Endothelin Receptor Antagonists in Kidney Disease

Abstract: Endothelin (ET) is found to be increased in kidney disease secondary to hyperglycemia, hypertension, acidosis, and the presence of insulin or proinflammatory cytokines. In this context, ET, via the endothelin receptor type A (ETA) activation, causes sustained vasoconstriction of the afferent arterioles that produces deleterious effects such as hyperfiltration, podocyte damage, proteinuria and, eventually, GFR decline. Therefore, endothelin receptor antagonists (ERAs) have been proposed as a therapeutic strategy to reduce proteinuria and slow the progression of kidney disease. Preclinical and clinical evidence has revealed that the administration of ERAs reduces kidney fibrosis, inflammation and proteinuria. Currently, the efficacy of many ERAs to treat kidney disease is being tested in randomized controlled trials; however, some of these, such as avosentan and atrasentan, were not commercialized due to the adverse events related to their use. Therefore, to take advantage of the protective properties of the ERAs, the use of ETA receptor-specific antagonists and/or combining them with sodium-glucose cotransporter 2 inhibitors (SGLT2i) has been proposed to prevent oedemas, the main ERAs-related deleterious effect. The use of a dual angiotensin-II type 1/endothelin receptor blocker (spartan) is also being evaluated to treat kidney disease. Here, we reviewed the main ERAs developed and the preclinical and clinical evidence of their kidney-protective effects. Additionally, we provided an overview of new strategies that have been proposed to integrate ERAs in kidney disease treatment. 

Keywords: endothelin; endothelin receptor antagonists (ERAs); atrasentan; spartan; kidney disease 

1. Introduction: The Endothelin System 

Endothelin (ET) is a 21-aminoacid polypeptide described as the major vasoconstrictor of the organism. It is produced mainly by endothelial cells, but also by cells of the renal system, such as the epithelial and mesangial cells [1]. Hickey et al. were the first to describe the existence of a molecule capable of causing capillary constriction produced by the endothelium in 1985 but it was not until 1988 that ET was identified [2,3]. The ET polypeptide is present in three isoforms: ET-1, ET-2, and ET-3, with ET-1 being the greatest vasoconstrictor and the only one found at the protein level in the kidney [4]. ET-2 and ET-3 differ from ET-1 in two and five residues of the N-terminal end, respectively (Table 1), which determines the differences on the receptor-binding affinity [5]. Moreover, ET-1 is mainly released by endothelial cells, while the intestine and the kidney produce ET-2 and the neural tissue releases ET-3; the three isoforms can act in a paracrine or autocrine manner [5]. 

Table 1. ET1, ET2 and ET3 amino acid sequences. ET is a 21-amino acid peptide present in three different isoforms in the organism: ET-1, ET-2 and ET-3 [5]; the differences in amino acid sequence between them are shown in bold. 

The action of ET is channeled through two membrane G-protein coupled receptors: Endothelin receptor A (ETA) and B (ETB). ETA is localized in vascular smooth muscle cells and presents more binding affinity for ET-1 and ET-2 than for ET-3, due to the differences in the amino acid sequences (Table 1). ETA activation induces a robust vasoconstrictor response and promotes cell proliferation and accumulation of the extracellular matrix. ETB is present in vascular smooth muscle cells and endothelial cells. The three ET isoforms present the same affinity for the ETB receptor, and its activation produces antiproliferative and antifibrotic effects, as well as the release of various vasodilator molecules [6]. Some experts have suggested an extended classification of the ET receptors, subdividing ETB into ETB1 and ETB2 to differentiate the receptors present on the endothelial cells and the ones present on the smooth muscle cells, respectively. Nonetheless, there is no pharmacological evidence that demonstrates a difference between the receptors expressed by these two cell types [7]. ET-1 binding to ETA causes G-proteins and phospholipase C (PLC) to join, leading to inositol triphosphate (IP3) and diacylglycerol (DAG) formation. Then, IP3 activates specific endoplasmic reticulum receptors to stimulate the release of stored Ca2+ causing a fast increase in intracellular Ca2+, which allows cell contraction and subsequent vasoconstriction. ET-1 activity through ETA receptors also involves other signaling pathways, such as the phospholipase D (PLD) or mitogen-activated protein kinase (MAPK) pathway, to carry out other physiological effects such as cell growth or mitogenesis [5]. Contrarily, ETB receptors produce their vasodilator effects through the activation of the nitric oxide synthases (NOS) system and the release of vasodilators as nitric oxide (NO) [8,9]. 

