Endothelin-1 Signaling in the Kidney: Recent Advances and Remaining Gaps

REVIEW Endothelin-1 signaling in the kidney: recent advances and remaining gaps Abigail J. Brooks, 1 María Del Carmen Gallego-L opez, 1,2 and Carmen De Miguel 1 1 Section of Cardio-Renal Physiology and Medicine, Division of Nephrology, Department of Medicine, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States and 2 Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain Abstract The involvement of endothelin-1 (ET-1) in the maintenance of kidney function as well as its role in renal pathophysiology has been appreciated for decades; however, there still exist important gaps in knowledge in our understanding of the mechanistic pathways activated by this system in the kidney. The purpose of this article is to review recent advances in the field, as well as to underscore areas that need more investigation, with an emphasis on the interplay of ET-1 with inflammation, sex differences, circadian rhythms of renal function, the most recent clinical trials involving the ET-1 system, and the interaction between microRNAs and the ET-1 system. circadian; clinical trials; endothelin; inflammation; sex differences INTRODUCTION Nearly 40 years ago, Yanagisawa et al. (1) discovered a 21amino acid protein in porcine aortic endothelial cells, consequently naming it endothelin. It quickly gained recognition as one of the most potent vasoactive peptides produced by the human body (1). Today, we know that the actions of endothelin-1 (ET-1) extend far beyond endothelial cells and vascular function modulation. Research has since revealed that ET-1 is produced by nearly every cell type in the body, including vascular, immune, and neuronal cells (2). This is especially true for the kidney, where ET-1 is found in particularly high concentrations, especially under diseased conditions (2–4). ET-1 levels can be influenced by a slew of chemical and physical stimuli, such as changes in the cellular environment (e.g., hypoxia, acidosis, and hyperglycemia) or the presence of other active factors (e.g., leptins, angiotensin II, and cytokines) (3–6). Given these influences, it follows that circulatory and tissue ET-1 levels fluctuate in the context of kidney disease, making it a critical driver of renal pathophysiology and a biomarker or predictor of kidney disease. The endothelin family consists of three structurally related peptides: ET-1, ET-2, and ET-3. These three peptides are composed of 21 amino acids, and ET-2 and ET-3 differ from ET-1 by two and six amino acids (aa), respectively (7). The three endothelins are synthesized as pre-pro-endothelin (212 aa) and then post-transcriptionally processed into inactive pro-endothelin (also known as big ET-1, with 39 aa) by endopeptidase cleavage of the signal peptide. Pro-endothelin is then cleaved by endothelin-converting enzyme into each of the 21 aa peptides: ET-1, ET-2, or ET-3 (7,8). The most extensively studied member of the family is by far ET-1, given that this peptide is the one with major effects on the cardiovascular system. This review paper is focused on ET-1 and the kidney. There are two G protein-coupled receptors for endothelin: the endothelin type A (ET A ) and endothelin type B (ET B ) receptors (3–5). Several subtypes of each of these receptors have also been suggested (2,9), but more evidence of their existence is still needed. All three isoforms of endothelin (ET-1, ET-2, and ET-3) bind to both receptors but with different affinities. The ET A receptor has equal affinity for ET-1 and ET-2 and much higher affinity for these isoforms than for ET-3 (ET A : ET-1 ¼ET-2 ET-3) (2,10). On the contrary, the ET B receptor binds ET-1, ET-2, and ET-3 with equal affinity (ET B :ET-1¼ET-2 ¼ET-3) (2,9,10). Like ET-1, the ET A and ET B receptors are distributed throughout the body, with expression of each receptor varying by tissue type (6). The expression ratio of ET A to ET B (ET A :ET B ) also varies throughout different regions of the kidney and even along segments of the nephron, explaining the diverse abilities of the endothelin system and its control of the fine-tuning ability of the kidney (11). Activation of each of the receptors often results in opposite physiological responses. Tubules in the kidney cortex and inner medulla are enriched in ET B receptors, whereas the tubules within the outer medulla express both ET A and ET B receptors (11, 12). When there is overactivation of the ET A receptor pathway, such as it happens during diabetes, hypertension, and other diseases associated with kidney disease, kidney hypertrophy, fibrosis, and inflammation occur. On the contrary, activation of the ET B receptor stimulates the production of nitric oxide and prostaglandins, the removal of ET-1 from Correspondence: C. De Miguel ([email protected]). Submitted 23 October 2024 / Revised 22 November 2024 / Accepted 21 April 2025 http://www.ajprenal.org 1931-857X/25 Copyright ©2025 The Authors. Licensed under Creative Commons Attribution CC-BY 4.0. Published by the American Physiological Society. F815 Am J Physiol Renal Physiol 328: F815–F827, 2025. First published April 24, 2025; doi:10.1152/ajprenal.00304.2024 Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. the circulation, and increases the excretion of water and sodium (11). Under normal conditions, ET-1 interacts with both receptors to regulate renal functions, such as hemodynamics, inflammation, electrolyte balance, and cell growth (2,5). However, in various disease states, there is exaggerated production of ET-1 coupled with a dysfunction of the ET B receptor that results in increased activation of the ET-1/ET A arm of the system, contributing to the progression of renal disease (Fig. 1). Although the involvement of ET-1 in the control of kidney function has been known for decades, the mechanisms that mediate its physiological and pathophysiological actions are not fully understood. The purpose of this article is to examine the role of ET-1 signaling in the kidney while also identifying areas that require further investigation, with a focus on the interplay of ET-1 with inflammation, sex differences, circadian rhythms of renal function, and the most recent clinical trials involving the ET-1 system. ET-1 AND KIDNEY INFLAMMATION Exaggerated inflammation is one of the hallmarks of kidney disease, with both the innate and adaptive arms of theimmunesystembeingimplicatedinthedevelopment and progression of different kidney diseases. The innate and adaptive arms of