Therapeutic potential of endothelin receptor antagonists for chronic proteinuric renal disease in humans

Biochimica et Biophysica Acta 1802 (2010) 1203–1213

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Review

Therapeutic potential of endothelin receptor antagonists for chronic proteinuric renal disease in humans Matthias Barton ⁎ Molecular Internal Medicine, University of Zurich, LTK Y 44 G 22, Winterthurer Strasse 190, CH-8057 Zürich, Switzerland

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Article history: Received 14 January 2010 Received in revised form 23 March 2010 Accepted 25 March 2010 Available online 30 March 2010 Keywords: Endothelin Glomerulosclerosis Aging Hypertension Injury Proteinuria Albuminuria Reversal Albuminuria Regression

a b s t r a c t Diabetes and arterial hypertension continue to be the main causes of chronic renal failure in 2010, with a rising prevalence in part due to the worldwide obesity epidemic. Proteinuria is a main feature of chronic renal disease and mediated by defects in the glomerular filtration barrier and is as a good predictor of cardiovascular events. Indeed, chronic renal disease due to glomerulosclerosis is one of the important risk factors for the development of coronary artery disease and stroke. Glomerulosclerosis develops in response to inflammatory activation and increased growth factor production. Preclinical and first preliminary clinical studies provide strong evidence that endogenous endothelin-1 (ET-1), a 21-amino-acid peptide with strong growth-promoting and vasoconstricting properties, plays a central role in the pathogenesis of proteinuria and glomerulosclerosis via activation of its ETA subtype receptor involving podocyte injury. These studies have not only shown that endothelin participates in the disease processes of hypertension and glomerulosclerosis but also that features of chronic renal disease such as proteinuria and glomerulosclerosis are reversible processes. Remarkably, the protective effects of endothelin receptors antagonists (ERAs) are present even on top of concomitant treatments with inhibitors of the renin–angiotensin system. This review discusses current evidence for a role of endothelin for proteinuric renal disease and podocyte injury in diabetes and arterial hypertension and reviews the current status of endothelin receptor antagonists as a potential new treatment option in renal medicine. © 2010 Elsevier B.V. All rights reserved.

1. Discovery and physiological relevance of endothelin Robert F. Furchgott (1916–2009) [1] made the seminal observation 30 years ago that vascular endothelial cells modulate vascular tone by releasing vasoactive factors [2]. Furchgott himself [3], as well as Ignarro et al. [4], later identified this factor as nitric oxide (NO). Subsequently, research in the early 1980s was concentrated on identifying new vasoactive molecules [1]. Already shortly after the initial discovery, it became clear that endothelial cells also release vasoconstrictor substances [5]. Work of several laboratories from the mid-1980s identified a potent vasoconstrictor substance released into the supernatant of endothelial cells [6,7]. The race in search of the identity and the sequence of the gene encoding for a 21-amino-acid peptide was on and a consortium of several Japanese laboratories led by Tomoh Masaki [8,9] identified the peptide named endothelin-1, which was published in Nature by Masashi Yanagisawa and colleagues in 1988 [10]. Endothelin-1 is the biologically predominant isoform of the endothelin peptide family, which also includes endothelin-2 and endothelin-3,

⁎ Tel.: +41 77 439 5554; fax: +41 44 635 6875. E-mail address: [email protected]. 0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.03.012

as well as 31- and 32-amino-acid isoforms of the peptide [11,12]. Endothelin binds to two receptors (ETA and ETB) that were identified shortly after discovery of the ligands [11,13]. ETA receptors primarily have vasoconstrictor and growth-promoting functions, whereas ETB receptors mediate mainly vasodilation and inhibition of growth and inflammation via nitric oxide and prostacyclin [11]. ETB receptors can therefore be regarded largely as “endogenous antagonistic receptors” counteracting the effects of the ETA receptor. In humans, endothelin represents the most potent and longlasting vasoconstrictor known and is about 100 times more potent than noradrenaline [12]. Endothelins (ETs) can be considered ubiquitously expressed stress-responsive regulators of cell function that act in a paracrine and autocrine fashion [13]. Endothelins exert a number of physiological functions, including embryonic development [14] and intestinal growth as well as natriuresis, the latter of which is maintained via activation of the ETB receptor [13,15], in the renal medulla. In principal cells of the collecting duct, activation of ETB receptors inhibits sodium and water transport [16]. ETB receptors also mediate clearance of endothelin from the circulation [17], and this effect appears to involve cross-talk with the ETA receptor, at least under physiological conditions [18]. In the vasculature and the kidney, via activation of its ETA receptors, endothelin has a basal (“tonic”) vasoconstricting role; endothelins also contribute to myocardial

