Interplay of the Circadian Clock and Endothelin System

Abstract

The peptide hormone endothelin-1 and its receptors are linked to several disease states. Pharmacological inhibition of this pathway has proven beneficial in pulmonary hypertension, yet its potential in other disease states remains to be realized. This review considers an often understudied aspect of endothelin biology, circadian rhythm regulation and how understanding the intersection between endothelin signaling and the circadian clock may be leveraged to realize the potential of endothelin-based therapeutics.

Introduction

Almost every living organism has evolved internal circadian clocks that regulate physiological processes to adapt to the various phases of a 24-h cycle. These internal circadian clocks are located in every cell of the body. In humans and other higher eukaryotes, the suprachiasmatic nucleus (SCN) in the brain, which is considered to be the central clock, uses light cues to determine the time of day. Other cell types throughout the body are considered peripheral clocks. Synchronization of the central and peripheral clocks is regulated by hormonal and neuronal signaling in response to light, food, and exercise cues. At a molecular level, the hormonal and neuronal signaling functions to regulate the expression of the core circadian clock transcription factors. In mammals, these transcription factors are BMAL1, CLOCK, CRY, and PER. In turn, these four transcription factors work in positive and negative feedback loops to regulate the expression of thousands of genes in a tissue-specific manner (FIGURE 1). BMAL1 and CLOCK form heterodimers and turn on transcription of genes, including the genes encoding PER and CRY. PER and CRY also form heterodimers but work in the negative arm of the feedback loop by inhibiting the actions of BMAL1/CLOCK. For more in-depth review on the molecular clock, see Partch et al. (51).

FIGURE 1.

Molecular mechanism of the circadian clock

At a molecular level, the circadian clock is controlled by the action of four core transcription factors. Briefly, BMAL1 and CLOCK form a heterodimer and bind to E-Box response elements within the promoters of target genes. BMAL1 and CLOCK promote transcription of many tissue-specific genes, like Edn1, in addition to the genes encoding clock factors PER and CRY. In a negative feedback loop, PER and CRY inhibit the actions of BMAL1 and CLOCK. Endothelin-1 (ET-1), the product of the Edn1 gene, acts on many different tissues and is involved in various physiological responses throughout the body. For a more in-depth review of the molecular circadian clock, see Partch et al. (51).

One gene that is regulated by the molecular circadian clock is the endothelin-1 (ET-1) gene, Edn1. Transcription of the Edn1 gene results in an mRNA that is translated into a 212-amino acid, preproET-1, which is cleaved by proteases located in the endoplasmic reticulum (4, 19, 71). The resulting 38-amino acid, big ET-1, is further processed by endothelin-converting enzymes into the active 21-amino acid ET-1 peptide hormone (60). ET-1 has an array of tissue-specific physiological effects through interactions with the endothelin A (ET_A) and B (ET_B) receptors. The endothelin receptors are expressed in a number of cells and tissues, including neurons, fibroblasts, endothelial cells, vascular smooth muscle cells, and various cell types within the kidney.

ET_A and ET_B have been shown to function in both opposing and synergistic actions. In the vasculature, ET-1 acts as the most potent vasoconstrictor produced in the body (71). Upon activation of ET_A receptors located within the vasculature of smooth muscle cells, the smooth muscle contracts, resulting in a vasoconstriction response and subsequent increase in blood pressure (BP) (24). Reports have suggested that ET_B receptors, which are located in smooth muscle cells, may also play a role in vasoconstriction, but the mechanism for this response is currently unknown. Running counter to these actions, ET_B receptors, located within the vasculature of endothelial cells, promote the release of nitric oxide (NO) and other vasodilators. The highest concentration of ET-1 is found in the collecting duct of the kidney and functions in the regulation of sodium handling (1, 7, 40, 42). ET_B receptors are highly expressed in this region of the kidney (37, 38), and upon activation, they promote sodium excretion primarily through inhibition of the epithelial Na channel (ENaC) via a NO-mediated pathway (62). ET_A receptors are also expressed in the collecting duct, but to a much lower extent than ET_B receptors (44, 69, 72). A collecting duct-specific KO of both ET_B and ET_A receptors in mice resulted in greater hypertension and Na retention than mice with only ET_B KO, suggesting ET_A receptors are also involved in the collecting duct natriuretic regulation (22).

