Chronic kidney disease (CKD) is common. A US population study suggested that >10% of the general adult population have an indicator of kidney damage: proteinuria, hematuria, and/or reduced GFR (1). Despite our best current treatments, progression to ESRD remains a major clinical and financial problem, and currently >1 million patients worldwide are on dialysis, with the number continuing to increase yearly. Medicare expenditure on dialysis totaled $14.8 billion in 2003 (2).
It is now widely recognized that cardiovascular disease (CVD) is strongly associated with CKD (1, 3) and constitutes one of its major causes of morbidity and mortality (1). Indeed, CKD has emerged as an important and powerful independent risk factor for CVD (1). As GFR declines, the risk for CVD increases, and patients with CKD that do not require dialysis are more likely to die from CVD than to develop ESRD (1). Furthermore, not only are individuals with CKD at increased risk for cardiovascular events, but also their outcome is worse than in those without CKD (4). Although the prevalence of traditional risk factors (e.g., diabetes, hypertension, dyslipidemia) in the CKD population is high, CVD events remain disproportionate to the underlying risk factor profile (1). Therefore, “nontraditional” risk factors, such as endothelial dysfunction, arterial stiffness, oxidative stress, and acute-phase inflammation, which may contribute to this excessive uremic cardiovascular risk, have become a major focus of interest.
There is an important unmet need for treatments that not only slow the rate of progression of renal impairment, delaying the onset of dialysis in CKD, but also improve the cardiovascular risk profile in these patients. Blockade of the endothelin (ET) system has emerged as one potential strategy. The ET system has been widely implicated in renal disease, including acute renal failure (5). However, the focus of this review is to examine our current understanding of the role that the ET system plays in CKD and whether inhibition of its actions might slow the progression of CKD and reduce the burden of CVD with which it is associated.
Biology of the ET System
First described by Yanagisawa et al. in 1988 (6), the ET system is a family of 21 amino acid peptides with powerful vasoconstrictor and pressor properties. Three different isopeptides, ET-1, ET-2, and ET-3, are known, each with distinct gene and tissue distributions (6–8). Of the three peptides, ET-1 is the major endothelial isoform and, in the human kidney, the only one that has been so far shown to be expressed at the protein level (9). Its main site of vascular production is the endothelial cell, but it is also produced by other cell types, including vascular smooth muscle cells and epicardial cells (10). Within the kidney, it is produced by glomerular epithelial and mesangial cells and renal tubular and medullary collecting duct cells (5). Furthermore, the renal medulla is not only an important site of ET-1 generation but also contains among the highest concentrations of immunoreactive ET-1 of any organ (11).
The gene product is the 212 amino acid prepro-ET-1, and regulation of ET synthesis occurs at the level of gene transcription. Enhanced generation occurs with a wide range of stimuli (12, 13). Those pertinent to CKD include other vasoactive hormones, such as angiotensin (AngII) and vasopressin (AVP), the cytokine IL-1, oxidized LDL, reduced extracellular pH, and cyclosporin A. In contrast, prostacyclin, nitric oxide (NO), and the natriuretic peptides all inhibit gene transcription. Prepro-ET-1 is cleaved to big ET-1 (38 amino acids), which is largely biologically inactive (14). ET-converting enzyme then catalyzes generation of the biologically active ET-1 and C-terminal fragment from big ET-1. Once synthesized, the secretion of mature ET-1 from endothelial cells is largely abluminal (15), toward the adjacent vascular smooth muscle, suggesting an autocrine or paracrine mechanism of action.
ET-1 Action in the Vascular System
ET-1 acts by binding to two distinct receptors, the ETA and ETB receptors (16, 17) (Figures 1 and 2). Within blood vessels, ETA receptors are found on smooth muscle cells, and their activation results in vasoconstriction. ETB receptors are also found on vascular smooth muscle cells (18), where they can mediate vasoconstriction, but are predominantly found on the vascular endothelium, where their activation results in vasodilation via prostacyclin and NO (19).
In addition to its vasodilatory function, the ETB receptor also acts as a clearance receptor for circulating ET-1. The half-life of ET-1 in the healthy circulation is approximately 1 min (20), with removal through receptor- and non–receptor-mediated mechanisms. ET-1 binds to ETB receptors, with subsequent ligand–receptor complex internalization and intracellular degradation accounting for the majority of clearance, particularly in the pulmonary circulation (21), although the splanchnic and renal circulations also contribute (12). Therefore, reductions in ETB numbers, or ETB receptor blockade, may reduce ET-1 clearance, increasing plasma concentrations without altering production. Importantly, because most ET-1 is released abluminally, plasma concentrations of ET-1 do not accurately reflect ET-1 production.
ET-1 Action in the Renal System
ET receptors are widely distributed within the human kidney, with the ETA subtype localized to vascular smooth muscle, notably in the glomeruli, vasa recta, and arcuate arteries, whereas ETB receptors are more numerous (ETB to ETA ratio 2:1) and more widespread, with a high concentration in the collecting system (9, 22). With respect to the renal system, ET-1 has a role in the paracrine/autocrine regulation of renal and intrarenal blood flow, glomerular hemodynamics, sodium and water homeostasis (23), and acid-base balance (24) (Figure 1). It is also clear that ET-1 has many other functions within the kidney (25). Evidence exists for renal and vascular ET-1 acting as two independent systems (26). After systemic infusion of radiolabeled ET-1, labeled compound makes up <% of total urinary ET-1 (27). Therefore, neither glomerular filtration nor tubular secretion of plasma ET-1 accounts for urinary ET-1, which is assumed therefore to be primarily of renal origin. Urinary excretion of ET-1 is therefore thought to reflect renal ET-1 production.
Defining the Role of ET in Physiology
ET-1 is a potent vasoconstrictor in vitro and pressor in whole animals (28). With respect to the kidney, exogenous ET-1 causes renal vasoconstriction (29). Indeed, the renal vasculature is more sensitive to the vasoconstricting effects of ET-1 than other vascular beds (30). Although exogenous ET-1 reduces total renal blood flow (RBF), a regional difference has been observed, with cortical vasoconstriction (31–33) and NO-dependent medullary vasodilation (31). Exogenous ET-1 has also been shown to cause constriction of afferent and efferent arterioles, with a greater effect on the former (34), and reduce filtration coefficient by mesangial cell contraction (25). In humans, a similar vasoconstrictor (14) and pressor response has been demonstrated (35), as well as renal vasoconstriction, a fall in total RBF, and a consequent reduction in GFR (36). As yet, there are no studies of the effects of ET-1 on intrarenal distribution of blood flow in humans.
With respect to renal tubular functions, there is now a substantial body of evidence supporting a role for ET-1 in the regulation of volume homeostasis. ET-1 inhibits the AVP-stimulated retention of water in inner medullary collecting duct cells in vitro (37), and extracellular sodium concentrations may regulate inner medullary collecting duct ET-1 production (23, 38). In addition, ET-1 seems to have a natriuretic role, at least in animals. ET-1, acting via ETB and NO, can inhibit chloride transport in the medullary thick ascending limb of Henle, thereby promoting natriuresis (39, 40). Picomolar ET-1, binding to ETB receptors, activates amiloride-sensitive sodium channels in distal tubular cells in vitro, although higher, nanomolar doses inhibit this channel by a non–ETB receptor–dependent mechanism (41). This has been supported by in vivo experiments in rats demonstrating natriuresis as a result of reduced sodium transport in the proximal and distal nephron segments in response to low-dose exogenous ET-1, with higher doses resulting in sodium retention as a result of glomerular vasoconstriction (42).
Agonist studies may not adequately represent the effects of a hormone, the actions of which are largely autocrine/paracrine, and inhibition of the production or actions of ET-1 may better define its physiologic and pathologic effects. In this respect, ET receptor antagonists have proved to be useful tools in defining the role of ET in health and disease. ET receptor antagonists are classified as ETA selective (e.g., the intravenous antagonist BQ-123) or ETB selective (e.g., the intravenous antagonist BQ-788), depending on their relative affinity for a receptor subtype, or nonselective (e.g., the oral drug bosentan) (12). It should be noted, however, that the distinction between selective and nonselective antagonists is not pharmacologically well defined. The so-called “nonselective” antagonists are still selective for the ETA receptor, but the ratio of ETA to ETB affinity is generally 10- to 100-fold selective for ETA over ETB, compared with 1000-fold or more for recent ETA selective agents (12).
With respect to systemic hemodynamics in healthy humans, selective ETA receptor antagonism is associated with vasodilation (43) and a reduction in BP (44), and selective ETB receptor antagonism is associated with vasoconstriction (43) and a pressor response (45). This suggests that endogenous ET-1 contributes to the maintenance of vascular tone and BP via the ETA receptor, and the balance of ETB receptor function favors activation of the endothelial over the vascular smooth muscle ETB receptor.
