First in man demonstration of direct endothelin-mediated natriuresis & diuresis

Abstract

Endothelin (ET) receptor antagonists are potentially novel therapeutic agents in chronic kidney disease and resistant hypertension but their use is complicated by sodium and water retention. In animal studies, this side effect arises from ET_B receptor blockade in the renal tubule. Previous attempts to determine whether this mechanism operates in humans have been confounded by the hemodynamic consequences of ET receptor stimulation / blockade.

We aimed to determine the effects of ET signalling on salt transport in the human nephron by administering sub-pressor doses of the ET-1 precursor, big ET-1. We conducted a 2-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. Following sodium restriction, subjects received either intravenous placebo or big ET-1, in escalating dose (up to 300pmol/min). This increased plasma concentration and urinary excretion of ET-1. Big ET-1 reduced heart rate (~8 beats/min) but did not otherwise affect systemic hemodynamics or glomerular filtration rate. Big ET-1 increased the fractional excretion of sodium (from 0.5 to 1.0%). It also increased free water clearance and tended to increase the abundance of the sodium-potassium-chloride co-transporter (NKCC2) in urinary extracellular vesicles.

Our protocol induced modest increases in circulating and urinary ET-1. Sodium and water excretion increased in the absence of significant hemodynamic perturbation, supporting a direct action of ET-1 on the renal tubule. Our data also suggest that sodium reabsorption is stimulated by ET-1 in the thick ascending limb and suppressed in the distal renal tubule. Fluid retention associated with ET receptor antagonist therapy may be circumvented by co-prescribing potassium-sparing diuretics.

Introduction

The endothelin (ET) peptide family was described by Yanagisawa in 1988.¹ They form an intricate signalling system, in which three mature peptides (ET-1, ET-2 and ET-3) interact with two receptors (ET_A and ET_B).^{2, 3} ET-1 is the main vascular endothelial species and the most powerful vasoconstrictor. It is generated from a precursor peptide, big ET-1, through proteolytic cleavage by two ET converting enzymes (ECE-1 & ECE-2, which exist in several isoforms) or other proteases.

The kidney is both a source and site of action of ET-1. Indeed the renal medulla contains the highest concentration of immunoreactive ET-1 in the body.⁴ Significant amounts of ET-1 are detectable in most renal cell types, probably acting as a paracrine/autocrine regulator of renal and intra-renal blood flow, glomerular hemodynamics and sodium and water transport.⁴ ET_A receptors are situated on vascular smooth muscle cells where they promote vasoconstriction and are thought to mediate many of the pathological effects of ET-1.⁵ In the kidney, ET_B receptors are expressed by the vascular endothelium, vascular smooth muscle and by tubular epithelial cells along the length of the nephron – with a particularly high density in the medullary collecting ducts.³ In vascular endothelium, ET_B receptor activation promotes vasodilation.² In the renal tubule, pre-clinical data suggest that ET_B receptors stimulate natriuresis and diuresis.^{3, 6} To date, this action has not been demonstrated in man.

Understanding the effect of ET signalling on renal salt and water transport is important because the system makes an attractive target for novel therapies in disorders of fluid-electrolyte homeostasis. An inability to appropriately excrete salt and water is a feature of a number of common conditions including chronic kidney disease (CKD), chronic liver disease, congestive heart failure and salt-sensitive hypertension. Importantly, salt and water retention is associated with both acute and chronic morbidity and mortality.^{7, 8} ET receptor antagonists (ERAs), a novel class of drug currently licensed for the treatment of pulmonary arterial hypertension and scleroderma digital ulcers,² are being investigated in a range of clinical conditions including diabetic nephropathy, heart failure and resistant hypertension.² However, salt and water retention is a common side effect of these agents and has led to the premature termination of a phase 3 clinical trial.⁹

