Endothelin Receptor Antagonists in Kidney Disease Ⅱ

4. Mechanisms of Renal Protection Mediated by ERAs 

ET, through the activation of its receptors, may be detrimental for the kidney, as it is involved in the progression of chronic kidney disease and other conditions such as diabetes [48]. Therefore, blockade of the ET receptors with ERAs has renal protective effects. ERAs protect the kidney by several mechanisms. First, this drug class has clear effects on glomerular hemodynamics [49–51]. ETA receptor antagonism improves blood pressure via vasodilatation and decreases proteinuria and the filtration fraction (ratio of glomerular filtration rate over renal plasma flow), providing renoprotective effects [14]. Moreover, ETA receptor blockade may improve endothelium-dependent relaxation and vasomotion [52–54]. There is no difference in terms of blood pressure reduction when comparing selective ETA receptor antagonists and mixed ETA/ETB receptor antagonists, which suggests that ETB receptor blockade does not change blood pressure. This also implies that combined ETA/ETB receptor antagonists and selective ETA receptor antagonists are similar in terms of their hypotensive effects and ETB receptor antagonists are not involved in this outcome [55]. Second, ERAs also produce effects on different renal cell types that express ET-1 or its receptors [56–58]. Podocytes are targets of ET-1 since they express ETA [59]. In this sense, several studies have been focused on the effects of ETA receptor antagonists on these cells. After treatment with ERAs, many studies have found a reduction in the podocyte injury, which lead to the stabilization of the glomerular and podocyte structure [60,61]. Exogenous ET-1 administration induced podocyte injury in rats, which could be prevented by ETA receptor blockade [61]. Also, in a hypertensive rat model, selective ETA receptor blockade restored podocyte injury and function. Further, ERAs ameliorate the structure of the glomerular basement membrane and have beneficial effects on glomerulosclerosis and proteinuria [62]. Mesangial cells produce ET-1, although in a much smaller proportion than endothelial cells. ET-1 produced by mesangial cells can act in an autocrine way by binding to ET receptors. Via ETA it results in the contraction of mesangial cells, cell proliferation and mesangial matrix accumulation [63,64]. These deleterious effects can be blocked using ERAs [65,66]. As mentioned before, ETB is expressed in all along the renal tubule but ETA expression in the proximal tubule and the descending Henle’s loop is low [56]. ETB receptor is responsible for the clearance of ET-1 and could have important implications since it modulates the presence of this vasoconstrictor [51]. Some studies reported that treatment with an ETB-selective receptor antagonist diminished ET-1 clearance, remaining in the plasma, and increasing the response to ET-1 leading to hypertension in some patients [67]. In addition to these effects, ET-1 can induce inflammation and fibrosis [16], since overexpression of ET-1 resulted in interstitial fibrosis in transgenic mice expressing human ET-1 [68] that can be reversed only by ETA-selective receptor antagonists [69].

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In summary, ETA-selective ERAs show a wide range of renoprotective effects especially by ameliorating blood pressure and modulating kidney hemodynamics although, as mentioned, ERAs have beneficial effects not mediated by its blood pressure-lowering capacity. ERAs can restore podocyte injury and its function; and improve mesangial matrix accumulation, inflammation, and fibrosis, eventually reducing glomerular permeability and proteinuria.