In the kidney, animal studies have suggested that both exogenous and endogenous ET-1–mediated reductions in total RBF are mediated via the ETA receptor (46, 47). Antagonist studies describe cortical vasoconstriction as ETA receptor mediated and medullary vasodilation as ETB receptor mediated (31, 32, 46). Furthermore, in vitro studies have shown that combined ETA/B receptor antagonism is required to abrogate fully the vasoconstricting effects of exogenous ET-1 on the afferent arteriole, suggesting that both ETA and vascular smooth muscle cell ETB receptors are involved. At the efferent arteriole, the effect of ET-1 is blocked by ETA receptor antagonism alone and enhanced by ETB receptor blockade, suggesting that ET-1 can modulate efferent arteriolar tone via the ETA receptor and that the balance of ETB receptor effects here is to produce vasodilation (34). The situation is less clear in healthy humans, where there are few studies. One study has demonstrated an increase in RBF after nonselective ET receptor blockade (48). Most, however, do not demonstrate an effect of selective ETA receptor blockade (49–53) or combined ETA/B receptor blockade (52) on basal renal hemodynamics, suggesting that ET-1 acting via the ETA receptor does not contribute to the maintenance of renal vascular tone in health. Selective and unopposed ETB receptor antagonism, however, can produce profound renal vasoconstriction, suggesting that ET-1–mediated tonic renal vasodilation via the ETB receptor is important (52).
Studies have suggested a natriuretic role for the tubular ETB receptor that is linked to NO generation. A potent inhibitory action of NO on tubular sodium reabsorption is well described (54). A rat model deficient in renal ETB receptors displays a salt-sensitive hypertension, with restoration of normal BP by amiloride, suggesting that the ETB receptor regulates sodium excretion at the epithelial sodium channel in collecting duct cells (55), and ETB antagonist-treated rats develop a sodium-dependent hypertension (56). In addition, in the face of acute ETB receptor blockade, pressure-natriuresis curves are shifted to the right such that a greater renal perfusion pressure is needed to excrete the same amount of sodium (57). Finally, administration of exogenous low-dose ET-1 to dogs in the presence of high-grade selective ETA receptor blockade results in renal vasodilation and natriuresis, presumably by unmasking an ETB receptor–mediated effect (58). Dissecting the different actions of the intrarenal ET system, however, has proved difficult, in part from an inability to discriminate between effects of ET-1 in vivo on the nephron and vasculature. To date, ET-1–associated natriuresis and diuresis have not been demonstrated in humans.
Defining the Role of ET in Pathophysiology
Hypertension
Initial evidence of a pressor action of ET-1 led to the suggestion that ET-1 might be implicated in hypertension (6). Production of vascular ET-1 is increased in some (e.g., the Dahl salt-sensitive and the stroke-prone spontaneously hypertensive rat) but not all animal models of hypertension (59). Models in which ET-1 production is increased (mostly but not exclusively salt-dependent types) are associated with increased vascular growth and a response to both selective and nonselective ET receptor antagonism that comprises not only a modest reduction in BP but also a marked regression of vascular growth (59).
In humans, ET-1 message and protein are increased in the vascular smooth muscle cells of hypertensive patients (60). Elevated plasma ET-1 concentrations, however, are not a consistent finding (59). High concentrations would seem, mostly, to be a feature of severe hypertension or indicative of the presence of complications or coexisting disease. Some (61) but not all (62) local studies with ET receptor antagonists have suggested increased vascular ET system activity in patients with hypertension compared with normotensive control subjects and a greater forearm vascular response to nonselective receptor antagonism compared with selective ETA antagonism, consistent with an increased importance of vascular smooth muscle vasoconstrictor ETB receptors in hypertension. In CKD, local administration of BQ-123 increases forearm blood flow (63). Systemic administration of BQ-123 (±BQ-788) to hypertensive patients with CKD showed that selective ETA receptor blockade produces a substantial reduction in BP (approximately 10 mmHg), whereas nonselective ETA/B receptor antagonism lowered BP to a lesser extent. In both cases, the reduction in BP was much greater than that in healthy control subjects (52), supporting an upregulation of the ET-1 system in CKD-associated hypertension. Only one major study has examined the longer term antihypertensive effects of ET receptor antagonism in humans. Bosentan, an orally available, nonselective ET receptor antagonist, reduced BP in patients with essential hypertension as much as did enalapril 20 mg (64). Importantly, this reduction was achieved without activation of the sympathetic nervous or renin-angiotensin system (RAS).
Altered intrarenal ET-1 production may contribute to hypertension (65, 66). Spontaneously hypertensive rats have reduced medullary ET-1 levels after the development of hypertension (67). More recently, Kohan et al. (66) successfully created an elegant tissue-specific knockout of the renal ET system. Mice that lack collecting duct expression of the ET-1 gene have reduced urinary ET-1. These animals are hypertensive and have an impaired ability to excrete a sodium load. It is interesting that these knockout mice excrete acute water loads less well than do wild-type mice and have a heightened physiologic response to AVP, consistent with an intrarenal role for ET-1 in blunting the response to AVP (68). Whereas plasma ET-1 concentrations are normal, urinary ET-1 excretion is reduced in hypertensive patients compared with healthy control subjects, suggesting that either renal ET-1 synthesis is reduced or breakdown is enhanced (69, 70). Therefore, renal ET-1 production or handling may be altered in hypertension, leading to inappropriate sodium and water retention and aiding the development and/or maintenance of hypertension.
Renal function may also influence the relationship between ET-1 and hypertension. First, as renal function declines, plasma ET-1 levels increase (70, 71). The effects of exogenous ET-1 on the renal vasculature are to cause vasoconstriction, activating the RAS and causing salt and water retention, both of which have the potential to raise BP. It remains to be seen whether the rise in ET-1 concentrations that is seen in CKD is due to biologically active or simply immunologically competent peptide, but infusion of exogenous ET-1 to bilaterally nephrectomized rats results in an increased plasma half-life of ET-1 and a prolonged rise in BP compared with sham-operated rats (72), consistent with the idea that elevated plasma ET-1 concentrations in CKD may cause hypertension. Second, there is an upregulation of renal ET-1 in CKD (73), as reflected by increased urinary ET-1 excretion (70). Third, there is a suggestion from an experimental model of nephritis associated with mesangial proliferation that the renal vasculature in this disease may be more sensitive to the vasoconstrictor effects of ET-1 than in normal kidneys (74). Therefore, an amplification of the renal vasoconstrictor effects of ET-1, promoting hypertension, could be envisaged in CKD.
Studies of ETB receptor knockout animals suggest the ETB receptors are important in protecting against hypertension. These animals exhibit a sodium-dependent hypertension that is attributed to an absence of tonic inhibition of the epithelial sodium channel in the distal nephron (55). It is interesting that ETB receptor–deficient mice show renal injury, an impaired ability to excrete a sodium load, and hypertension that persists when they are cross-transplanted with wild-type kidneys, suggesting that it may be not only renal but also extrarenal ETB receptors that play a protective role against hypertension (75).
Endothelial Dysfunction and Atherosclerosis
The endothelium is a crucial regulator of vascular tone (76), and its function is impaired, both in hypertension and in groups who are at risk for hypertension, with a shift toward reduced vasodilation, associated with a proinflammatory and prothrombotic state. Endothelial dysfunction (ED) is also a widely recognized feature of CKD (76), is recognized to be a key early determinant in the progression to atherosclerosis, and is now well established to be independently associated with increased cardiovascular risk (77). Mechanisms that participate in ED include reduced NO generation, oxidative stress, and upregulation of inflammatory mediators (76). Animal models of ED across a number of animal species have shown that antagonism of the ET system, predominantly with selective ETA receptor antagonists, improves NO-mediated endothelial function (78–80), suggesting that ET-1, acting via ETA receptors, is involved in the pathogenesis of ED. Treatment with selective ETA receptor antagonism also improves endothelial function in the coronary vessels of patients with atherosclerosis, again suggesting that ET-1, acting via ETA receptors, is involved in the pathogenesis of ED in these patients (81).
The ET system is also implicated in the development of atherosclerosis. In smooth muscle cells and foamy macrophages in atherosclerotic models, both ETA and ETB receptors are highly expressed (82). Increased expression of ET-1 and ET-converting enzyme is seen in human arteries at different stages of atherosclerosis (60, 83), and high levels of ET-1 have been found in human atherosclerotic lesions (60, 83–85). Furthermore, plasma ET-1 concentrations correlate positively with the degree of atherosclerosis present (84). Importantly, not only is restoration of the impaired activity of the NO system and, hence, improvement in endothelial function seen after ET receptor antagonism in a range of animal models of atherosclerosis (78, 79, 82), but so too is a reversal of atherosclerotic lesion development. Therefore, ET antagonists reduce the activity of the ET system, increase NO bioavailability, improve endothelial function, and slow the progression of atherosclerosis. To date, there are no therapeutic studies on endothelial function or atheroma progression with ET antagonism in patients with CKD.