It has been hypothesized that blockade of ET_A receptors confers clinical benefit (reducing blood pressure (BP), proteinuria and renal inflammation) whilst ‘off-target’ blockade of ET_B in the renal tubule induces deleterious sodium and water retention.¹⁰ This hypothesis is supported by data from animal models, in which low doses of ET-1 promote natriuresis and diuresis in the absence of significant hemodynamic change, by stimulating ET_B receptors in the renal tubule.¹¹ However, a direct natriuretic action of ET-1 has not yet been demonstrated in man. Previous studies have been confounded by the changes in systemic hemodynamics induced by ET receptor agonism and antagonism. For example, the administration of exogenous ET-1 produces profound retention of salt and water accompanied by systemic and renal vasoconstriction,^{12, 13} whilst ET_A blockade in CKD induces a natriuresis and an increase in renal blood flow.¹⁴ It is not possible to easily differentiate the indirect effect of any hemodynamic changes on renal salt and water excretion from direct effects on renal tubular cell function.

The aim of this study was to demonstrate a direct natriuretic effect of ET receptor activation in man. We administered incremental doses of intravenous big ET-1 to healthy volunteers. We used big ET-1 (as opposed to ET-1), as this would more closely reflect normal physiology, with ET-1 being generated only in those tissues expressing ECE. We hypothesized that big ET-1 would lead to a gradual increase in natriuresis and free water clearance (FWC) in the absence of significant hemodynamic changes.

Methods (also see Supplementary Information)

Subjects

This was a two-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. The study was carried out with the approval of the local research ethics committee and the written informed consent of each subject. The investigations conformed to the principles outlined in the Declaration of Helsinki.

Male and female subjects were recruited from the community between September 2006 and January 2007. To be eligible for inclusion, subjects had to be between 18 and 80 years of age with no past medical history documented by their primary care physician and prescribed no regular medications. Other inclusion criteria were a body mass index <30kg/m², BP <140/90mmHg, normal biochemical parameters and a clear urinalysis.

Study protocol

Subjects were asked to adhere to a standardized diet (avoiding high salt-containing foods) for 3 days prior to each study day and to complete two 24-hour urine collections during this time, the second completing on the morning of the study. These were to assess daily sodium intake. Subjects abstained from alcohol, caffeine and smoking for 48 hours prior to each study phase and, apart from a light breakfast on the study day, remained fasted throughout each study phase.

All studies were performed at the same time of day in a quiet, temperature-controlled room. The protocol for each study day is summarised in Supplementary figure S1. In brief, after an initial 500ml bolus of 5% dextrose to initiate diuresis, a maintenance infusion (300ml/h) continued throughout the study. Following a 1.5 hours equilibration period, baseline measurements were made over the next half hour, after which placebo or big ET-1 (Clinalfa^®) were administered. Systemic hemodynamic and renal responses were then followed for a period of 2.5 hours after the infusions were commenced. BP, cardiac output, (cardiac index) CI and heart rate were recorded throughout the study by well-validated non-invasive automated techniques^{15, 16} every 15min, and urine was collected every 30min by spontaneous voiding whilst standing. The doses of big ET-1 used here were based on previous studies.^{17, 18}

Alongside systemic hemodynamic and renal responses, pulse wave velocity (PWV), the gold standard for measurement of arterial stiffness,¹⁹ was measured every 30min by the foot-to-foot wave velocity method using the SphygmoCor^® system (SphygmoCor^® Mx, AtCor Medical, Sydney, Australia, version 6.31), in which a high-fidelity micromanometer (SPC-301, Millar Instruments, Texas, USA) was used to determine carotid-femoral PWV.

Sample collection & analysis

Samples of venous blood were collected every 30min into EDTA tubes (Sarstedt) for measurement of plasma ET-1 and osmolality, and into plain tubes (Sarstedt) for serum creatinine and sodium. Additionally, after measuring urine volume, 20ml aliquots from each voiding were collected into plain tubes for the measurement of urinary creatinine, sodium and osmolality. For urine ET-1, a 20ml aliquot of urine was collected into plain tubes with 2.5ml of 50% acetic acid. Blood samples were centrifuged immediately at 2500g for 20min at 4°C. All samples were stored at -80°C until analysis.

