# EVATRAN (The Effect of Eplerenone on the Evolution of Vasculopathy in Renal Transplant Patients): study protocol for a cross-over randomized controlled trial

Arnaud Simon, Nicolas Girerd, Frédéric Jaisser, Erwan Bozec, Xavier Lepage, Ludovic Mercklé, Patrick Rossignol, Luc Frimat, Sophie Girerd

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## To cite this version:

Arnaud Simon, Nicolas Girerd, Frédéric Jaisser, Erwan Bozec, Xavier Lepage, et al., EVATRAN (The Effect of Eplerenone on the Evolution of Vasculopathy in Renal Transplant Patients): study protocol for a cross-over randomized controlled trial. Trials, 2025, 26 (1), pp.479. 10.1186/s13063-025-09187-w. hal-05363318

HAL Id: hal-05363318

https://hal.univ-lorraine.fr/hal-05363318v1

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STUDY PROTOCOL

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# EVATRAN (The Effect of Eplerenone on the Evolution of Vasculopathy in Renal Transplant Patients): study protocol for a cross-over randomized controlled trial

Arnaud Simon¹, Nicolas Girerd² ID, Frédéric Jaisser²,³, Erwan Bozec², Xavier Lepage², Ludovic Mercklé², Patrick Rossignol², Luc Frimat¹ and Sophie Girerd¹,²*

**Abstract**

**Background** Arterial stiffness is a major prognostic factor of cardiovascular (CV) morbi-mortality in kidney transplant (KT) patients. Preclinical studies have demonstrated that the vascular toxicity of calcineurin inhibitors (CNI) is mediated through the activation of the mineralocorticoid receptor (MR) in vascular smooth muscle cells. Additionally, the role of MR in contributing to arterial stiffness is well documented in non-transplanted individuals. This study aims to investigate the impact of MR antagonist treatment on the progression of arterial stiffness in KT patients on cyclosporine.

**Methods** This is a randomized, open-label, single-center, cross-over trial involving 36 stable KT patients who have been transplanted for at least 1 year and are maintained on cyclosporine therapy. After a 4-week run-in period, participants will be randomly assigned to one of two groups. Group A will receive eplerenone at a dose of 50 mg daily for 6 months, followed by a 6-month period without treatment. There will be an 8-week washout phase between the treatment and non-treatment periods. Group B will start with 6 months without treatment, followed by the same 8-week washout phase, and then receive eplerenone for 6 months. The primary outcome is to assess the effect of 6 months of eplerenone on arterial stiffness, measured through pulse wave velocity (PWV) using the Sphygmocor® method at the start and end of each treatment period. Secondary outcomes will include changes in (1) central and peripheral blood pressure profiles, (2) intima-media thickness, (3) left ventricular mass, (4) biomarkers of oxidative stress and endothelial dysfunction, and (5) renal graft function markers, such as proteinuria and creatinine levels. Additionally, the incidence of hyperkalemia and episodes of acute renal failure will be monitored.

**Discussion** CNI are a key component of immunosuppressive therapy in KT patients. By limiting vascular toxicity through MR blockade, CV risk in these patients may be reduced.

**Trial registration** ClinicalTrials.gov identifier: NCT04450953. EudraCT number 2019-004243-74.

**Keywords** Eplerenone, Mineralocorticoid antagonist, Pulse wave velocity, Arterial stiffness, Cyclosporine, Calcineurin inhibitor, Kidney transplant, Cardiovascular

<footnote>*Correspondence:
Sophie Girerd
s.girerd@chru-nancy.fr
Full list of author information is available at the end of the article</footnote>
**Background**

Cardiovascular (CV) mortality is the leading cause of death in kidney transplant (KT) patients [1]. Although transplant patients have better CV outcomes than those on dialysis, their CV mortality remains higher compared to the general population [2].

Pulse wave velocity (PWV), a marker of arterial stiffness, is a key prognostic factor for CV events and mortality post-KT. In a retrospective cohort study by Dahle et al., which included 1040 kidney transplant patients with PWV measurements at 8 weeks post-transplant, each 1 m/s increase in PWV was associated with higher all-cause mortality (HR 1.36, 95% CI 1.14 to 1.62, *p*=0.001) [3]. Additionally, several studies indicate that PWV is linked to an increased risk of CV events up to 5 years after transplantation [4–6]. This underscores the importance of managing arterial stiffness progression in these patients.

Data on the evolution of PWV indicate a decrease in PWV during the first year after KT. A meta-analysis of 12 studies, involving measurements from 473 renal transplant recipients between 6 months and 1 year post-transplant, revealed a reduction in central PWV of 1.20 m/s [7]. Several other studies also support a decrease in PWV during the first year after KT [8–14]. Data on the long-term evolution of post-transplant PWV are limited and more controversial. Few studies have examined PWV beyond 1 year, and those that have suggest a subsequent increase in PWV [15].