Many physiological processes related to the regulation of ET-1 production, i.e., sodium excretion and urine volume, exhibit 24-h circadian rhythms (9). In this review, we examine clinical and preclinical evidence of ET-1 circadian rhythms, potential molecular mechanisms regulating circadian rhythms of ET-1, and the clinical implications of ET-1 circadian rhythms. For a more comprehensive review on endothelin-signaling regulation, others have published excellent review articles on the subject (31, 73).

Clinical Evidence for ET Rhythms

Most physiological functions exhibit circadian rhythms in humans. Loss of these rhythms leads to detrimental effects. For example, BP exhibits a circadian rhythm with a peak in BP during our active period (daytime) and >10% dip in BP during our inactive period (nighttime). Individuals with blunted circadian rhythms of BP, such as those with nondipping hypertension, have a greater risk for the development of chronic kidney disease, stroke, and cardiovascular disease (11). ET-1 rhythms may have a role in the maintenance of physiological rhythms. In humans, circulating ET-1 exhibits a circadian rhythm with an increase in the morning, and then a peak in the afternoon (68). Herold et al. (28) showed evidence for an 8-h cycle of ET-1 plasma levels in a group of seven individuals, with peak concentrations occurring around 6 AM, 2 PM, and 10 PM. Moreover, the ET-1 peaks appeared to align with other physiological parameters, including serum sodium levels and BP. Hwang et al. (32) showed that urinary ET-1 (in the form of ET-1 like immunoreactive peptide) excretion correlated closely with urinary sodium excretion in both normotensive and hypertensive subjects. The group further commented that urinary sodium excretion closely matched the circadian pattern of BP measured in the subjects and, thus, provided some of the first evidence for a circadian pattern of ET-1 excretion. Elherik et al. (18) measured plasma NO and ET-1 levels in adult males and found peak ET-1 levels at hours 20 and 8. Additionally, they measured an acetylcholine (ACh) response via laser-Doppler imaging of the forearm skin, and levels correlated significantly with both ET-1 and NO levels. These studies not only show ET-1 functioning in a circadian manner, but also link ET-1 actions to important physiological parameters that also exhibit circadian patterns, including BP and sodium excretion.

The circadian variation of ET-1, specifically the morning increase, has been demonstrated in various studies and further supports a connection between ET-1 and the increased incidence of cardiovascular events around this time. Li et al. (46) demonstrated that plasma ET-1 levels were significantly increased in the morning compared with the afternoon in patients with stable angina, whereas the ischemic threshold in these same patients was lower in the morning. The researchers concluded that the circadian variation of ET-1 may be playing a role in the decreased ischemic threshold in these patients with coronary heart disease. In a recent study, 21 healthy middle-aged adult participants were placed in constant dim light over a 5-day period, and the participants were exposed to cycles of 2 h and 40 min sleep time followed by 2 h and 40 min of wake time (68). Various physiological responses, including plasma ET-1 concentration, were monitored. The forced desynchrony protocol removed exogenous circadian light cues, which allowed the researchers to examine whether vascular endothelial function (VEF) was affected by endogenous circadian regulation and other relevant biomarkers, including ET-1. By measuring rhythms of flow-mediated dilation (FMD) and low-flow-mediated constriction, the group concluded that VEF was regulated by endogenous circadian function. Similar to the FMD rhythms, plasma ET-1 maintained a circadian rhythm, increasing in the morning, during which time, there is an increased risk for adverse cardiovascular events.

Differences in BP phenotypes among different ethnicities have been linked to ET-1. A longitudinal study consisting of 351 participants looked at the effect of ethnic discrimination and the ET-1/Lys198Asn T-allele between European-Americans and African-Americans on BP and dipping status (23). It was observed that African-Americans who carried the Lys198Asn T-allele and scored higher on the everyday discrimination scale were at an increased risk for developing a nondipping BP phenotype, as well as increased diastolic BP. Cooper et al. previously showed that plasma ET-1 levels were increased in black adults who experienced higher levels of ethnic discrimination when compared with white adults, at a baseline measurement and regardless of socioeconomic status (8).