Plasma and urine ET-1 were determined by ELISA (R&D systems). The mean recovery of ET-1 was >95%. The intra- and inter-assay variations were 4% and 6%, respectively. The cross-reactivity of the assay was 23% for ET-2, 0.5% for ET-3, and there was no cross-reactivity with big ET-1. Plasma and urine sodium concentrations were measured using an ion-selective electrode. Urine calcium concentration was measured by flame photometry (BWB Technologies UK). Plasma and serum osmolality was measured by freezing point depression using a standard osmometer. Plasma vasopressin concentration was determined by ELISA (Enzo labs). The concentration of total urinary nitrite and nitrate (NOx) was measured by colorimetric assay (780001; Cayman Chemicals, Ann Arbor, MI, USA).

Preparation of urinary extracellular vesicles (uEVs)

In five subjects, uEVs were prepared by ultracentrifugation as previously described.^{20, 21} Urine samples were taken from the collection made between 90 and 120min following treatment with placebo or big ET-1. This was the time point at which maximal natriuresis and FWC were seen.

Immunoblotting

Immunoblot analyses were conducted with the experimenter blinded to the treatment received. The primary antibodies were rabbit anti-AQP2 (AB3274, Millipore, 1:600), sheep anti-NKCC2 (DSTT Dundee, 1:10000) and rabbit anti-NCC (AB3553, Millipore, 1:1000); the secondary antibodies were HRP-conjugated goat anti-rabbit-Ig (sc-2301, Santa-Cruz, 1:2000) and HRP-conjugated donkey anti-sheep Ig (A3415, Sigma, 1:20000). Immunoblot analyses were conducted with the experimenter blinded to the treatment received (hence, the lack of systematic lane order). In the densitometry analysis, band density was divided by the time taken to collect the urine sample used for uEV preparation so that each result represents the abundance of antigen excreted per unit time.

Data analysis

Data were stored and analyzed in Graph Pad Prism, version 6.0 (Graph-Pad Software Inc. San Diego, California). Systolic and diastolic BP at each time point was calculated as the mean of 2 recordings. Mean arterial pressure (MAP) was calculated as diastolic BP + 1/3 pulse pressure. Bioimpedance data at each time point were calculated as the mean of 4 recordings, each the average of 15 consecutive heart beats. Data were corrected for body surface area to give CI, for direct comparison between subjects. Systemic vascular resistance index (SVRI) was calculated by dividing MAP by CI and expressed in dyne/s/m²/cm⁵/100. Urinary sodium excretion (UNaV) and ET-1 excretion were calculated as (urinary sodium x urinary flow rate) and (urinary ET-1 x urinary flow rate), respectively. The fractional excretion of sodium (FeNa) and ET-1 (FeET-1) were calculated as [(urine sodium/serum sodium x serum creatinine/urine creatinine) x 100]% and [(urine ET-1/plasma ET-1 x serum creatinine/urine creatinine) x 100]%, respectively. FWC was calculated as [urine flow rate x 1-urine osmolality/plasma osmolality].

Statistical analysis

Baseline hemodynamic data were calculated as the mean of the 2 time points that immediately preceded administration of the study drug. For urine data, only one baseline measurement was used immediately prior to drug dosing. Hemodynamic and urine results are expressed as mean ± SEM change from baseline for drug and placebo. Statistical analysis was performed on untransformed data. Responses were examined by repeated measures ANOVA, and Bonferroni correction was used to assess significance at specific time points. Statistical significance was taken at the 5% level.

Results

All 10 subjects fully completed the placebo and big ET-1 phases of the study without adverse events. All subjects had similar baseline 24-hour urinary sodium excretion on each study day. Subject demographics and baseline parameters are shown in Table 1.

Table 1. Baseline study participant data.

Values are given as mean of baseline pre-treatment periods over the 2-study days ± SD (range). BMI: body mass index; bpm: beats per minute; CI: cardiac index; DBP: diastolic BP; SBP: PWV: pulse wave velocity; systolic BP; SVRI: systemic vascular resistance index.