In the kidney, ET has an essential role in blood flow and glomerular filtration regulation and in water–sodium, and acid–base balances. ETA and ETB are expressed on the glomerular podocytes, mesangial cells, and on the afferent and efferent arterioles. Regarding the tubular compartment, ETB is expressed in all the regions in the renal tubule while ETA is scarcely expressed on the proximal tubule and the descending Henle’s loop [10]. In physiological conditions, ET-1 through ETA produces vasoconstriction of the afferent arteriole, reducing blood flow and, consequently, the glomerular filtration rate (GFR). Contrarily, the activation of ETB induces vasodilation, antiproliferative effects, and ET-1 depuration [8,9]. In pathological conditions, such as diabetes or hypertension, the concentration of ET-1 is increased because of hyperglycemia, acidosis, and the presence of insulin, angiotensin II, and proinflammatory cytokines, which causes sustained vasoconstriction. This may contribute to deleterious effects such as hyperfiltration (mainly in early diabetic nephropathy or incipient obesity-related kidney disease [11–13]) or podocyte damage and, eventually, proteinuria and GFR decline (Figure 1) [14]. 

The endothelin receptor antagonists (ERA) are postulated as a therapeutic strategy to reduce proteinuria and delay the progression of GFR decline [14]. Promising results using ERAs in kidney disease have been obtained in recent years. The purpose of this review is to provide an overview of the main ERAs and of the mechanisms by which these drugs protect the kidney with a special focus on the results obtained in updated experimental studies and randomized clinical trials. Additionally, we provide a glance of the main novel approaches to introduce ERAs for kidney disease prevention.

Figure 1. Scheme of the endothelin system. ET-1 acts through its binding to ETA and ETB producing opposite effects in the kidney. The effects caused by the activation of ETA are shown in red and the effects of ETB activation are shown in blue. In pathological conditions, hyperglycemia, acidosis and the presence of insulin, angiotensin II and proinflammatory cytokines cause the increase of ET-1 concentration, which produces deleterious effects on renal function, such as vasoconstriction and endothelial damage, inflammation, fibrosis, podocyte damage or albuminuria.

2. Methods

We searched PubMed, Scopus, and Google Academic during November-December 2022 using the following search terms (alone of combined) to find publications related to the endothelin system and the endothelin receptor antagonists in experimental and human kidney disease: “endothelin or ET”, “endothelin receptor antagonists or ERA”, “endothelin receptor A or ETA”, “endothelin receptor B or ETB”, “kidney”, “kidney disease”, “chronic kidney disease”, ”kidney injury”, “experimental models”, “mice”, ”rat”, “podocytes”, “randomized clinical trials”. We critically reviewed the reports found and selected the relevant studies to construct the review text. For the sections “5. Preclinical experimental evidence of ERAs protective effects on kidney damage” and “6. Randomized controlled trials (RCTs) using ERAs for prevention of kidney disease progression”, we mainly focused on the works published in the last five years not to overlap previous reviews on the topic. The complete literature review strategy is available from the authors upon request. Further, we consulted https://clinicaltrials.gov during November–December 2022 to obtain information regarding unpublished ongoing clinical trials that we have started using the corresponding National Clinical Trial (NCT) number. 