the immune system work in a coordinated manner to mount the inflammatory response. Among the major effector cells of the innate immune system are neutrophils, macrophages, and dendritic cells (DCs) (Fig. 2). These innate immune cells are the first to respond to an insult and are in charge of activating the adaptive arm of the inflammatory response, which is primarily made of T and B lymphocytes, mounting cell-mediated responses (T cells) or antibody-mediated responses (B cells). Growing evidence implicates ET-1 as a crucial player in the inflammatory response, with some reports even referring to it as a “profibrotic cytokine”(13,14). In clinical and experimental settings, elevated levels of ET-1 in plasma are directly associated with an overactive inflammatory response (15), leading to renal injury and subsequent end-organ damage (16,17). One of the immunomodulatory effects of ET-1 is its ability to induce the production of various proinflammatory cytokines, such as TNF-a, IL-1b, and IL-6 (18–20). Notably, these cytokines, in turn, also promote the production of ET-1, creating a positive feedback loop that drives the overactivation of the immune system (18,19). Similar interactions have been observed with transcription factors like NF-κB(18,19). In addition, ET-1’s impact on vascular permeability represents another critical immunomodulatory function within the endothelin system (14), leading to exaggerated extravasation of inflammatory cells into organs, including the kidney, Kidney homeostasis Kidney dysfunction VS. ETBETA ET-1 ET-1 ETA ETB disease Maintenance of: Hemodynamics Immune response Electrolyte balance Cell metabolism ROS Vasoconstriction Fibrosis Na + retention dysfunction overactivation Figure 1. Summary of the main functions of endothelin-1 (ET-1) in the kidney during homeostasis and during kidney dysfunction. ET A , endothelin type A; ET B , endothelin type B; Na þ , sodium; ROS, reactive oxygen species. Figure created with BioRender. ET-1 Innate immune response Adaptive immune response Neutrophils Macrophages Dendritic cells B cells T cells ETB driven ? ETB driven ETA driven ETA driven ? Adhesion Chemotaxis/transmigration Cytokine/ROS production Chemokinesis Clearance of ET-1 Vascular contractility Fibrosis/ROS production Maturation/activity Antigen presentation Survival Cytokine production Activation Proliferation Further investigation is required Figure 2. Summary of our knowledge of the actions of ET-1 on immune cells of the innate and adaptive immune response, as well as what ET-1 receptors have been reported to mediate the actions of ET-1 in each of these immune cell types. Yellow boxes highlight the cell types where information is very scarce or still unclear. Figure created with BioRender. ET-1, endothelin-1; ET A , endothelin type A; ET B ,endothelintypeB;ROS, reactive oxygen species. ENDOTHELIN SIGNALING IN THE KIDNEY F816 AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. and granting ET-1 both direct and indirect control over the inflammatory processes within the kidney. Studies using ET A blockers have consistently demonstrated their protective effects, showing reductions in proinflammatory markers and decreased infiltration of immune cells in kidney tissue, ultimately preserving renal structure and function. Saleh et al. (21) illustrated the direct effects of ET-1 by infusing ET-1 into Sprague-Dawley rats, which resulted in a prominent increase in chemoattractants and adhesion molecules (ICAM-1 and MCP-1), nephrin excretion, and renal immune cell infiltration (macrophages and T cells). These effects occurred without an increase in blood pressure, thereby highlighting ET-1’s modulation of renal inflammation outside of its known hemodynamic and blood pressure-rising effects. Specific inhibition of the ET A receptor attenuated all of these ET-1-induced effects, suggesting that all were mediated by the ET A receptor. Similar renal protective effects of ET A blockade were reported in type 1 diabetic mice, further confirming the role of the ET-1/ET A axis in promoting proinflammatory activity during kidney disease (22). Dysfunction of the ET B receptor, with concomitant excessive activation of the ET A receptor, is also associated with increased proinflammatory markers, greater immune cell infiltration into the kidneys, and more severe renal damage during salt-sensitive hypertension (23). Moreover, a study comparing ET A -specific antagonism with dual ET A /ET B antagonism in a mouse model of sickle cell nephropathy showed that blocking both ET-1 receptors was less effective than blocking ET A alone in preventing kidney inflammation and preserving renal structure and function (24). Hypertension (16,21,23,25), diabetes (26–28), and sickle cell disease (24,29,30) frequently exhibit elevated ET A receptor expression, concurrent ET B receptor downregulation, and an overactive immune response in the kidney, further implicating the ET-1/ET A receptor axis in kidney pathophysiology (31). Studies on hypertensive kidney disease (16,21,23), IgA nephropathy (32), acute kidney injury (33,34), and diabetic nephropathy (26–28) all underscore the critical role of the ET-1/ET A axis in these conditions as well as other kidney diseases (35,36). As a result, the endothelin system has emerged as a key player in immune dysregulation during kidney disease and a promising therapeutic target. Importantly, immune cells like macrophages (37–39), dendritic cells (40), and T cells (41) not only produce ET-1 but also express ET A and ET B receptors on their surface (41–44). This suggests that both the innate and adaptive immune systems are stimulated by ET-1 through autocrine and paracrine signaling pathways and are responsive to changes in circulating ET-1 levels, as seen in many disease states. Czopek et al. (45) explored this relationship in three different experimental models of hypertension: ET-1 infusion, angiotensin II infusion, and high-salt feeding. Their study used a macrophage-specificET B knockdown model in mice, demonstrating that macrophages play a significant role in the uptake and clearance of ET-1. Mice with diminished ET B expression on macrophages showed a greater pressor response to ET-1, higher circulating ET-1 levels, and a more pronounced increase in blood pressure in response to hypertensive stimuli compared with transgenic controls, although these studies did not evaluate kidney damage in this mouse model. Similar findings were obtained in vitro using human bone marrow-derived macrophages (45). In addition, in vitro studies demonstrated that pharmacological blockade of the ET A receptor dampens the activation of human immune cells, such as dendritic cells (DCs) and T cells, implying a crucial role of ET A signaling for their biological function (46–49). In one study, human monocytes cultured with a specificET A receptor blocker exhibited reduced DC maturation, lower levels of the proinflammatory cytokine IL-12, diminished ability of these DCs to stimulate T cells, and increased DC apoptosis (40). In contrast, blocking the ET B receptor in DCs resulted in increased cell survival and maturation (40). Further supporting these findings, studies conducted by Elisa et al. (41) confirmed the presence of ET-1 receptors on immune cells and described how the endothelin system modulates immune cell adaptation to various stimuli. They demonstrated that immune cells from both the innate and adaptive immune systems in humans express both ET-1 receptors, but that T cells and neutrophils exhibit greater expression of ET A receptor than ET B receptor in patients with systemic sclerosis compared with healthy individuals. These findings demonstrated that the proinflammatory actions of ET-1 in these particular immune cell subsets were mediated by engagement of the ET A receptor in this autoimmune disease. In addition, they also demonstrated that selective or dual blockade of endothelin receptors modulated cytokine release and chemotaxis, suggesting that the proinflammatory and profibrotic actions of ET-1 occur through a synergistic action on ET A and ET B receptors, at least in systemic sclerosis. Therefore, a dual receptor blockade strategy is likely to better control inflammation and fibrosis than a selective receptor blockade (41). Observations have also been made regarding increased ET-1 production by monocytes as a direct result of close interactions with activated T cells, suggesting that ET-1 release by the immune system, independent of vascular sources, may primarily occur through adaptive immune responses (50). These findings highlight the dangerous positive feedback loops within the ET-1 system and provide compelling evidence for its role in various diseases, including kidney diseases. In addition, it gives rise to the hypothesis that altered ET A -ET B receptor expression patterns present on immune cells such as T lymphocytes and monocytes under disease conditions can influence various negative pathologies. However, this was not the case for B cells. Although B lymphocytes have been shown to express endothelin receptors to a similar, or even greater, extent as other immune cells, knowledge regarding the impact of the endothelin system on B-cell function is extremely limited, with little to no substantial research available (41), especially when related to their possible role in the development and progression of kidney disease. Figure 2 summarizes our current understanding of how elevated levels of ET-1 affect the different immune cell types and which ET-1 receptor subtype has been implicated in the inflammatory effects. We also highlight the particular immune cell subsets where more investigation is needed. Taken together, all of these recent studies underscore the dynamic role of the endothelin system in regulating inflammatory responses. The ET-1-inflammatory axis contributes to the development, function, and activation of various ENDOTHELIN SIGNALING IN THE KIDNEY AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org F817 Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. immune cell types, with the specific involvement of ET A or ET B receptors depending on the cell type. However, knowledge of the endothelin system within the context of immune cells remains quite limited, particularly when related to its involvement in kidney physiology and pathophysiology. This raises a large gap in the knowledge of the interplay between ET-1 and the inflammatory response during kidney disease. Current understanding relies heavily on work done in vitro, with few studies being conducted in vivo, limiting our understanding of valuable physiological and pathophysiological factors within this system. Of the limited in vivo studies performed in human patients, reports are mostly observational, providing correlative, rather than mechanistic, evidence of ET-1’s role (28,41,51,52). In addition, the selection criteria of these studies cause the research to have a narrowed view by focusing on the fully developed disease state rather than a deeper understanding of how this interaction may drive the development and/or progression of various renal diseases. Although the endothelin system has been highlighted in various recent encouraging clinical trials as a promising potential therapeutic target, extensive work is left to be done to fully appreciate the immunomodulatory effects of ET-1 within renal pathophysiology to fully understand its potential pivotal role during kidney disease development and progression. SEX DIFFERENCES IN THE RENAL ET-1 SYSTEM Sex differences in the endothelin signaling pathway have been demonstrated in several organs, including the lung (53, 54), vasculature (54–56), and the liver (57). Many of the studies in the literature are in the setting of hypertension, given the critical role that ET-1 plays in the development and progression of this disease, as well as in the associated kidney damage. There are also important differences in the production of ET-1 between males and females during hypertension, with the presence of exaggerated circulating levels of ET-1 in men compared with women with hypertension (51). Aging is another factor that affects circulating levels of ET-1 (58–61), indeed leading to increasing plasma levels in older healthy women compared with younger women (52). Dissimilar expression of ET-1 receptors has also been found and linked to the male-female differences that exist in kidney function (Fig. 3). As mentioned earlier, the location of ET A and ET B receptors within the kidney is not homogenous. Moreover, differences in the expression levels of these receptors have also been reported in males and females, which explains, for instance, the differences observed between the sexes in renal sodium handling. ET-1-induced natriuresis is mainly mediated through activation of ET B receptors (11); however, activation of the medullary ET A receptor facilitates the ET-1-mediated natriuresis only in females (62). Radioligand-binding studies demonstrated that male rats have greater expression