2013 Dahl Lecture AHA Council for High Blood Pressure Research

The suggestion that the peptide, endothelin (ET)-1, contributes to the development of hypertension was clearly evident from the first in vivo characterization of ET-1. In the ganglion-blocked rat, Yanagisawa first reported that a 1 nmol/kg intravenous injection of ET-1 produced a transient decrease in blood pressure lasting less than 30 seconds followed by a sustained hypertension.¹ These findings stimulated an incredible response in new research especially within the pharmaceutical industry including some efforts to target the ET system for the treatment of hypertension. Two receptor subtypes, ET_A and ET_B, mediate the opposing actions of ET-1. We now know that the slow and sustained hypertensive response is mediated primarily by ET_A receptor activation on vascular smooth muscle. Importantly, the hypotension is due to endothelial-dependent relaxation mediated by the ET_B receptor. The purpose of this review is to highlight some of the more important aspects of the ET system as it relates to the physiological and pathophysiological role in the regulation of blood pressure and development of hypertension. It should be noted that the ET_A and ET_B receptor systems are important in craniofacial and enteric nerve development, respectively, so the reader is referred elsewhere for more details on these topics.^{2, 3}

Complexities of the ET system

There are a several unique aspects of the ET system that are worth mentioning because of their unique nature compared to more classical peptide/G-protein linked receptor systems. Probably the most unusual aspect of this system that has made it difficult to study through the years is the irreversible binding of the endogenous ligand to the receptor.⁴ This binding is believed to account in large measure for the prolonged vasoconstrictor actions of ET-1 mediated by the ET_A receptor. Importantly, the same ligand binding behavior exists for the ET_B receptor such that loss of ET_B receptor activity results in exaggerated ET_A dependent contraction. Somewhat surprisingly, this irreversible binding does not prevent ET_A selective antagonists from reversing the contractile effects of ET_A activity. In isolated vascular smooth muscle, contraction can be rapidly reversed, but then is restored once the antagonist (and agonist) is washed from the muscle bath.⁵

One must also consider that much of what we have learned about the ET system, especially in the early years, is based on application of exogenous ligand to in vitro or in vivo systems, the latter typically being intravenous or intra-arterial infusion. While useful information has been gained from this approach, it has also misled us in our efforts to define the physiological role of endogenous ET-1 because endogenous ET-1 does not first enter the broader systemic circulation, but rather, is confined to a paracrine or autocrine role.⁶ It is uniformly accepted that endogenous ET-1 is released from endothelial cells primarily towards the basolateral side of the cell and does not function as a classic circulating hormone. Given the irreversible nature of ET-1 binding, there can be considerable endogenous ET-1 binding without any change in circulating ET-1 levels. Therefore, plasma ET-1 measurements are not considered a reliable refection of ET-1 production, but could be a reflection of ET_B receptor availability as explained below.

Loss of ET_B receptor activity, whether by administration of specific receptor antagonists or through genetic deletion or mutation results in significant increases in plasma ET-1 levels even without noting any change in ET-1 gene expression.^{7, 8} This led to initial descriptions of the ET_B receptor as a “clearance” receptor. However, the extent to which ET_B receptors clear ET-1 compared to functional effects that oppose ET_A receptor activity has not been clearly established. Furthermore, given that ET_A receptors also bind to ET-1 in an irreversible fashion, one must also consider these receptors in the “clearance” of ET-1 from the circulation even though most studies do not show any increase in circulating ET-1 in conjunction with ET_A receptor blockade.⁹

Although not universally observed, there have been reports of increased plasma ET-1 following specific ET_A receptor blockade, such as in mineralocorticoid-induced hypertension or loss of functional ET_B receptor activity.^{8, 10} The most likely explanation is that such increases are only observed when there is insufficient ET_B receptor available to manage endogenous ET-1 production. Within the general circulation, it is fairly clear that ET_B receptors are predominant. This may seem counterintuitive given the powerful vasoconstriction seen with exogenous ET-1 administration, but more revealing information may be gleaned when considering experiments in animals that have examined selective ET_A versus selective ET_B receptor blockade in otherwise normal animals.¹¹ ET_A selective blockade has little effect on baseline blood pressure. In contrast, when an ET_B antagonist is given, a robust increase in blood pressure is quite evident.