The mechanisms underlying arterial stiffness in kidney recipients are multifaceted. They include the pre-transplant history of the patients, such as age, duration of chronic renal failure and dialysis, and comorbidities like hypertension or diabetes. Post-transplant factors also contribute, particularly cardiovascular risk factors associated with immunosuppressive therapy, especially calcineurin inhibitors (CNIs), among which cyclosporine is the most widely used. Since the 1980s, CNI have been a cornerstone of immunosuppressive therapy in organ transplantation. However, they can cause vascular toxicity, either directly through harmful effects on the vascular wall or indirectly by promoting hypertension, diabetes, and dyslipidemia. Moreover, nearly 15% of patients develop post-transplant diabetes (NODAT), primarily due to KT-related factors, particularly immunosuppressive treatments like tacrolimus and corticosteroids [16].

CNI are likely to play a significant role in the persistence of increased arterial stiffness in KT patients, even after renal function has been restored [17]. Additionally, several studies have reported an improvement in arterial stiffness after discontinuation of CNI in favor of alternative treatments, such as mTOR inhibitors [18, 19].

Preclinical studies have shown that the vascular toxicity of cyclosporine is mediated through the activation of the mineralocorticoid receptor (MR) in vascular smooth muscle cells (VSMC) [20, 21]. Amador et al. investigated this by using mice with selective MR deletion in VSMCs and endothelial cells [20]. Their findings suggest that cyclosporine's vascular toxicity is primarily driven by MR activation in VSMCs, which become hyper-activated following cyclosporine administration. This activation leads to protein phosphorylation that contributes to vasoconstriction and the activation of calcium channels, increasing arterial resistance. This study underscores the detrimental role of MR activation in cyclosporine-induced vascular toxicity.

**Hypothesis**

Given that cyclosporine induces MR activation in VSMCs, which may contribute to persistent or worsening arterial stiffness after initial improvements following KT, we hypothesize that eplerenone, as a MR antagonist, could mitigate these vascular effects. This trial aims to investigate, for the first time, the impact of a MR antagonist on the progression of arterial stiffness in KT recipients undergoing cyclosporine therapy.

**Methods/design**

**Study design**

As detailed in version 2.0 (dated 22/06/2022) of the protocol, this study is a prospective, monocentric, randomized crossover blinded endpoint study (PROBE) conducted at Nancy University Hospital. Renal transplant patients on cyclosporine who meet the inclusion criteria will be eligible for participation. Following a run-in phase (25 mg/day of eplerenone for 2 weeks, increased to 50 mg/day for two additional weeks if tolerated), a washout period was introduced to ensure that baseline assessments reflect the untreated state. Patients are then randomly assigned (1:1) to one of two groups: group A receives eplerenone (50 mg/day) for 6 months followed by a 6-month period without eplerenone (with a washout period in between), while group B receives the same sequence in reverse.

**Participants**

**Inclusion criteria**

Graft recipients aged over 50 will be eligible if they meet the following conditions:

* Received kidney transplant at least 1 year prior

* Currently on cyclosporine therapy

* Clinically and biologically stable for at least 3 months (no treatment changes affecting blood pressure, except for adjustments in immunosuppressive ther-
apy dosage; no acute rejection episodes within the past 3 months)

* Estimated GFR ≥ 30 mL/min/1.73 m² (calculated using the CKD-EPI formula)

* Systolic blood pressure (SBP) ≥ 110 mmHg, regardless of antihypertensive therapy

* Signed informed consent for study participation, obtained by the investigators, who are practicing nephrologists directly involved in the care of kidney transplant patients

## Exclusion criteria

Graft recipients will be excluded if they present any of the following conditions:

* Documented serum potassium (kalemia) ≥ 5 mmol/L within the past 15 days

* Current treatment with a mineralocorticoid receptor antagonist

* Current treatment with potassium-sparing diuretics

* Current treatment with digoxin

* Blood bicarbonate level < 20 mmol/L, with or without supplementation, within the past 15 days

* Mandatory indication for combined use of ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs), though each is permitted separately

* Known hypersensitivity or allergy to eplerenone or its excipients

* Severe liver failure

* Current treatment with CYP 3A4 inhibitors (e.g., itraconazole, ketoconazole, ritonavir, nelfinavir, clarithromycin, telithromycin, nefazodone)

* Contraindication to sodium polystyrene sulfonate

* Participation in other interventional research studies

* Women planning pregnancy within the next 15 months

* Women of childbearing age not using highly effective contraception

* Persons listed in Articles L. 1121-5, L. 1121-7, and L. 1121-8 of the Public Health Code

* Individuals deprived of liberty by judicial or administrative decision

* Individuals under psychiatric care as per Articles L. 3212-1 and L. 3213-1

Inclusion and exclusion criteria are verified at the time of study entry, i.e., prior to the run-in period.

## Participant recruitment

Recruitment is performed by the investigators during routine follow-up visits of kidney transplant patients at

the transplant center, ensuring direct access to the target population.

Eligible patients who meet the inclusion and exclusion criteria will be contacted by phone to explain the study, and an information letter will be sent by mail. During a routine post-transplant follow-up visit, patients will receive oral information from their nephrologist, who will answer any questions they may have about the study. Prior to the first study visit, the investigator will provide detailed information about the study and obtain the patient's free, informed, and written consent.