of ET A receptors in inner medullary collecting ducts compared with female rats (63) and that the response to ET-1 was exaggerated in males compared with females. Similar expression of ET B receptors in this region of the kidney was found between the sexes. These observations suggest that greater activation of ET A receptors in the inner medulla of the kidney in males may be responsible for the dysregulation of sodium natriuresis and the elevated sodium retention that has been reported in this sex. Our group has also demonstrated that ET-1 signaling in the kidney is a crucial driver in the sex differences that can be found in the acclimation to increased salt consumption (64). We demonstrated that during a 5-day challenge with increased sodium intake, female rats have an enhanced capacity to excrete excess sodium compared with males and acclimate to the salt challenge in only 1 day, whereas males take between 3 and 5 days to return to balance (64). This series of studies also showed that the ET-1 system is highly activated in the female kidney under baseline conditions (64). Further studies by Nasci et al. (65) demonstrated that dual inhibition of ET A and ET B receptors eliminates the sex difference in acclimation to a high-salt diet by promoting natriuresis in male rats. Although the field has made strides in starting to understand the role that the ET-1 system plays in kidney physiology in both sexes during high-salt consumption and hypertension, more research is warranted. ovx Natriuresis mediated by inner medulla ET B ET A ET A : ET B ratio ET A : ET B ratio Hypertension plasma ET-1 Inner medulla ET B no change in ET B ADPKD plasma ET-1 kidney ET-1 ET A , ET B ??? AKI kidney ET-1 ET A inhibition survival mortality Figure 3. Table summarizing reports in the literature regarding sex differences in the ET-1 system, focusing on participants in maintenance of natriuresis as well as in some diseases like hypertension, autosomal-dominant polycystic kidney disease (ADPKD), and acute kidney injury (AKI). ET-1, endothelin-1; ET A , endothelin type A; ET B , endothelin type B; Ovx, ovariectomized. Figure created with BioRender. ENDOTHELIN SIGNALING IN THE KIDNEY F818 AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. Growing evidence supports the involvement of ET-1 in the development of kidney disease. Human studies using ET A receptor antagonists (described in depth in the following sections of this article) as well as studies using transgenic animal models demonstrate a direct connection of an activated ET-1 system and the kidney injury that is associated with ischemia-reperfusion (33), acute kidney injury (31,66), or sepsis (26), for instance. Similarly, overactivation of the ET-1 system is also associated with the development of kidney damage in hypertension (5,23,25), diabetes (16,27,28), polycystic kidney disease (35), IgA nephropathy (32), or sickle cell nephropathy (24,30), to name a few pathologies. Notably, the susceptibility to kidney injury is different in males and females. However, few studies in the literature, especially those of a preclinical nature, use females or report the data separated by sex. Thus, there is a big gap in knowledge of whether the same pathophysiological processes are mediated by ET-1 in the male and female kidney. We will next highlight several studies that shed light on the differential role that ET-1 may play in the progression of kidney disease in males and females. Men with autosomaldominant polycystic kidney disease (ADPKD) have a faster progression of kidney disease than females (36,67,68). Plasma levels of ET-1 in patients with ADPKD are higher in men than women (69,70), and ET-1 was found in the fluid within cysts in human kidneys (69), suggesting a direct involvement of ET-1 in the development of this kidney disease. Similar to reports in humans, the Han:SPRD rat model of polycystic kidney disease showed greater levels of ET-1 in the kidney in both males and females compared with noncystic rats of the same sex, but females had lower ET-1 levels than their male counterparts (35). Ovariectomy of these cystic females resulted in levels of kidney ET-1 as high as those observed in males, suggesting that the attenuated ET-1 levels in cystic females are governed by estrogen (35). More information is needed regarding how activation of each of the endothelin receptors may be different between the sexes in this disease setting, as this would possibly open new therapeutic avenues for these patients. Similarly, reports of the interplay between the ET-1 system, sex differences, and acute kidney injury are scarce, possibly due to the reported resistance that females show to the development of the same degree of ischemia-reperfusion injury as males when their kidneys are clamped experimentally (71– 73). In studies where male rats underwent orchidectomy and then ischemia-reperfusion, M€ uller et al. (34)reportedthatthe extent of kidney damage was then comparable with that present in female rats undergoing the same ischemia-reperfusion protocol. This group reported that expression of ET-1 in the kidney was prominently greater in males than females after ischemia-reperfusion, and further, that blockade of the ET A receptor before ischemia-reperfusion improved survival in males but, on the contrary, increased mortality in females (34). These results suggest that ET-1 might be a critical mediator in maintaining the sex differences in kidney damage following renal ischemia. Although this study did not further investigate the kidney expression of ET A and ET B receptors, others have reported an increased affinity of ET-1 for its receptors after renal ischemia-reperfusion (74). Given the reported differences in endothelin receptors in males and females in other diseases, if there are differences between the sexes in the expression and activity of these receptors within the kidney in this setting, as well as possible regional differences, need to be investigated. As mentioned in ET-1 AND KIDNEY INFLAMMATION, immune cells and inflammatory mediators are important in the progression of kidney disease. Important sex differences in the kidney inflammation that is present during disease have been demonstrated in hypertension (42–44) and lupus erythematosus (75), among other diseases. As we also mentioned, immune cells express ET A and ET B receptors and produce ET-1 upon activation. However, if the sex differences in the progression of kidney disease are due to differential activation of the