ET_B receptors function to protect from over-activity of the ET_A receptor by removing ET-1 from the circulation as well as stimulating the production of endothelial dependent relaxing factors such as nitric oxide and prostacyclin. Indeed, the hypertension produced by reduced ET_B receptor function can be mitigated with an ET_A antagonist.¹¹ However, this does not suggest that the only role for ET_B receptors is to clear ET-1 from the plasma because loss of ET_B receptor induced hypertension is highly salt dependent as discussed in detail below.

The actions of ET-1 in the kidney have been a major focus of our laboratory and others. When considering renal ET-1, there is considerable evidence that urinary ET-1 is derived exclusively from intrarenal production and not derived from the circulation.⁷ First of all, as already stated, both ET_A and ET_B receptors bind ET-1 irreversibly and so it would be difficult to traverse the entire renal circulation without being cleared. Second, intravenous infusion of radiolabeled ET-1 results in accumulation within vascular structures, but not epithelial cells or urine.¹² While ET-1 is synthesized by most all segments of the nephron, principal cells of the inner medullary collecting duct far exceed production from other cell types in the kidney. Selective gene deletion of ET-1 from collecting duct cells reduce urinary ET-1 excretion by at least 50%, suggesting that the majority of urinary ET-1 is from this cell type. Furthermore, while plasma levels of ET-1 are influenced by receptor availability, receptor antagonists do not influence urinary ET-1 excretion suggesting that the urinary ET-1 had limited exposure to ET_A or ET_B receptors or that receptor antagonists do not have access to receptors on the luminal surface of tubular epithelium.^{9, 11}

Industry response to ET-1 discovery

It did not take long after the discovery of ET-1 for a number of companies to develop immunoassays for measurement of ET-1 peptide concentrations in plasma, urine and tissues. One of the problems with many of these initial assays is that they had varying degrees of sensitivity and cross-reactivity with the less biologically active peptides (e.g., ET-3, big ET-1, etc.). Nonetheless, there were reports of elevated plasma ET-1 levels in hypertensive subjects. Of note, these levels were even higher in African American subjects.¹³

The time to develop highly selective, potent, and orally active ET_A or combined ET_A/ET_B receptor antagonists was incredibly fast, only a few years after the discovery of ET-1.^14–16 The first antagonists were cyclic pentapeptides discovered through natural product screening. The most common of these peptides was BQ-123, which is highly selective for the ET_A receptor, while another peptide, BQ-788, has clear ET_B selectivity. Both of these commercially available peptide antagonists have a very high degree of receptor specificity, but are limited in their use because of a lack of oral bioavailability. However, it did not take long for the development of orally active small molecular weight organic compounds with variable degrees of selectivity for the two receptors. Bosentan was the first such compound to be developed and has been used for a number of years in the treatment of pulmonary hypertension.¹⁷ Developed by Actelion, bosentan is referred to as a mixed or combined antagonist because it has similar affinity for both ET_A and ET_B receptors. In October 2013, Actelion received approval from the US FDA for marketing their next-generation antagonist, macitentan, for pulmonary hypertension that is again a mixed antagonist, but chemically modified to be more lipophilic and thus concentrate in tissues more effectively. The only other antagonist approved for human use in the United States is ambrisentan,¹⁸ a product of Gilead Life Sciences, which has a fairly high degree of ET_A receptor selectivity. A number of other companies have developed similar compounds targeting other cardiovascular-related disease, but most of the effort to develop these compounds has been abandoned due to lack of efficacy and concern over waning patent life, side effects, lack of efficacy and the high cost of new trials. Nonetheless, there remains some interest in these antagonists for disorders beyond pulmonary hypertension including essential hypertension and various forms of nephropathy, in particular, diabetic nephropathy.

ET antagonists in human hypertension

The most significant study during the initial years of development with these drugs was that published by Krum et al. in the New England Journal of Medicine.¹⁹ In this study, 293 subjects with identified hypertension were required to withdraw from all current medication for a four-week period, then were treated with one of three doses of bosentan. An additional subgroup was given the angiotensin converting enzyme inhibitor, enalapril, as a comparator. Interestingly, the degree of blood pressure lowering was identical between bosentan and enalapril. However, Actelion did not pursue use of bosentan as an anti-hypertensive agent further because the company decided to develop the drug for use in primary pulmonary hypertension.