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function, bronchial tone, and vascular smooth muscle cell proliferation, the latter of which is an important factor by which endothelin facilitates the development of vascular disease in hypertension and atherosclerosis (reviewed in Refs. [11,13]). In addition, endothelin controls immune function and inflammation [19,20], glucose metabolism, and metabolic functions in adipose tissue and liver [13]. 2. Proteinuric renal disease due to glomerulosclerosis Chronic renal disease can be both cause and consequence of chronically elevated arterial blood pressure. Other contributing factors accelerating the development of renal disease that are frequently coexisting with hypertension are diabetes due to obesity, hyperinsulinemia, high cholesterol plasma levels, and aging [21]. Sclerosis of the glomerulus involves glomerular obsolence with fibrosis of the tuft and podocyte injury. Renal failure itself leads to hypertension (secondary renal–parenchymal hypertension) and aggravates coexisting risk factors such as dyslipidemia because of increased loss of lipoproteins due to a defective glomerular filtration barrier and compensatory upregulation of hepatic lipoprotein synthesis [22]. Between the early 1990s and 2002, the incidence of chronic renal failure in the United States has almost doubled [23]. Numbers are still increasing, and similar developments have been reported elsewhere in the world [24–26]. The main cause of deaths in patients with severe chronic renal disease are cardiovascular complications such as myocardial infarction or stroke due to cerebrovascular disease [27–29]. Chronic renal disease also represents a major economic challenge to health systems worldwide as the health costs related to chronic renal disease have quadrupled [23]. In view of the anticipated growth of the world population from 6 to 9 billion within the next 40 years [30], the economic burden related to chronic renal disease can be expected to increase even further. Importantly, the prevalence of obesity in Western countries such as Germany is highest among individuals aged 50 to 70 years [31], an age group in which comorbidities such a arterial hypertension, albuminuria, and diabetes are frequently present [32,33]. 3. Arterial hypertension Treatment of arterial hypertension and the prevention of endorgan damage associated with it remain a major task yet to be accomplished in clinical medicine. In many patients suffering from arterial hypertension, the disease is either insufficiently treated or even undiagnosed [34,35], creating a substantial disease burden. The major clinical complications related to arterial hypertension are myocardial infarction, stroke, and renal disease [27,36], which form the number one cause of mortality and disability in Western countries. Many of the patients with so-called essential hypertension develop the disease due to genetic predisposition [34,35]; however, an increasing number of patients is being diagnosed with hypertension due to secondary conditions such as obesity, renal failure, or menopause [27,37–39]. Hypertension due to obesity, which is associated with renal injury [40,41], is increasingly seen in school children [39,42,43]. Other conditions such as Conn's disease or pheochromocytoma represent only a small proportion of secondary causes of hypertension [44]. Although hypertension often, over many years, goes unnoticed without symptoms [34,35], chronic elevation of blood pressure leads to a number of changes within the body, causing substantial organ damage in the arterial wall, the myocardium, the kidney, the brain, and the eyes, among others [45]. The most prominent feature of arterial hypertension is an increased vascular resistance leading to structural changes in the vascular wall [46]. Increased blood pressure results in hypertrophy of the medial smooth muscle layer and causes abnormal vasomotion promoting vasoconstriction [47]. Specifically,

hypertension is associated with an attenuation of nitric oxidedependent vasodilation due to increased oxidative stress [48] and an increased sensitivity to vasoconstrictors such as norepinephrine or angiotensin II and, in particular, increased formation and activity of endothelin-1 [49–51]. Despite continued advances in antihypertensive therapy, many hypertensives with or without chronic kidney disease are insufficiently treated or show little response to antihypertensive treatment, even when treated with a combination of several antihypertensive drugs [52]. Blood pressure in patients with uncontrolled arterial “essential” hypertension – even under triple antihypertensive therapy – is considered as “therapy-resistant hypertension” [53,54]. Therapy-resistant hypertension is particularly frequent in Blacks, Hispanics, patients with obesity, and postmenopausal women [38,54,55]. It appears that salt sensitivity and thus salt intake play an important role in the pathogenesis of resistant hypertension [56]. Early on, endothelin was suspected to play a role in arterial hypertension since surgical removal of an endothelinproducing hemangioendothelioma resulted in normalization of blood pressure in a hypertensive patient [57]. However, endothelial cells are not the only cells producing endothelin; endothelin-1 is also produced by vascular smooth muscle cells, renal mesangial cells, glomerular podocytes, white blood cells, and others (reviewed in Ref. [21]). Interestingly, endothelial cell-specific overexpression of endothelin-1 causes vascular remodeling and abnormal changes in function even independent of blood pressure [58], in line with the pressure-independent effects of systemic endothelin-1 overexpression in other organs involved in cardiovascular homeostasis [59]. 4. Renal disease and glomerulosclerosis The kidney plays a central role in the regulation of arterial blood pressure by controlling blood volume, release of vasoactive substances, and blood flow [60]. Therefore, renal disease can be considered an important secondary cause of hypertension [61]. Over the past 20 years, research has generated robust evidence that endothelin plays a central role in kidney function and renal disease progression [21,22,60,62]. Endogenous renal endothelin controls water and sodium excretion and acid–base balance and maintains normal renal cell growth and tonic vasoconstriction (reviewed in Refs. [63,64]). However, under disease conditions or in the presence of conditions such as arterial hypertension or diabetes, endothelin production and activity increase and thereby promote the development of glomeruloslerosis [22,62]. Endothelin-dependent mechanisms contribute to proteinuria and glomerular injury and particularly involve injury of glomerular podocytes (Fig. 1) and activation of fibrogenic pathways in mesangial cells (reviewed in Ref. [65]). Endothelin directly promotes glomerular production of collagen and fibronectin [66]. Renal disease, hypertension, and diabetes develop at a faster rate in Blacks than in other ethnic groups [55]. Black individuals even if normotensive have higher risk for these diseases than Caucasians and also carry a higher risk of developing hypertension [67]. Interestingly, circulating levels of ET-1 are much higher in Blacks than in other ethnic groups [67], with Blacks being particularly susceptible to development of hypertension [55]. The fact that circulating ET-1 levels substantially fall upon successful antihypertensive therapy in hypertensive Blacks points at a possible causal involvement of the endothelin system in this disease [68]. 5. Mechanisms of endothelin-dependent renal injury A common denominator of all fibro-proliferative diseases are inflammatory responses in the diseased organ [69,70]. The increased formation of pro-inflammatory and fibrogenic peptides such as angiotensin II appears to play an essential role in this process [71–73]. Renal endothelin also activates pathways that have been independently associated with the physiopathology of hypertensive renal disease,