5. Preclinical Experimental Evidence of ERAs Protective Effects on Kidney Damage 

In recent years, several preclinical studies have investigated the effects of ERAs using different experimental models. The first studies on cultured mesangial cells showed that ET produced cellular contraction, hypertrophy, and extracellular matrix production [70,71]. These effects were reversed using ERAs [66], as happened in hypertensive rats [72]. Experiments using stroke-prone spontaneously hypertensive rats (SHRSP) demonstrated that the ETA receptor blockade provided renal protection by normalizing the expression of growth factors, diminishing extracellular matrix proteins, and reducing metalloproteinase-2 (MMP- 2) activity [66]. Spires et al. studied the effect of atrasentan (an ETA receptor antagonist, Table 2) in streptozotocin-treated Dahl salt-sensitive (STZ-SS) and type 2 diabetic (T2DN) rats. Both rat models showed increased levels of ET-1 during the progression of the renal disease. Atrasentan diminished glomerular injury and renal fibrosis in both models but only reduced arterial pressure and proteinuria in STZ-SS. This could be explained by differences in the severity of the kidney injury of these models [73]. In any case, it illustrates that improvement of kidney damage is possible without changing arterial pressure and/or proteinuria. In this sense, Harvey et al. [74], demonstrated that ETA blocking with atrasentan (but not ETB blocking) improved the integrity and viability of cultured podocytes submitted to hypoxia to mimic chronic renovascular disease [74]. In this line, Dolinina et al. tested BQ-788 (an ETB receptor antagonist, Table 2) and JKC-301 (an ETA receptor antagonist) in Sprague-Dawley rats where glomerular permeability was induced by administration of ET-1. The study demonstrated that the glomerular hyperfiltration improvement was dependent on ETA receptors since ETA receptor blockade ameliorated glomerular hyperfiltration but not ETB receptor blockade [75]. Similar results were obtained in a study where the effects of BQ-788 and atrasentan were compared in uni-nephrectomized Sprague-Dawley rats on a high-sodium diet (HS/UNX) and in spontaneously hypertensive rats (SHR). Both hypertensive rat models showed altered nitric oxide levels, possibly related to ETA receptor hyperactivity. Also, in the HS/UNX model ETA receptor blockade reduced blood pressure and decreased renal excretion, while ETB receptor blockade did not alter blood pressure or renal excretion. The SHR model showed a reduction in blood pressure after treatment with atrasentan. This comparison confirmed, as well, the dependence of blood pressure and renal hemodynamics on ETA receptors, since ETB receptor antagonists did not modify renal hemodynamics [76]. Indeed, ETA-selective ERAs have clear renoprotective effects. In this sense sitaxentan (an ETA receptor antagonist, Table 2) improved kidney function and tubular atrophy in a rat model of chronic interstitial nephritis induced by adenine. Further, sitaxentan in combination with cinacalcet (an allosteric modulator of the calcium-sensing receptor), increased renal angiotensin-converting enzyme 2 (ACE2) expression, which is protective for the kidney, and normalized urinary calcium loss [77]. Similar results were obtained in the study of Caires et al. who tested bosentan and macitentan (Table 2) in normotensive and hypertensive rats with cyclosporin A (CsA)-induced kidney damage. CsA is nephrotoxic and has a vasoconstrictive effect, which was partially reversed only by bosentan. However, both, bosentan and macitentan were able to improve the hemodynamic changes induced by CsA in hypertensive rats by decreasing blood pressure. Furthermore, bosentan and macitentan reduced the generation of reactive oxygen species produced by CsA. Thus, the ERAs used in these experiments had similar effects although bosentan seemed to be better at reversing the hemodynamic changes [78]. Ambrisentan (an ETA receptor antagonist, Table 2) and bosentan showed similar kidney protective effects in an ischemia-reperfusion rat model in terms of reduction of kidney apoptosis, tissue damage and inflammation probably mediated by an increase of nitric oxide levels. Another study using the normotensive Wistar Kyoto (WKY) rats compared the effects of macitentan and sitaxentan. The results of these experiments revealed that sitaxentan prevented sunitinib-induced hypertension in the same manner as macitentan indicating that the increase in blood pressure was mediated by ETA receptors. Sitaxentan also improved albuminuria and diminished prostacyclin levels [79].