A number of years later, Gilead Sciences, Inc., sponsored several clinical trials targeting resistant hypertension with their highly selective ET_A antagonist, darusentan.²⁰ This trial included subjects with resistant hypertension as defined by in adequate blood pressure control despite treatment with 3 distinct anti-hypertensive medications. After 14 weeks of treatment, the darusentan treated group had an average of about 10 mmHg lower 24-hr ambulatory blood pressure compared to the placebo group, an effect that was highly significant (Figure 1). Unfortunately, the primary end point in the trial was clinic blood pressure that was not statistically different from the placebo group. So, even though there is a consensus among hypertension specialists that ambulatory blood pressure is a more reliable measure of hypertension and predictor of complications, the company decided not to pursue development of this drug.

Figure 1.

Change in mean 24-hr systolic blood pressure in subjects with resistant hypertension treated with either placebo, darusentan (DAR), or guanfacine (GUAN) for 14 weeks in addition to at least three other types of anti-hypertensive medications (from Bakris et al.²⁰)

ET-1 control of sodium and water excretion

Due to the incredible potency and prolonged vasoconstrictor effects of exogenous ET-1, for a long time, investigators believed that the physiological role of ET-1 must be related to this effect. This is why one of the first studies to carefully examine renal effects of ET-1 was largely ignored as a probable artifact. Time has proven that the study by Schnermann et al published in 1992 to be highly relevant.²¹ In anesthetized rats, these investigators infused a relatively high dose of ET-1 and observed a reduction in urine flow rate along with a decrease in GFR. However, when infusion was switched to a low dose that did not reduce GFR, there was an increase in urine flow rate without any associated increase in systemic arterial pressure. These are the first indications that ET-1 may function to promote the excretion of water.

Several studies have shown that urinary ET-1 excretion is positively correlated with sodium excretion as well as other pro-natriuretic systems such as nitric oxide and cGMP.⁷ In 2000, work by Plato and Garvin demonstrated that ET-1 can directly inhibit Cl⁻ flux in isolated thick ascending limb segments indicating a direct effect independent of any hemodynamic influence.²² These investigators demonstrated that this is mediated by ET_B receptor dependent NO production. In isolated split open collecting ducts, work from Stockand’s lab demonstrated a direct effect of ET-1 via the ET_B receptor to reduce the open probability of epithelial sodium channels.²³ These investigators went on show that mice lacking the ET_B receptor in the collecting duct do not have suppressed ENaC activity when placed on a high salt diet.²⁴ These findings provide evidence not only that ET-1 suppress ENaC activity, but actually participates in the regulation of activity in response to changes in dietary salt intake.

Kohan and colleagues published a series of studies demonstrating that the collecting duct ET-1/ET_B autocrine pathway is important for regulating blood pressure.^25–28 Using a now common cre-lox approach, these investigators knocked out ET-1 from the collecting duct, which resulted in an elevated baseline blood pressure and exaggerated hypertensive response to a high salt diet (Figure 2). Loss of collecting duct ET-1 also reduced urinary NO metabolite excretion. They went on to show that ET-1 appears to function in an autocrine manner since knocking out the ET_B receptor or both ET_A and ET_B receptors from the collecting duct results in a similar salt-sensitive hypertensive phenotype.

Figure 2.

Systolic blood pressure measured by telemetry in collecting duct specific knockout (KO) mice in response to being placed on a high salt diet (adapted from a series of papers from Kohan’s laboratory^25–28).

ET-1 stimulates NO release from cultured collecting duct cells, an effect that can be blocked by a specific inhibitor of NOS1 (sometimes called neuronal or nNOS).²⁹ This led our laboratory to conduct studies examining intramedullary ET_B agonist infusion which revealed that stimulation of ET_B receptors with the selective agonist, sarafotoxin 6c, produced a natriuresis that could also be blocked by specific NOS1 inhibition.³⁰ These findings clearly suggested that ET_B inhibition of sodium reabsorption in the collecting duct was mediated by NOS1 generated NO. However, more definitive evidence came from a recent study by Hyndman et al. who showed that similar to the ET-1 or ET_B receptor gene deletion studies, knockout of NOS1 specifically from the collecting duct also led to reduced NO excretion and a salt sensitive blood pressure phenotype.³¹ Stockand and colleagues have gone on to show that ET-1 does not reduce ENaC activity in collecting ducts from collecting duct specific NOS1 knockout mice (unpublished observations, manuscript under revision).