Randomization will be performed using a centralized, computer-generated sequence (1:1 allocation, non-stratified), managed through the dedicated randomization software of the Nancy Clinical Investigation Center CIC. The randomization list will be generated and implemented by the CIC data manager, ensuring allocation concealment. Investigators will have controlled access to the system to randomize eligible patients.

## Intervention

### Timing of administration of the study treatment

The study treatment will be dispensed by the hospital pharmacy at visit 1, marking the start of the run-in phase for all patients. During the randomized period, patients in group A will receive eplerenone 50 mg/day as a single oral dose for 24 weeks. Eplerenone will be dispensed at visits 4 and 5 (V4 and V5). This will be followed by an 8-week washout period, after which there will be a 24-week eplerenone-free period until the end of the study.

Patients in group B will receive eplerenone during the second 24-week period, with the medication dispensed at visits 7 and 8 (V7 and V8) (see Fig. 1).

### Posology change

During the run-in phase, the dosage of eplerenone will be adjusted as follows: initially, 25 mg/day will be administered for the first 2 weeks. If the following conditions are met, the dosage will be increased to 50 mg/day for the subsequent 2 weeks:

* Stability of creatinine levels (no increase of more than 25% compared to baseline)

* Serum potassium levels do not exceed 5.5 mmol/L

* Absence of hypotension

Only patients who successfully tolerated 50 mg/day of eplerenone for at least 2 weeks during the run-in phase were eligible for randomization.

After randomization, if serum potassium levels increase to 5.5–6 mmol/L or creatinine levels rise by 30–50%, the dosage of eplerenone will be reduced to
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Figure of enrollment, intervention, and evaluation</figcaption></figure>

25 mg/day. In cases of severe hyperkalemia (>6 mmol/L) or a creatinine elevation beyond 50%, eplerenone will be discontinued.

After randomization, eplerenone may also be discontinued if:

* Persistent orthostatic hypotension occurs despite adjustments to other antihypertensive medications according to predefined protocols

* There is a suspected allergy to eplerenone or its excipients

* Acute renal failure develops due to intercurrent events

## Premature discontinuation of the study

Patients who do not meet the safety criteria during the run-in phase, or who are not randomized for any other reason, will have their participation in the study discontinued. The investigator may also terminate a patient's participation for any safety-related concerns. In the event of premature discontinuation, patients will continue to be monitored according to standard care practices and established transplant guidelines.

## Appropriate administration of treatment units

At the end of each eplerenone treatment period, participants will be asked to return the remaining medication blisters to the hospital pharmacy to verify treatment

adherence. The remaining tablets will be collected to assess patient compliance.

## Concomitant medication

During the study, patients must not receive potassium-sparing diuretics (such as amiloride, potassium canrenoate, spironolactone, or triamterene) or any oral potassium supplements. The combination of eplerenone with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARBs) is permitted, but a dual blockade with both ACEi and ARBs is an exclusion criterion. Significant pharmacokinetic interactions may occur with CYP3A4 inhibitors, including ritonavir-boosted protease inhibitors, cobicistat, azole antifungals, macrolides, amiodarone, diltiazem, verapamil, nefazodone, and grapefruit; therefore, the use of CYP3A4 inhibitors is contraindicated. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) are contraindicated due to the risk of hyperkalemia. As the impact of eplerenone on patients under cyclosporine is the focus of this study, the combination of eplerenone and cyclosporine is permitted. Family doctors will be informed and instructed not to introduce or increase the dosage of any medication known to induce hyperkalemia or hypotension, or any drugs that are contraindicated during the study period.

## Study outcomes

The primary endpoint is the change in pulse wave velocity (PWV, in m/s), adjusted for blood pressure, measured using Sphygmocor® after 24 weeks of treatment
with eplerenone. The PWV measurement will be per-
formed according to standard methods using Sphygmo-
cor® Analysis. Sphygmocor®-acquired PWV data will be
preferred for the final analysis unless signal quality does
not permit accurate measurement. In such cases, PWV
data acquired using Complior® Analyse will be used. The
operator will be blinded to the randomization arm.

The carotido-femoral distance (L), sterno-femoral dis-
tance, and sterno-carotid distance are initially estimated
with a tape measure. Arterial landmarks are identified
at the sites of femoral and carotid pulse palpation, as
well as at the sternal notch. The aorto-femoral distance,
which accounts for the anatomical configuration of the
aortic arch, is approximated by subtracting the distance
between the sternal notch and the femoral measurement
site (inguinal arch) from the distance between the carotid
measurement site and the sternal notch.

The propagation time (*t*, in ms) of the carotido-femo-
ral pulse wave is measured by the Sphygmocor® device
with a probe placed sequentially at the carotid and femo-
ral pulses. Complior® Analyse measures the propagation
time using two sensors positioned simultaneously at the
carotid and femoral pulses.

The carotido-femoral PWV (m/s) is calculated by both
devices according to the ratio between the direct carot-
ido-femoral distance (L) *0.8 and the carotido-femoral
propagation time (t) using the formula: PWV = L*0.8/(t).
The PWV will be measured at the beginning and end of
each period for both groups (V4, V6, V7, V9).