It is important to note that several studies in the literature did not find evidence for circadian variation of plasma ET-1 levels in healthy subjects. A study designed to compare absolute, as well as circadian ET-1 levels between healthy controls and systemic scleroderma patients, did not find evidence for daily ET-1 fluctuations (47). ET-1 was significantly higher in scleroderma patients compared with controls. Samples in this study were collected every 6 h for a 24-h period from patients and healthy controls of both sexes, ranging in age from 22 to 62. A later study demonstrated higher plasma ET-1 levels in stroke patients compared with healthy controls, but circadian variation in plasma ET-1 was not observed in either group (20). Samples were collected every 2 to 4 h over a 24-h period. Study subjects in the stroke group and health controls ranged in age from 45 to 83 and included near-equal numbers of men and women. A 1996 study from Kanai et al. (36) showed that there was minimal variation in plasma and urinary ET-1 levels in nine male and four female volunteers. Urine and plasma samples were collected every 6 h from 8 AM to 2 AM The lack of evidence for circadian variation in plasma ET-1 levels in these studies could be due to differences in sampling or processing compared with the other studies discussed above. The studies referenced here all used different assays to measure ET-1 and reported varying levels of cross reactivity with the ET-2 and ET-3 isoforms. A lack of specificity to ET-1 could be a contributing factor for why these studies did not detect rhythms in ET-1. Heterogeneity in the subject populations may also contribute to these effects.

Evidence From Preclinical Literature

Like humans, laboratory animals exhibit circadian rhythms of physiological functions. BP, urinary electrolyte excretion, heart rate, and body temperature are all physiological outputs known to exhibit ∼24-h rhythms in animal models ranging from mice to primates (52, 56). Many of these rhythmic functions have also been shown to be regulated by the actions of the endothelin system. In this section, we summarize preclinical studies, which have revealed a relationship between the endothelin system, the circadian clock, and physiological outputs.

Various studies have demonstrated circadian rhythms in Edn1 mRNA levels. Particularly compelling are data from unbiased approaches, including RNA seq and microarray studies. One such data collection, CircaDB, a database of circadian clock-controlled gene expression rhythms, indicated that the Edn1 gene exhibited a circadian rhythm of expression in the male C57BL/6J mouse lung, kidney, brown adipose, cerebellum, and brain stem (FIGURE 2A) (54). In all of the tissues, peak Edn1 expression was observed during the mouse active period. Additionally, endothelin receptor mRNA (Ednra and Ednrb) exhibited tissue-specific circadian rhythms of expression (FIGURE 2, B AND C). Interestingly, in the kidney, the rhythm of Ednra mRNA is similar to that of Edn1; however, Ednrb expression peaks during the inactive period. Unfortunately, no such database like CircaDB currently exists for circadian gene expression in female mice. However, we have shown in the female kidney that Edn1 mRNA levels are higher at midnight, the midpoint of the mouse active period, compared with noon in the renal cortex and medulla (15), which correlates with available data from males. In situ hybridization revealed Edn1 expression rhythms in the SCN of male Wistar rats (26). Additionally, the same study used Northern blots to show variation of Edn1 expression over a 24-h period in whole brain, heart, and lung tissue. Similar to the CircaDB data from mice, Edn1 mRNA expression was observed to peak during the rat active period. Rhythms of Edn1 expression have also been observed in heart tissue of male C57BL/6J mice (75). In the same study, the administration of a Western diet for 4 wk to in ApoE−/− mice (a mouse model of atherosclerosis) caused the Edn1 mRNA expression rhythm to be flipped, with peak expression during the inactive period. Rhythmicity of Edn1 expression has also been observed in epithelial tissue from colons of male mice (45). Colon Edn1 expression was increased after fasting the mice and refeeding not only decreased Edn1 expression, but also significantly reduced Edn1 expression rhythms. These studies demonstrate circadian rhythms of Edn1 at a transcriptional level, which can be altered via feeding cues known to also regulate the circadian clock.

FIGURE 2.