Knowing that salt intake is a powerful stimulus of ET-1 production in the kidney, we now have a fairly clear picture of the physiological role of ET-1, that is, to facilitate the excretion of salt and water (see discussion below). This effect is primarily through and autocrine action of ET-1 stimulating ET_B dependent NOS1 dependent NO in the collecting duct, but may also involved thick ascending limb NOS3 activation as well as effects within the renal microcirculation.

High salt diet stimulates renal ET-1

As has been reported many times, a high salt diet increases renal medullary ET-1 production (Figure 3).⁷ However, the mechanisms by which this stimulus occurs in not completely clear. Herrera and Garvin showed that renal medullary interstitial osmolarity in rats on a high salt diet is elevated.³² They also observed that increasing osmolarity in the culture media stimulates ET-1 release from isolated thick ascending limbs. This raises the question also of whether increased tubular fluid flow and/or sodium delivery to the distal nephron could be a stimulus for ET-1 production and thus facilitating sodium excretion. In cultured collecting duct cells, Kohan has reported that extracellular sodium can stimulate ET-1 production.³³ In a more intact preparation, Boesen infused hypertonic NaCl into the renal medullary interstitium and reported that higher osmolarity increased urinary excretion rate of ET-1.³⁴

Figure 3.

Hypothetical scheme for ET-1 and ET_A and ET_B receptor involvement in the physiological response to high salt. Sites where angiotensin II may interfere to induce salt-sensitivity are indicated by the red asterisk.

Renal hemodynamics

The renal circulation, similar to most vascular beds, contains both ET_A and ET_B receptors. Intravenous infusion of exogenous ET-1 reveals a profound vasoconstrictor action that appears to be primarily mediated by ET_A receptors, but does involve ET_B-dependent constriction at higher doses.^{35, 36} Endothelial-dependent vasodilation mediated by the ET_B receptor is evident, although the prolonged vasoconstrictor effects of ET-1 over-ride this effect when exogenous peptide is administered.⁷ There also appears to be a heterogeneous distribution of ET_A and ET_B receptors over the length of the renal arterial system as revealed in isolated vascular preparations.³⁷ In general, the relative proportion of ET_A to ET_B receptors decreases as one moves along the renal arterial tree, with more prominent ET_B-dependent vasodilation being more evident in the efferent arteriole. ET_B dependent vasoconstriction is only seen at higher concentrations of ET-1 suggesting weaker binding affinity for ET_B receptors on vascular smooth muscle. Very little is known about the renal hemodynamic effects of ET-1 in humans. There have been few studies to evaluate the receptor specific effects of exogenous ET-1 on renal hemodynamics in humans. Kaasjager et al. demonstrated that ET-1 produced a profound decrease in renal plasma flow and GFR, while the ET_B selective ligand, ET-3, had no effect, thus suggesting a predominance of the ET_A receptor in humans.³⁸

Over 10 years ago, our laboratory observed that intravenous infusion of the ET-1 precursor, big ET-1, reduces renal cortical blood flow in rats, while medullary blood flow remains relatively unchanged.³⁹ Knowing that the renal medulla is where ET-1 and ET_B receptor expression is most prevalent, we hypothesized that the actions of ET-1 within the renal medullary circulation could function to influence renal medullary control of salt and water excretion. Infusion of big ET-1 into the renal medulla of rats on a high salt diet resulted in a significant decrease in renal cortical blood flow as in rats on a normal salt diet. However, renal medullary blood flow was actually increased by systemic infusion of big ET-1 in rats on a high salt diet. Once again, ET-1 would appear to be behaving as a pro-natriuretic factor.

Our collaborators have further investigated the influence of salt diet on renal hemodynamics in a series of preliminary, unpublished experiments. Inscho and colleagues have observed that a high salt diet reduces renal blood flow autoregulatory efficiency whether it is in vivo or in the in vitro blood perfused juxtamedullary nephron preparation. Autoregulation was normalized in preparations where the ET_B receptor was inhibited consistent with ET-1 also having important hemodynamic effects under conditions of chronic elevations in salt intake. In other words, efforts to maintain a high GFR under conditions of high salt intake help to facilitate high tubular flow rates and elevated renal medullary perfusion, both of which are pro-natriuretic and complement the renal tubular actions of this system.