Arterial Stiffness
Arterial stiffness is an important independent predictor of all-cause and cardiovascular mortality in patients with ESRD (86). Moreover, a therapeutic trial in patients with ESRD by Guerin et al. (87) showed that after BP reduction, cardiovascular survival was observed mainly in patients who also displayed a reduction in arterial stiffness (87). Epidemiologic studies indicate that there is an increased cardiovascular risk early on in CKD, but there are as yet few data that show how early arterial stiffness develops (88). Increased arterial stiffness results in a selective elevation of pulse pressure, causing deleterious consequences for the heart. Through an elevation of central systolic BP, arterial stiffness enhances left ventricular load, favoring development of cardiac hypertrophy, and through reduction of central diastolic BP, it decreases coronary perfusion pressure, contributing to myocardial ischemia (89).
Arterial stiffness is linked to ED (89), and the two conditions commonly coexist in patients who are at increased risk for CVD. A number of interventions that reduce arterial stiffness also improve endothelial function (89). To date, few studies have addressed the relationship between these two markers of CVD after treatment and none in patients with CKD. However, there now is evidence from both animal and human studies that the endothelium is an important regulator of arterial stiffness. Basal endogenous NO generation decreases arterial stiffness in animals (90) and humans (91–93). By contrast, ET-1, at concentrations similar to those observed in the plasma of patients with CKD, caused an increase in arterial stiffness that can be blocked by concomitant administration of an ETA receptor antagonist (94). Furthermore, endogenous ET-1 was shown recently to increase arterial stiffness (95). Therefore, in ED, where NO is downregulated and ET-1 is upregulated, the balance will likely shift in favor of increased arterial stiffness. Clinical studies of the effects of ET receptor antagonism on arterial stiffness in CKD will clearly be of great interest.
Oxidative Stress and Inflammation
Oxidative stress and inflammation are well documented in ESRD (96, 97). Indeed, they are very common even with mild renal insufficiency (98). Oxidative stress is characterized by an imbalance between exposure to free radicals, principally derived from oxygen, and antioxidant defenses. As in ED, there is loss of NO availability in states of oxidative stress, and the close relationship among increased oxidative stress, reduced NO availability, and subsequent cardiovascular events is well established (99). In addition, there is now mounting evidence, although scarce human data, supporting the hypothesis that at least some of the injurious effects of ET-1 on the vasculature are mediated via an increase in oxidative stress and that ET system blockade may be of use in reducing this (100, 101). Indeed, in DOCA-salt hypertension, the ET-1–promoted production of reactive oxygen species, the principal mediators of oxidative stress, is ETA receptor mediated, and selective ETA receptor blockade normalizes the ED found in this model (102), independent of changes in BP. Data in CKD are scarce at present, and it remains unclear whether oxidative stress is a cause or a consequence of renal insufficiency.
Reactive oxygen species likely promote the development of atherosclerosis through a number of mechanisms (103), including ED, increased production of proinflammatory cytokines such as IL-6, and acute-phase proteins such as C-reactive protein (CRP) (104). IL-6 and CRP are both independent predictors of cardiovascular events and mortality (105). Recent evidence suggests that a reduction in kidney function per se may be associated with an inflammatory response in both mild (106) and advanced (107) kidney disease, and a number of studies have shown that CRP predicts all-cause and cardiovascular mortality in dialysis patients (108). In addition to being an important prognostic marker for CVD, CRP may contribute to the atherosclerotic process mainly through the impairment of endothelial function (109, 110). Furthermore, CRP has been shown to decrease the activity of the NO system (111) and to potentiate the release of ET-1 (104). ET receptor antagonism has been shown to attenuate the proatherogenic effects of CRP in vitro (104), consistent with anti-inflammatory and antiatherogenic actions.
Defining the Role of ET in Renal Pathophysiology
CKD and Renal Hemodynamics
There are only few studies in animal models of CKD. Nevertheless, these show that ET receptor antagonism improves RBF and preserves GFR. Nonselective ETA/B receptor antagonism with bosentan can prevent the renal vasoconstriction that is seen in the early phases of streptozocin-induced diabetes (112), and selective ETA receptor antagonism can preserve GFR and RBF during acute (113) and chronic cyclosporin A administration (114). In addition, in the Dahl salt-sensitive hypertensive rat, where a reduced RBF and GFR are observed after a high-salt diet, systemic ETA/B receptor antagonism tended to increase and intrarenal interstitial infusion significantly increased RBF and GFR (115).
In patients with CKD, selective ETA receptor antagonism produces an increase in RBF and a decrease in renovascular resistance (52), suggesting that ET-1 acting via ETA receptors is involved in the increased renovascular tone. These changes are accompanied by a fall in effective filtration fraction (EFF), suggesting that ET-1, acting via ETA receptors, exerts a preferential efferent arteriolar vasoconstrictive effect, raising the possibility that ET-1 promotes hyperfiltration with its consequent potential for renal injury. The renal hemodynamic effects of selective ETA receptor antagonism can be abolished by concomitant administration of an ETB receptor antagonist, and, as in health, selective blockade of the ETB receptor produced renal vasoconstriction (52). Notably, in these studies, selective ETB receptor antagonism increased renal vascular resistance by twice as much (approximately 20 to 30%) as systemic vascular resistance (approximately 10 to 15%), suggesting that tonic ETB receptor–mediated renal vasodilation plays a key role in opposing renal vasoconstriction. This is likely to be of particular importance in CKD, where baseline renal vascular resistance is high. The renal hemodynamic changes in these studies are consistent with other work in patients with CKD, in whom nonselective ET receptor blockade reduces EFF while maintaining GFR (56), and in patients who have diabetes with albuminuria, in whom selective ETA receptor antagonism reduced both BP and urinary protein excretion (116, 117).
Proteinuria
Significant proteinuria has emerged as a powerful predictor of renal disease progression (118), and proteinuria reduction is an important strategy to retard or prevent renal functional loss (118, 119). In addition, proteinuria is no longer simply a renal risk factor. Alongside the concept of CKD as a global vascular disease state is emerging the global cardiovascular risk that is associated with proteinuria. Albuminuria is incrementally associated with increased cardiovascular risk in both individuals with preexisting risk (e.g., hypertensive patients) (120) and individuals with no known risk factor (121). This is true even in the presence of normal renal function (122). Furthermore, at least in hypertension, reduction of albuminuria confers cardiovascular protection (120).
Upregulation of the renal ET system exacerbates proteinuria. Through its hemodynamic effects, ET-1 causes glomerular capillary hypertension, an increase in glomerular permeability, and excessive protein filtration (123). A reduction in microalbuminuria in patients with diabetes was shown recently after chronic selective ETA receptor blockade (116, 117). Furthermore, the reductions in EFF that were observed after acute selective ETA receptor antagonism in patients with CKD were accompanied by a reduction in proteinuria (52), suggesting that one mechanism for the antiproteinuric effect of ETA receptor antagonism in CKD may relate to alterations in glomerular hemodynamics with, potentially, a fall in glomerular capillary perfusion pressure. This would produce a situation analogous to that seen with angiotensin-converting enzyme (ACE) inhibitors (118), in which case ET antagonists might be expected to be renoprotective and improve long-term renal outcome.
The development of proteinuria is also associated with damage to the renal podocyte (124), the highly specialized glomerular epithelial cell that helps to maintain an intact filtration barrier under normal conditions. Recent in vitro studies suggested that podocytes undergo phenotypic changes that resemble de-differentiation as a result of exposure to exuberant amounts of protein (125). In parallel with these changes, there was increased ET-1 production by the podocyte, which was dependent, at least partly, on the cytoskeletal rearrangements that were brought about by excess protein exposure. In the same model, administration of exogenous ET-1 brought about similar podocyte cytoskeletal changes. Therefore, the authors concluded that podocyte-derived ET-1 acting in an autocrine and paracrine manner promotes further podocyte ultrastructural degeneration and hence its own production, both of these contributing to a further breakdown in the glomerular filtration barrier. These data are in line with in vivo evidence in a murine model of protein overload that displays increased renal ET-1 production alongside the development of podocyte structural damage (126). Whether damage to the podocyte is the primary event that leads to subsequent proteinuria or vice versa remains unclear. Nevertheless, it is not unreasonable to envisage a series of events with initial ET-1–mediated glomerular hypertension exposing podocytes to unusually large amounts of protein and so leading to their de-differentiation and production of ET-1. This podocyte-derived ET-1 then could exacerbate glomerular hypertension and lead to further podocyte de-differentiation, thereby setting up a vicious cycle.
Clinical studies are supportive of a role for the ET system in proteinuric nephropathies. Patients with chronic glomerulonephritis and proteinuria displayed a rise in renal ET-1 and tubular ETB receptor expression that increased with higher degrees of proteinuria (127), and patients who were exposed to selective ETA receptor antagonism both in the acute (52) and the chronic (117) setting showed a significant reduction in proteinuria.