Circadian expression of the endothelin system in mouse tissues

A. Expression of endothelin-1 mRNA (Edn1) in male mice exhibits a circadian rhythm in various peripheral tissues apart from the suprachiasmatic nucleus. Relative circadian expression peaks during the mouse active period in lung, kidney, brown fat, cerebellum, and brain stem. B. Expression of endothelin receptor A mRNA (Ednra) exhibits a circadian pattern in lung, kidney, brown adipose, and heart. Peak expression in the lung occurs during the inactive phase, whereas expression in other tissues peaks during the active period. C. Expression of endothelin receptor B mRNA (Ednrb) exhibits a circadian pattern in liver and kidney. Peak expression in the liver occurs during the active period, whereas expression in the kidney peaks during the inactive period. This data were compiled using CircaDB, a public database of clock-controlled gene expression in male C57BL/6J mice (54). As described by Zhang et al. (74), 6-wk-old male C57BL/6J acquired from Jackson Laboratories were placed into constant darkness to study endogenous rhythms. Starting at circadian time18 after placement in constant darkness, three mice were euthanized every 2 h, for 48 h. Gray bars represent the mouse active period, while white bars represent the mouse inactive period.

Several animal studies have also shown that ET-1 peptide levels exhibit circadian rhythms (FIGURE 3). Plasma ET-1 peptide peaks during the active period vin rats (64). Circulating levels of ET-1 are regulated at the level of transcription and at the level of protein. Notably, ET_B receptor expression and activity within the lung, liver, and kidney are involved in plasma ET-1 peptide clearance (3, 17, 21). Urinary ET-1 peptide measurements are commonly used to determine production of kidney ET-1. Rats and mice exhibit diurnal rhythms of urinary ET-1, with more urinary ET-1 in the active phase compared with the inactive phase (13, 64). The diurnal rhythm of urinary ET-1 is not observed in rats without functional ET_B receptors, suggesting that rhythms in urinary ET-1 are, in part, dependent on clearance by ET_B receptors (63). Importantly, these rhythms in ET-1 peptide production are associated with rhythms in sodium handling.

FIGURE 3.

Circadian rhythms of circulating endothelin-1 (ET-1) in mice and humans

The molecular circadian clock receives cues from the environment, such as light from the sun, which stimulates action by the circadian clock transcription factors. These transcription factors regulate the expression of thousands of genes, including the ET-1 gene. Studies referenced in this review have demonstrated that both expression and circulating levels of ET-1 peptide exhibit circadian rhythms. Although the specific peaks of circulating ET-1 peptide vary within the studies, overall, circulating levels of ET-1 peak during the active period. Mice are nocturnal, meaning their active period is during the night or when lights are off (gray bars). The human active period is during the daytime or when lights are on (white bars). Circulating levels of ET-1 peak during the mouse active period (nighttime) and during the human active period (daytime). Understanding how these rhythms in circulating ET-1 levels relate to regulation of rhythms in physiological outputs, such as BP rhythms, will be important for treatment of disorders in which rhythms are disturbed, i.e., nondipping hypertension.

Preclinical data from animal models support a relationship between ET-1, sodium handling, and BP. Work done by Speed et al. (63) showed that ET-1 via the ET_B receptor plays an important role in modulating the circadian rhythm of BP under high-salt conditions. Following administration of a high-salt diet, ET_B-deficient rats developed salt-sensitive hypertension and an increase in amplitude of mean arterial pressure rhythms compared with control rats. ET_B-deficient rats also showed an increase in skin sodium and water content in the active phase relative to the inactive phase. These data suggest that the actions of ET-1 via the ET_B receptors appear to be involved in the regulation of skin sodium content and BP diurnal rhythms.

ET-1 through the ET_B receptor promotes natriuresis by inhibition of the epithelial sodium channel (ENaC) (6, 33). If ET-1 were regulated by the circadian clock, it would be expected that alterations in the endothelin system would result in disruption of sodium-handling rhythms. Indeed, ET_B-deficient rats exhibited alterations in the timing of sodium excretion compared with rats with the receptor (35). ET_B-deficient rats given a salt load at the beginning of the inactive period did not excrete the majority of the salt load until 12–24 h later, whereas control rats were able to excrete the majority of the salt load within the first 12 h postadministration. Mistiming of salt excretion in male ET_B-deficient rats was also observed when the salt load was given at the beginning of the rat active period. Administration of the ET_A receptor antagonist, ABT-627, improved the timing of salt excretion in these rats, suggesting that the altered natriuretic response rhythms in the ET_B-deficient rats is, in part, due to ET_A action.

