and ACE.[350] Several such inhibitors are available including mixanpril (S21402), CGS30440, aladotril, MDL 100173, sampatrilat, and omapatrilat. All these compounds show potent activity to inhibit ACE and NEP, although the degree of potency against the individual enzymes does vary among compounds. Omapatrilat, the most clinically advanced VPI, has similar potency against both NEP and ACE (NEP Ki=9 nmol/L; ACE Ki=6 nmol/L).[383] Unfortunately, in initial clinical studies focused on hypertension and heart failure, a high incidence of angioedema, a potentially life-threatening complication, occurred with omapatrilat. It is not clear that this represents a side effect of all VPIs, especially given the differences in potencies for ACE versus NEP that exist. However, this life-threatening side effect has significant impaired development of this new class of agents for the time being.

Vasopeptidase Inhibitors and Renal Disease.

In experimental models of renal disease, the VPIs offer some hope in terms of achieving more aggressive blood pressure targets. In the diabetic hypertensive rat, the VPI S21402 had increased efficacy on blood pressure in diabetic SHR, compared with ACE or NEP inhibition alone over a 4-week period.[384] Urinary albumin excretion rate was lower in both diabetic and nondiabetic SHR treated with the dual NEP/ACE inhibitor. In a long-term study in diabetic SHR, another VPI, omapatrilat, conferred renoprotection in a dose-dependent manner, in association with a reduction in expression of TGF-β and β-inducible gene-H3, a TGF-b-dependent matrix protein.[385]

This dual ACE/NEP inhibition has also been reported by several groups to attenuate the progression of renal injury in the model of subtotal nephrectomy (STNx). [386] [387] Overall, these findings suggest that vasopeptidase inhibition may be a therapeutic approach for retarding progression of renal injury, in which reducing both blood pressure and proteinuria are considered important targets.

There are no clinical studies that have specifically assessed VPIs in diabetic or nondiabetic renal disease. However, if clinical studies produce results similar to those in animal models, this new class of drugs may have a major effect in reducing the number of patients that progresses to end-stage renal failure.


The NP system includes ANP, BNP, CNP, and dendroaspis natriuretic peptide. ANP and BNP are similar in their ability to act as endogenous antagonists of the RAS to cause natriuresis and diuresis, vasodilation, and suppression of the sympathetic nervous system, as well as inhibiting cell growth and reducing secretion of aldosterone and renin. Although less is known about the actions of the other NPs, CNP has been demonstrated to be a potent vasodilator in arteries and veins and to inhibit mitogenesis, migration, and growth of vascular smooth muscle cells. NPs mediate their effects through binding to high-affinity receptors, NPR-A, B, and C. The NPs play an important role in the regulation of cardiovascular, renal, and endocrine function. Therapeutic approach to increase endogenous NP levels by inhibition of the enzymatic degradation by NEP has been proposed, and novel compounds that simultaneously inhibit both NEP and ACE, the VPIs, were developed. Unfortunately, preclinical and early clinical trials with omapatrilat in hypertension and heart failure were disappointing, partly because of the potentially life-threatening side effect, angioedema, with this agent.


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