Parameter	n = 10
Age, years	31 ± 10 (20 – 49)
Sex (M/F)	8/2
BMI, kg/m2	23.7 ± 1.9 (20.4 – 27.0)
SBP, mmHg	123 ± 9 (112 – 134)
DBP, mmHg	63 ± 9 (49 – 72)
Pulse, bpm	63 ± 8 (48 – 72)
SVRI, dyne/s/m2/cm5	1796 ± 355 (1420 – 2400)
CI, l/min1/m2	4.4 ± 0.8 (3.5 – 5.3)
PWV, m/s	5.4 ± 0.9 (4.1 – 6.5)
Serum creatinine, mg/dl	0.92 ± 0.11 (0.77 – 1.09)
24h urine sodium, mmol/d	89 ± 15 (56 – 105)
Plasma ET-1, pg/ml	1.2 ± 0.4 (0.3 – 1.6)
Urine ET-1 excretion, pg/min	0.87 ± 0.42 (0.27 – 1.42)

Plasma & urinary ET-1

Placebo was not associated with any changes in plasma or urinary ET-1 (Figures 1A-1D). Infusion of big ET-1 led to a ~1.5-fold increase in circulating ET-1 but only after the highest dose (Figure 1A). Plasma ET-1 gradually fell after the infusion of big ET-1 stopped. In parallel with this increase in circulating ET-1 there was a gradual ~2.5-fold rise in urinary ET-1 excretion from 0.78 to 1.97pg/min (Figure 1B). There were 4-fold increases in fractional excretion of ET-1 (FeET-1) from 0.6 to 2.4% (Figure 1C) and urine ET-1/creat from 0.05 to 0.20pg/µmol (Figure 1D).

Figure 1. Change in plasma and urinary ET-1.

Change from baseline ± SEM in plasma ET-1 (A), urinary ET-1 excretion (B), fractional excretion of ET-1 (FeET-1) (C) and urine ET-1/creat following treatment with placebo (blue line) and big ET-1 (red line). **p <0.01, ***p <0.001 and ****p <0.0001 for placebo vs. big ET-1 (ANOVA plus Bonferroni correction for significance at specific time points).

Systemic hemodynamics

Neither placebo nor big ET-1, at any of the 3 doses infused, were associated with changes in systolic or diastolic BP, SVRI or CI (Figures 2A-2D). Whereas placebo was not associated in any change in heart rate, infusion of big ET-1 led to an early and sustained fall in heart rate over the course of the study – a maximal fall of ~8bpm (Figure 3A). In keeping with the lack of change in BP, there was no change in PWV with either placebo or big ET-1 (Figure 3B).

Figure 2. Changes in systemic hemodynamics.

Change from baseline ± SEM in systolic BP (SBP) (A), diastolic BP (DBP) (B), systemic vascular resistance index (SVRI) (C) and cardiac index (CI) (D) following treatment with placebo (blue line) and big ET-1 (red line).

Figure 3. Changes in heart rate and arterial stiffness.

Change from baseline ± SEM in heart rate (A) and pulse wave velocity (PWV) (B) following treatment with placebo (blue line) and big ET-1 (red line). *p <0.05 for placebo vs. big ET-1 (ANOVA plus Bonferroni correction for significance at specific time points).

Renal responses

Compared to placebo, big ET-1 infusion was associated with a gradual increase in UNaV (Figure 4A). This was maximal at 120min, 30min after the completion of the highest dose of big ET-1, and equated to ~40µmol/min. There was no change in creatinine clearance, plasma concentration of cystatin C or potassium excretion with either placebo or big ET-1 over the time course of the study (Supplementary figures S2A-S2C).

Figure 4. Changes in renal responses.

Change from baseline ± SEM in urinary sodium clearance (UNaV) (A), fractional excretion of sodium (FeNa) (B), free water clearance (FWC) (C) and urinary excretion of nitric oxide metabolites (UNOxV) (D) following treatment with placebo (blue line) and big ET-1 (red line). *p <0.05, **p <0.01 and ****p <0.0001 for placebo vs. big ET-1 (ANOVA plus Bonferroni correction for significance at specific time points).