CKD Progression
Excess protein filtration at the glomerulus leads to increased tubular reabsorption. This can activate tubular-dependent pathways of interstitial inflammation and fibrosis, with progressive renal scarring (128). Studies suggest a link between upregulation of the renal ET system and tubular protein reabsorption. Exposure of proximal tubular cells in vitro to a protein load leads to a dose-dependent increase in ET-1 production (129). This phenomenon is not associated exclusively with albumin but may be seen with other proteins, such as IgG and transferrin (129). Animals that ingest dietary acid have increased accumulation of interstitial ET-1, suggesting that the consequent enhanced renal ET-1 production, possibly by renal tubular and/or renal endothelial cells, is abluminally secreted (130). Within the interstitium, ET-1 has the capacity to bind to interstitial fibroblasts and promote their proliferation and to generate extracellular matrix (131). Furthermore, ET-1 is chemotactic for blood monocytes (132), leading to secretion of proinflammatory cytokines and growth factors, events that could contribute to interstitial remodeling and scarring. Hence, a potential pathway may be envisaged whereby ET-1 could link proteinuria to interstitial fibrosis.
In the remnant kidney model, renal ET-1 gene expression and urinary ET-1 excretion correlate with degree of proteinuria and extent of renal damage (73). Also, transgenic animal studies in which renal ET pathways are upregulated display renal tubulointerstitial lesions independent of changes in BP (133). These BP-independent effects of ET are supported by antagonist studies in which ET receptor antagonists led to a slowing of progressive renal damage, even in the absence of BP modification (134). Nonselective ETA/B receptor antagonism can reduce the increase in collagen and extracellular matrix deposition that is seen in rats that are treated with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, independent of BP changes, and can also reduce collagen I gene activity to normal levels, suggesting that ET-1 promotes renal fibrosis via activation of this gene (135). ET receptor antagonists have also been shown to attenuate the progression of renal insufficiency in a rat remnant kidney model (136). Although nonselective ETA/B receptor antagonists have produced positive results (137), the effect is greater with selective ETA receptor antagonism (138). Indeed, concomitant administration of an ETB receptor antagonist can abolish the beneficial effects of ETA receptor antagonism (139). In patients with nephrotic syndrome as a result of focal and segmental glomerulosclerosis, plasma and urinary ET-1 concentrations are significantly higher than those in healthy control subjects (140), and nephrotic patients who show a reduction in proteinuria with immunosuppressive therapy also show reductions in urinary ET-1 excretion (141).
Blockade of the ET System and RAS: A Potential Synergism
ET-1 and AngII are powerful vasoconstrictors involved in the regulation of vascular tone, and there is considerable evidence for an interaction between the ET and RAS (142). AngII increases ET-1 transcription and secretion in vitro in a variety of cell types, including endothelial and vascular smooth muscle cells (143, 144). ACE inhibitors also reduce renal ET-1 formation. Rats with reduced renal mass show a parallel fall in proteinuria, vascular and glomerular prepro-ET-1 mRNA, and ET-1 peptide after RAS blockade with losartan and captopril (145). Chronic ACE inhibitor treatment in animal models of glomerulosclerosis (146) and immune-mediated glomerulonephritis (147) leads to a reduction in proteinuria as well as a fall in renal ET-1 mRNA and protein expression.
It is interesting that animal data have suggested that concomitant acute blockade of the RAS and ET system produces greater hemodynamic changes than those seen with blockade of either system alone (148–152). Also, many clinical studies using ET receptor antagonists in patients with heart failure demonstrated major additional hemodynamic effects (153, 154) in patients who already were receiving ACE inhibitors. Synergism with respect to acute systemic hemodynamic effects between ETA receptor antagonists and angiotensin receptor type 1 antagonists (ARB) (51) or ACE inhibitors (53) has been demonstrated in humans. More recently, the combination of ET receptor antagonism and ACE inhibition has been shown to improve endothelial function (155, 156).
With respect to the kidneys, when ETA receptor antagonism is given in the presence of ACE inhibition in healthy subjects, contrary to a lack of effect of ETA receptor antagonism alone, an increase in RBF and natriuresis is observed, an effect that seems to be both ETB dependent and NO mediated (53). Although it is tempting to attribute the increase in sodium excretion to the activity of an unblocked tubular ETB receptor, it is possible that the natriuresis is entirely a consequence of the renal vasodilation and so essentially a hemodynamic effect. This would likely be the case if the intrarenal changes in RBF—of an ETB-mediated increase in medullary blood flow—that are seen in animal models also occur in humans. Further human studies are needed to characterize the role of the renal ETB receptor and the interaction between ET and AngII in CKD, in which there is increased activity in the RAS, and in a setting where many patients are already treated with ACE inhibitors.
ET Antagonism as a Treatment Strategy in CKD
ET-1 plays a role in the maintenance of BP and arterial stiffness. It also contributes to ED, oxidative stress, and vascular inflammation, with additional longer term effects on vascular remodeling. In animals, ET receptor antagonists have been shown to reduce BP, improve arterial stiffness and ED, and retard the progression of atherosclerosis. Some of these observations are confirmed by clinical studies. However, studies in CKD are fairly limited but suggest that, in addition to having a beneficial effect on systemic hemodynamics, ET receptor antagonists improve renal function and may potentially reduce renal disease progression.
The question of whether selective or nonselective receptor blockade should be used is probably disease specific and in CKD remains to be clarified. However, preliminary evidence in patients with CKD suggests that both selective ETA and nonselective ETA/B receptor blockade reduces BP but that selective ETA blockade has additional desirable effects on renal hemodynamics (52). From the current evidence base, concomitant ETB receptor blockade seems at best to offer no advantage over selective ETA antagonism and may potentially reduce the benefits. Further studies are needed to discern the theoretical beneficial effects of an unblocked ETB receptor in terms of natriuresis, diuresis, and glomerular hemodynamics.
Currently, a phase III trial is in progress to explore the potential of a selective ETA receptor antagonist, avosentan, in diabetic nephropathy (157), but more clinical data are essential to advancing our broader understanding of the role of ET receptor antagonism not only as a potential renoprotective therapy in CKD but also as a treatment for the CVD with which it is associated.
;)
Figure 1: The role of endothelin-1 in chronic kidney disease (CKD) and cardiovascular disease. ET, endothelin. Illustration by Josh Gramling—Gramling Medical Illustration.
Figure 2: Actions of endothelin-1. Except where indicated, most of the data are drawn from studies in animals. This table shows the receptor responsible for each action but, particularly in the case of ETA receptor–mediated actions, does not exclude a small contribution from the ETB receptor. CyA, cyclosporine; RVR, renal vascular resistance. Illustration by Josh Gramling—Gramling Medical Illustration.
Published online ahead of print. Publication date available at www.jasn.org.