3. The Endothelin Receptor Antagonists

Endothelin receptor antagonists (ERAs) are drugs that block the endothelin receptors, preventing the endothelin action. There exist different types of ERAs, which can be distinguished by their affinity for binding to ETA or ETB. In some cases, these antagonists do not present selectivity and are able to interact with both receptors. The selectivity of an ERA for each receptor subtype is determined by a competition binding assay against [125I]-ET-1 that allows to calculation the equilibrium dissociation constant of each compound to both receptors, ETA, and ETB. To establish a selectivity threshold, in 2006, Maguire and Davenport et al. [15] proposed that an ERA should present more than 100-fold selectivity for ETA or ETB to be considered selective for one or the other receptor. Those with less than 100-fold selectivity should be classified as non-selective or mixed antagonists [15]. The main ERAs are summarized in Table 2.

The majority of the antagonists here described are still under investigation in ongoing clinical trials, while others are not used in clinical practice because of lack of efficacy or due to the presence of adverse events related to their use that compromise the safety of patients. Therefore, to take advantage of these compounds with clear beneficial effects (Table 2), therapeutic approaches under study are the combination of ERAs with other nephroprotective drugs such as sodium-glucose cotransporter 2 inhibitors (SGLT2i) and the use of dual drugs such as sparsentan that blocks at the same time angiotensin-II type 1 and endothelin receptors. 

3.1. ETA-Selective Receptor Antagonists 

The binding of ET-1 to ETA, in pathologic conditions, can lead to vasoconstriction, inflammation, cellular injury, fibrosis, and, finally to proteinuria and loss of renal function [31]. To counteract these effects, several selective ETA-receptor antagonists have been developed as potential therapeutic agents. Some of them are currently used to treat pulmonary arterial hypertension (ambrisentan and macitentan), meanwhile, others are still under study in ongoing randomized clinical studies. To date, none of the ETA-selective receptor antagonists have been approved to treat kidney disease, despite their demonstrated kidney protective effects (Table 2).

BQ-123 was the first ETA selective antagonist peptide isolated that derived from Streptomyces misakiensis fermentation products. It has been used in investigations in both animals and humans where the molecule reduced glomerular permeability [20]. Darusentan (LU 135252) is a selective endothelin receptor antagonist, with high affinity towards ETA receptor [17], with a ratio of relative selectivity of 170:1 ETA:ETB [32]. It is derived from the optimization of two initial lead structures (LU 110896 and LU 110897) found in a screening of the library of human recombinant ETA receptors [33]. Darusentan was promising because reduced blood pressure in resistant hypertension patients in early clinical studies but unfortunately failed to achieve efficacy in phase III clinical studies [32]. Other examples of ETA selective antagonists are sitaxentan, ambrisentan, avosentan, atrasentan, macitentan and zibotentan (Table 2). Currently, sitaxentan, ambrisentan and macitentan are approved to treat pulmonary arterial hypertension [21,34,35] while avosentan, atrasentan and zibotentan have been proposed as therapeutic agents in kidney disease [19,24,36]. Macitentan is a sulfamide with high affinity for ETA that has been used in pulmonary arterial hypertension since its approval in 2013 [37,38]. It belongs to the next generation of antagonists, as it was developed following the structural basis of bosentan [20], but with improvements such as a prolonged receptor binding capacity and better pharmacodynamics and pharmacokinetics [37,38]. In vivo, macitentan is metabolized by cytochrome P450 3A4 (CYP3A4) into an active metabolite, which is called aprocitentan (a non-selective ERA, see below) [26]. Avosentan is a selective ETA inhibitor that presents ~500-fold selectivity for ETA over ETB receptors [39]. It was developed for the treatment of diabetic nephropathy. A study performed by Wenzel et al. in 2009 [23] demonstrated that the addition of avosentan to the standard of care antihypertensive therapy with RAS blockers produced additional antiproteinuric effects in diabetic nephropathy patients. However, a second study to test long-term treatment with avosentan was stopped prematurely because of safety concerns [36]. Atrasentan is an oral selective ETA inhibitor with a selective ETA: ETB blockade ratio of 1200:1 [40] and 1800-fold selectivity for ETA [39]. In patients with diabetes and chronic kidney disease, atrasentan reduces the risk of renal events and albuminuria [19,22,41]. Currently, it is under study in an ongoing phase 2 clinical trial to evaluate the efficacy and safety of atrasentan in patients with proteinuric glomerular diseases (AFFINITY: Atrasentan in Patients with Proteinuric Glomerular Diseases—NCT04573920). Finally, zibotentan is a selective ETA antagonist, which shows a potent affinity to this specific receptor [42]. In a recent clinical trial, it has been demonstrated that zibotentan could be beneficial for the treatment of systemic sclerosis-associated chronic kidney disease because its effects in the improvement of estimated GFR and the absence of increased endothelin serum levels during treatment [24].