As UNaV may be affected by more subtle changes in intra-renal hemodynamics that may not be reflected by the creatinine clearance, we calculated FeNa as a measure of tubular sodium handling. Placebo was not associated with any change in FeNa over the time course of the study. By comparison, big ET-1 led to a marked natriuresis with a doubling of FeNa from 0.5 to 1.0% (Figure 4B). Placebo did not affect FWC. However, FWC did increase with big ET-1, but only after the highest dose – an increase from 4.1 to 5.5ml/min (Figure 4C). Interestingly, there was a positive correlation between the maximal change in FeET-1 and the maximal increase in FWC (r = 0.83, p =0.003) such that those subjects showing the greatest increase in renal ET-1 production had the greater increase in FWC (Supplementary figure S3A). There was a trend for this association between FeET-1 and FeNa (r = 0.64, p =0.05, Supplementary figure S3B). The summed urinary excretion of nitrates and nitrites (UNOxV) increased following the administration of big ET-1 (Figure 4D), compatible with increased renal production of nitric oxide.

In a subset of subjects (n = 5), we explored the molecular mechanisms responsible for the increased FWC induced by big ET-1. Big ET-1 tended to increase the plasma concentration of vasopressin (Figure 5A). There was also a trend to an increase in the abundance of aquaporin 2 (AQP2) and the bumetanide-sensitive sodium-potassium-chloride co-transporter (NKCC2) (Figure 5B). There was no significant difference in the abundance of the thiazide-sensitive sodium-chloride co-transporter (NCC) (Figure 5B).

Figure 5. Molecular mechanisms regulating free water clearance.

Change from baseline ± SEM in plasma concentration of vasopressin (A). Effect of big ET-1 on the abundance of AQP2, NKCC2 and NCC in uEVs, assessed by immunoblot (B). For AQP2, bands were detected at 28-35 and 40-55kDa, corresponding to the non-glycosylated and glycosylated forms, respectively. Urine samples from subject number 5 were not available in sufficient quantity to prepare uEVs for the NCC blot.

Additionally, we performed a more detailed clearance analysis in this subset. Thus, subjects were maintained in hypotonic diuresis, and in this context the fractional delivery of sodium to the thick ascending limb (TALH) can be estimated by [CH₂O + CNa]/100ml GFR, and the fraction of this sodium load that is reabsorbed in the diluting segment (approximating to the TALH) by [CH₂O/(CH₂O + CNa) ×100]%.^{22, 23} The effects of big ET-1 on these parameters are shown in Supplementary figure S4. Big ET-1 increased the measure of sodium reabsorption in the diluting segment, but had no effect on the estimate of fractional sodium delivery to the TALH. Urinary calcium excretion was lower following big ET-1 but this did not reach significance: 0.103 ± 0.006 vs. 0.083 ± 0.009 mg per min (placebo vs. big ET-1; p = 0.078).

Finally, as we were unable to discriminate between ET_A and ET_B mediated effects in the current study and in order to highlight the clinical importance of our findings we analysed FWC in one of our previous studies.²⁴ Here, in both healthy volunteers and those with CKD, we have demonstrated that selective ET_B receptor antagonism, but not selective ET_A or mixed ET_A/B blockade, diminishes the capacity to clear free water (Supplementary figure S5).

Discussion

In this study we have shown, for the first time in man, that big ET-1 stimulates renal salt excretion. This is independent of changes in systemic hemodynamics, arterial stiffness and eGFR and so most likely reflects a direct action of ET-1 on the renal tubule.

The effects of big ET-1 are likely mediated through its intra-renal conversion to ET-1

It is unlikely that the effects seen in our study are due to big ET-1 directly, as radio-ligand binding studies have established that big ET-1 does not bind to either ET_A or ET_B receptors, nor does it have any other recognized binding site.²⁵ Furthermore, the hemodynamic effects seen following big ET-1 infusion in man fully depend on its conversion to ET-1.²⁶ In the current study, infusion of big ET-1 led to a large rise in circulating ET-1 in keeping with vascular conversion as seen previously.²⁷ Furthermore, given that once formed ET-1 is likely to bind to its receptors from which it dissociates slowly, plasma ET-1 concentration is probably an underestimate of its production. The exact site of the vascular conversion of big ET-1 to ET-1 remains unclear but is unlikely to be in whole human blood²⁸ or in the vascular endothelium.²⁹ The extracellular surface of the smooth muscle cell has been identified as a plausible location for this conversion.³⁰