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Fig. 1. Schematic representation of the actions and interactions of ET-1 (yellow) between the glomerular capillary basement membrane (GBM, red), the glomerular endothelial cells (GEC, blue), the glomerular podocytes (grey), and the slit diaphragm (green). The figure is based on a concept of intracellular glomerular communication initially put forward by Kalluri [94], and features signaling mechanisms (proposed by the author (1)–(7)) through which ET-1 contributes to podocyte and glomerular injury. ET-1 is released from cells on both sides of the GBM, namely glomerular endothelial cells (GEC) and podocytes. The newly formed ET-1 then interacts with different cellular components of the glomerular capillary. ET-1 released from GEC then act on the GBM (1), with the slit diaphragm (2), or with the podocyte (3). ET-1 release and signaling may also occur in the reverse direction. Finally, ET-1 released from podocytes may interact with the GBM and vice versa (4). Within the podocyte, ET-1 activates endothelin ETA receptors (ETA–R) and promotes glomerular injury and sclerosis through MAPKs p38 and p44/p42 pathways (5); ET-1 also stimulates the growth promoter and cyclin-dependent kinase-inhibitor p21waf/cip1, and pro-inflammatory NF-kappa B (6). ET-1 causes disruption of the F-actin cytoskeleton (7) and promotes dysfunction of the slit diaphragm (green), via activation of the Rho-kinase and PI3-kinase pathways.

such as formation of reactive oxygen species production and inflammation [69,74]. Endothelin itself stimulates the formation of angiotensin II by increasing the activity of angiotensin-converting enzyme [75,76]. Because angiotensin AT1 receptor blockade inhibits endothelin-1 production in vivo [77], and beneficial effects of AT1 receptor blockade are independent of podocyte AT1 receptor expression, as demonstrated by Matsusake et al. [78], it is well possible that angiotensin-mediated renal and podocyte injury is directly mediated via endothelin [79]. Interestingly, in experimental obesity, ACE in the kidney becomes activated, which can be completely prevented by ETA receptor antagonists [80], suggesting angiotensin-converting enzyme (ACE)-inhibitor-like functions of these drugs in physiopathologies such as obesity. Obesity-associated glomerulopathy is characterized by focal–segmental glomerulosclerosis (FSGS) and appears to be reversible following successful weight loss [40,41]. On the other hand, angiotensin II, the main effector peptide of the renin–angiotensin system, activates renal endothelin production [79] by partly pressure-independent mechanisms (Fig. 2) and causes renal inflammation [71–73]. The direct or indirect trophic effects of endothelin promote mesangial cell proliferation as well as hypertrophy of the glomerular basement membrane (GBM) (Fig. 1). GBM hypertrophy is indirectly mediated via injury of glomerular epithelial cells and an important indicator of injury of the glomerulus [81,82]. Other important factors that aggravate glomerular injury via endothelin are hyperglycemia, salt (sodium)-sensitive hypertension (Fig. 3) [83,84], or high levels of aldosterone (reviewed in Refs. [22,85]). It is possible if not likely that a main common determinant of the detrimental changes mediated by endothelin is inflammation, which plays a crucial role for glomerulosclerosis, and can be inhibited by ETA receptor antagonists [19,20]. Such a notion would be also supported by the findings of a pressureindependent, antiproteinuric effect of ETA receptor antagonists in proliferative nephritis, which – like angiotensin-II-induced renal injury [71–73] – is characterized by glomerular inflammation [86]. Indeed, Pollock's group has recently demonstrated using the ETA receptor antagonist atrasentan that endothelin blockade indeed has anti-inflammatory effects in the diabetic kidney [87], effects that have also been suggested by transplantation studies [88]. Finally, dietary protein intake may also aggravate renal injury in an endothelindependent fashion as suggested by Wesson and colleagues [89]. Taken together, data from preclinical prevention studies and transgenic animal models provide convincing evidence for a critical involvement

of the endothelin pathway in the pathogenesis of proteinuria and glomerulosclerosis likely in part due to its pro-inflammatory activity [20]. This makes endothelin receptor antagonists promising candidates for the clinical treatment of proteinuric renal disease [22,90,91]. 6. Podocytes—glomerular gatekeepers and their regulation by endothelin receptors Podocytes are glomerular epithelial cells that regulate the protein filter function of the glomerulus and its slit diaphragm [74,81,82] (Fig. 1). Wiggins recently proposed podocytopathy as a general element of all glomerular diseases and emphasized the need for

Fig. 2. Regulation of endothelin-1 peptide concentrations by angiotensin II in kidney and arteries in vivo and the effects of endothelin receptor blockade in a model of experimental hypertension. Shown are endothelin peptide levels (pg ET-1/g tissue wet weight) in normotonsive WKY control rats (open bars), hypertensive angiotensin IIinfused rats (filled bars), and hypertensive angiotensin-II-infused rats concomitantly treated with the ETA-selective endothelin receptor antagonist darusentan (hatched bars). Systolic blood pressure was increased in angiotensin-II-infused rats (mean value: 190 mm Hg) and reduced in angiotensin-II–darusentan-treated rats (mean value: 172 mm Hg). ET-1 peptide levels were 19 ± 2 pg/g in the kidney (left panel, n = 5) and 44 ± 8 pg/g n the aorta (right panel, n = 7) of control animals. Angiotensin II treatment increased ET-1 peptide levels about threefold in both kidney and arteries, which was completely prevented by the ETA antagonist (n = 5–7/group) despite that blood pressure levels were still in the hypertensive range. *p b 0.05 vs. control, †p b 0.05 vs. Ang II. Figure reproduced from reference [79] with permission of the publisher.

