Apelin and Copeptin Levels in Patients With Chronic SIAD Treated With Empagliflozin

Abstract Background Empagliflozin increases sodium levels in patients with a chronic syndrome of inappropriate antidiuresis (SIAD), and dapagliflozin increases apelin levels in patients with diabetes mellitus. Exogenous apelin increases sodium levels in rats with SIAD. We aimed to investigate whether an increase in plasma apelin concentration may contribute to the efficacy of empagliflozin in SIAD. Methods Post hoc secondary analysis of a double-blind, crossover, placebo-controlled trial performed from December 2017 to August 2021 at the University Hospital Basel, Switzerland, investigating the effect of 4-week treatment with empagliflozin 25 mg/day as compared to placebo in 14 outpatients with chronic SIAD (NCT03202667). The objective was to investigate the effect of empagliflozin on plasma apelin and copeptin concentrations and their ratio. Results Fourteen patients, 50% female, with a median [interquartile range] age of 72 years [65–77] were analyzed. Median apelin concentration was 956 pmol/L [853, 1038] at baseline. Median [interquartile range] apelin relative changes were +11% [0.7, 21] and +8% [−5, 25] (P = .672) at the end of the placebo and empagliflozin phases, respectively. Median copeptin concentration was 2.6 [2.2, 4.5] pmol/L at baseline and had a relative change of +5 [−2. 11]% and +25% [10, 28] (P = .047) over the placebo and empagliflozin phases, respectively. Conclusion Empagliflozin did not lead to significant changes in apelin or the apelin/copeptin ratio in patients with chronic SIAD but led to an increase in copeptin. This suggests that the efficacy of empagliflozin in SIAD is independent of apelin and is not blunted by the adaptative increase in copeptin.

Hyponatremia is the most common electrolyte disorder encountered in clinical practice [1] and is associated with increased morbidity and mortality [2].The syndrome of inappropriate antidiuresis is one of the most common causes of hyponatremia [3].It is characterized by hypotonic euvolemic hyponatremia and hemodynamically and osmotically inappropriate increased arginine vasopressin (AVP) activity.This results in the inability to produce dilute urine and in free water retention [3].
Apelin is an endogenous peptide hormone produced in several organs and tissues, such as the brain, the heart, the vascular endothelium, and the kidneys [4][5][6], and is involved in cardiovascular regulation and energy metabolism as well as water homeostasis [7][8][9][10].The latter is regulated by apelin and AVP in opposing ways: apelin promotes diuresis and vasodilatation whereas AVP promotes antidiuresis and vasoconstriction [11].This functional crosstalk is mirrored by the colocalization of both hormones and their respective receptors.AVP and apelin are secreted from neurosecretory cells of the hypothalamic supraoptic and paraventricular nuclei and apelin receptors [12] and arginine vasopressin receptors (AVPR)1a/1b are expressed by magnocellular AVP neurons [13].In the kidneys, apelin receptors are found in the glomeruli and in all nephron segments including the collecting duct where AVPR2 are also expressed [14,15].In states of water depletion and hyperosmolality, AVP is released by the posterior pituitary.It then binds to AVPR2 on principal cells in the renal collecting duct, activating the cAMP/protein kinase A pathway and leading to insertion of aquaporin 2 in the apical membrane and free water retention [16].In a state of hypoosmolality, apelin acts in opposition by inhibiting the secretion of AVP from the posterior pituitary gland [17], increasing renal blood flow, and negating the effect of AVP on the kidneys [18].In lactating rats, intravenous apelin administration reduces aquaporin 2 insertion in the apical membrane of the collecting duct cells by inhibiting the intracellular production of cAMP and calcium influx, thus increasing diuresis and reducing urine osmolality [19].
Patients with syndrome of inappropriate antidiuresis (SIAD) have an altered balance between apelin and copeptin with disproportionately increased levels of copeptin, an equimolar surrogate marker of AVP [20], while levels of apelin are only slightly increased [21].An abnormal apelin/vasopressin balance may thus contribute to water retention in patients with SIAD [21].In support of this, a study in rats with SIAD showed that reestablishing a physiological apelin/copeptin ratio with the administration of exogenous apelin increases urinary output and plasma sodium concentration [22].Whether this approach is also effective in patients with SIAD is currently being investigated in a double-blind placebo-controlled crossover study (NCT06277336).
We demonstrated in 2 randomized double-blind placebocontrolled trials that the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin is a promising treatment option for patients with SIAD [23,24].One month of treatment with empagliflozin led to a sodium increase of 4 mmol/L in comparison to placebo [24].Others have shown that the SGLT2 inhibitor dapagliflozin increased apelin levels by up to 18% in patients with type 2 diabetes mellitus and heart failure [25].To our knowledge, the effect of SGLT2 inhibitors on the concentration of apelin and apelin/copeptin ratio in patients with SIAD is unknown.We hypothesized that SGLT2 inhibitors increase apelin levels in SIAD.To investigate this, we performed a secondary analysis of our crossover trial in outpatients with chronic SIAD [24] and aimed to characterize the course of apelin levels and of the apelin/copeptin ratio during 4 weeks of treatment with empagliflozin.