References
1. Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B, Hamm LL, McCullough PA, Kasiske BL, Kelepouris E, Klag MJ, Parfrey P, Pfeffer M, Raij L, Spinosa DJ, Wilson PW: Kidney disease as a risk factor for development of cardiovascular disease: A statement from the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Circulation 108 : 2154 –2169, 2003
2. US Renal Data System: USRDS 2005 Annual Data Report: Atlas of End-Stage Renal Disease in the United States, Bethesda, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2005
3. Foley RN, Murray AM, Li S, Herzog CA, McBean AM, Eggers PW, Collins AJ: Chronic kidney disease and the risk for cardiovascular disease, renal replacement, and death in the United States Medicare population, 1998 to 1999. J Am Soc Nephrol 16 : 489 –495, 2005
4. Anavekar NS, McMurray JJ, Velazquez EJ, Solomon SD, Kober L, Rouleau JL, White HD, Nordlander R, Maggioni A, Dickstein K, Zelenkofske S, Leimberger JD, Califf RM, Pfeffer MA: Relation between renal dysfunction and cardiovascular outcomes after myocardial infarction. N Engl J Med 351 : 1285 –1295, 2004
5. Kohan DE: Endothelins in the normal and diseased kidney. Am J Kidney Dis 29 : 2 –26, 1997
6. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332 : 411 –415, 1988
7. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T: The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 86 : 2863 –2867, 1989
8. Arinami T, Ishikawa M, Inoue A, Yanagisawa M, Masaki T, Yoshida MC, Hamaguchi H: Chromosomal assignments of the human endothelin family genes: The endothelin-1 gene (EDN1) to 6p23–p24, the endothelin-2 gene (EDN2) to 1p34, and the endothelin-3 gene (EDN3) to 20q13.2-q13.3. Am J Hum Genet 48 : 990 –996, 1991
9. Karet FE, Davenport AP: Localization of endothelin peptides in human kidney. Kidney Int 49 : 382 –387, 1996
10. Eid H, de Bold ML, Chen JH, de Bold AJ: Epicardial mesothelial cells synthesize and release endothelin. J Cardiovasc Pharmacol 24 : 715 –720, 1994
11. Morita S, Kitamura K, Yamamoto Y, Eto T, Osada Y, Sumiyoshi A, Koono M, Tanaka K: Immunoreactive endothelin in human kidney. Ann Clin Biochem 28 : 267 –271, 1991
12. Attina T, Camidge R, Newby DE, Webb DJ: Endothelin antagonism in pulmonary hypertension, heart failure, and beyond. Heart 91 : 825 –831, 2005
13. Wesson DE, Simoni J, Green DF: Reduced extracellular pH increases endothelin-1 secretion by human renal microvascular endothelial cells. J Clin Invest 101 : 578 –583, 1998
14. Haynes WG, Webb DJ: Contribution of endogenous generation of endothelin-1 to basal vascular tone. Lancet 344 : 852 –854, 1994
15. Yoshimoto S, Ishizaki Y, Sasaki T, Murota S: Effect of carbon dioxide and oxygen on endothelin production by cultured porcine cerebral endothelial cells. Stroke 22 : 378 –383, 1991
16. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S: Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348 : 730 –732, 1990
17. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T: Cloning of a cDNA encoding a non-isopeptide selective subtype of the endothelin receptor. Nature 348 : 732 –735, 1990
18. Davenport AP, O’Reilly G, Molenaar P, Maguire JJ, Kuc RE, Sharkey A, Bacon CR, Ferro A: Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020: Evidence for expression of ETB receptors in human vascular smooth muscle. J Cardiovasc Pharmacol 22[Suppl 8] : S22 –S25, 1993
19. DeNucci G, Thomas R, D’Orleans-Juste P, Antunes E, Walder C, Warner TD, Vane JR: Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A 85 : 9797 –9800, 1988
20. Gasic S, Wagner OF, Vierhapper H, Nowotny P, Waldhausl W: Regional haemodynamic effects and clearance of endothelin-1 in humans: Renal and peripheral tissues may contribute to overall disposal of the peptide. J Cardiovasc Pharmacol 19 : 176 –180, 1992
21. Dupuis J, Stewart DJ, Cernacek P, Gosselin G: Human pulmonary circulation is an important site for both clearance and production of endothelin-1. Circulation 94 : 1278 –1284, 1996
22. Kuc R, Davenport AP: Comparison of endothelin-A and endothelin-B receptor distribution visualized by radioligand binding versus immunocytochemical localization using subtype selective antisera. J Cardiovasc Pharmacol 44[Suppl 1] : S224 –S226, 2004
23. Kohan DE, Padilla E: Osmolar regulation of endothelin-1 production by rat inner medullary collecting duct. J Clin Invest 91 : 1235 –1240, 1993
24. Wesson DE: Endogenous endothelins mediate increased acidification in remnant kidneys. J Am Soc Nephrol 12 : 1826 –1835, 2001
25. Sorokin A, Kohan, DE: Physiology and pathology of endothelin-1 in renal mesangium. Am J Physiol Renal Physiol 285 : F579 –F589, 2003
26. Serneri GGN, Modesti PA, Cecioni I, Biagini D, Migliorini A, Costolo A, Colella A, Naldoni A, Paoletti P: Plasma endothelin and renal endothelin are two distinct systems involved in volume homeostasis. Am J Physiol 268 : H1829 –H1837, 1995
27. Benigni A, Perico N, Gaspari F, Zoja C, Bellizzi M, Gabanelli M, Remuzzi G: Increased renal endothelin production in rats with reduced renal mass. Am J Physiol 260 : F331 –F339, 1991
28. Yanagisawa M, Inoue A, Ishikawa T, Kasuya Y, Kimura S, Kumagaye S, Nakajima K, Watanabe TX, Sakakibara S, Goto K, Masaki T: Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci U S A 85 : 6964 –6967, 1988
29. Chou SY, Porush JG: Renal actions of endothelin-1 and endothelin-3: Interactions with the prostaglandin system and nitric oxide. Am J Kidney Dis 26 : 116 –123, 1995
30. Pernow J, Franco-Cereceda A, Matran R, Lundberg JM: Effect of endothelin-1 on regional vascular resistance in the pig. J Cardiovasc Pharmacol 13[Suppl 16] : S205 –S206, 1989
31. Rubinstein I, Gurbanov K, Hoffman A, Better OS, Winaver J: Differential effect of endothelin-1 on renal regional blood flow: Role of nitric oxide. J Cardiovasc Pharmacol 26 : S208 –S210, 1995
32. Gurbanov K, Rubinstein I, Hoffman A, Abassi Z, Better OS, Winaver J: Differential regulation of renal regional blood flow by endothelin-1. Am J Physiol 271 : F1166 –F1172, 1996
33. Denton KM, Shweta A, Finkelstein L, Flower RL, Evans RG: Effect of endothelin-1 on regional kidney blood flow and renal arteriole calibre in rabbits. Clin Exp Pharmacol Physiol 31 : 494 –501, 2004
34. Inscho EW, Imig JD, Cook AK, Pollock, DM: ET(A) and ET(B) receptors differentially modulate afferent and efferent arteriolar responses to endothelin. Br J Pharmacol 146 : 1019 –1026, 2005
35. Sorensen SS, Madsen JK, Pedersen EB: Systemic and renal effects of intravenous infusion of endothelin-1 in healthy human volunteers. Am J Physiol 266 : F411 –F418, 1994
36. Rabelink TJ, Kaasjager KAH, Boer P, Stroes EG, Braam B, Koomans HA: Effects of endothelin-1 on renal function in humans. Implications for physiology and pathophysiology. Kidney Int 46 : 376 –381, 1994
37. Tomita K, Nonoguchi H, Terada Y, Marumo F: Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat. Am J Physiol 264 : F690 –F696, 1993
38. Yang T, Terada Y, Nonoguchi H, Ujiie K, Tomita K, Marumo F: Effect of hyperosmolality on production and mRNA expression of ET-1 in inner medullary collecting duct. Am J Physiol 264 : F684 –F689, 1993
39. Plato CF, Pollock DM, Garvin JL: Endothelin inhibits thick ascending limb chloride flux via ET(B) receptor-mediated NO release. Am J Physiol Renal Physiol 279 : F326 –F333, 2000
40. Herrera M, Garvin JL: Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb. Am J Physiol Renal Physiol 287 : F231 –F235, 2004
41. Gallego MS, Ling BN: Regulation of amiloride-sensitive Na+ channels by endothelin-1 in distal nephron cells. Am J Physiol 271 : F451 –F460, 1996
42. Harris PJ, Zhuo J, Mendelsohn FA, Skinner SL: Haemodynamic and renal tubular effects of low doses of endothelin in anaesthetized rats. J Physiol 433 : 25 –39, 1991
43. Verhaar MC, Strachan FE, Newby DE, Cruden NL, Koomans HA, Rabelink TJ, Webb DJ: Endothelin-A receptor antagonist-mediated vasodilatation is attenuated by inhibition of nitric oxide synthesis and by endothelin-B receptor blockade. Circulation 97 : 752 –756, 1998