Secondary outcomes include measurements of:

1. Central systolic blood pressure, central diastolic blood pressure, central pulsed pressure, index of increase measured by Sphygmocor® (Atcor Medical) applanation tonometry

2. Peripheral systolic blood pressure, peripheral dias-
tolic blood pressure, pulsed pressure (peripheral
systolic blood pressure – peripheral diastolic blood
pressure)

3. Intima-media thickness measured by Echotracking (ART.LAB, ESAOTE®)

4. Left ventricular mass measured by cardiac ultra-
sonography

5. Biological markers of oxidative stress (plasma iso-
prostane and malondialdehyde (MDA))

6. Biological markers of endothelial dysfunction (endothelin, soluble endothelium selectin (sE-selectin), von Willebrand factor)

7. Graft function as measured by blood creatinine (in µmol/L) with estimation of glomerular filtration rate (eGFR in mL/min/1.73 m²) according to the CKD-EPI formula, as well as proteinuria as measured by the ratio proteinuria/creatininuria (in mg/g)

8. The occurrence and frequency of hyperkalemia dur-
ing follow-up, defined by potassium levels as follows:
5.0–5.49 mmol/L, 5.5–6.0 mmol/L, and >6.0 mmol/L

9. The risk of acute renal failure defined as an increase in creatinine of more than 50%

Study visits and run-in phase

For patients meeting the eligibility criteria at the inclu-
sion visit, the run-in period will start the day following
enrollment, following this sequence (see Fig. 2):

1. Administration of eplerenone: 25 mg per day (one tablet) for 2 weeks.

2. Telephone contact with the medical investigator: at 2 weeks (visit 2, V2) to assess tolerance. If clinical and biological tolerance is confirmed, the dosage will be increased to 50 mg/day.

3. Follow-up contact or consultation: at 4 weeks (visit 3, V3) to evaluate tolerance and compliance at the 50 mg/day dosage.

Tolerance to eplerenone will be assessed by weekly creatinine and potassium measurements, performed in certified private medical laboratories where participants attend for blood sampling. Only patients who exhibit a creatinine increase of 30% or less (from the start of the run-in phase) and have a potassium level of <5.5 mmol/L while receiving eplerenone 50 mg daily for 2 weeks, and who have not used any potassium binders, will be randomized.

As shown in Fig. 3, patients will attend regular visits that include a medical interview, clinical examination, automated blood pressure measurement, and standard biological samples (V1, V4, V5, V6, V7, V8, and V9). During the run-in phase, visits 2 and 3 will be conducted by telephone. Primary and secondary endpoints (criteria 1 to 6) will be assessed at the beginning and end of each period during visits V4, V6, V7, and V9 at the Nancy Clinical Investigation Center, University Hospital of Nancy.

As patients are directly followed by their treating physi-
cians within the transplant center, participant retention is
expected to be maximal.

Adverse effects

A moderate and transient increase in potassium lev-
els and creatinine is expected after starting eplerenone.
These levels will be monitored weekly during the run-in
period. It is important to note that if a potassium binder
is required or if eplerenone needs to be discontinued
during this phase, the patient will be ineligible for rand-
omization. The investigator will contact patients 14 days
<table><caption>**Fig. 2** Enrollment and run-in period</caption><thead><tr><th rowspan="3">TIME POINT</th><th colspan="5">RUN IN PERIOD</th><th rowspan="3">Wash out</th></tr><tr><th>Enrolment</th><th colspan="3">Visit 2</th><th rowspan="2">Visit 3</th></tr><tr><th>Visit 1</th><th>- 8 W</th><th>- 7 W</th><th>- 6 W</th><th>- 5 W</th><th>- 4 W</th><th>4 W</th></tr></thead><tbody><tr><td><strong>ENROLMENT</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Eligibility screen</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Informed consent</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Physical exam</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Medical history</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Kalemia, creatinine, bicarbonate, Level of cyclosporine</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- proteinuria / creatininuria ratio</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Beta HCG blood test</td><td>X</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>RUN IN PERIOD</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Eplerenone 25 mg/day</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Eplerenone 50 mg/day</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- Kalemia, creatinine, bicarbonate</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td></tr><tr><td>- Level of cyclosporine</td><td></td><td></td><td></td><td>X</td><td>X</td><td></td></tr><tr><td>- Blood pressure self measurment</td><td></td><td></td><td></td><td>X</td><td>X</td><td></td></tr></tbody></table>

after the initiation of treatment (visit 2) and again 14 days after any dosage increase (visit 3) to collect self-measured blood pressure readings and to assess any symptoms related to hypotension.

After randomization, blood potassium and creatinine levels will be monitored at least once a month. Serious adverse events (SAEs) will be recorded according to Good Clinical Practice (GCP) standards and in compliance with French regulations. Suspected unexpected SAEs or reactions must be reported by the attending physician to the sponsor immediately upon discovery. Investigators are responsible for documenting all adverse events, whether serious or non-serious, in the electronic case report form (e-CRF) throughout the patient's follow-up. Adverse events will be recorded until the end of the study. Eplerenone has a short half-life (approximately 4 h), allowing for prompt discontinuation in the event of an unanticipated adverse event.