Study Design and Participants
This is a post hoc secondary analysis of a prospective randomized, crossover, double-blind, placebo-controlled trial performed at the University Hospital Basel, Switzerland from December 2017 to August 2021.The competent ethics committee (EKNZ 2017-00701) and the national agency for the authorization and supervision of therapeutic products (Swissmedics 2017DR2127) approved the study protocol and study medication.The study was registered at ClinicalTrials.gov(NCT03202667) and complied with the principles of the Declaration of Helsinki.
The investigational medical products consisted of encapsulated empagliflozin 25 mg and matched to a visually corresponding placebo.Patients were randomly allocated to 28 days treatment with empagliflozin followed by 28 days treatment with placebo or vice versa.Further treatment included limitation of daily fluid intake to ≤1.5 L/day.Further details can be found in the initial report of the study [24].

Laboratory Markers
Fasting blood samples were drawn between 8:00 and 11:00 AM, and fresh serum and plasma aliquots were stored at −80 °C until batch analysis.Apelin was measured using a commercial enzyme-linked immunosorbent assay kit (Phoenix Pharmaceuticals Cat# EK-057-23, RRID:AB_3096967) with a lower limit of detection 0.07 ng/mL and intra-and interassay variation of <10% and <15%, respectively.Copeptin was measured with the CE certified automated immunoassay BRAHMS Copeptin proAVP assay (Thermo Fisher Scientific B.R.A.H.M.S Cat# 857050N, RRID:AB_3073917) with a coefficient of variation within-laboratory precision of approximately 9.8% and a coefficient of variation for reproducibility of 7%.Baseline values correspond to the first visit of the first treatment phase.The apelin/copeptin ratio was calculated by dividing the apelin by the copeptin concentration.

Study Objectives and Outcomes
The primary objective was to investigate the effect of empagliflozin on apelin concentration.Secondary objectives were to investigate the effect of empagliflozin on copeptin levels and on the apelin/copeptin ratio as well as to identify predictors for changes in apelin.
The primary outcome was the absolute change in plasma apelin concentration (pmol/L) at the end of the empagliflozin phase compared to the end of the placebo phase.Secondary outcomes were the relative and absolute changes in apelin concentration, copeptin concentrations, and apelin/copeptin ratio after 1 and 4 weeks of treatment; the relationship between apelin and sodium concentrations; and the adjusted relationship between empagliflozin and the changes at week 4 in apelin concentrations, copeptin concentrations, and their ratio.