ET and the sympathetic nervous system

Attention to the renal sympathetic nerves in control of blood pressure has taken something of a roller coaster ride over the past century, but in recent years, renewed interest has sprung from promising clinical trials using new methods for renal nerve ablation. Involvement of the ET-1 system with central and peripheral nervous system components has received very limited attention, but represent a fertile area for future investigation. One of the most notable observations developed from studies where the ET_B receptor gene was knocked out of mice resulting in a lethal phenotype.⁴⁰ The lethality is due to the lack of enteric nervous system development, thus leading to aganglianosis and megacolon characteristic of Hirshsprung’s disease. Indeed, the lack of a functional ET_B receptor has been identified in this patient population.

Studies of ET_A and ET_B receptor function in the sympathetic nervous system have been limited to only a few groups of investigators despite clear functional involvement. A series of studies from investigators at Michigan State University (Kreulen, Gilligan, Watts and Fink) has demonstrated functional ET_B receptors in sympathetic ganglia that account for hypertension produced by chronic infusion of an ET_B selective agonist.^41–43 Studies from our own laboratory also support a sympatho-activation role for ET_B receptors in the hypertensive response to acute stress. Using acute (3 min) air jet stress in restrained rats, we observed that combined ET_A/ET_B receptor blockade, but not ET_A selective blockade severely inhibited the pressor response in Dahl salt-sensitive rats.⁴⁴ In contrast, under conditions of ET_B receptor deficiency where ET-1 levels are chronically elevated, ET_A receptors function to inhibit acute stress-induced pressor responses.⁴⁴ In both human and animal studies, acute stress results in transient increases in circulating ET-1 within 1–2 minutes suggesting either a rapid release of ET-1 or a reduction in ET_B receptor availability, or both.^{13, 45}

Our group has reported that African Americans have higher plasma concentrations of ET-1 suggesting a relative reduction in ET receptor number or availability, and most likely the ET_B receptor given their more pronounced influence on circulating ET-1 levels and the higher level of salt-sensitivity in the African American population.¹³ We have also reported differences in the response to acute environmental stress among races that could possibly be explained by differences in ET_A or ET_B receptor function. The pressor response to acute stress is more pronounced in obese subjects carrying a specific polymorphism in the ET-1 gene suggesting that a combination of genetic and environmental factors may play a role in ET-1 involvement in acute blood pressure responses mediated by the sympathetic nervous system, but specific mechanisms have yet to be clarified.⁴⁶ Furthermore, whether the actions within the sympathetic nervous system have any relation to the overall physiological role in regulating fluid-electrolyte balance is unknown.

Another area where ET_A and ET_B receptors may also influence sodium excretion is by modulating the activity of renal sensory nerves within the renal pelvis. Kopp et al have shown that ET-1 enhances the activation of renal sensory nerves in rats on a high sodium diet via ET_B receptors. In contrast, on a low sodium diet, ET-1 suppresses the activation of renal sensory nerves by stimulation of ET_A receptors.⁴⁷ The effect on renal sensory nerves is important since they have a profound influence on renal efferent nerve activity.

Skin as a buffer for extracellular sodium

A very recent, and very provocative hypothesis has recently emerged in the past few years regarding fluid-electrolyte homeostasis. This is the concept that sodium can be stored in the interstitial matrix, in particular, within the skin as a means of sodium conservation. This work, primarily driven by Jens Titze at Vanderbilt University, suggests that sodium can be “stored” and then cleared through the lymphatic system to serve as a secondary clearance system for sodium in the body.⁴⁸ Deposition of sodium in the interstitial space requires activation of a series of inflammatory signaling systems, such as monocyte chemoattractant protein-1 and intracellular cell adhesion molecule-1.⁴⁹ These are the same factors activated by sub-pressor doses of ET-1.⁵⁰ While the role of ET-1 in this storage mechanism has yet to be determined, it is compelling that ET-1 is increased in extra-renal tissues in response to high sodium intake.⁵¹ In cross-transplantation studies, Ohkita et al. provided evidence that extra-renal ET-1 plays a significant role in the cardiovascular response to increases in dietary sodium intake.⁵² Our working hypothesis for on-going experiments is that ET-1 functions as a physiological modulator in movement of sodium through extra-renal tissue in a fashion similar to that in the kidney.

