Inhibition of Calcium Release-Activated Calcium Channels to Treat Acute Kidney Injury: Design and Rationale of the KOURAGE Study a Randomized Trial Open Access

Acute kidney injury (AKI) is a syndrome characterized by a sudden decrease in the glomerular filtration rate, clinically recognized by an increase in serum creatinine and/or a decrease in urine output [1]. AKI occurs in up to 20% of hospitalized patients in high income countries and its development heralds a risk of mortality that increases with more severe AKI stages [2], and critically ill patients with AKI who require kidney replacement therapy (KRT) have in-hospital mortality rates of ≥50% [3]. Classification of the Kidney Disease Improving Global Outcomes (KDIGO) stages of AKI is provided in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000546863). Additionally, patients with AKI are twice as likely to require short-term or long-term care following hospital discharge than patients without AKI [3]. AKI often leads to the development of pulmonary edema, both cardiogenic (“high pressure”) and/or non-cardiogenic (“low pressure”), caused by pulmonary capillary injury and leak. In the setting of AKI, cardiogenic pulmonary edema typically results from fluid retention and volume overload in the setting of impaired kidney function, potentially complicated by new or worsening impairment of left ventricular function related to retained solutes and the uremic milieu. Non-cardiogenic pulmonary edema is often seen in conjunction with the hyperinflammatory state, which may be caused by the same insult that caused AKI (e.g., sepsis).

Conversely, pulmonary capillary permeability may be precipitated or exacerbated by distant organ injury, including AKI. Specifically, isolated AKI leads to the increased production of the transcription factor NF-kB, as well as the increased production and release of inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and IL-17; these mediators are thought to cause distant organ injury (including acute lung injury and myocardial apoptosis). Furthermore, markedly elevated serum concentrations of IL-17 were noted in critically ill patients with AKI stages 2 and 3 compared to levels in patients without AKI, and serum IL-17 concentrations were directly associated with in-hospital mortality and major adverse kidney events (MAKE) [4].

The injurious link between the kidney and lung is bidirectional (Fig. 1). Among patients with acute respiratory distress syndrome (ARDS), two-thirds develop AKI, and half progress to KDIGO stage 3 AKI. The development of stage 2 or 3 AKI in a patient with ARDS portends a 30-day mortality ≥50% [5]. In the setting of ARDS, AKI may develop in part because of the effects of positive pressure mechanical ventilation on renal blood flow and renal compensatory neurohormonal mechanisms, elevated pulmonary pressures with resulting venous congestion and renal edema, and the systemic release of inflammatory cytokines from the lung. The synthesis and release of cytokines such as IL-17, IL-6, and TNFα that ultimately damage renal tubules are induced by sepsis as well as extracellular nucleotides that are released by injured pulmonary epithelial cells [6]. Taken together, there is evidence of substantial injurious lung and kidney “crosstalk” that may necessitate management strategies to alleviate clinical or subclinical injury in both organ systems. The current randomized, placebo-controlled phase 2 trial will evaluate the safety and efficacy of Auxora™ in patients with KDIGO stage 2 or 3 AKI and AHRF, with a primary efficacy endpoint of days alive, ventilator-free, and KRT-free from the start of the first infusion of study drug through day 30.

KOURAGE (ClinicalTrials.gov identifier: NCT06374797) is a double blind, randomized, placebo-controlled study (Fig. 2) that will be conducted at up to 40 sites in the USA and Ireland. The study will be conducted in accordance with the ICH Harmonized Guideline for Good Clinical Practice, applicable local regulations, and the ethical principles laid down in the Declaration of Helsinki. Written informed consent to participate in the study will be provided by each patient or legally authorized representative prior to screening.

The study will enroll approximately 150 adult patients recruited across the USA and Ireland with severe AKI (KDIGO stages 2 and 3) and AHRF, defined as a PaO₂/FiO₂ (P/F) ≤ 300) in patients receiving invasive or noninvasive mechanical ventilation, or treated with high flow nasal cannula at a minimum flow rate ≥ 30 L/min. Severe AKI (KDIGO stages 2–3) is defined as a doubling or more of the serum creatinine concentration from baseline, or oliguria defined by urine output <0.5 mL/kg/h for ≥ 12 h. The P/F ratio will be determined by either an arterial blood gas or imputed from the oxygen saturation (SpO₂) recorded using pulse oximetry (online suppl. Table S2) [7]. Patient enrollment criteria are provided in Table 1.

After eligibility and informed consent have been confirmed, patients will be screened. Table 2 outlines all assessments that will be performed during screening. Following screening, patients will be randomized 1:1 to receive either Auxora (n = 75) or a matching placebo (n = 75), using a computer-generated randomization scheme accessed through an interactive voice/web response system.

Study drug infusion should commence within 24 h of the patient or their legal representative signing the consent forms. Patients will receive a total of five doses of study drug, as outlined in Table 3 and Figure 2. Dosing will be based on actual body weight at the time of hospitalization, if available, or the recorded weight closest to the time of hospitalization. The upper limit of the volume of Auxora or placebo administered will be 156.25 mL.

Study assessments will be conducted according to online supplementary Table S3. Each patient’s respiratory status will be assessed daily using an 8-point ordinal scale (Table 4). Assessments of the level of vasopressor support, amount of urine output, and the use of RRT will also be performed daily using the Renal and CV Ordinal (RENCON) scale The RENCON scale consists of a numerical scale from 1 to 7 together with an alphabetical subscale from a to c (Table 5). In addition, the estimated glomerular filtration rate (eGFR) will be determined at day 90 using both serum creatinine and cystatin C concentrations.