immune ET-1 system is unclear and is an active area of investigation in the field. Undoubtedly, more research in this area is needed and forthcoming. The role that sex hormones play in modulating the ET-1 system also needs further investigation. Particularly, the interplay between these steroids and ET-1 in the control of kidney function in health and disease is understudied. Estrogen has been proven to suppress the renal production of ET-1 and the ischemia-reperfusion injury that occurs during acute kidney injury (76). Hormone replacement therapy during menopause also decreases plasma levels of ET-1 in humans (77) and after ovariectomy in rats (78). Interestingly, both estrogen and ET-1 receptor antagonism result in vascular protection in experimental animals, suggesting an interplay betweenestrogenandtheET-1system(79–81). Progesterone has also been implicated in the regulation of the expression of ET A receptor, resulting in increased kidney expression of this receptor in ovariectomized mice (82). These studies demonstrated that there is a conserved progesterone-responsive site upstream of the Ednra promoter (gene that encodes for ET A receptor) and that a GATA2 site also needs to be engaged to stimulate the progesterone-induced expression of the ET A receptor (82). Treatment with progesterone also ameliorates ET-1-mediated vasoconstriction but to a lesser extent than estrogen does (80). Aligning with these studies, Gohar et al. (83) also reported that surgical removal of the ovaries in rats resultsinenhancedexpressionofbothET A and ET B receptors in the kidney inner medulla, whereas both receptors are remarkably decreased in the cortex. Furthermore, cotreatment with estrogen and progesterone prevents the upregulation of ET A and ET B in the inner medulla. However, treatment with estrogen alone did not revert the downregulation of these receptors in the kidney cortex seen with ovariectomy (83). These findings demonstrate that ovarian hormones modulate the endothelin system. Given that females are more prone to cardiovascular and kidney disease after menopause, the interaction between ovarian hormones and the ET-1 system may play an important role in maintaining female kidney health. On the contrary, less is known about the interplay between androgens and the endothelin system. Studies in female-tomale sex transition suggest that testosterone treatment augments the production of ET-1, since these patients show elevations of plasma ET-1 levels (70). In turn, ET-1 is also known to promote the production of testosterone in a cancer cell line (84), and other studies have also reported that androgens can upregulate ET-1 production in the kidney (85), promote renal vasoconstriction, and induce renal fibrosis (86). Orchiectomy attenuated the increased renal levels of ET-1, suggesting an ENDOTHELIN SIGNALING IN THE KIDNEY AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org F819 Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. interaction of male sex hormones with the ET-1 system (85). However, further studies are needed to clarify this relationship in the setting of kidney disease. Taken together, the current evidence in the literature suggests that sex hormones may be important in the modulation of the ET-1 system in the kidney, but we need more investigation into the molecular mechanisms that may be at play in each sex. Similarly, future studies should aim to expand our knowledge of the sex hormone effects on the ET-1 system in the vasculature versus the tubular system within the kidney. ET-1 AND CIRCADIAN CONTROL OF KIDNEY FUNCTION Circadian clocks in the kidney are recognized as important controllers of kidney physiology, and active research efforts in this field are undergoing to better define the mechanisms by which derailment of circadian rhythms leads to renal dysfunction. A succession of positive and negative feedback loops controlled by four critical circadian proteins (CLOCK, BMAL1, PER, and CRY) makes the molecular circadian clock, which is present in almost every cell type in the body. The molecular clock in peripheral tissues, like the kidney, is regulated by cues such as food and water intake, core body temperature, physical activity, and other environmental factors that still need to be determined (87–91). It is also important to note that the genes encoding for these key circadian transcription factors are highly conserved between mice and humans (92). In turn, and in a tissue-specific manner, CLOCK, BMAL1, PER, and CRY regulate the expression of thousands of genes, as reviewed by Partch et al. (88), Takahashi (89), and Douma et al. (93). One of the factors that is important for kidney function and is under circadian regulation is ET-1 (94). Expression of Edn1 as well as the genes encoding for the ET A and ET B receptors (Ednra and Ednrb, respectively) follows a circadian rhythm, with enhanced kidney expression of Edn1 and Ednra during the active period (daytime for humans and nighttime for rodents) and downregulation during the inactive period. Interestingly, gene expression of the ET B receptor follows an opposite pattern, with increased expression during the inactive phase and decreased expression during the active period (91,93,94). Seminal in vitro studies by Gumz et al. (95) demonstrated that expression of Per1 and Edn1 in inner medullary collecting ducts increases with treatment with aldosterone. Further studies by the same group determined that the transcriptional modulation of Edn1 expression by aldosterone in rat kidney and a variety of murine cell lines is under the control of the mineralocorticoid receptor (96,97). Other studies also demonstrated that the time-dependent regulation of ET-1 in the kidney is controlled by PER1 (98), as knockdown of this gene in cortical collecting ducts led to a remarkable upregulation of the expression of ET-1 in these cells (99). Global knockout of PER1 has yielded contradictory results in mice, mainly depending on the genetic background of these animals. Knockout of PER1 in 129/Sv mice was reported to be protective against the development of salt-sensitive hypertension (99,100), whereas the same genetic manipulation in the saltresistant C57Bl6 background resulted in hypertension in response to high salt feeding, although this behavior was only observed in males, not females (101). In contrast, specific knockout of PER1 in the distal nephron and collecting duct led to elevated sodium retention and increases in blood pressure after high salt plus