44. Spratt JCS, Goddard J, Patel N, Strachan FE, Rankin AJ, Webb DJ: Systemic ETA receptor antagonism with BQ-123 blocks ET-1 induced forearm vasoconstriction and decreases peripheral vascular resistance in healthy men. Br J Pharmacol 134 : 648 –654, 2001
45. Strachan FE, Spratt JC, Wilkinson IB, Johnston N, Gray GA, Webb DJ: Systemic blockade of the endothelin-B receptor increases peripheral resistance in healthy men. Hypertension 33 : 581 –585, 1999
46. Evans RG, Madden AC, Oliver JJ, Lewis TV: Effects of ET(A)- and ET(B)-receptor antagonists on regional kidney blood flow, and responses to intravenous endothelin-1, in anaesthetized rabbits. J Hypertens 19 : 1789 –1799, 2001
47. Abassi Z, Francis B, Wessale J, Ovcharenko E, Winaver J, Hoffman A: Effects of endothelin receptors ET(A) and ET(B) blockade on renal haemodynamics in normal rats and in rats with experimental congestive heart failure. Clin Sci (Lond) 103 Suppl 48 : 245S –248S, 2002
48. Freed MI, Wilson DE, Thompson KA, Harris RZ, Ilson BE, Jorkasky DK: Pharmacokinetics and pharmacodynamics of SB 209670, an endothelin receptor antagonist: Effects on the regulation of renal vascular tone. Clin Pharmacol Ther 65 : 473 –482, 1999
49. Schmetter L, Dallinger S, Bobr B, Selenko N, Eichler H-G, Woltz M: Systemic and renal effects of an ETA receptor subtype-specific antagonist in healthy subjects. Br J Pharmacol 124 : 930 –934, 1998
50. Montanari A, Biggi A, Carra N, Fasoli E, Calzolari M, Corsini F, Perinotto P, Novarini A: Endothelin-A blockade attenuates systemic and renal hemodynamic effects of L-NAME in humans. Hypertension 35 : 518 –523, 2000
51. Montanari A, Carra N, Perinotto P, Iori V, Fasoli E, Biggi A, Novarini A: Renal hemodynamic control by endothelin and nitric oxide under angiotensin II blockade in man. Hypertension 39 : 715 –720, 2002
52. Goddard J, Johnston NR, Hand MF, Cumming AD, Rabelink TJ, Rankin AJ, Webb DJ: Endothelin-A receptor antagonism reduces blood pressure and increases renal blood flow in hypertensive patients with chronic renal failure: A comparison of selective and combined endothelin receptor blockade. Circulation 109 : 1186 –1193, 2004
53. Goddard J, Eckhart C, Johnston NR, Cumming AD, Rankin AJ, Webb DJ: Endothelin A receptor antagonism and angiotensin-converting enzyme inhibition are synergistic via an endothelin B receptor-mediated and nitric oxide-dependent mechanism. J Am Soc Nephrol 15 : 2601 –2610, 2004
54. Wilcox SC: L-Arginine-nitric oxide pathway. In: The Kidney, Vol. 1 , edited by Seldin DW, Giebisch G, Philadelphia, Lippincott Williams & Wilkins, 2000 , pp 849 –871
55. Gariepy CE, Ohuchi T, Williams SC, Richardson JA, Yanagisawa M: Salt-sensitive hypertension in endothelin-B receptor-deficient rats. J Clin Invest 105 : 925 –933, 2000
56. Webb DJ, Monge JC, Rabelink TJ, Yanagisawa M: Endothelin: New discoveries and rapid progress in the clinic. Trend Pharmacol Sci 19 : 5 –8, 1998
57. Vassileva I, Mountain C, Pollock DM: Functional role of ETB receptors in the renal medulla. Hypertension 41 : 1359 –1363, 2003
58. Brooks DP, DePalma PD, Pullen M, Elliot JD, Ohlstein EH, Nambi P: SB 234551, a novel endothelin-A receptor antagonist, unmasks endothelin-induced renal vasodilatation in the dog. J Cardiovasc Pharmacol 31[Suppl 1] : S339 –S341, 1998
59. Schiffrin EL: Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens 14 : 83S –89S, 2001
60. Rossi GP, Colonna S, Pavan E, Albertin G, Della Rocca F, Gerosa G, Casarotto D, Sartore S, Pauletto P, Pessina AC: Endothelin-1 and its mRNA in the wall layers of human arteries ex vivo. Circulation 99 : 1147 –1155, 1999
61. Cardillo C, Kilcoyne CM, Waclawiw M, Cannon RO 3rd, Panza JA: Role of endothelin in the increased vascular tone of patients with essential hypertension. Hypertension 33 : 753 –758, 1999
62. Martin P, Ninio D, Krum H: Effect of endothelin blockade on basal and stimulated forearm blood flow in patients with essential hypertension. Hypertension 39 : 821 –824, 2002
63. Hand MF, Haynes WG, Webb DJ: Reduced endogenous endothelin-1-mediated vascular tone in chronic renal failure. Kidney Int 55 : 613 –620, 1999
64. Krum H, Viskoper RJ, Lacourciere Y, Budde M, Charlon V: The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. N Engl J Med 338 : 784 –790, 1998
65. Kohan DE: Autocrine role of endothelin in rat inner medullary collecting duct: Inhibition of AVP-induced cAMP accumulation. J Cardiovasc Pharmacol 22[Suppl 8] : S174 –S179, 1993
66. Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE: Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 114 : 504 –511, 2004
67. Hughes AK, Cline RC, Kohan DE: Alterations in renal endothelin-1 production in the spontaneously hypertensive rat. Hypertension 20 : 666 –673, 1992
68. Ge Y, Ahn D, Stricklett PK, Hughes AK, Yanagisawa M, Verbalis JG, Kohan DE: Collecting duct-specific knockout of endothelin-1 alters vasopressin regulation of urine osmolality. Am J Physiol Renal Physiol 288 : F912 –F920, 2005
69. Hoffman A, Grossman E, Goldstein DS, Gill JRJ, Keiser H: Urinary excretion rate of endothelin-1 in patients with essential hypertension and salt-sensitivity. Kidney Int 45 : 556 –560, 1994
70. Zoccali C, Leonardis D, Parlongo S, Mallamaci F, Postorino M: Urinary and plasma endothelin-1 in essential hypertension and in hypertension secondary to renoparenchymal disease. Nephrol Dial Transplant 10 : 1320 –1323, 1995
71. Koyama H, Tabata T, Nishzawa Y, Inoue T, Morii H, Yamaji T: Plasma endothelin levels in patients with uraemia. Lancet 333 : 991 –992, 1989
72. Kohno M, Murakawa K, Yasunari K, Yokokawa K, Horio T, Kurihara N, Takeda T: Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metab Clin Exp 38 : 712 –713, 1989
73. Orisio S, Benigni A, Bruzzi I, Corna D, Perico N, Zoja C, Benatti L, Remuzzi G: Renal endothelin gene expression is increased in remnant kidney and correlates with disease progression. Kidney Int 43 : 354 –358, 1993
74. Kanai H, Okuda S, Kiyama S, Tomooka S, Hirakata H, Fujishima M: Effects of endothelin and angiotensin II on renal haemodynamics in experimental mesangial proliferative nephritis. Nephron 64 : 609 –614, 1993
75. Ohkita M, Wang Y, Nguyen ND, Tsai YH, Williams SC, Wiseman RC, Killen PD, Li S, Yanagisawa M, Gariepy CE: Extrarenal ETB plays a significant role in controlling cardiovascular responses to high dietary sodium in rats. Hypertension 45 : 940 –946, 2005
76. Endemann DH, Schiffrin EL: Endothelial dysfunction. J Am Soc Nephrol 15 : 1983 –1992, 2004
77. Lerman A, Zeiher AM: Endothelial function: Cardiac events. Circulation 111 : 363 –368, 2005
78. Barton M, Haudenschild CC, D’Uscio LV, Shaw S, Munter K, Luscher TF: Endothelin ET(A) receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A 95 : 14367 –14372, 1998
79. Best PJM, McKenna CJ, Hasdai D, Holmes DRJ, Lerman A: Chronic endothelin receptor antagonism preserves coronary endothelial function in experimental hypercholesterolaemia. Circulation 99 : 1747 –1752, 1999
80. Bauersachs J, Fraccarollo D, Galuppo P, Widder J, Ertl G: Endothelin-receptor blockade improves endothelial vasomotor dysfunction in heart failure. Cardiovasc Res 47 : 142 –149, 2000
81. Halcox JP, Nour KR, Zalos G, Quyyumi AA: Coronary vasodilation and improvement in endothelial dysfunction with endothelin ET(A) receptor blockade. Circ Res 89 : 969 –976, 2001
82. Kowala MC, Rose PM, Stein PD, Goller N, Recce R, Beyer S, Valentine M, Barton D, Durham SK: Selective blockade of the endothelin su btype A receptor decreases early atherosclerosis in hamsters fed cholesterol. Am J Pathol 146 : 819 –826, 1995
83. Ihling C, Szombathy T, Bohrmann B, Brockhaus M, Schaefer HE, Loeffler BM: Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis. Circulation 104 : 864 –869, 2001
84. Lerman A, Edwards BS, Hallett JW, Heublein DM, Sandberg SM, Burnett JC Jr: Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N Engl J Med 325 : 997 –1001, 1991
85. Timm M, Kaski JC, Dashwood MR: Endothelin-like immunoreactivity in atherosclerotic human coronary arteries. J Cardiovasc Pharmacol 26[Suppl 3] : S442 –S444, 1995
86. Blacher J, Guerin AP, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness on survival in end-stage renal disease. Circulation 99 : 2434 –2439, 1999