Animal studies have also suggested that the time-dependent effects of the endothelin system are sex-specific. Unlike male ET_B-deficient rats that had altered natriuresis rhythms, female ET_B-deficient rats were better able to maintain natriuresis rhythms following a salt load that was given during the rat active period (35). Ovarian hormones can regulate expression of the ENaC subunits, which may be one reason for the observed sex differences. Estrogens have also been shown to regulate the expression of the Period circadian clock genes (49). Indeed, sex differences in natriuresis and ET-1 rhythms are observed in PER1 KO mice. Male C57BL/6 global PER1 KO mice challenged with a dietary treatment model of salt-sensitive hypertension develop a nondipping hypertension phenotype associated with an altered circadian rhythm of sodium excretion (14, 61). Additionally, male C57BL/6 PER1 KO mice exhibited altered rhythms of urinary ET-1, resulting from a significant increase of urinary ET-1 during the mouse inactive period (13). Interestingly, female global PER1 KO mice did not exhibit significant differences in rhythms of BP, sodium excretion, or urinary ET-1 compared with WT female mice (15). These data suggest that the regulation of ET-1 by the circadian clock occurs via a sex-dependent mechanism.

Limited data are available concerning ET-1 rhythms in animal models of pathophysiology. One study used the 1 kidney-1 clip (1K-1C) rat model of arterial hypertension (AH), which induces significant sodium retention, reduction in GFR, higher BP, reduced heart period (HP), and reduced baroreflex sensitivity (BRS) (39). Male 1K-1C AH rats exhibited changes in circadian rhythms of BP, HP, and BRS compared with sham male rats. The peak, or acrophase, of BP during the 1K-1C AH rats active period was shifted and was associated with a shift in the acrophase of HP and BRS during the rat inactive period. Plasma ET-1 acrophase in the 1K-1C male rats shifted to later in the active period, similar to the BP rhythms. It was also observed that the lowest plasma ET-1 levels coincided with the lowest BP values in 1K-1C rats. The relationship between alterations in BP and ET-1 rhythms must be studied further to deduce the role of circadian regulation of ET-1 on the development of pathophysiological conditions. Additionally, time-of-day needs to be taken into consideration as a biological variable within preclinical studies to enhance reproducibility and rigor.

Molecular Mechanism of Circadian Regulation of ET-1

Our laboratory became interested in the molecular regulation of circadian ET-1 rhythms after observing that in response to aldosterone, the sodium and water regulating mineralocorticoid, mouse inner medullary collecting duct cells (mIMCD-3) significantly upregulated the transcription of both Per1 and Edn1 (25). Knockdown of Per1 mRNA in collecting duct cells by siRNA resulted in the upregulation of Edn1 mRNA in addition to Cav1 and Ube2e3, all of which are negative regulators of ENaC (66). Our laboratory studied the effects of global PER1 KO in two different strains of male mice; 129/Sv mice, known to be salt-sensitive, and C57BL/6J mice, known to be resistant to dietary challenges. Global KO of PER1 in 129/Sv male mice resulted in significantly lower BP compared with WT littermates during both the active and inactive periods (66). The lower BP in the 129/Sv PER1 KO male mice was associated with a significant increase in renal ET-1 production compared with WT mice. Conversely, KO of PER1 in C57BL/6J mice resulted in nondipping hypertension, changes in the night-day ratio of sodium and ET-1 excretion, and increased Edn1 mRNA and ET-1 peptide expression (13, 14, 61). While the PER1-dependent BP phenotypes differ between the C57BL/6J and 129/Sv mouse strains, one thing that is consistent is that knockout of PER1 results in an upregulation of ET-1 at the level of mRNA and protein, suggesting that PER1 is a negative regulator of ET-1 production.