desoxycorticosterone pivalate treatment. This is an experimental hypertension model that mimics the low renin and high aldosterone levels that are seen in humans with salt-sensitive hypertension (102). Importantly, global and distal nephron-specificknockoutof PER1 resulted in elevated circulating and kidney ET-1 levels (99,102), suggesting that the effects seen in blood pressure maybeinpartmediatedbyactionsofET-1.Additionalstudies showed that global knockout of PER1 in the Dahl salt-sensitive background exacerbates hypertension and renal injury in response to a high-salt diet (103). These contrasting results in different strains and species demonstrate the importance of PER1 in the control of renal function. However, they also relay the complicated interaction between circadian rhythms and renal physiology and highlight the need for more investigation into the molecular mechanisms (including those mediated by ET-1) that may be interacting in these settings. Not only is ET-1 controlled by the circadian clock, but other studies also demonstrate that ET-1 may be involved in the circadian control of renal function. Elegant studies performed by Johnston et al. (104) discovered that the ET B receptor in rats facilitates the diurnal sodium excretion capacity in response to an acute load and that the magnitude of these effects was time-dependent and sex-dependent. They demonstrated that the impairment of the natriuretic response caused by ET B dysfunction in rats was much more severe when the acute load of sodium was given at the beginning of the inactive period than when given before the active period. Because the diurnal response was attenuated in females, they also evidenced that the diurnal control of natriuresis by ET-1 exhibits sex differences (104). Other evidence in the literature strongly suggests that the ET B receptor also plays an important role in regulating the circadian pattern of BMAL1 expression in the kidney. Using ET B -deficient rats, Speed et al. (105) demonstrated that high salt feeding induces a phase shift in gene expression of Bmal1 in the kidney and that this shift is ET-1/ ET B receptor axis dependent. They showed that these effects were specific to the inner medulla, as the dysfunction of the ET B receptor did not induce differences in Bmal1 expression in the cortex under the same conditions (105). These findings suggest that the regulation of clock genes by renal ET-1, particularly Bmal1, may be responsible for the diurnal patterns that exist in the renal handling of sodium. Further studies using collecting duct-specificBmal1 knockout mice demonstrated similar renal levels of ET-1 between the genotypes, suggesting that collecting duct BMAL1 does not affect the ET1 production by the inner medulla (106). More investigation on the relationship between the different clock proteins and ET-1 is needed, as well as further insight into where in the kidney and which specific kidney functions are modulated by the close relationship between diurnal rhythms and the endothelin system. Future studies in this field will be instrumental in the prevention of kidney and cardiovascular disease in populations that regularly face circadian misalignments, such as those employees with constant schedule shifts or those who regularly change time zones. ENDOTHELIN SIGNALING IN THE KIDNEY F820 AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. CLINICAL ADVANCES TARGETING THE ET-1 SYSTEM The discovery of endothelin and its role in regulating blood pressure sparked significant interest, as hypertension was already recognized as a major public health issue in the 1980s (107). Bosentan, the first Food and Drug Administration (FDA)-approved endothelin receptor antagonist (ERA), was developed to treat pulmonary arterial hypertension. Although it showed cardiovascular and renal benefits, some patients experienced hepatotoxicity, prompting further refinement of this drug class (108). As indicated throughout this review, elevated rates of ET-1 interacting with its ET A receptor can lead to vasoconstriction, inflammation, apoptosis, and fibrosis, all of which drive proteinuria and, eventually, permanent renal impairment. A Study of Cardiovascular Events iN Diabetes (ASCEND) trial represented the first attempt at repurposing an ERA (avosentan) for treating kidney disease specifically (109). In conjunction with the current standard of care [i.e., Angiotensin Converting Enzyme (ACE) inhibitors or angiotensin receptor blockers], the small-scale study Reducing Albuminuria in Diabetes and Also Renal (RADAR) demonstrated the ability of a selective ET A antagonist to not only have antihypertensive effects but also reduce proteinuria in type 2 diabetic patients with diabetic nephropathy (110). Unfortunately, this success was short-lived as a follow-up study investigating long-term effects was halted prematurely due to cardiovascular side effects relating to fluid overload (110). The Study of Diabetic Nephropathy with At Rasentan (SONAR) trial, testing atrasentan in diabetic kidney disease, aimed to mitigate these risks by introducing an enrichment phase to eliminate high-risk patients (111,112). Thus, only patients who did not demonstrate fluid retention and had reductions in proteinuria during that 6 wk enrichment period were admitted in the double-blind long-term study (112,113). Despite improved renal outcomes, cardiovascular risks remained elevated, although they were less prominent in the treatment group (111,112). Fortunately, recent advances in the development of sodium-glucose cotransporter-2 (SGLT2) inhibitors give revived hope to the implementation of ET A antagonists in kidney disease. SGLT2 inhibitors have been shown to possess diuretic effects and increase hemoglobin levels, combating fluid overload driven by the ET A -selective antagonists (114). The Zibotentan in combination with dapagliflozin compared with dapagliflozin in patients with chronic kidney disease (ZENITH-CKD) trial was specifically designed to analyze this new combinatory approach in type 2 diabetic patients with CKD (115). Completed as of 2023, ZENITH-CKD found zibotentan (ET A -selective antagonist) plus dapagliflozin (SGLT2 inhibitor) to be more effective in reducing albuminuria compared with dapagliflozin plus placebo while minimizing fluid retention in these patients (114). A crossover study, named Zwolle Outpatient Diabetes project Integrating Available Care (ZODIAC) (https://www.clinicaltrials.gov; NCT05570305), is currently ongoing to further investigate