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Fig. 3. Effects of hypertension induced by dietary salt and effects of endothelin receptor blockade on structural vascular and renal injury in a model of salt-sensitive hypertension (Dahl hypertension). Histologic sections of the thoracic aorta (upper panels, A–C) or renal cortex (lower panels, D–F) are shown. Left panels (A, D) are from control animals on a lowsodium diet, middle panels (B,E) are from animals kept on a high-sodium (4%) diet for 2 months, and right panels (C, F) are from animals on a high-sodium diet concomitantly treated with the endothelin ETA receptor antagonist darusentan. Upper panels: representative aortic sections from the study animals (H&E, original magnification: 63×). Vascular hypertrophy in hypertensive animals is associated with increased media thickness and intralamellar widening, which is inhibited by concomitant Darusentan treatment. Lower panels: representative renal histology in Dahl salt-sensitive rats (periodic acid Schiff , original magnification ×100). Salt-induced hypertension induced severe glomerulosclerosis (E) that was largely prevented by ETA antagonist darusentan (F). Figures reproduced from M. Barton, L.V. d'Uscio, S. Shaw, P. Meyer, P. Moreau, T.F. Lüscher. ETA receptor blockade prevents increased tissue endothelin-1, vascular hypertrophy and endothelial dysfunction in salt-sensitive hypertension. Hypertension 31, 499–504, 1998. With permission of the publisher and the American Heart Association, and from Barton M, Vos I, Shaw S, Boer P, d'Uscio LV, Grone HJ, Rabelink TJ, Lattmann T, Moreau P, Luscher TF. Dysfunctional renal nitric oxide synthase as a determinant of salt-sensitive hypertension: mechanisms of renal artery endothelial dysfunction and role of endothelin for vascular hypertrophy and glomerulosclerosis. J Am Soc Nephrol 2000; 11: 835-45, with permission of the publisher and the American Society of Nephrology.

clinical efforts toward prevention and preservation of these cells [92]. Podocytes contain endothelin binding sites, and human podocytes express a fully functional endothelin system, including endothelin receptors, prepro-endothelin-1, and endothelin-converting enzyme-1 [22,93]. The actin cytoskeleton is essential for podocyte function and structural integrity [94]. Angiotensin II, which is a strong inductor or renal inflammation [71–73] and of renal endothelin production in vivo (Fig. 2), promotes podocyte actin cytoskeleton disruption and albumin permeability and promotes podocyte apoptosis in vitro [22] and in vivo [95]; virtually identical effects on podocyte actin cytoskeleton disruption have been observed following exposure to endothelin [96]. The first observation that endothelin could be indeed involved in the podocyte injury came from experiments in human podocytes demonstrating that injury due to puromycin aminonucleoside, which causes glomerulosclerosis in vivo [97,98], can be prevented by using different selective ETA receptor antagonists, BQ123 or darusentan, or by gene silencing of the ETA receptor [93]. Interestingly, protein loading of podocytes induces endothelin formation and hence increases glomerular permselectivity for proteins, an effect that is regulated in an ETA-dependent manner (reviewed in Ref. [22]). Moreover, endothelin appears to be a crucial mediator of podocyte injury as pretreatment with ETA receptor antagonists prevents actin cytoskeleton disruption after in vitro injury by puromycin aminonucleoside [93], with similar effects seen after angiotensin AT 1 -angiotensin receptor antagonist treatment (reviewed in Ref. [22]). Consequently, endothelin has been implicated

in renal injury due to excessive protein load for which ETA receptordependent mechanisms have been described (reviewed in Ref. [22]). 7. Endothelin antagonist treatment in diabetic nephropathy: preclinical data Hyperglycemia as metabolic consequence of diabetes stimulates endothelin production and is associated with activation of renal growth factors and mesangial cell proliferation (reviewed in Ref. [99]). Urinary endothelin excretion is increased in patients with diabetic nephropathy or with primary focal–segmental glomerulosclerosis (reviewed in Ref. [22]). Podocyte depletion is also present in diabetic or diabetic-obese (db/db) obese rodents lacking the leptin receptor (reviewed in Ref. [22]). A number of studies have investigated the therapeutic potential of endothelin receptor antagonists for the treatment of diabetic nephropathy due to insulindependent or type 2 diabetes (reviewed in Ref. [22]). Importantly, obesity is often associated with diabetes and characterized by insulin resistance with high circulating levels of insulin, which itself is a potent stimulus of endothelin production in vitro and in vivo [100,101]. Thus, high local tissue or circulating levels of insulin will further increase endothelin production. A high glucose milieu seen in prediabetes or diabetes also causes actin cytoskeleton disassembly in podocytes (Fig. 1) as has been observed with angiotensin II and endothelin-1, and podocyte depletion and apoptosis appear to require ROS-dependent mechanisms (reviewed in Refs. [22,90]). While in the