Circadian clocks can undergo a phase resetting to adapt to a new environment. Both Per1 and Per2 transcription are thought to be critical in starting the phase resetting process. By monitoring Per2:Luciferase expression in Rat-1 fibroblasts, Nakahata et al. (48) identified ET-1 as an entertainment cue for circadian rhythms. Treatment of Rat-1 fibroblasts with ET-1 peptide induced a significant upregulation of Per1 and Per2 transcription (70). At the protein level, PER1 and PER2 were found to be present in high quantities within the nucleus just 1.5 h after ET-1 treatment. The same study showed similar ET-1-induced changes in circadian clock gene expression using mouse embryonic fibroblasts. Evidence of ET-1 as an entrainment cue combined with the work from our laboratory demonstrating PER1 regulation of ET-1 expression indicates that there may be a reciprocal regulation between the endothelin system and the circadian clock.

It is currently unknown how ET-1 acts as an entrainment cue for the molecular clock, but it was shown to induce rat Per2 transactivation through a G protein (Gq/11)-mediated pathway via Ca²⁺ mobilization in Rat-1 cells (67). The endothelin receptors are GPCRs known to couple with Gq/11 (29). Following ET-1 treatment, Bmal1 expression peaks after and cycles antiphase of Per1/2 expression (70). Interestingly, a high-salt diet not only induced ET-1 expression, but also resulted in a significant phase shift of Bmal1 expression in rat kidney inner medulla (64). The induced shift in Bmal1 expression was not seen in the kidneys of ET_B-deficient rats. These results suggest that ET_B actions in the kidney regulate the circadian clock response.

Melatonin is another hormone known to regulate and synchronize the circadian clock. Studying the role of melatonin in circadian regulation revealed that melatonin lowered ET-1 levels in HUVEC cells exposed to increased pressure in culture, to simulate hypertension (58). ET-1 levels were lower 18 and 24 h after melatonin treatment compared with vehicle-treated cells. Angiotensin II levels also decreased at 18 h postmelatonin treatment, whereas NO and eNOS levels increased at all timepoints (6, 12, 18, and 24 h posttreatment) compared with vehicle-treated cells. This study demonstrates that melatonin may restore circadian rhythms of multiple vasoactive molecules which, in turn, could affect BP rhythms. These data suggest that melatonin could be beneficial in hypertension, which is consistent with a randomized, double-blind crossover study examining the effect of nighttime melatonin administration in humans (57). Scheer at al. reported reduced BP and increased BP rhythms in response to repeated melatonin administration before sleep.

Regulation of the Edn1 gene is quite complex even though the gene contains only five exons (65). Our laboratory recently identified a long noncoding (lnc) RNA arising from the Edn1 locus, which we termed Edn1-AS (16). This lncRNA appears to be a natural antisense transcript (NAT) that is expressed in a number of different human cell lines, as well as the human kidney. Using synchronized human kidney proximal tubule cells (HK-2), we showed that Edn1-AS exhibits circadian variation in vitro. Although many NATs exert a negative regulation on the sense mRNA from their respective locus, we found that Edn1-AS expression correlates with that of Edn1. An area within the predicted promoter of Edn1-AS contains a putative circadian clock-mediated regulatory element (E-box), as well as a glucocorticoid response element. CRISPR (clustered regularly interspaced short palindromic repeats)-mediated deletion of this region in HK-2 cells resulted in increased expression of Edn1-AS and increased levels of secreted ET-1 peptide. Since Edn1-AS exhibits circadian rhythms of expression and contains binding sites for circadian transcription factors, Edn1-AS regulation may play a role in the maintenance of ET-1 rhythms. The mechanism of Edn1-AS regulation of ET-1 production remains to be determined but may involve the interaction of Edn1-AS with miRNAs that are known to regulate Edn1 mRNA levels (34).

Clinical Implications

Regulation of fluid and electrolyte transport, the immune system, and pulmonary function are just some of the known roles of the ET-1 peptide. Consistent with ET-1 exhibiting a large number of tissue-specific functions, dysregulation of ET-1 has been implicated in the pathogenesis of a variety of diseases. Additionally, clinical data have shown that circadian disruptions have distinct effects on physiology and pathophysiology. The preclinical data and mechanistic studies presented in this review suggest that the ET-1 system and the circadian clock system are intertwined. Dissecting the ET-1/circadian clock relationship could have implications for clinical treatment and management of a number of diseases.