The effects of ERAs have also been studied on top of RAS blockers, the current standard of care for many chronic kidney diseases [80–82]. Atrasentan combined with losartan (an angiotensin-II type 1 receptor blocker) improved podocyte number and structure and decreased proteinuria in BTBR ob/ob mice [83] similar to what happens in humans [19]. Other studies combined two RAS blockers (trandolapril and losartan) with atrasentan in a rat model of chronic kidney disease, showing an additional beneficial effect of the combination of ERAs with RAS blockers. This combination of drugs increased the survival rate and reduced proteinuria and renal glomerular damage [84]. Also, Gagliardini et al., combined avosentan (an ETA receptor antagonist, Table 2) with lisinopril (an angiotensin-converting enzyme inhibitor) in uni nephrectomized streptozotocin-induced diabetic rats. Combined therapy was able to improve proteinuria, protect from glomerular and tubulointerstitial damage, restore podocyte number, nephrin levels and glomerular permeability. The combination also improved the deleterious changes in the peritubular capillaries and renal interstitial blood perfusion, which could lead to amelioration of the tubular function [85]. Preclinical studies in mice and rats using spartan, a new dual AT1/ETA receptor antagonist with affinity to ETA and angiotensin II (type 1) receptors (Table 2), showed that this dual inhibition protects the glomeruli from podocyte loss and podocyte foot effacement. The effects also include maintenance of the glomerulus basement membrane, glomerular glycocalyx integrity and reduction of blood pressure [86,87]. In addition, studies using multiphoton microscopy imaging in Confetti mice with focal segmental glomerulosclerosis induced by transient receptor potential channel 6 (TRPC6) overexpression, showed greater preservation of the kidney function in mice treated with spartan in comparison with the mice that received no drug or losartan [88]. ERAs have also been studied in combination with sodium-glucose type 2 cotransporter (SGLT2) inhibitors (SGLT2i) in several diabetic mice models because of the potential for SGLT2i to reduce the volume overload induced by ERAs. Atrasentan combined with dapagliflozin (an SGLT2i) did not improve albuminuria, glomerular filtration rate, kidney inflammation or fibrosis but ameliorated of glomerulosclerosis and podocyte injury in a mouse model of type 2 diabetic kidney disease. This suggests that the dual therapy approach can have therapeutic potential [89]. A recent study of Vergara, A. et al. [90] tested the capacity of an SGLT2 inhibitor (empagliflozin) and/or an ERA (atrasentan) on top of RAS blockade with ramipril to protect the diabetic kidney in experimental diabetic nephropathy using db/db mice. This study revealed that triple therapy with empagliflozin, atrasentan and ramipril maintained the impact of each therapy alone and added to organ protection. Empagliflozin combined with ramipril or in triple therapy with atrasentan ameliorated hyperfiltration, but only the triple combination exerted greater protection against podocyte loss. The combined therapy not only protected against kidney injury but also provided cardiac protection in terms of a decrease of cardiomyocyte hypertrophy. Additionally, the add-on triple therapy further enhanced the intrarenal ACE2/Angiotensin(1-7)/Mas protective arm of the RAS. These data suggest that triple therapy with empagliflozin, atrasentan and ramipril have a synergistic cardiorenal protective effect in experimental diabetic nephropathy. Thus, the combination with RAS blockers and/or SGLT2i may promote the use of the ERAs in clinical practice as it has shown add-on effects in experimental models and has the potential to mitigate adverse events produced by ERAs in monotherapy. This therapeutic approach is currently being evaluated in randomized controlled trials. 

6. Randomized Controlled Trials (RCTs) Using ERAs for Prevention of Kidney Disease Progression

The largest trials testing ERAs have been performed in type 2 diabetic patients (Table 3). In these studies, ERAs have been shown to reduce albuminuria and slightly decrease blood pressure [19,23]. The effect of selective endothelin antagonists on albuminuria is consistent across different studies, obtaining a 30–40% reduction in urine albumin-to-creatinine ratio (UACR) in the groups that received the active treatment. However, blood pressure reduction is moderate and shows different results between RCTs. Overall, selective ERAs seem to reduce 3–5 mmHg of both systolic and diastolic blood pressure (SBP and DBP, respectively). The effects on BP vary among the employed ERA, with the greatest reductions described for atrasentan (9.9 mmHg reduction in SBP and 4.6 mmHg reduction in DBP) [91]. Nevertheless, the latter study included patients with resistant hypertension, which may have contributed to the larger differences in the active treatment arms [91]. In addition, the SONAR study showed that BP reduction is more evident when initiating the treatment and becomes milder after chronic treatment [19]. Regarding GFR preservation, selective ERAs have displayed protective effects or no effect among the different RCTs performed to date. The SONAR trial, which treated responder patients (patients that showed a decrease in UACR of at least 30% with no substantial fluid retention during the enrichment period) for a median follow-up of 2.2 years, showed that 0.75 mg of atrasentan on top of the RAS blockade was able to preserve 0.65 mL/min/1.73 m2 of GFR and to prevent the doubling of serum creatinine during the treatment period [19]. In the same line, in patients with systemic sclerosis, zibotentan was able to preserve 4.3 mL/min/1.73 m2 of GFR after 6.5 months of treatment [24]. The only ERA that showed a significant decrease in GFR that could be related to the type of patients included and the greater BP reduction was darusentan [91].