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normal kidney only senescent podocytes are shed, even viable podocytes become shed in diabetic nephropathy [102,103]. Podocytes shedding into the urine has also been observed in patients with FSGS and with pre-ecclampsia [104,105]. Most recent work suggests a causal role for endothelin in the shedding process as recombinant endothelin triggers nephrin shedding from podocytes, an effect that can be prevented by blocking ETA receptors [106]. This suggests the possibility that hyperglycemia-induced renal injury contributes to glomerular podocyte loss via activation of endothelin. The loss of viable podocytes into the urine might provide a new and rather sensitive marker of early glomerular injury [103]. Urinary loss of podocytes has also been observed in diabetic rodents and in patients with obesity-associated diabetes diagnosed with obesity-associated glomerulopathy [107], which pathologically is characterized by focal– segmental glomerulosclerosis (FSGS) [40,41]. Also in insulin-dependent type-1 diabetes in experimental models such as non-obese diabetic mice, which spontaneously develops autoimmune diabetes, up-regulation of the podocyte protein nephrin is present even before the development of diabetes [108]. This supports the hypothesis that the podocyte becomes a target of diabetic injury already very early in the course of the disease. Blockade of endothelin receptors delays the onset of autoimmune diabetes [109], consistent with the known immunomodulatory functions of endothelin [19,20]. A number of experimental prevention studies have shown an involvement of endothelin in diabetic nephropathy; however, in many studies, partial or even complete normalization of blood pressure was observed [110–112]. Interestingly, in those studies where little or no changes in blood pressure were observed, effects of endothelin blockade also did not affect glomerular filtration rate. Studies by Hocher and coworkers have demonstrated that the ETA receptor antagonist darusentan reduces proteinuria and glomerulosclerosis in type 1 diabetes [113], results that are supported by several studies from Amann's laboratory [110,111,114,115]. The latter investigators found that in insulin-treated streptozotocindiabetic rats, darusentan was as effective as ACE inhibition in reducing glomerulosclerosis, while ACE inhibition showed greater effect in reducing blood pressure, proteinuria, and glomerular volume [110,115]. Nevertheless, only ETA receptor blockade using darusentan, but not ACE inhibition, completely restored the number of glomeruli per kidney and increased the number podocytes per glomerulus, suggesting that endothelin blockade has unique and specific glomeruloprotective effects [110]. Possible mechanisms involved in regeneration have been most recently reported by Moeller's group [116,117] and by Benigni's group [112], who reported that podocytes are recruited from parietal glomerular epithelial cells via migration onto the glomerular tuft and contribute to re-structuring the capillary architecture, respectively. In a study by Amann and colleagues, increased podocyte expression of endothelin was observed in diabetic hypertensive animals [110]; darusentan, an ETA receptor antagonist, reduced podocyte injury and proteinuria despite negligible effects on arterial blood pressure [110]. Benigni's group recently reported similar effects using the non-selective compound avosentan as an endothelin antagonist [112]. In their recent study, the investigators even found reversal of glomerulosclerosis and proteinuria, and additive effects were noted in animals already treated with an ACE inhibitor [112]. Avosentan improved capillary architecture and reduced inflammation [112]. While it has been recently proposed that anti-inflammatory and indirect antioxidant effects also contribute to the antiproteinuric and anti-sclerotic effects of endothelin blockade in diabetes [19,20,87], it is currently unclear whether and to what degree the ETB receptor is important for the nephroprotective effects of endothelin blockade [118,119]; data from studies in diabetic renin-dependent hypertensive rats indicate a lack of protective effect of the non-selective endothelin antagonist bosentan [120]. In another study, however, using the avosentan, an antagonist with low receptor selectivity, the investi-

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gators found that the nephroprotective effect of avosentan in diabetic atherosclerotic mice was comparable to that of the ACE inhibitor quinapril, an effect that was associated with a reduction in atherosclerosis and inflammatory activation [121]. 8. Endothelin antagonist therapy for hypertensive nephropathy: preclinical data Within a few years after the discovery of endothelin, Benigni et al. reported reductions in proteinuria and glomerulosclerosis after selective blockade of ETA receptors in a renal mass reduction rat model of hypertensive nephropathy [122]. A role for endothelin in renal disease was further supported by work by Hocher and colleagues showing that systemic overexpression of the human prepro-endothelin gene [123] causes glomerulosclerosis, even in the absence for changes in blood pressure. Numerous preclinical studies have since confirmed the renoprotective effects and shown that blockade of ETA receptors potently inhibit the growth-promoting effects of endothelin in conditions such as hypertensive nephropathy (Figs. 2 and 3), as well as largely normotensive conditions such as diabetes, obesity, or aging (reviewed in Refs. [21,22,62,91]). Chatziantoniou and colleagues reported reversal of vascular fibrosis and collagen deposition in a model of NO-deficient hypertension after endothelin blockade [124]. Experimental prevention studies with only very few exceptions found renoprotective effects of ERA treatment that were associated with a partial reduction in blood pressure. Several forms of hypertension (angiotensin-II-dependent, renin-dependent, salt-loaded renin-dependent; aldosterone-induced, genetically salt-sensitive, DOCA-salt induced) were studied (reviewed in Ref. [85]), and as long as ETA receptors were blocked, renoprotection was achieved [11] (Fig. 3). Work by Vanecková and colleagues even indicates that endothelin blockade maintains its profound antiproteinuric effects even in the presence of malignant hypertension and that ERA treatment was associated with a reduction in podocyte injury [125]. The importance of these studies is due to the fact that treatment was begun after hypertension had been established and that beneficial effects on podocytes were seen even in the absence of glomerulosclerosis, i.e., at an early stage of renal disease. Vanecková and colleagues also found that in hypertensive rats, podocyte injury preceded proteinuria [125]. The selective antagonist atrasentan but not the non-selective antagonist bosentan prevented podocyte injury and substantially reduced proteinuria despite the fact that animals were still markedly hypertensive [125]. Podocyte injury before development of glomerulosclerosis was also observed in Dahl hypertensive rats, which was sensitive to mineralocorticoid receptor blockade [126]. Importantly, unlike endothelin blockade or mineralocorticoid receptor blockade, antihypertensive treatment with hydralazine had no effect on podocyte injury, again indicating that the nephroprotective effects are specific and largely pressure-independent [126]. This would again be supported by the development of glomerulosclerosis in the absence of hypertension in experimental animals that overexpress human endothelin [123] and by the beneficial effects of endothelin blockade in experimental diabetes discussed above. 9. Endothelin antagonists for focal–segmental glomerulosclerosis: preclinical data Focal–segmental glomerulosclerosis has been termed a “podocyte disease” [92,127,128], and only a limited number of therapies are available [129]. The signaling mechanisms are complex; however, a role for nephrin/PI3 kinase signaling has been recently identified [130] (Fig. 1). We have previously investigated the effects of endothelin receptor antagonism using darusentan, an ETA-selective drug, on established focal–segmental glomerulosclerosis and proteinuria in a model of aging [93]. In these studies, which were the first