3.2. ETB-Selective Receptor Antagonists

Few ETB-selective antagonists have been developed. This can be explained by the fact that when endothelin binds ETB, it triggers beneficial effects such as vasodilation; hence inhibiting the action of ETB may not be a suitable therapeutic strategy. In addition, ETB-selective antagonists are usually less potent than ETA-selective agonists [20]. However, some small molecules have been developed to block ETB, such as non-peptide RO468443 which displays 2000-fold ETB selectivity [43], and A192621 [20]. Nevertheless, the most important ETB-selective antagonist is BQ-788, which was described for the first time by Ishikawa et al. in 1994. BQ-788 has been studied in combination with BQ-123, an ETA-selective ERA, and it causes the reduction of glomerular permeability to albumin but does not add to the effect of BQ-123 in monotherapy [39]. In cancer, BQ-788 inhibits cell growth and induces the death of melanoma cells, both in vivo and in vitro [26]. 

3.3. Non-Selective Endothelin Receptor Antagonists

Some ERAs can interact with either ETA or ETB receptors. Some of the non-selective ERAs are bosentan, tezosentan and aprocitentan. Bosentan is a non-peptide derivative dual endothelin receptor antagonist with affinity to both receptors ETA and ETB, but with barely higher affinity towards ETA (ETA:ETB 20:1) [22,35,40]. Currently, it is used in the treatment of pulmonary arterial hypertension [34] and in pediatric idiopathic pulmonary hypertension [22]. Bosentan decreases vascular resistance, resulting in an increasing cardiac output without disrupting the heart rate. It also plays a role in the inhibition of endothelial cell proliferation [25]. Tezosentan is a dual endothelin receptor antagonist with a selectivity ratio of 30:1 ETA:ETB [44]. It was developed for the treatment of heart failure and preclinical studies have shown that bosentan improves hemodynamics and renal function in rats [26] but does not improve dyspnoea or reduce the risk of cardiovascular events [26]. Aprocitentan (ACT-132577) is a dual inhibitor of ETA/ETB with a selective ratio of 1:16 [45–47]. It belongs to the sulfonamide class of molecules and is obtained by oxidative depropylation from macitentan [45–47]. In the PRECISION clinical trial, finished in 2022, aprocitentan lowered blood pressure in patients with resistant hypertension [27]. 

3.4. Other Types of Endothelin Receptor Antagonists 

Spartan (BMS-346567) is a dual endothelin receptor/angiotensin-II type 1 receptor antagonist (DEARA) which presents high affinity for ETA (~1000-fold). It was created by combining structural elements of both irbesartan, an angiotensin II type 1 receptor antagonist, and biphenyl sulfonamide, an endothelin receptor antagonist. Thus, sparsentan blocks at the same time the RAS and the endothelin system reason why it is expected to show additional renoprotective effects. Sparsentan reduces blood pressure in hypertensive patients [28]. The antiproteinuric and the possible nephroprotective effects of spartan are currently studied in focal segmental glomerulosclerosis (DUPLEX study) [29] and IgA Nephropathy (PROTECT study; NCT03762850) patients in an ongoing phase 3 clinical trials. 

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