ET-1 interaction with angiotensin II

One of the early pre-clinical observations with ET antagonists in models of systemic hypertension is their effectiveness seemed to be somewhat limited to salt-dependent forms of hypertension including mineralocorticoid and angiotensin II infused models, but not spontaneously hypertensive rats (SHR). Chronic angiotensin II (Ang II) produces a prolonged, sustained hypertension that can be completely prevented or reversed by either ET_A selective or ET_A/ET_B receptor antagonists.^53–55 This appears to be a direct effect of Ang II on ET-1 production as demonstrated in cultured endothelial cells.⁵⁶

Given the important role of the ET_B receptor in mediating sodium excretion, we hypothesized that the ET_B receptor system was dysfunctional in Ang II dependent hypertension leaving the ET_A receptor unchecked in contributing to blood pressure elevation as a means of facilitating pressure natriuresis by the kidney. As discussed above, intramedullary infusion of the ET_B selective agonist, sarafotoxin 6c (S6c) in the rat results in a natriuretic response. In rats that were chronically infused with Ang II, the natriuretic response to S6c was absent.⁵⁷ The lack of a response to intramedullary S6c infusion appears most likely due to an effect of Ang II to reduce ET_B receptor expression as evidenced by reduced ET_B-specific binding in membrane preparations from the inner medullar of Ang II infused rats. These studies are consistent with an overall autocrine/paracrine function in the inner medulla that is impaired during Ang II exposure and can explain, at least to some degree, the salt-sensitivity associated with this model (Figure 4).

Figure 4.

Scheme depicting the autocrine and paracrine actions of ET-1 within the renal inner medulla. Used with permission.⁷

To determine whether the effect on the ET_B natriuretic pathway is a function of physiological regulation by endogenous Ang II alone or whether this effect is perhaps a consequence of hypertension induced injury, we conducted similar intramedullary S6c infusion experiments in rats with elevated endogenous Ang II by feeding them a low sodium diet for one week.⁵⁸ Interestingly, the natriuretic response to S6c was absent in animals maintained on a low sodium diet. In addition, in animals given a low sodium diet along with the AT1 receptor antagonist, candesartan, the natriuretic response to S6c was restored. These findings provide clear evidence that Ang II is an important physiological regulator of ET_B dependent natriuresis.

Another interesting aspect of this relationship between the ET and Ang II systems is that female rats appear to be slightly different from males. First of all, it is well known that Ang II infusion does not produce nearly as strong of a hypertensive response in females compared to males. In addition, we observed that chronic Ang II does not completely block ET_B dependent natriuresis as it does in the males.⁵⁷ Evidence to date also suggests that in the rat, the ET_A receptor can contribute to some of the natriuretic response to ET-1, but more work needs to be done in determine whether this occurs in humans and whether this could explain some of the fluid retention issues reported in humans taking ET_A antagonists.

Summary

The accumulation of evidence over the past 10–20 years has led to a fairly clear picture that one of the major physiological roles of ET-1 is to participate in the regulation of fluid and electrolyte balance and that derangements of this system lead to salt sensitive hypertension. This role covers a full range of aspects that were not covered in this particular review, but all point towards a wide range of actions within the kidney, vasculature, and even the peripheral nervous system (Figure 5).

Figure 5.

Potential pathways depicting the overall physiological role of ET-1 to function as a pro-natriuretic factor to regulate the sodium excretion (UNaV). The three pathways on the right are well established with sympatho-regulation and skin Na⁺ buffering await further confirmation.

The fundamental mechanism for ET-1 participation in hypertension appears to be a loss of epithelial ET_B receptor function in the kidney and perhaps beyond to potentially account for loss of endothelial dependent NO production. This results in an inappropriate level of ET_A receptor activation that, while facilitating natriuresis, results in elevated renal perfusion pressure and risk for hypertensive end organ damage. The rationale for use of ET_A receptor antagonists in resistant hypertension is quite strong, but issues related to drug development and risk for side effects have led to the major pharmaceutical houses to withdraw interest in this area. Indeed, fluid retention is reported as the most prominent complication, especially in patients with some level of renal dysfunction.^{59, 60} It is not completely clear whether this is related to some degree of ET_B receptor blockade even with ET_A specific antagonists or it could involve ET_A dependent effects on fluid-electrolyte handling that have heretofore been underestimated. There also remain additional therapeutic targets within the spectrum of hypertension including that induced by tyrosine kinase inhibitors.⁶¹ Nonetheless, promising studies related to the use of these antagonists in diabetic nephropathy may help aid in our understanding how and when these drugs may be more effectively prescribed for cardiovascular related disease and perhaps provide a means of managing the fluid retention.⁶²