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demonstration of structural and functional reversal of proteinuric renal disease by ERAs under normotensive conditions [93] and without changes in blood pressure, we found that 4 weeks of treatment with darusentan reversed not only proteinuria by about 50% but also glomerulosclerosis (Fig. 4). The reversal effect was associated with reduced glomerular expression of MMP-9, a newly identified marker of glomerulosclerosis-associated podocyte injury [131]. The anti-sclerotic and antiproteinuric effects were also

independent of renal blood flow and glomerular filtration rate. Electron microscopy studies revealed that treatment also reversed podocyte injury, which was characterized by vacuolar degeneration of podocyte cell bodies and foot process fusion (Fig. 4C) [93]. These changes in the aging kidney, which were recently identified to be at least in part mediated by podocyte autophagy (autophagy-related 5 gene) [132], were completely reversed after darusentan treatment (Fig. 4D and F) [93], strongly suggesting that ERA treatment affects

Fig. 4. Histological sections and electron micrographs demonstrating reversal of established glomerulosclerosis and podocyte injury following pharmacological blockade of ETA receptors. Aged rats, which had spontaneously developed focal–segmental glomerulosclerosis, were treated with darusentan or placebo for 4 weeks. (A) In untreated rats, moderate mesangial matrix expansion and hypertrophy of podocytes with enlarged nuclei, prominent nucleoles, and an increased number of intracytoplasmatic vesicles are present, indicating podocyte injury and cell activation. (B) After treatment with the ETA receptor-selective antagonist darusentan, glomerular injury is markedly reduced, and mesangial matrix expansion and podocyte hypertrophy and activation are much milder due to partial healing of the previously injured glomerulus and podocytes. Representative transmission electron micrographs of podocytes and glomerular basement membranes from untreated (C, E) and darusentan-treated aged kidneys (D, F). In untreated animals, glomerular basement membrane hypertrophy and podocyte injury and detachment are evident. (C) High-power micrograph, demonstrating thickening of glomerular capillary basement membrane with podocyte detachment. Injury of podocytes is characterized by hypertrophy, inclusion of cytoplasmatic absorption droplets due to vacuolar degeneration involving autophagy [132], and diffuse effacement of foot processes (E). Darusentan treatment improved attachment of the podocyte to the basement membrane and reversal of glomerular capillary hypertrophy (D). The high-power micrograph shows podocyte recovery after darusentan treatment. Endothelin blockade is associated with a reduction of podocyte injury and disappearance of absorption droplets/vacuolar degeneration. Scale bar (A, B), 50 μm. Figures reproduced from J. Ortmann, K. Amann, R.P. Brandes, M. Kretzler, K. Münter, N. Parekh, T. Traupe, M. Lange, T. Lattmann, M. Barton. Role of podocytes for the reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition. Hypertension 2004; 44: 974–981. With permission of the publisher and the American Heart Association.

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podocyte autophagy mechanisms. Electron microscopy also showed normalization of the thickness of the previously hypertrophied glomerular basement membrane (Fig. 4D and F), the thickness of which is proportional to the degree of disease and which is dependent on podocyte-derived matrix proteins [81] (Fig. 1). These data were the first demonstration that endothelin represents a previously unidentified factor regulating the architecture of the glomerular capillary [93]; this has been recently strengthened by work from Benigni's laboratory reporting similar findings, albeit using a different endothelin antagonist [112]. The mechanisms involved in disease regression appear to involve changes in podocyte function and structure (Fig. 5) as well as capillary restructuring/ angiogenesis, which has been shown to be involved in renal repair. These effects possibly occur via signaling through VEGF-mediated angiogenesis, which mediates its effects through NO and/or via accelerated formation of neo-vessels. Since ETA receptor blockade not only increases bioactivity of NO [133], a strong stimulus of angiogenesis, the L-arginine/NO pathway is likely to play a role in glomerular repair following endothelin blockade. Whether and to what extent endothelin inhibition underlies the antiproteinuric effects of drugs other that ET receptor antagonists is not fully understood. However, recent work from Benigni's and Remuzzi's groups suggests that endothelin antagonists indeed promote capillary (re-)growth in a non-aging model [112] and that treatment with antiproteinuric drugs actually leads to re-population of podocytes in the aging kidney [134]. Currently, ACE inhibitors and ARBs are the only antiproteinuric drugs in clinical use, although antiproteinuric effects have also been described for other drugs and treatments, including inhibitors of HMG CoA reductase (statins) and certain calcium antagonists (reviewed in Refs. [22,135,136]). Established glomerulosclerosis was also reversed following chronic NO deficiency (which is also a feature of aging [136,137]) and effects were independent of blood pressure [138,139]. These experimental data further support the notion that the renoprotective effects of endothelin antagonists are likely to be mostly if not completely independent of systemic hemodynamics and that these drugs represent a promising way to interfere with and even reverse established proteinuria and glomerulosclerosis [22,90].