It seems likely that the increase in urinary ET-1 excretion was due to the intra-renal conversion of big ET-1 to ET-1 for two reasons. First, this can be predicted from the existing literature. Isotope studies in animals³¹ and clearance studies in man³² have shown that ET-1 in the urine is likely to be generated within the kidney (rather than being filtered or secreted into the urinary space from the plasma). ECE isoforms are expressed in the renal microvascular and the tubular epithelium.^{3, 33} Second, urine ET-1 excretion (measured as net excretion or as fractional excretion of the filtered load) increased before any detectable rise in circulating ET-1 (i.e. when big ET-1 was infused at lower doses). This short time course also suggests that ET-1 was likely to be generated from the intra-renal conversion of big ET-1 to mature peptide rather than de novo generation. Our data are consistent with a previous smaller study in healthy man that suggested an increase in renal ET-1 production following big ET-1 infusion.³⁴

Lack of significant hemodynamic effect

Importantly, big ET-1 did not affect systemic hemodynamics at any of the doses given. There was no detectable change in systolic or diastolic BP. There was no evidence of systemic vasoconstriction, given the lack of change in systemic vascular resistance or cardiac output. We did observe a fall in heart rate, the magnitude of which was similar to that seen in earlier studies where big ET-1 has been infused in human subjects.^{18, 34} This is unlikely to be a baroreceptor reflex since BP remained constant. Thus, this negative chronotropy likely reflects a direct effect of ET-1 on the heart, an effect previously reported in pre-clinical studies.³⁵ We observed no change in creatinine clearance or plasma cystatin C, both estimates of GFR, over the time course of the study, suggesting that there was no significant change in renal hemodynamics at the doses of big ET-1 used. For comparison, a previous study that infused big ET-1 into healthy humans and found significant changes in systemic and renal hemodynamics used a dose that was ~2-fold greater than the highest dose used here (~600pmol given intravenously over 20min compared to 300pmol given over 30min here).³⁴ Others have administered a similar dose of big ET-1 to that used in the current study to patients with end-stage renal disease and this did indeed increase BP (and reduce splanchnic blood flow) but this likely reflects the fact that patients with renal disease have an activated ET system and are more sensitive to its effects.^{4, 24}

However, we cannot exclude regional changes in renal cortical or medullary blood flow (which are not readily assessable in man). Such changes are recognized following infusion of ET-1 in animals^{36, 37} and may affect tubular handling of salt and water. Indeed, we found that the excretion of NO metabolites was increased following big ET-1. In rodent models, ET-1 has been shown to increase renal NO production (through its action on ET_B receptors in the collecting duct),³⁸ which elicits diuresis and natriuresis.^{39, 40} These effects of NO may be mediated, in part, by changes in regional blood flow within the kidney.⁴¹

Natriuretic effect of big ET-1

As hypothesized, big ET-1 infusion led to a significant natriuresis as shown by the increases in both UNaV and FeNa of ~40% from baseline. If this were maintained over a 24-hour period it would amount to ~60mmol of sodium excreted in the urine and is broadly equivalent to the natriuresis elicited by a single 25mg dose of spironolactone.⁴² Our subjects were healthy and relatively sodium restricted (with a sodium intake of ~90mmol Na or ~5g NaCl per day, around 50% of that contained in a standard Western); it is unclear whether the natriuretic effect of ET-1 would be different on a high salt diet, or in patient populations in which there is activation of the renin-angiotensin-aldosterone system (RAAS), such as in congestive heart failure, CKD or liver cirrhosis. This would be of particular interest, because maximizing renal salt and water excretion is an important focus of clinical management in these patient groups.