## Sample size and power calculation

We estimate that the study intervention will result in a decrease in pulse wave velocity (PWV) by 0.2 m/s, which corresponds to 25% of the difference in PWV

observed between patients on cyclosporine and those who switched from cyclosporine to mTor inhibitors [19]. Ignace et al. reported that the variance of the observed PWV difference between pre-kidney transplantation (KT) and 3 months post-KT was 50% greater than the observed difference [9]. This estimate is conservative, as the standard deviations of PWV differences in their study ranged from 50 to 100% of the mean difference (–5.5%±2.2% in younger patients and 2.1%±1.9% in older patients). To detect a PWV difference of 0.2 m/s with a variance of 0.3 m/s, an alpha risk of 5%, and a beta risk of 10% (power of 90%), 24 patients are needed. Anticipating a 10% randomization rate for non-analyzable patients and a 20% rate of failure to complete the run-in phase, a total of 36 patients should be recruited.

## Statistical analysis

The primary outcome will be analyzed using a bilateral paired *t*-test to compare the pulse wave velocity (PWV) at the beginning and end of each study period. The normality of the distribution of the PWV changes (deltas) between the two periods will be assessed. If the distribution is not normal, a Wilcoxon signed-rank
<table><caption>**Fig. 3 Randomization, timing of administration of the study treatment, and primary outcome measurement**</caption><thead><tr><th rowspan="3">TIME POINT</th><th colspan="8">STUDY PERIOD</th><th rowspan="3">End of study</th></tr><tr><th colspan="2">Wash out</th><th colspan="3">Allocation</th><th colspan="3">Wash out</th></tr><tr><th>4 W</th><th>Visit 4</th><th>Visit 5</th><th>Visit 6</th><th>8 W</th><th>Visit 7</th><th>Visit 8</th><th>Visit 9</th></tr></thead><tbody><tr><td>ALLOCATION</td><td colspan="8">X</td></tr><tr><td>INTERVENTION</td><td colspan="8"></td></tr><tr><td>- Group A : Eplerenone 50 mg/day</td><td colspan="8"></td></tr><tr><td>- Group B : Eplerenone 50 mg/day</td><td colspan="8"></td></tr><tr><td>ASSESMENT</td><td colspan="8"></td></tr><tr><td>- Medical history, physical exam</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td></tr><tr><td>- Blood pressure</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td></tr><tr><td>- Creatinine, bicarbonate, kalemia</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td></tr><tr><td>- Level of cyclosporine</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td></tr><tr><td>- Kalemia (group B)</td><td></td><td></td><td></td><td></td><td>X</td><td></td><td></td><td></td></tr><tr><td>- Beta HCG blood test</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td><td></td></tr><tr><td>- Proteinuria/creatinuria ratio</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td></tr><tr><td>Blood and urine sample biobanking</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td></tr><tr><td>Pulse wave velocity (primary end point)</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td></tr><tr><td>Intima-media thickness</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td></tr><tr><td>Peripheral and central blood pressure, index of increase</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td></tr><tr><td>Cardiac ultrasonography</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td></tr><tr><td>Adverse event</td><td colspan="8"></td></tr></tbody></table>

test will be employed. Additionally, the interaction between the sequence of interventions and the effect of the treatment will be examined using a linear or non-linear mixed-effects model, incorporating a random effect for the patient.

For secondary outcomes with continuous measures, hypotheses will be tested using a bilateral paired t-test if the distribution of deltas is normal, or a Wilcoxon signed-rank test if it is not. Differences in proportions for dichotomous variables will be evaluated using a bilateral McNemar test, while variables with three or more levels will be analyzed using a chi-square test. No interim analysis is planned. Analyses will be conducted using an intention-to-treat approach, with a per-protocol sensitivity analysis performed as a secondary step.

## Ethical issues

### Ethics committee approval

This study follows the principles outlined in the Declaration of Helsinki and complies with Good Clinical Practice (GCP). The trial has been approved by the French health authorities (Agence Nationale de Sécurité du Médicament et des Produits de Santé, ANSM) and by the ethics committees (Comités de Protection des Personnes, CPP).

## Information and consent forms

Patients are included based on written information and by signing an informed consent form (see Additional file 2). The informed consent form clearly states that patients can withdraw from the study at any time without affecting their current or future medical treatment.

All planned analyses of biological samples are included in the initial informed consent signed by participants.
## Interim analyses and study termination rules

An independent Data Safety and Monitoring Board (DSMB) consisting of four international experts in kidney transplantation and biostatistics will oversee the safety of the trial and monitor research progress in accordance with the protocol. Serious adverse events (SAEs) will be reviewed during DSMB sessions, with the board blinded to the randomization arm. No interim analyses are planned. The DSMB has the authority to terminate the trial early for the following reasons: detection of adverse events, poor data quality, inadequate implementation, fraud, or new information that would render the trial unnecessary, futile, or unethical.