87. Guerin AP, Blacher J, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness attenuation on survival of patients in end-stage renal failure. Circulation 103 : 987 –992, 2001
88. Wang MC, Tsai WC, Chen JY, Huang JJ: Stepwise increase in arterial stiffness corresponding with the stages of chronic kidney disease. Am J Kidney Dis 45 : 494 –501, 2005
89. Oliver JJ, Webb DJ: Noninvasive assessment of arterial stiffness and risk of atherosclerotic events. Arterioscler Thromb Vasc Biol 23 : 554 –566, 2003
90. Wilkinson IB, Qasem A, McEniery CM, Webb DJ, Avolio AP, Cockcroft JR: Nitric oxide regulates local arterial distensibility in vivo. Circulation 105 : 213 –217, 2002
91. Schmitt M, Avolio A, Qasem A, McEniery CM, Butlin M, Wilkinson IB, Cockcroft JR: Basal NO locally modulates human iliac artery function in vivo. Hypertension 46 : 227 –231, 2005
92. Kinlay S, Creager MA, Fukumoto M, Hikita H, Fang JC, Selwyn AP, Ganz P: Endothelium-derived nitric oxide regulates arterial elasticity in human arteries in vivo. Hypertension 38 : 1049 –1053, 2001
93. Wilkinson IB, MacCallum H, Cockcroft JR, Webb DJ: Inhibition of basal nitric oxide synthesis increases aortic augmentation index and pulse wave velocity in vivo. Br J Clin Pharmacol 53 : 189 –192, 2002
94. Vuurmans TJ, Boer P, Koomans HA: Effects of endothelin-1 and endothelin-1 receptor blockade on cardiac output, aortic pressure, and pulse wave velocity in humans. Hypertension 41 : 1253 –1258, 2003
95. McEniery CM, Qasem A, Schmitt M, Avolio AP, Cockcroft JR, Wilkinson IB: Endothelin-1 regulates arterial pulse wave velocity in vivo. J Am Coll Cardiol 42 : 1975 –1981, 2003
96. Stenvinkel P, Pecoits-Filho R, Lindholm B: Coronary artery disease in end-stage renal disease: No longer a simple plumbing problem. J Am Soc Nephrol 14 : 1927 –1939, 2003
97. Arici M, Walls J: End-stage renal disease, atherosclerosis, and cardiovascular mortality: Is C-reactive protein the missing link? Kidney Int 59 : 407 –414, 2001
98. Oberg BP, McMenamin E, Lucas FL, McMonagle E, Morrow J, Ikizler TA, Himmelfarb J: Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int 65 : 1009 –1016, 2004
99. Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T: Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 104 : 2673 –2678, 2001
100. Kaehler J, Sill B, Koester R, Mittmann C, Orzechowski HD, Muenzel T, Meinertz, T: Endothelin-1 mRNA and protein in vascular wall cells is increased by reactive oxygen species. Clin Sci (Lond) 103[Suppl 48] : 176S –178S, 2002
101. Amiri F, Virdis A, Neves MF, Iglarz M, Seidah NG, Touyz RM, Reudelhuber TL, Schiffrin EL: Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction. Circulation 110 : 2233 –2240, 2004
102. Callera GE, Touyz RM, Teixeira SA, Muscara MN, Carvalho MH, Fortes ZB, Nigro D, Schiffrin EL, Tostes RC: ETA receptor blockade decreases vascular superoxide generation in DOCA-salt hypertension. Hypertension 42 : 811 –817, 2003
103. Landmesser U, Harrison DG: Oxidant stress as a marker for cardiovascular events: Ox marks the spot. Circulation 104 : 2638 –2640, 2001
104. Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA: Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation 105 : 1890 –1896, 2002
105. Ridker PM: High-sensitivity C-reactive protein: Potential adjunct for global risk assessment in the primary prevention of cardiovascular disease. Circulation 103 : 1813 –1818, 2001
106. Shlipak MG, Fried LF, Crump C, Bleyer AJ, Manolio TA, Tracy RP, Furberg CD, Psaty BM: Elevations of inflammatory and procoagulant biomarkers in elderly persons with renal insufficiency. Circulation 107 : 87 –92, 2003
107. Pecoits-Filho R, Heimburger O, Barany P, Suliman M, Fehrman-Ekholm I, Lindholm B, Stenvinkel P: Associations between circulating inflammatory markers and residual renal function in CRF patients. Am J Kidney Dis 41 : 1212 –1218, 2003
108. Yeun JY, Levine RA, Mantadilok V, Kaysen GA: C-Reactive protein predicts all-cause and cardiovascular mortality in hemodialysis patients. Am J Kidney Dis 35 : 469 –476, 2000
109. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, Dhillon B, Weisel RD, Li RK, Mickle DA, Stewart DJ: A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 106 : 913 –919, 2002
110. Verma S, Kuliszewski MA, Li SH, Szmitko PE, Zucco L, Wang CH, Badiwala MV, Mickle DA, Weisel RD, Fedak PW, Stewart DJ, Kutryk MJ: C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: Further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 109 : 2058 –2067, 2004
111. Venugopal SK, Devaraj S, Yuhanna I, Shaul P, Jialal I: Demonstration that C-reactive protein decreases eNOS expression and bioactivity in human aortic endothelial cells. Circulation 106 : 1439 –1441, 2002
112. Ding SS, Qiu C, Hess P, Xi JF, Zheng N, Clozel M: Chronic endothelin receptor blockade prevents both early hyperfiltration and late overt diabetic nephropathy in the rat. J Cardiovasc Pharmacol 42 : 48 –54, 2003
113. Fogo A, Hellings SE, Inagami T, Kon V: Endothelin receptor antagonism is protective in in vivo acute cyclosporine toxicity. Kidney Int 42 : 770 –774, 1992
114. Hunley TE, Fogo A, Iwasaki S, Kon V: Endothelin A receptor mediates functional but not structural damage in chronic cyclosporine nephrotoxicity. J Am Soc Nephrol 5 : 1718 –1723, 1995
115. Kassab S, Miller MT, Novak J, Reckelhoff J, Clower B, Granger JP: Endothelin-A receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats. Hypertension 31 : 397 –402, 1998
116. Honing MLH, Bouter PK, Ballard DE, Padley P, Morrison PJ, Rabelink, TJ: ABT-627, a selective ETA-receptor antagonist, reduces proteinuria in patients with diabetes mellitus. In: Regulation of Vascular Tone in Humans by Endothelium-Derived Mediatorsm [Doctoral thesis]. University of Utrecht, 2000 , pp 89 –102
117. Wenzel RR, Mann J, Jurgens C, Yildrim I, Bruck H, Philipp T, Mitchell, A: The ETA-selective antagonist SPP301 on top of standard treatment reduces urinary albumin excretion rate in patients with diabetic nephropathy [Abstract]. J Am Soc Nephrol 16 : 58A , 2005
118. Jafar TH, Stark PC, Schmid CH, Landa M, Maschio G, de Jong PE, de Zeeuw D, Shahinfar S, Toto R, Levey AS: Progression of chronic kidney disease: The role of blood pressure control, proteinuria, and angiotensin-converting enzyme inhibition: A patient-level meta-analysis. Ann Intern Med 139 : 244 –252, 2003
119. Ruggenenti P, Perna A, Remuzzi G: Retarding progression of chronic renal disease: The neglected issue of residual proteinuria. Kidney Int 63 : 2254 –2261, 2003
120. Ibsen H, Olsen MH, Wachtell K, Borch-Johnsen K, Lindholm LH, Mogensen CE, Dahlof B, Devereux RB, de Faire U, Fyhrquist F, Julius S, Kjeldsen SE, Lederballe-Pedersen O, Nieminen MS, Omvik P, Oparil S, Wan Y: Reduction in albuminuria translates to reduction in cardiovascular events in hypertensive patients: Losartan Intervention for Endpoint Reduction in Hypertension Study. Hypertension 45 : 198 –202, 2005
121. Wang TJ, Evans JC, Meigs JB, Rifai N, Fox CS, D’Agostino RB, Levy D, Vasan RS: Low-grade albuminuria and the risks of hypertension and blood pressure progression. Circulation 111 : 1370 –1376, 2005
122. Freedman BI, Langefeld CD, Lohman KK, Bowden DW, Carr JJ, Rich SS, Wagenknecht LE: Relationship between albuminuria and cardiovascular disease in type 2 diabetes. J Am Soc Nephrol 16 : 2156 –2161, 2005
123. Benigni A: Defining the role of endothelins in renal pathophysiology on the basis of selective and unselective endothelin receptor antagonist studies. Curr Opin Nephrol Hypertens 4 : 349 –353, 1995
124. Kriz W, Gretz N, Lemley KV: Progression of glomerular diseases: Is the podocyte the culprit? Kidney Int 54 : 687 –697, 1998
125. Morigi M, Buelli S, Angioletti S, Zanchi C, Longaretti L, Zoja C, Galbusera M, Gastoldi S, Mundel P, Remuzzi G, Benigni A: In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: Implication for permselective dysfunction of chronic nephropathies. Am J Pathol 166 : 1309 –1320, 2005