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10. Current status of clinical studies in renal patients using endothelin receptor antagonists Currently, several drug companies are evaluating endothelin receptor antagonists (ERAs) as a new treatment option in patients with proteinuric renal disease. One of the side effects common to the ERA class of drugs – which appears to be transient and responds well to diuretic therapy – is the early formation of peripheral edema. The mechanism underlying edema formation is still not clear and may be particular to humans as it has not been reported to occur in animals. A comprehensive review of completed clinical trials that were ongoing at the time (2005) has been published [140]. The most recent developments with regard to the use of ERAs in clinical studies in renal patients have taken place only in the past 2 years and will therefore be discussed here. 10.1. Atrasentan/ABT-627/Xinlay™ (Abbott) Atrasentan is a non-sulfonamide carboxylic acid, ETA-selective antagonist with high selectivity. The first clinical evidence for antiproteinuric activities of the new ERA class of drugs was presented already a decade ago by Rabelink's group, who reported different degrees of reversal of proteinuria in 10 patients with type 1 diabetes treated with the ETA antagonist atrasentan for 12 weeks [141,142]. The effects were seen on top of standard RAAS blockade with an AT1 receptor antagonist or/and ACE inhibitor. In some of the patients treatment also had blood pressure-lowering effects. This initial observation of antiproteinuric effects and the underlying mechanisms is now being investigated in a clinical trial in diabetic patients with proteinuria that is being performed by Abbott Laboratories [143]. 10.2. Avosentan/SPP 301Ro-67-0656 (Speedel/Novartis) Avosentan is a sulfonamide-based ERA with low receptor selectivity that was licensed from Roche to Speedel Pharmaceuticals (now Novartis). Unlike as claimed by investigators who participated in the Speedel clinical trials [144,145], avosentan does not have ETA selectivity as compared with other ERAs, a notion also supported by

Fig. 5. Proposed concept of renal disease regression after inhibition of endothelin action through blockade of endothelin ETA receptors. Left: glomerular renal injury with damage of podocytes (dark green) and formation of fibrotic, “sclerotic” tissue (yellow) resulting in proteinuria (“injury”). Right: glomerulosclerosis and subsequent proteinuria can be reversed by ETA antagonist treatment if renal structural injury is less than severe. Disease regression is accompanied by improvements of glomerular architecture and regeneration of podocytes (dark green) and reversal of hypertrophy of the basement membrane of the glomerular capillary (“regression”).

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most recent experimental in vivo studies [146]. In a dose-finding trial, Wenzel et al. found substantial reductions in albuminuria in 286 patients with type 2 diabetic nephropathy of moderate severity after 12 weeks of treatment with avosentan [144]. Avosentan, like bosentan, belongs to the sulfonamide class of drugs, which are contra-indicated in patients with impaired liver function. Interestingly, avosentan treatment also had a cholesterol-lowering effect, which was also seen in patients treated with atrasentan [141,142]. As with atrasentan, the antiproteinuric effects were obtained on top of standard antiproteinuric therapy with ACE inhibition/angiotensin receptor blockade, indicating an additional and independent beneficial effect of endothelin inhibition. The ASCEND (Avosentan on Doubling of Serum Creatinine, End-stage Renal Disease and Death in Diabetic Nephropathy) phase III clinical trial was stopped due to severe drug-related side effects, including severe heart failure and deaths [145,147,148]. These patients, which had advanced renal failure with average eGFRs of 33 ml/min, were followed for 3 and 6 months, and again substantial reductions in albuminuria were noted that were similar at both doses used [145,148]. Treatment effects were independent of systemic blood pressure, suggesting that the structural effects of the ETA antagonist treatment on the kidney predominate with regard to its antiproteinuric effects. It should be pointed out that the average BMI of the study patients was above 30 kg/m2, that is, patients were obese and had advanced renal disease [148]. Obesity represents an additional and thus aggravating risk factor for glomerulosclerosis in these patients [149] and also leads to fatty liver disease and hypertension; it remains currently unknown in addition to the high doses of avosentan used to what degree the high morbidity of the study patients has contributed to the outcomes of the this study. Also, it is very likely – since no data are available on the lowest effective dose – that a lower dose of the drug might have reduced the rate of adverse events. After Speedel Pharmaceuticals was taken over by Novartis in 2008, the fate of avosentan is unknown. 10.3. Bosentan/Ro 47-0203/Tracleer™ (Actelion) The sulfonamide-based, non-selective endothelin receptor antagonist bosentan developed by Roche Pharmaceuticals and licensed to Actelion is currently being investigated for the potential indication as antiproteinuric treatment in a small clinical trial in patients with diabetic nephropathy [150]. As with the studies conducted by Abbott Laboratories and Speedel Pharmaceuticals, treatment is performed on top of RAAS inhibition. Doses will be up-titrated and investigated in a small number of 35 patients, the study duration will be 16 weeks, and completion of the study is expected within the year 2010 [150]. 10.4. Darusentan/LU135252 (Gilead Sciences) Darusentan is a non-sulfonamide, propanoic acid ETA receptor antagonist formerly developed by Knoll AG, Germany. Focal–segmental glomerulosclerosis is a feature of hypertensive nephropathy in patients with hypertension [61,149,151], which is associated with accelerated development of renal disease due to salt sensitivity or obesity [149]. Though endothelin blockade has been investigated as a novel means to interfere with clinical hypertension since the late 1990s (reviewed in Ref. [152]) and a strong role for endothelin in the disease had been proposed [153], resistant hypertension had not been specifically addressed in clinical studies. Blockade of ETA receptors using darusentan has been recently shown to effectively reduce blood pressure in a phase II dose-finding trial in patients with resistant hypertension treated with two antihypertensive drugs and a diuretic [154]. Whether darusentan treatment in humans also exerts any effects on reversing proteinuria or glomerulosclerosis as demonstrated in animals studies [93,155] (Fig. 3) or whether it has an impact on morbidity or mortality in such patients remains to be shown. In a recently completed phase III clinical trial [156], darusentan was