## Data quality and regulatory issues

### Monitoring of the study

The sponsor of the study, Nancy University Hospital (Research Department, Nancy University Hospital, Rue du Morvan, 54500 Vandoeuvre-les-Nancy; phone: +33 3.83.15.52.85, fax: +33 3.83.15.74.51), will oversee the study through audits and quality control measures to ensure adherence to the protocol and that data is collected accurately, completely, and in a timely manner. Amendments to the study trial will be handled and communicated to the investigation by the sponsor. All adverse events will be reported to the sponsor's pharmacovigilance, as well as to the ANSM and CPP for any unexpected serious adverse events. The sponsor will provide an annual safety report to the French regulatory agencies and ethics committees.

In accordance with French law, participants are covered by the sponsor's insurance (Nancy University Hospital), which provides compensation in case of harm related to trial participation. No specific ancillary or post-trial care is planned beyond routine clinical follow-up.

### Data management of the study

Study data will be collected using electronic case report forms (e-CRFs) created with Ennov Clinical® Electronic Data Capture (EDC) software v7.5. Remote access to the e-CRFs will be available via a web browser to authorized users only, each with specific rights and personal login credentials. Data quality will be maintained through programmed inconsistency tests. The database will be locked once all discrepancies have been resolved. Data will be provided to the statistical team in SAS 9.4 (SAS Institute, Cary, NC, USA) format for analysis, under the guidance of the trial's methodologist. In light of the mono-center nature of the trial, a data monitoring committee was not created, and the data management was performed by one person (LM).

Participant data will be de-identified and securely stored in e-CRFs and subsequent databases, with access limited to authorized personnel within the sponsor's secure informatic infrastructure. Data will be made available upon reasonable request to the sponsor and principal investigator, provided the steering committee approves the scientific purpose of the query and the sponsor validates it in compliance with applicable regulations.

Participant data will be de-identified using a unique study code. The identification key will be stored separately in a secured, access-restricted file, and all analyses will be performed on de-identified datasets.

### Authorship eligibility

The manuscript will be written entirely by the investigators, ensuring that all authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship. No professional writers will be employed in the preparation of the manuscript.

### Dissemination policy

Trial results will be communicated through peer-reviewed publications and submitted to the ANSM. Results will also be posted on ClinicalTrials.gov (in addition to all World Health Organization Trial Registration items—aligning with the 2.0 version of the protocol, dated 22/06/2022) to ensure transparency and accessibility for participants, healthcare professionals, and the public.

## Discussion

### Challenge of CNI treatment in the current epidemiological context of kidney transplantation

Over the past decades, the number of KT recipients has risen, along with the average age of both recipients and donors (https://rams.agence-biomedecine.fr/greffe-renale-0). Arterial stiffness, partly attributed to the toxicity of CNIs, significantly contributes to CV mortality among these patients [3]. CV death remains the leading cause of mortality among KT patients, with a risk that remains significantly higher compared to the general population [22, 23].

Although arterial stiffness appears to decrease within the first year after KT [4, 7] some studies suggest that this initial improvement may be followed by subsequent stiffening. Kim et al. observed that 52.4% of patients at 1 year post-transplant had arterial stiffness (PWV) levels comparable to those before KT, which remain higher than those of the general population [4]. Desjardins et al. found that while the stiffness index—a marker of arterial stiffness—initially decreased within the first 3
months after transplantation, it increased again at 6 and 24 months [24]. Age-related changes likely continue to play a significant role, but the long-term deterioration in arterial stiffness suggests the influence of additional risk factors associated with KT. Several studies indicate that CNIs may have a detrimental effect on arterial stiffness [5, 17].

In recent decades, the average age of cadaveric kid-
ney donors has significantly increased (https://rams.
agence-biomedecine.fr/greffe-renale-0) with a rise in
the number of expanded-criteria kidney donors (ECD).
Delahousse et al. [25] reported that donor age is an inde-
pendent factor affecting PWV at 1 year in KT recipients
from deceased donors. Specifically, PWV was found to be
1 m/s higher in recipients of kidneys from donors over
the age of 53, the majority of whom were ECD donors.

Finally, the increasing age of KT recipients and the
associated burden of comorbidities, enhancing CV
prognosis in these patients, presents a significant pub-
lic health challenge. This elevated risk underscores the
importance of targeted CV risk management strategies in
the specific population of older KT recipients [22, 23].

Choice of inclusion criteria

Cyclosporine remains widely used in transplantation despite tacrolimus being the preferred CNI in Europe and the USA. Cyclosporine may be particularly beneficial for patients at low immunological risk but at high risk for post-transplant diabetes, such as obese individuals. The DIRECT trial demonstrated that the incidence of NODAT 6 months post-KT was significantly lower in patients treated with cyclosporine compared to those treated with tacrolimus (26.0% versus 33.6%, *p* = 0.046) [16]. However, cyclosporine appears to have greater vascular toxicity than tacrolimus [17, 26]. Preclinical models have shown that cyclosporine directly affects mineralocorticoid receptor in vascular smooth muscle cells in mice [20].