Statistical Analysis
Baseline characteristics are summarized using descriptive statistics.Discrete variables are expressed as frequencies [percentage (%) and number of patients (n)].Continuous variables are expressed as median and interquartile range (IQR).Changes in apelin and copeptin are represented graphically with boxplots.
Absolute and relative changes in apelin, copeptin, and their ratio were compared using a paired Wilcoxon signed-rank test.The relationships between apelin, apelin/copeptin ratio, and sodium concentrations were investigated by computing Spearman correlation coefficients.The association between changes in apelin, copeptin, and their ratio and empagliflozin was further investigated by fitting a linear mixed-effects model [26,27] with changes in the laboratory parameters as the outcome variable, patients as the random effect, and the following fixed effects: the respective baseline value, age, sex, and treatment with an angiotensin converting enzyme (ACE) inhibitor or angiotensin II receptor blocker (ARB).As there was no evidence for either carryover or sequence effect, treatment sequence and study phase were not included in the final models.A linear mixed regression model was used to analyze changes in sodium levels, for which details are found in the main report of this study [24].
All analyses were performed using the statistical program R (version 4.2.3 [28]).A two-sided significance level of 0.05 was used for every analysis and there was no correction for multiple testing.

Discussion
Our analysis has 2 main findings.First, 4 weeks treatment with empagliflozin had no effect on plasma apelin concentration.Second, copeptin-the surrogate marker of AVP-increased by a median of 25% upon empagliflozin, but this increase did not correlate with changes in plasma sodium.To our knowledge, we provide the first cross-over data on the effect of empagliflozin on apelin, copeptin, and apelin/ copeptin ratio in patients with chronic SIAD.Apelin inhibits AVP pituitary secretion and AVP-mediated antidiuresis and thus acts as its antagonist in salt and water homeostasis [18].A cross-sectional study by Blanchard et al showed a higher copeptin to apelin ratio despite higher apelin concentrations in patients with SIAD as compared to healthy controls [21], suggesting not only increased AVP activity but also insufficiently increased apelin contributes to antidiuresis in these patients.In their study, apelin levels were more than 3 times lower and apelin/copeptin ratio about 8 times lower than the baseline values of our cohort.This is probably due to the discrepancy of the assays used in the 2 studies [30].Sex differences in apelin levels remain elusive.One study in healthy volunteers and patients with chronic kidney disease (CKD) found 60% higher plasma apelin concentrations in women than in men [15], and another study showed that female patients with osteoarthritis had higher apelin levels than men [31].In contrast, we did not find a sex difference in SIAD patients, and Blanchard and colleagues also found comparable apelin levels in healthy men and women of more than 60 years of age (age group of our cohort).In comparison, apelin concentrations were higher in younger men than in younger women in their study [21].
The therapeutic potential of increasing apelin levels and thus restoring a physiological apelin to copeptin ratio is supported by a recent preclinical study, in which the exogenous subcutaneous administration of a long-acting apelin-17 analogue in rats with SIAD increased urinary output and plasma sodium concentrations [22].A recent uncontrolled intervention study in 153 patients with type 2 diabetes mellitus and heart failure showed that 6 months of treatment with the SGLT2 inhibitor dapagliflozin led to an 18% increase in apelin levels [25].Furthermore, colocalization of apelin, its receptor, and SLGT2 has been demonstrated in the human kidney [15].We therefore hypothesized that a similar increase in apelin could occur in patients with SIAD treated with empagliflozin and could thus contribute to augment diuresis in a glucose-independent manner.Contrary to our hypothesis, we found no difference in apelin levels at the end of the 4-week treatment with empagliflozin as compared to the 4-week treatment with placebo (Fig. 1).These differing findings could originate from the use of different assays and the difference in treatment duration and the administered Median [interquartile range] concentration of plasma apelin concentrations, plasma copeptin, and their ratio at baseline and over the 2 treatment phases.The apelin/ copeptin ratio was calculated by dividing apelin concentration by copeptin concentration for each patient.SGLT2 inhibitor.In addition, the increase in apelin in patients with heart failure might have been elicited by improved cardiac function [25].Moreover, our hypothesis that patients with higher apelin concentrations would have higher sodium concentrations at baseline and that a greater increase in apelin during treatment would lead to a greater sodium increase was also not confirmed by our data.