superior in reducing sitting systolic and diastolic blood pressure compared with another antihypertensive drug and darusentan treatment met several of the secondary endpoints (that included changes in 24-h blood pressure, reaching blood pressure goal levels after 14 weeks, and changes in estimated GFR). Importantly, at the highest dose, darusentan treatment reduced urinary albumin excretion in patients with renal disease by almost 60%. Yet Gilead Sciences in December 2009 announced to discontinue the development of darusentan for the treatment of resistant arterial hypertension and its related complications [156]. 10.5. Sitaxentan/TBC11251/Thelin™ (Pfizer) Sitaxentan is a sulfonamide-based ETA receptor antagonist with high receptor selectivity developed by Texas Biotechnology/Encysive, which was acquired by Pfizer in early 2008. Sitaxentan has been recently investigated in patients with proteinuric non-diabetic renal disease of different etiologies, including focal–segmental glomerulosclerois. Sitaxentan is currently being investigated in a small clinical with 27 patients and a treatment duration of 6 weeks, and results will be compared to the calcium-antagonist nifedipin [157]. Some of the results have been published demonstrating that the antiproteinuric effect of sitaxentan is also present in non-diabetic renal patients [157,158]. 11. What the future holds There are convincing preclinical and clinical data suggesting that endothelin receptor blockade could become a novel treatment paradigm to interfere with and even reverse proteinuric renal disease in patients. Endothelin receptor blockers appear to offer therapeutic efficacy to inhibit proteinuric renal disease even on top of currently approved antiproteinuric medications (Fig. 5). Thus, this class of drugs could represent an urgently needed next step in renal medicine for the treatment of patients with chronic proteinuric renal disease [159]. As genetic factors related to the components of the endothelin system could influence the susceptibility to develop proteinuria in diabetics, further pharmacogenomic studies are warranted. An issue relevant to therapy in humans will also be to determine whether ETA receptor selectivity represents a therapeutic advantage over non-selective compounds. This is particularly important since the selectivity of certain compounds may not be sufficiently high enough to avoid blockade of ETB receptors, which may be an important determinant of drug-related side effects such as edema. Clinical investigators should determine factors such as careful drug dosing to avoid toxicity effects and should select patients carefully taking into account the comorbidities and avoiding treatment of those that are severely ill, as learned from the ASCEND trial [22,145,148]. In addition, future basic science studies should address whether and in which way the antiproteinuric effect of ET receptor blockade are different from those of ACEIs or ARBs. Also, an unsolved question is whether the use of ERAs might perhaps be even superior to ACEIs or ARBs in certain forms of renal disease. Finally, it remains to be shown whether receptor selectivity for the ETA receptor will translate into an improved clinical outcome such as survival or need for renal transplantation, and whether and how side affects seen in the early phases of ERA treatment (i.e., peripheral edema) can be avoided or controlled. Certain renal patient subgroups could have greater benefit from ERA treatment than others, also, effects and side effects are likely to differ between the two main classes of ERAs, the sulfmonamidederived and the propanoic acid-derived ERA compounds [21]. There is, however, reason to believe that 20 years after the discovery of endothelin, the new ERA class of drugs could provide us with new renal therapies that might allow us to halt or even reverse proteinuric renal disease in patients [21,119,159] (Fig. 5).

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Acknowledgments I thank colleagues and collaborators of many years who have contributed to the studies discussed here, particularly Kerstin Amann (University of Erlangen), Ralf Brandes (University of Frankfurt), Livius d'Uscio (Mayo Medical School), Matthias Kretzler (University of Michigan), Thomas Lattmann (Triemli Hospital Zurich), Pierre Moreau (Université de Montréal), Klaus Münter (Bayer AG), and Sidney Shaw (University of Bern). I acknowledge the work of all colleagues in the field and apologize to those whose work was not cited because of space limitations. This work was supported by the Swiss National Science Foundation (grants nos. 3200-108258 and K33KO-122504).

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