the use of zibotentan plus dapagliflozin and validate the synergistic effect of blocking these two pathways that was noted in the ZENITH-CKD trial. As evident by the wide capabilities of the endothelin system, ERAs are not limited to treating diabetic nephropathy but have shown promise across a range of kidney diseases. Although many kidney pathologies present with elevated proteinuria, not all are complicated by cardiovascular comorbidities, indicating a high likelihood of success in these patient populations. In focal segmental glomerulosclerosis (FSGS), the development of a treatment strategy for chronic kidney disease–mineral and bone disorder by a mUltilateral mechanism of ETelcalcetide hydrochloride (DUET) trial tested the novel dual antagonist sparsentan, which targets both the ET A receptor and the angiotensin II type 1 receptor (116). The DUET trial proved sparsentan to be much more effective than irbesartan in reducing proteinuria and achieving higher levels of remission (116). Hoping to become the first drug approved for FSGS, the success of this dual antagonist is being further evaluated in the DUPLEX trial (Dual Endothelin Receptor and Angiotensin Receptor Blocker, on Renal Outcomes in Patients with Primary FSGS), which has already demonstrated promising long-term results (116–118), including reduction in proteinuria at 108 wk of treatment despite no improvements in estimated glomerular filtration rate (eGFR). A similar approach was taken in IgA nephropathy with the PROTECT trial (Study of the Effect and Safety of Sparsentan in the Treatment of Patients With IgA Nephropathy), where sparsentan also outperformed irbesartan, with continued improvements in proteinuria reduction over time (119,120). Enhanced eGFR preservation over time further supported sparsentan’sefficacy, leading to its FDA-accelerated approval for treatment of IgA nephropathy in 2023 (119,121). In addition, atrasentan (an ET A -selective antagonist) is also under investigation in the ALIGN trial for IgA nephropathy (Atrasentan in patients with IgAN; https://www.clinicaltrials.gov; NCT04573478), where it has already demonstrated a 36.1% reduction in proteinuria (P<0.0001), offering another promising treatment option for this kidney disease (122,123). More recently, the PRECISION study (dual endothelin antagonist aprocitentan for resistant hypertension) (124) assessed the effectiveness of the dual ET A and ET B receptor antagonist aprocitentan against resistant hypertension. This phase 3 trial used a blinded, randomized, and parallel-group study design in hospitals/research centers in four continents (Europe, North America, Asia, and Australia). They demonstrated that 4 wk of treatment with aprocitentan resulted in a decrease of 15 mmHg in systolic blood pressure compared with patients receiving a placebo. These findings were confirmed with 24 h ambulatory blood pressure measurement. Interestingly, the amelioration in blood pressure was the greatest in older patients. Although the difference in blood pressure between the treatment and placebo groups decreased once the treatment was withdrawn, the investigators still reported a difference of 6 mmHg between the groups 4 wk after ceasing treatment. Moreover, they also described a protective effect on kidney function, as those patients receiving aprocitentan also underwent a decrease in the urine albumin-creatinine ratio. As in other endothelin antagonism trials, PRECISION investigators also reported mild-to-moderate fluid retention in 9%– 18% of patients receiving aprocitentan, with patients with chronic kidney disease stage 3–4 being the most affected. Regardless, the findings in this study are very promising, especially for those patients who suffer from resistant hypertension despite being on three antihypertensive medications. In ENDOTHELIN SIGNALING IN THE KIDNEY AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org F821 Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. Table 1. Summary of clinical trials to date targeting the ET-1 system in kidney disease Trial Drug, Mechanism Disease Inclusion Criteria Primary Outcome Measure (s) Status Findings ASCEND (2005) (109) Avosentan, ET A /ET B antagonist Diabetic nephropathy Type 2 diabetes, UACR 35 mg/mmol, serum creatinine ¼1.2–3.0 mg/dL Time to doubling of serum creatinine and ESRD Terminated - Reduced proteinuria - Increased CVD events RADAR (2011) (110) Atrasentan, ET A antagonist Diabetic nephropathy Type 2 diabetes, eGFR 30–75 mL/min, UACR ¼34–396 mg/mmol Change in UACR Completed - Reduced proteinuria - Slowed eGFR decline - Increased CVD events SONAR (2013) (113–115) Atrasentan, ET A antagonist Diabetic nephropathy Type 2 diabetes, eGFR 25–75 mL/min, UACR ¼34–565 mg/mmol Change in serum creatinine or ESRD Terminated - Improved renal health - Increased CVD events ZENITH-CKD (2021) (115) Zibotentan, ET A antagonist Diabetic nephropathy Type 2 diabetes, CKD (eGFR 20 mL/min and UACR ¼150– 5,000 mg/g) Change in UACR from baseline Completed - Significant reduction in UACR ZODIAC (2022) (NCT05570305) Zibotentan, ET A antagonist Diabetic nephropathy Type 2 diabetes, CKD (eGFR 30 mL/min and UACR ¼100– 3,500 mg/g) Change in albuminuria from baseline Ongoing - Results not yet available DUET (2014) (116) Sparsentan, dual ET A and angiotensin II type 1 receptor blocker FSGS FSGS diagnosis, Up/C 1.0 g/g, eGFR >30 mL/min Percent change in Up/C Completed - Reduced proteinuria - Slowed eGFR decline DUPLEX (2018) (117,118) (NCT03493685) Sparsentan, dual ET A and angiotensin II type 1 receptor blocker FSGS FSGS diagnosis, Up/C 1.5 g/g, eGFR 30 mL/min Slope of eGFR, percentage of participants achieving partial remission Ongoing (Preliminary) -Reduced proteinuria -Slowed eGFR decline PROTECT (2018) (121–123) (NCT03762850) Sparsentan, dual ET A and angiotensin II type 1 receptor blocker IgA nephropathy IgA nephropathy diagnosis, proteinuria 1 g/day, eGFR 30 mL/ min Change in Up/C from baseline Ongoing, FDA approved (2023) - Reduced proteinuria - Stabilized eGFR ALIGN (2020) (122,123) (NCT06072326) Atrasentan, ET A antagonist IgA nephropathy IgA nephropathy diagnosis, proteinuria 1 g/day, eGFR 30 mL/ min Change in Up/C from baseline Ongoing (Preliminary) -Reduced proteinuria PRECISION (2022) (124) Aprocitentan, dual ET A and ET B antagonist Resistant hypertension SBP 140 mmHg despite taking three hypertensive medications Change in unattended office SBP from baseline to week 4 and from withdrawal baseline to week 40; decrease in UACR Completed - Reduced blood pressure - Improved renal health CKD: chronic kidney disease; eGFR: estimated glomerular filtration rate; ESRD: end-stage renal disease; ET A , endothelin type A; ET B , endothelin type B; FSGS: focal segmental glomerulosclerosis; NCT: National Clinical Trial number; SBP: systolic blood pressure; UACR: urine albumin/creatinine ratio; Up/C: urine protein/creatinine ratio. Boldface indicates preliminary data from these studies. ENDOTHELIN SIGNALING IN THE KIDNEY F822 AJP-Renal Physiol doi:10.1152/ajprenal.00304.2024 www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal at Univ De Sevilla (193.147.173.203) on October 15, 2025. fact, in March 2024, aprocitentan became the first oral antihypertensive with a novel mechanism of action to be approved by the US FDA to manage systemic hypertension in over 40 years. Although the use of ERAs is still relatively limited, new earlystage clinical trials are exploring renoprotective benefits in various conditions [e.g., hepatorenal syndrome (125), type 1 diabetes (ASPIRE, https://www.clinicaltrials.gov; NCT06072326), and ischemic kidney injury (NCT04450095)], which is congruent withtherateatwhichbasicsciencestudiescontinuetoidentify the endothelin system as a potential therapeutic target in various pathologies [e.g., sickle cell disease (24,29), obesity (126), and acute kidney injury (66,127)]. All these findings are highly promising and may expand the therapeutic scope of ERAs to many other kidney diseases. However, further research is crucial to fully understand how the endothelin system influences electrolyte balance so that strategies can be developed to minimize adverse effects like fluid retention, which remain a concern in this drug class. Importantly, emphasis on studying possible sex dimorphisms regarding the effects of ERAs is also needed in the field. Table 1 summarizes all the clinical trials that we covered in this section. Regulation of ET-1 by miRNAs As mentioned, ET-1 is produced by nearly every cell type in the human body and can act on many different tissues, making it an attractive therapeutic target for a wide range of pathologies. However, this broad activity of ET-1 also poses challenges for precise modulation. Micro RNAs (miRNAs), endogenous single-stranded small molecular RNA, have been proposed as a potential solution as they can significantly modulate ET-1 transcription and do so in a tissue-specific manner (7,128). The inhibition process of protein translation by miRNAs occurs through imperfect base pairing between miRNAs and mRNAs, a process that frequently targets the 30untranslated region, which constitutes 50% of ET-1 mRNA and is highly conserved among mammals (129, 130). A study by Jacobs et al. (128) predicted that the human Edn1 gene has multiple possible target sequences for many miRNAs, thus highlighting the importance of these molecules in modulating the genetic expression of ET-1. Although numerous in vitro and in silico studies have demonstrated successful inhibition of ET-1 expression by this approach (129,131–135), it has yet to be validated in full physiological systems. These studies focus on the use of miRNAs such as miR-199, R-125a/b-5p, miR-1, and miR-133a in the context of C2C12 cells, rhabdomyosarcoma cells, and various types of endothelial cells (131–135). The absence of kidney-specific studies highlights a crucial need for further investigation into this ET-1-concentrated organ system. However, given that the addition of miRNAs results in decreased ET-1 expression, promising avenues for understanding the pathophysiology of ET-1 dysregulation are now available. As miRNA-based therapies gain attention, many believe that these tools may not only enhance our understanding of ET1-related diseases but also help alleviate symptoms caused by endothelin system dysregulation, thus giving credit to the hypothesis that miRNAs could be the solution to mitigating many of the adverse effects seen in clinical trials using global endothelin receptor antagonists. If ET-1 is able to regulate the expression of certain miRNAs and promote kidney damage in disease settings, is also understudied, needs to be clarified and further investigated. CONCLUSIONS/PERSPECTIVES Despite the known role of ET-1 in the kidney, there still remain important gaps of knowledge in this field. In this review, we have highlighted exciting areas of active research that will undoubtedly expand our understanding of the importance of endothelin in the kidney. For instance, we know very little about the endothelin system’s modulatory actions on immune cell responses both systemically and within the kidney during pathological states. Another area that needs further investigation is whether the endothelin-1 system is similarly involved in the pathological processes that mediate acute and chronic kidney disease in both sexes. This includes further research into the interplay between male and female sex hormones and the endothelin system. In addition, the two-way relationship between diurnal rhythms and ET-1 needs to be better understood, as it can identify new therapeutic approaches for those populations that undergo frequent schedule changes and higher risk of cardiorenal disease due to their occupations. A deeper understanding of ET-1 signaling in the kidney, both during health and disease, will open avenues for more precise and successful therapeutics to prevent or attenuate the development or progression of kidney disease. As we highlight in this review, there are many open opportunities for future and exciting research in the field of ET-1 and kidney disease. GRANTS This work was supported in part by Deep South KUH PRIME (Grant Nos. U2C DK133422 and TL1 DK139566) and UAB AMC21 scholarship (to A.J.B.); University of Seville pre-doctoral fellowship for research and teaching personnel VI-PPITUS (to M.D.C.G.-L.); Diabetes Research Connection Pilot funding and the Louisiana Center for Advancing Underrepresented Scientists Careers in Health, Nutrition, Obesity, and Disparities Research (LAUNCHED) Pilot and Feasibility Grant U24 DK132740 (to C.D.M.). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS A.J.B., M.D.C.G.-L., and C.D.M. prepared figures; A.J.B. and C.D.M. drafted manuscript; A.J.B., M.D.C.G.-L., and C.D.M. edited and revised manuscript; A.J.B., M.D.C.G.-L., and C.D.M. approved final version of manuscript. REFERENCES 1. 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. doi:10.1038/332411a0. 2. 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