When analyzing major renal events, only ASCEND and SONAR trials were designed to find differences in a primary composite kidney outcome [19,36]. The ASCEND trial had to be prematurely stopped because an increased number of deaths due to cardiovascular causes in the group of patients receiving the active treatment [36]. As death was included within the main composite outcome, the study was unable to find significant differences between the groups receiving avosentan and the group receiving placebo. The increased number of CV deaths was also linked to an increased number of adverse events: fluid overload, heart failure and anemia. However, if we only consider end-stage renal disease (ESRD) or doubling of serum creatinine as events, the group of patients receiving avosentan showed a lower risk compared to those treated with placebo (HR 0.63, 95%CI: 0.42–0.95). To overcome the evident adverse events related to the inhibition of ETA receptor-mediated sodium and water excretion, the SONAR trial only included responder patients who did not show adverse events during an initial enrichment period [19]. In the latter study, atrasentan was able to reduce the number of renal events when compared to placebo. Nevertheless, previously described adverse events such as fluid overload, heart failure or anemia were again more frequent in the group treated with atrasentan. In this line, the addition of the new renoprotective SGLT2i to the treatment with ERAs in type 2 diabetic patients could prevent the development of fluid retention or anemia, as the former drug class has diuretic effects and increases hemoglobin levels [94,95]. A recent post-hoc analysis of patients receiving atrasentan and SGLT2i in the SONAR trial revealed that weight increase (a surrogate marker of fluid overload) was reduced in patients receiving both atrasentan and SGLT2i [96]. New trials with prespecified kidney outcomes that evaluate the synergistic effects of the combination will shed light upon the future of ERAs in the treatment of chronic kidney disease. The currently ongoing ZENITH-CKD trial (NCT04724837), for example, will evaluate the efficacy of the combination of zibotentan and dapagliflozin in the treatment of CKD.

Additionally, the use of ERAs is being extended to kidney diseases with albuminuria such as primary FSGS or IgA nephropathy, where the existence of previous cardiovascular comorbidities is less frequent and the risk of adverse events also lower. Spartan, a dual angiotensin II type 1 and endothelin type A (ETA) receptor antagonist was already tested in the DUET trial that included patients with primary focal segmental glomerulosclerosis (FSGS) [97]. After eight weeks of treatment, spartan obtained greater reductions in proteinuria, and was superior to irbesartan, achieving partial remission of the disease (28% vs. 9%). The promising effects of spartan will be further confirmed by the ongoing trials on primary FSGS (DUPLEX study, NCT03493685) and IgA nephropathy (PROTECT and SPARTAN studies, NCT03762850 and NCT04663204, respectively). The ALIGN (NCT04573478) will also give insights about the impact of the combination of atrasentan and RAS blockade for the treatment of IgA nephropathy

7. Conclusions 

As reviewed here, the concentration of ET-1 is increased in pathological conditions, such as diabetes or hypertension, causing sustained vasoconstriction that ultimately leads to kidney damage. The ERAs show clear renoprotective effects in preclinical experimental models and in humans mainly by hemodynamic effects but also by restoring podocyte injury, reducing mesangial matrix accumulation, fibrosis and inflammation which reduces glomerular permeability and proteinuria. However, the use of ERAs in clinical practice to prevent kidney disease is narrow because some ERAs failed to demonstrate efficacy in phase III randomized clinical trials and/or produced adverse events such as oedemas. To overcome these limitations, the combination of ERAs with SGLT2i has been proposed as well as the use of the dual angiotensin-II type 1/endothelin receptor blockers. The utility of these therapeutic approaches to treat kidney disease is currently being tested in ongoing randomized controlled trials.

Author Contributions: Writing—original draft preparation; I.M.-D., N.M., C.L.-C. and A.V. Writing— review and editing; F.J.Á., P.W.B., A.V., C.J.-C. and M.J.S.; Figure design: A.V. All authors have read and agreed to the published version of the manuscript. 

Funding: The authors have funding from Fondo de Investigación Sanitaria-Feder, ISCIII, PI21/01292, RICORS RD21/0005/0031, and Marató TV3 421/C/2020, Marató TV3 759/U/2020, Marató TV3 215/C/2021. 

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable. 

Data Availability Statement: Not applicable. 

Conflicts of Interest: F.J.Á. and P.W.B. are employees of Travere Therapeutics. A.V. reports personal fees from MUNDIPHARMA, and non-financial support from MUNDIPHARMA, SANOFI and NOVO NORDISK outside this work. C.J.C. declares travel support and a research grant from TRAVERE THERAPEUTICS outside this work. M.J.S. reports grants from BOEHRINGER, personal fees from NOVO NORDISK, JANSSEN, BOEHRINGER ASTRAZENECA, FRESENIUS, MUNDIPHARMA, PFIZER, ICU, GE Healthcare BAYER, TRAVERE THERAPEUTICS and VIFOR, and non-financial support from ELI LILLY and ESTEVE outside this work. 

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