In animal models, ET-1 inhibits sodium reabsorption along the length of the renal tubule.^{3, 43} Our data strongly suggest that the natriuretic effect of big ET-1 is not localised to the TALH. Indeed, clearance data [CH₂O/(CH₂O + CNa) point to increased sodium reabsorption in this segment, consistent with the ~35% increase in FWC and the trend to reduced urinary calcium excretion.^{22, 23}

These functional data are supported by our molecular data showing that the abundance of NKCC2 on uEVs was increased by big ET-1 (and furthermore that the observed increase in AQP2 expression occurred in a direction that would oppose free water clearance). Enhanced transport in the TALH sits at odds with the established literature; ET-1 has been consistently shown to inhibit NKCC2 activity (via ET_B receptors acting through NO-dependent and NO-independent pathways).^{3, 44} This apparent conflict may be explained by our finding that big ET-1 increased the concentration of circulating vasopressin (another established ET-1 effect).⁴⁵ Vasopressin increases the renal expression of both NKCC2 and AQP2 and we speculate that this indirect effect of big ET-1 over-rides any direct effect of ET receptor activation in the TALH. As overall FeNa increased, if sodium reabsorption were no different in the proximal tubule or DCT and stimulated in the TALH, then sodium reabsorption must have been suppressed elsewhere in the nephron (i.e. connecting tubule or collecting ducts). We attempted to assess the abundance of ENaC subunits in urinary vesicles but were unable to generate detectable bands on Western blot (presumably due to the low abundance of antigen in urine samples that were rendered dilute by the water-loading protocol).

ET_B receptors are likely to mediate aquauresis and natriuresis

Pre-clinical studies have demonstrated that big ET-1⁴⁶ and ET-1³ promote natriuresis and diuresis through an ET_B receptor-mediated, NO-dependent, mechanism. In the present study, we did not attempt to directly differentiate between ET_A and ET_B-mediated effects. However, in a separate cohort of healthy volunteers and patients with CKD we have demonstrated that ET_B blockade diminishes the capacity to clear free water. Thus, the results of our present study are consistent with big ET-1 acting (after conversion to ET-1) on ET_B receptors to increase free water clearance. Furthermore our finding of increased excretion of NO metabolites supports an ET_B-mediated effect.

Perspectives

In healthy volunteers, sub-pressor doses of big ET-1 induced natriuresis and aquauresis, effects likely mediated through through renal tubular ET_B receptors (after intra-renal conversion to mature ET-1). Our clearance and molecular data suggest that sodium reabsorption was stimulated by ET-1 in the TALH, but inhibited in distal nephron segments. Taken together with the pre-clinical literature, our data support the potential use of highly selective ET_A receptor antagonists in clinical conditions associated with salt and water retention. In line with this, pre-clinical studies in congestive heart failure supported a selective ET_A blocking approach.⁵ Long-term clinical studies, however, have been disappointing. However, all have used non-selective antagonists or modestly ET_A selective antagonists at doses that probably block the ET_B receptor. Therefore, it may well be that a truly ET_A selective approach has not yet been studied. Fortunately, studies using a selective approach in CKD have been encouraging^{14, 47} and the outcomes of a large phase 3 study (SONAR) are eagerly awaited.⁴⁸ As an alternative to selective ET receptor blockade, our data provide a rationale for testing potassium-sparing diuretics (rather than loop diuretics) in an attempt to ameliorate the fluid retention associated with ET receptor antagonists. Although this strategy has not, to our knowledge, been tested prospectively, a post-hoc analysis of the ARIES trial found that co-prescription of spironolactone with ambrisentan was associated with better outcomes in pulmonary arterial hypertension.⁴⁹

Novelty & Significance.

What is new?

• This is the first study to explore the effects of big ET-1 on renal sodium and water handling in man.

• Our study protocol allowed us to determine the effects of big ET-1 on the kidney without significant hemodynamic perturbation.

What is relevant?

• Big ET-1 infusion elicited a clinically significant natriuresis and diuresis.

• Renal clearance data and a molecular analysis of sodium transporter expression in urinary extracellular vesicles suggest that big ET-1 inhibits sodium reabsorption through ENaC in the collecting ducts.

• Endothelin receptor antagonists have been associated with edema and heart failure in clinical trials. Our results suggest this adverse effect is a consequence of stimulated sodium reabsorption in the collecting ducts.

Summary.

In healthy, infusion of big endothelin-1 elicited a natriuresis (via inhibition of sodium reabsorption in the renal collecting ducts). Blockade of this pathway may account for the edema and heart failure observed with the clinical use of endothelin receptor antagonists. Our data support the use of potassium-sparing diuretics as a therapeutic strategy to ameliorate fluid retention associated with these agents.