Only KT patients over 50 years of age will be included
in this study. Research indicates that in the general popu-
lation, the increase in arterial stiffness with age becomes
more pronounced after 50 years, suggesting that pulse
wave velocity (PWV) is a more sensitive marker of age-
related arterial stiffness in individuals over this age [27].

Thus, we chose to include only KT patients who are
beyond 1 year post-transplantation. During the first year
after KT, changes in PWV can be influenced by various
confounding factors, such as improved blood pressure
control, correction of renal failure (uremia), and the reso-
lution of chronic overload and inflammation associated
with dialysis. While there is limited data on the long-
term evolution of aortic stiffness post-transplant, a study
by Desjardins et al. suggests an initial reduction in stiff-
ness index 3 months after KT, followed by a subsequent

increase at 6 and 24 months [24]. Additionally, using
eplerenone is likely safer after 1 year, once renal function
has stabilized, thereby reducing the risk of acute renal
failure related to rejection.

Justification of the choice of MRA, dosing and timing
of drug administration

Among the available MRAs, we chose to administer
eplerenone in this study due to its superior selectivity for
the mineralocorticoid receptor compared to spironolac-
tone [28]. This selectivity reduces the risk of certain side
effects, such as gynecomastia.

We decided to administer eplerenone at a dosage of 50 mg/day. Randomized controlled trials (RCTs) in heart failure after myocardial infarction have used similar dosing regimens: 25 mg/day of spironolactone in the RALES study [29] and 25–50 mg/day of eplerenone in the EPHE-SUS [30] and EMPHASIS-HF studies [31]. Thus, administering eplerenone twice daily should be both effective and safe (see dedicated section).

Eplerenone will be administered for 6 months in each
group during the study period. A randomized trial dem-
onstrated a reduction in pulse wave velocity (PWV) with
linagliptin after 6 months, independent of blood pres-
sure, in diabetic patients [32]. London et al. randomized
40 hypertensive hemodialysis patients to a 4-month
treatment with either nitrendipine or placebo, show-
ing a significant reduction in carotido-femoral PWV
(-2.15 m/s [95% CI -3.6; -0.71], p < 0.01) [33]. Therefore,
it is reasonable to expect that administering eplerenone
for 6 months could similarly lead to a reduction in PWV.

Justification of the primary outcome

We selected carotid-femoral PWV measurement as the
primary endpoint. Numerous studies have underscored
the importance of arterial stiffness as a risk factor for CV
disease in the general population [34, 35]. Additionally,
PWV has been identified as an independent risk factor
for CV mortality in various populations, including hyper-
tensive patients [36] and those with chronic renal failure
[37, 38]. Arterial stiffening also independently predicts
CV events in KT patients, as demonstrated by several
prospective studies [5, 6].

Carotid-femoral PWV is considered the gold standard for assessing arterial stiffness, according to the European Expert Consensus on Arterial Stiffness (2006) [39] due to its extensive epidemiological evidence supporting its predictive value for CV events and its relatively simple technical requirements. Among various measures of arterial stiffness, only carotid-femoral PWV, which reflects the stiffness of the central arteries, has demonstrated a predictive value for morbidity and mortality.
## Safety concerns

The potential risks of eplerenone in KT patients include hyperkalemia and acute renal failure. However, the limited data available suggest that eplerenone is generally safe for use in this population.

Firstly, in a study of 31 renal transplant patients who had been on cyclosporine for over a year with an estimated GFR of 30 to 50 mL/min, and 61% of whom were treated with ACE inhibitors or ARBs, Bertocchio et al. reported that eplerenone administered at a dose of 25 mg/day for 8 weeks presented a low risk of hyperkalemia (>6 mmol/L) were reported in this study. Additionally, a clinical trial involving pediatric renal transplant patients with an estimated GFR greater than 60 mL/min investigated the impact of 25 mg/day of eplerenone versus placebo over 6 months. The study focused on the progression of allograft nephropathy as confirmed by biopsy [41]. The authors reported a higher estimated GFR at the end of the follow-up period in the 13 children receiving the MR antagonist compared to the 10 children on placebo, although the difference was not statistically significant due to limited statistical power. These results suggest a potential nephroprotective effect of MR antagonists in renal transplant patients. Additionally, the authors noted that there were no significant variations in potassium levels with this dosage regimen.

Finally, Mortensen et al. recently reported results from the SPIREN RCT, which included 188 kidney transplant (KT) patients randomized to receive either spironolactone (25 to 50 mg/day) or placebo for 3 years [42]. Plasma potassium levels increased significantly in the spironolactone group, with increases ranging from 0.3 [95% CI, 0.2 to 0.4] to 0.4 [95% CI, 0.2 to 0.5] mEq/L. This increase was of moderate clinical significance, and no adverse events specifically related to hyperkalemia were reported. However, four patients required a reduction in their study drug dosage due to hyperkalemia (potassium levels > 5.5 mEq/L), and all of these cases occurred in the spironolactone group.