SIAD is characterized by an increase in AVP activity, which either relies on increased entopic or ectopic AVP secretion or increased AVP renal sensitivity depending on its etiology [32,33].Measurement of the AVP-surrogate marker copeptin is the gold standard to diagnose AVP-deficiency [34,35] but has negligeable diagnostic value in hyponatremia because of an important overlap between etiologies, confounders increasing copeptin levels like stress and illness, and the high variability of copeptin levels in SIAD [36][37][38].The latter is the reason why median baseline copeptin levels in our analysis (2.6 pmol/L) were much lower than in hospitalized patients with profound SIAD in 1 of our previous observational studies (11.9 pmol/L) [36].The lower copeptin levels might also be due to the milder hyponatremia.However, we found no correlation between baseline copeptin and baseline sodium concentrations.Moreover, a third of the cohort had a drug-induced SIAD, in which drugs can directly stimulate AVPR2 and in which therefore, copeptin levels are expected to be low [33].We observed an increase in copeptin at the end of the 4-week treatment with empagliflozin (Fig. 1), which we interpret as a hemodynamic AVP response secondary to glucose-induced osmotic diuresis.Importantly, we found no correlation between copeptin increase and sodium increase; therefore this increase in copeptin does not seem to blunt treatment efficacy.An increase in copeptin as an adaptive response to SGLT2 inhibitors is consistent with the findings of our first 4-day placebo-controlled trial in hospitalized patients with SIAD [23,39] and previous studies in type 1 and type 2 diabetic patients under SGLT2-inhibitor treatment [40,41].
The apelin system (apelin and its receptor) is currently being investigated as a possible treatment target for CKD and its associated cardiovascular diseases (ClinicalTrials.gov:NCT03956576) as it counteracts the renin-angiotensinaldosterone system (RAAS), reduces blood pressure, and has an anti-inflammatory and antifibrotic effect [10,42].Whether an apelin agonist could offer additional benefits to current renoprotective treatments such as RAAS-and SGLT2-inhibitiors would be of great interest.We observed a tendency toward a greater apelin change with empagliflozin in SIAD patients already treated with RAAS inhibitors.One could hypothesize that combining RAAS and SGLT2 inhibition could offer additional synergistic benefits through an increase in apelin.Dual RAAS and SGLT2 inhibitor therapy has already demonstrated benefits in terms of disease progression and survival in patients with CKD and albuminuria [43].It is known that treatment with SGLT2 inhibitors has a plethora of favorable effects (pleiotropic effects) [44]: cardiorenal protection, blood pressure and albuminuria reduction, lipid profile alteration, effects in myocardial metabolism [45], and even modulation of inflammatory response in acute myocardial infarction [46].Further studies are needed to confirm the effect of a double inhibition of RAAS and SGLT2 on the apelin system and whether there is a resulting apelin increase associated with the positive effects of this combination therapy.Furthermore, clinical studies are needed to show whether a combination of RAAS inhibition, SGLT2 inhibition, and apelin agonism have an additive, clinically relevant effect in patients with cardiovascular disease and CKD.
Our analysis has several limitations.First, our sample size was modest and not powered for the current analysis.Second, the 4-week treatment might not have been long enough to lead to significant changes in apelin.Nevertheless, the main strength of our study relies in its cross-over and double-blind placebo-controlled design, providing a high internal validity in a difficult cohort of multimorbid and polymedicated patients.

Conclusion
Treatment with empagliflozin in patients with chronic SIAD did not lead to significant changes in plasma apelin concentrations but led to an increase in copeptin, which did not blunt its efficacy in increasing sodium levels.Our findings do not support an empagliflozin-mediated increase in apelin to be involved in its efficacy in treating SIAD.Despite apelin not being influenced by SGLT2 inhibition in this setting, its therapeutic potential in treating SIAD remains intact and should be investigated in further research, including its changes following the use of other SIAD treatments, particularly vaptans, given the interplay between AVP and apelin.The ongoing double-blind placebo-controlled trial (ESCAPE Trial, ClinicalTrials.gov:NCT06277336) will provide additional insights into the effect of apelin administration in SIAD.

Figure 1 .
Figure 1.Changes in apelin and copeptin by treatment.Changes in laboratory parameters were computed by calculating the relative change from baseline to week 1 and 4 respectively.Values above the dashed line represent an increase and values below the dashed line a decrease from baseline.