126. Benigni A, Corna D, Zoja C, Longaretti L, Gagliardini E, Perico N, Coffman TM, Remuzzi G: Targeted deletion of angiotensin II type 1A receptor does not protect mice from progressive nephropathy of overload proteinuria. J Am Soc Nephrol 15 : 2666 –2674, 2004
127. Lehrke I, Waldherr R, Ritz E, Wagner J: Renal endothelin-1 and endothelin receptor type B expression in glomerular diseases with proteinuria. J Am Soc Nephrol 12 : 2321 –2329, 2001
128. Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies. N Engl J Med 339 : 1448 –1456, 1998
129. Zoja C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, Ronco P, Remuzzi G: Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 26 : 934 –941, 1995
130. Wesson DE: Endogenous endothelins mediate increased distal tubule acidification induced by dietary acid in rats. J Clin Invest 99 : 2203 –2211, 1997
131. Ong AC, Jowett TP, Firth JD, Burton S, Kitamura M, Fine LG: Human tubular-derived endothelin in the paracrine regulation of renal interstitial fibroblast function. Exp Nephrol 2 : 134 , 1994
132. Achmad TH, Rao GS: Chemotaxis of human blood monocytes toward endothelin-1 and the influence of calcium channel blockers. Biochem Biophys Res Commun 189 : 994 –1000, 1992
133. Hocher B, Thone-Reineke C, Rohmeiss P, Schmager F, Slowinski T, Burst V, Siegmund F, Quertermous T, Bauer C, Neumayer HH, Schleuning WD, Theuring F: Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension. J Clin Invest 99 : 1380 –1389, 1997
134. Benigni A, Remuzzi G: Endothelin antagonists. Lancet 353 : 133 –138, 1999
135. Boffa JJ, Tharaux PL, Dussaule JC, Chatziantoniou C: Regression of renal vascular fibrosis by endothelin receptor antagonism. Hypertension 37 : 490 –496, 2001
136. Brochu E, Lacasse S, Moreau C, Lebel M, Kingma I, Grose JH, Lariviere R: Endothelin ET(A) receptor blockade prevents the progression of renal failure and hypertension in uraemic rats. Nephrol Dial Transplant 14 : 1881 –1888, 1999
137. Nabokov A, Amann K, Wagner J, Gehlen F, Munter K, Ritz E: Influence of specific and non-specific endothelin receptor antagonists on renal morphology in rats with surgical renal ablation. Nephrol Dial Transplant 11 : 514 –520, 1996
138. Wolf SC, Brehm BR, Gaschler F, Brehm S, Klaussner M, Smykowski J, Amann K, Osswald H, Erley CM, Risler T: Protective effects of endothelin antagonists in chronic renal failure. Nephrol Dial Transplant 14[Suppl 4] : 29 –30, 1999
139. Shimizu T, Hata S, Kuroda T, Mihara S, Fujimoto M: Different roles of two types of endothelin receptors in partial ablation-induced chronic renal failure in rats. Eur J Pharmacol 381 : 39 –49, 1999
140. Chen HC, Guh JY, Chang JM, Tsai JC, Hwang SJ, Lai YH: Plasma and urinary endothelin-1 in focal segmental glomerulosclerosis. J Clin Lab Anal 15 : 59 –63, 2001
141. Vlachojannis J, Tsakas S, Petropoulou C, Kurz P: Increased renal excretion of endothelin-1 in nephrotic patients. Nephrol Dial Transplant 12 : 470 –473, 1997
142. Rossi GP, Sacchetto A, Cesari M, Pessina AC: Interactions between the endothelin-1 and the renin-angiotensin-aldosterone system. Cardiovasc Res 43 : 300 –307, 1999
143. Imai T, Hirata Y, Emori T, Yanagisawa M, Masaki T, Marumo F: Induction of endothelin-1 gene by angiotensin and vasopressin in endothelial cells. Hypertension 19 : 753 –757, 1992
144. Kohno M, Horio T, Ikeda M, Yokokawa K, Fuki T, Yasunari K, Kurihara N, Takeda T: Angiotensin II stimulates endothelin-1 secretion in cultured rat mesangial cells. Kidney Int 42 : 860 –866, 1992
145. Lariviere R, Lebel M, Kingma I, Grose JH, Boucher D: Effects of losartan and captopril on endothelin-1 production in blood vessels and glomeruli of rats with reduced renal mass. Am J Hypertens 11 : 989 –997, 1998
146. Remuzzi A, Benigni A, Malanchini B, Bruzzi I, Foglieni C, Remuzzi G: ACE inhibition prevents renal failure and death in uninephrectomized MWF/Ztm rats. Kidney Int 47 : 1319 –1326, 1995
147. Ruiz-Ortega M, Gomez-Garre D, Liu XH, Blanco J, Largo R, Egido J: Quinapril decreases renal endothelin-1 expression and synthesis in a normotensive model of immune-complex nephritis. J Am Soc Nephrol 8 : 756 –768, 1997
148. Benigni A, Corna D, Maffi R, Benedetti G, Zoja C, Remuzzi G: Renoprotective effect of contemporary blocking of angiotensin II and endothelin-1 in rats with membranous nephropathy. Kidney Int 54 : 353 –359, 1998
149. Berthold H, Munter K, Just A, Kirchheim HR, Ehmke H: Contribution of endothelin to renal vascular tone and autoregulation in the conscious dog. Am J Physiol 279 : F417 –F424, 1999
150. Amann K, Simonaviciene A, Medwedewa T, Koch A, Orth S, Gross ML, Haas C, Kuhlmann A, Linz W, Scholkens B, Ritz E: Blood pressure-independent additive effects of pharmacologic blockade of the renin-angiotensin and endothelin systems on progression in a low-renin model of renal damage. J Am Soc Nephrol 12 : 2572 –2584, 2001
151. Bauersachs J, Fraccarollo D, Schafer A, Ertl G: Angiotensin-converting enzyme inhibition and endothelin antagonism for endothelial dysfunction in heart failure: Mono or combination therapy. J Cardiovasc Pharmacol 40 : 594 –600, 2002
152. Mulder P, Boujedaini H, Richard V, Henry JP, Renet S, Munter K, Thuillez C: Long-term survival and hemodynamics after endothelin-A receptor antagonism and angiotensin-converting enzyme inhibition in rats with chronic heart failure: Monotherapy versus combination therapy. Circulation 106 : 1159 –1164, 2002
153. Cowburn PJ, Cleland JGF, McArthur JD, MacLean MR, McMurray JJV, Dargie HJ: Short-term haemodynamic effects of BQ-123, a selective endothelin ETA-receptor antagonist, in chronic heart failure. Lancet 352 : 201 –202, 1998
154. Sutsch G, Kiowski W, Yan X-W, Hunziker P, Christen S, Strobel W, Kim, J-H, Rickenbacher P, Bertel O: Short-term oral endothelin-receptor antagonist therapy in conventionally treated patients with symptomatic severe chronic heart failure. Circulation 98 : 2262 –2268, 1998
155. Ko L, Maitland A, Fedak PW, Dumont AS, Badiwala M, Lovren F, Triggle CR, Anderson TJ, Rao V, Verma S: Endothelin blockade potentiates endothelial protective effects of ACE inhibitors in saphenous veins. Ann Thorac Surg 73 : 1185 –1188, 2002
156. Bohm F, Beltran E, Pernow J: Endothelin receptor blockade improves endothelial function in atherosclerotic patients on angiotensin converting enzyme inhibition. J Intern Med 257 : 263 –271, 2005
157. To Determine the Effects of Avosentan on Doubling of Serum Creatinine, End-Stage Renal Disease and Death in Diabetic Nephropathy. Available:
http://www.clinicaltrials.gov/ct/show/NCT00120328. Accessed February 28, 2006
158. Fukuroda T, Fujikawa T, Ozakai S, Ishikawa K, Yano M, Nishikibe M: Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Commun 199 : 1461 –1465, 1994
159. Willette RN, Anderson KM, Nelson AH, Olzinski AR, Woods T, Coatney RW, Aiyar N, Ohlstein EH, Barone FC: Enrasentan improves survival, limits left ventricular remodeling, and preserves myocardial performance in hypertensive cardiac hypertrophy and dysfunction. J Cardiovasc Pharmacol 38 : 606 –617, 2001
160. Ebihara I, Nakamura T, Tomino Y, Koide H: Effect of a specific endothelin receptor A antagonist and an angiotensin-converting enzyme inhibitor on glomerular mRNA levels for extracellular matrix components, metalloproteinases (MMP) and a tissue inhibitor of MMP in aminonucleoside nephrosis. Nephrol Dial Transplant 12 : 1001 –1006, 1997
161. Gomez-Garre D, Largo R, Liu XH, Gutierrez S, Lopez-Armada MJ, Palacios I, Egido J: An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis. Kidney Int 50 : 962 –972, 1996
162. Nakamura T, Ebihara I, Tomino Y, Koide H: Effect of a specific endothelin A receptor antagonist on murine lupus nephritis. Kidney Int 47 : 481 –489, 1995
163. Rothermund L, Traupe T, Dieterich M, Kossmehl P, Yagil C, Yagil Y, Kreutz R: Nephroprotective effects of the endothelin ET(A) receptor antagonist darusentan in salt-sensitive genetic hypertension. Eur J Pharmacol 468 : 209 –216, 2003
164. Suga S, Yasui N, Yoshihara F, Horio T, Kawano Y, Kangawa K, Johnson RJ: Endothelin A receptor blockade and endothelin B receptor blockade improve hypokalemic nephropathy by different mechanisms. J Am Soc Nephrol 14 : 397 –406, 2003