Therefore, current literature suggests that the increase in potassium and creatinine levels due to MRAs in KT patients is generally small. In the present study, EVATRAN, potassium and creatinine levels will be closely monitored throughout the study period, with appropriate corrective measures implemented if hyperkalemia or acute renal failure occurs. Additionally, the run-in phase will help exclude patients for whom the risk of side effects appears to be too high.

## Clinical implications

We hypothesize that eplerenone, by blocking the mineralocorticoid receptor (MR), will reduce arterial stiffness

in KT recipients under cyclosporine beyond 1 year post-transplant. Identifying an intervention that can effectively reduce CV morbidity and mortality is a crucial challenge in kidney transplantation.

Rodriguez et al. systematically reviewed studies evaluating pharmacologic interventions in patients with end-stage renal disease to assess their effects on pulse wave velocity (PWV) [43]. They reported that management of hypertension with calcium channel blockers may reduce aortic stiffness versus placebo, but the quality of evidence was very low. However, there are not any clinical trials in transplant recipients aiming to assess whether therapeutic interventions would reduce arterial stiffness.

CNIs remain the first-line therapeutic class used in KT due to their efficacy in preventing rejection and their lower cost compared to alternatives such as belatacept. If MR antagonists can mitigate the vascular toxicity associated with cyclosporine, this could lead to a reconsideration of cyclosporine's role in transplantation. Although tacrolimus is more effective in preventing rejection, it is associated with an increased risk of de novo diabetes after transplantation. Consequently, cyclosporine may be particularly beneficial for patients at high risk of diabetes, such as those who are obese, but who have low to moderate immunological risk.

## Conclusion

This study aims to evaluate the impact of treatment with a mineralocorticoid receptor antagonist on the progression of arterial stiffness in renal transplant recipients under cyclosporine. If a beneficial effect of eplerenone on arterial stiffness is demonstrated in this study, it could pave the way for a larger multi-center clinical trial to further explore its therapeutic potential in this population.

## Trial status

The necessary regulatory authorizations have been obtained. Recruitment began in November 2021. The last patient was included in September 2024. The last follow-up is expected on November 15, 2025.

## Abbreviations

<table><tr><td>CV</td><td>Cardiovascular</td></tr><tr><td>MR</td><td>Mineralocorticoid receptor</td></tr><tr><td>VSMC</td><td>Vascular smooth muscle cells</td></tr><tr><td>CNI</td><td>Calcineurin inhibitors</td></tr><tr><td>PWV</td><td>Pulse wave velocity</td></tr><tr><td>NODAT</td><td>New onset diabetes after transplantation</td></tr><tr><td>CIC</td><td>Clinical Investigation Center</td></tr><tr><td>CKD</td><td>Chronic kidney disease</td></tr><tr><td>ACEi</td><td>Angiotensin converting enzyme inhibitor</td></tr><tr><td>ARBs</td><td>Angiotensin receptor blockers</td></tr><tr><td>SAE</td><td>Serious adverse event</td></tr><tr><td>CRF</td><td>Case report form</td></tr><tr><td>DSMB</td><td>Data Safety and Monitoring Board</td></tr><tr><td>RCTs</td><td>Randomized controlled trials</td></tr><tr><td>ECD</td><td>Expanded-criteria kidney donors</td></tr></table>
**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s13063-025-09187-w.

Additional file 1: SPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents.

Additional file 2: Informed consent materials.

**Acknowledgements**

This work was supported by INI-CRCT. The authors thank the CRB Lorrain for the EVATRAN biobank management.

**Authors' contributions**

AS participated in the design of the study, is co-principal investigator of the study, and contributed to the drafting of the manuscript. SG participated in the design of the study, is co-principal investigator of the study, and contributed to the drafting of the manuscript. NG participated in the design of the study (statistical analysis) and contributed to the drafting of the manuscript. FJ participated in the design of the study and contributed to the revising of the manuscript. EB participated in the design of the study (primary outcome measurement) and contributed to the drafting of the manuscript. XL participated in the design of the study and contributed to the drafting of the manuscript. LM participated in the design of the study (randomization and data management) and contributed to the revising of the manuscript. PR participated in the design of the study and contributed to the revising of the manuscript. LF contributed to the revising of the manuscript. All authors read and approved the final manuscript.

**Funding**

The EVATRAN study was supported by a grant from the French Ministry of Health (Contrat de Programme de Recherche Clinique mai 2019). The authors are supported by a public grant overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-15-RHU-0004), and GEENAGE Impact Lorraine Université d'Excellence (reference: ANR-15-IDEX-04-LUE).

**Data availability**

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

**Declarations**

**Ethics approval and consent to participate**

The trial has been approved by the French regulatory agency (Agence nationale de la sécurité du medicament et des produits de santé: ANSM) and the appropriate ethics committee (Comité de Protection des Personnes: CPP). Written informed consent will be obtained from all participants by investigators.

**Consent for publication**

All authors have read and approved the final submitted version of this manuscript.

**Competing interests**

FJ received a research grant from Bayer and AstraZeneca. NG received honoraria from AstraZeneca, Bayer, Boehringer, Lilly, Novo Nordisk, Novartis, and Roche Diagnostics. The remaining authors declare that they have no competing interests.

**Author details**

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