Elevated levels of ET-1 have been observed in chronic obstructive pulmonary disease (COPD) and asthma events. Evaluation of the night and day variations of circulating ET-1 levels in patients with COPD revealed that patients with COPD who experienced nocturnal hemoglobin desaturation had higher daytime ET-1 levels compared with COPD patients without nocturnal hemoglobin desaturation (50). Additionally, this subset of patients experienced higher plasma ET-1 levels at nighttime compared with daytime. Asthma patients had higher hemoglobin saturation and lower ET-1 levels than COPD patients. As ET-1 is a known vasoconstrictor, the increase in nighttime ET-1 could restrict coronary circulation and contribute to COPD nocturnal hemoglobin desaturation and consequent comorbidities.

Obstructive sleep apnea (OSA) affects patients’ circadian rhythms and ET-1 levels. In a prospective study, researchers measured plasma ET-1, BP, and other related physiological parameters in patients with OSA before and after continuous positive airway pressure therapy (53). They showed that ET-1 levels and MAP were significantly increased in OSA patients before therapy but decreased after 5 h of therapy, whereas in control patients ET-1 levels were unchanged and BP measurements were opposite to those seen in OSA patients. An interventional clinical trial, which started in August 2014 and completed in March 2020, sought to determine how the circadian clock and behavior may be contributing to blood pressure control in a cohort of patients with OSA (59). The results of this study are not yet available.

The clinical use of ET-1 receptor blockers, specifically endothelin receptor antagonists, has been shown to be effective in managing high BP in patients with chronic kidney disease, diabetes, and pulmonary hypertension (5). Despite this, their use by clinicians is not widespread due to concern of potential adverse effects, including fluid retention and in some cases, liver toxicity (30, 43). The kidneys are made up of mainly ET_B receptors, with the minority ET_A receptors concentrated in the renal smooth muscle vasculature (5). ET_A-receptor antagonists are proving to be a vital tool for clinicians because of their ability to specifically block the vasoconstrictive and, consequently, detrimental effects of ET-1 action via ET_A. Consistent with preclinical results demonstrating a connection between ET-1 and BP rhythms, Dhaun et al. (12) showed that ET_A blockade in chronic kidney disease (CKD) patients resulted in improved BP rhythms. More specifically, administration of an ET_A receptor antagonist restored the BP dip during the rest phase. Although additional studies must be pursued, this study identifies a role for ET_A antagonists in the maintenance of BP rhythm and further shows potential as an effective treatment option in CKD patients.

Differences between males and females exist in the pathology, presentation, and treatment of disease. For example, females tend to be more prone to the development of pulmonary arterial hypertension and CKD, whereas males have a higher prevalence of severe CKD and progression to ESRD (2, 10, 27). Polderman et al. (55) showed that females have a lower plasma ET-1 concentration than their male counterparts, and pregnant women had even lower plasma ET-1 concentration compared with nonpregnant women. Females also appear to be protected from the negative effects of ET_A receptor action compared with males (41). Future studies examining the relationship between ET-1 and pathophysiology in both sexes will be critical in determining the clinical significance of ET-1 circadian rhythms. Additionally, studying both sexes could provide significant insight into the development and administration of drugs targeting the endothelin system.

The studies presented here reveal an interplay of regulation between ET-1, the circadian clock, and pathological conditions. Circadian disruption is known to increase the risk for cardiovascular disease, cancer, and metabolic syndromes. Furthermore, circadian disruption is observed in many disease states, including CKD. Dysregulation of ET-1 has also been observed in various pathophysiological states. The question remains whether or not dysregulation of ET-1 rhythms promotes the development of disease and/or whether disease states cause disruption of ET-1 rhythms, leading to further clinical implications. More studies are needed to understand this relationship. Additionally, the connection between endothelin signaling and the circadian clock across the lifespan is an area ripe for future study. Moving forward, it will be important for future preclinical and clinical studies to report time of day in which samples are collected and to consider time of day as a biological variable. By reporting the time of day of sample collection and/or drug administration, more analyses can be performed examining the role of circadian rhythms, such as those seen in the endothelin system, on the development, and treatment of pathological conditions.