The primary efficacy endpoint is the number of days alive, ventilator-free and KRT-free from initiation of study drug infusion through day 30. Secondary efficacy endpoints (Table 6) are MAKE 90-1: ≥ 25% decline in eGFR from baseline, incident KRT, and all-cause mortality at 90 days; MAKE 90-2: ≥ 35% decline in eGFR from baseline, incident KRT, and all-cause mortality at 90 days; the proportion of patients alive at day 30; the proportion of patients alive at day 90; the number of days alive and ventilator-free from start of first infusion of study drug through day 30; the number of days alive and KRT-free from start of first infusion of study drug through day 30; the proportion of patients recovered from AHRF through day 30 as categorized by an 8 point ordinal scale; the proportion of patients receiving KRT at day 30; and the proportion of patients receiving KRT at day 90.

Safety will be monitored by assessing adverse events (AEs) along with clinical laboratory tests (hematology and chemistries), and vital signs (including temperature, heart rate, systolic and diastolic blood pressures, and respiratory rate). All AEs will be recorded starting after randomization through day 90.

Patients may request to discontinue administration of the study drug at any time. Investigators may also discontinue administration of the study drug to a patient because of an AE or change in medical status that raises a safety concern about the patient receiving additional doses of the study drug. Treatment discontinuation will be defined as an investigator or patient discontinuing administration of study drug before all five doses are administered, even if the patient remains in hospital. Patients who do not receive all five doses because the treating physician discharged them from the hospital will not be considered to have treatment discontinuation.

We will strongly encourage that AKI be managed according to the KDIGO 2012 guidelines [1], which recommend maintaining adequate organ perfusion, avoiding volume overload, avoiding hyperglycemia, discontinuing nephrotoxic agents, and adjusting dosing of renally excreted medications. If a patient develops worsening complications of AKI (e.g., fluid overload, hyperkalemia, acidemia), KRT should be considered. As detailed in online supplementary Tables S4 and S5, we will strongly encourage that acute hypoxemic respiratory failure/ARDS be managed according to the 2023 European Society of Intensive Care Medicine major recommendations [8].

Patients randomized in this study will receive conservative IV fluid strategies such as that described in the Fluid and Catheter Treatment Trial (FACTT) LITE [9]. Patients will receive pharmacological prophylaxis as per local standard of care to prevent the development of venous thromboembolic disease. Nonsteroidal anti-inflammatory drugs, renin-angiotensin-aldosterone system inhibitors, and sodium-glucose cotransporter 2 inhibitors will generally be avoided during the 90-day study period. We will record the name, dose, and frequency of concomitant medications administered during the study.

To estimate the sample size, we utilized a parallel, 2-group design (with a hierarchical composite endpoint) to test whether the win ratio is different from 1 (H₀: WR = 1 versus H₁: WR≠1, where WR is the win ratio: P_ᴡɪɴ/P_ʟᴏss). To determine the level of statistical significance, we will incorporate a 2-sided, natural-log-based, win ratio Z-test, with a type I error rate (α) of 0.05. The probability of a tie is assumed to be 0.25. To detect a win ratio of 2 (P_ᴡɪɴ = 0.5, P_ʟᴏss = 0.25) with 80% power, 75 patients will be required in each group (150 patients in total).

Stratification factors will include use/non-use of invasive mechanical ventilation and stage 3 AKI versus stage 2 AKI.

Efficacy analyses will be performed on the modified intent-to-treat population (mITT), which will consist of all randomized patients who received one or more doses of study drug. Safety analyses will also be performed on the mITT data set.

The primary efficacy analysis will apply the win-ratio method [10] and the Finkelstein-Schoenfeld method [11] to the primary endpoints. The win-ratio method allocates all Auxora and placebo pairs to the components that comprise the primary endpoint in a hierarchical fashion and within each stratum of use of invasive mechanical ventilation and stage 3 AKI.

Categories (a) and (c) represent Auxora wins based on all-cause mortality and days ventilator-free, and KRT-free. Similarly, categories (b) and (d) represent placebo wins. Category (e) represents ties, pairs in which patients were not able to be differentiated.

• (a)

  Death on placebo, but alive on Auxora at day 30

• (b)

  Death on Auxora, but alive on placebo at day 30

• (e)

  Death on placebo and death on Auxora at day 30

  • If alive on placebo and alive on Auxora at day 30

• (c)

  Greater number of days alive, ventilator-free, and KRT-free on Auxora

• (d)

  Greater number of days alive, ventilator-free, and KRT-free on Placebo

• (e)

  Equal number of days alive, ventilator-free, and KRT-free

We will calculate the overall win ratio by adding (a) + (c) for all strata and dividing it by the sum of (b) + (d) across all strata. We will summarize the win ratio and 95% confidence intervals between Auxora and placebo treatment groups and calculate p values based on the Finkelstein-Schoenfeld method [11].

We will summarize the proportion of patients who experience discrete endpoints (e.g., MAKE 90-1, MAKE 90-2) by treatment group, and compare proportions using a Cochran-Mantel-Haenszel (CMH) test stratified by use of invasive mechanical ventilation and by stage 3 AKI. We will calculate an odds ratio and corresponding 95% confidence interval with logistic regression, with the treatment group as the independent variable stratified by the same factors.

We will summarize days alive and ventilator-free and days alive and KRT-free from initiation of study drug through day 30 using mean ± standard deviation or median with 10%, 90% range, and compare these values using analysis of variance, with treatment group, use of invasive mechanical ventilation, and stage 3 AKI as fixed effects in the model. We will also summarize days alive and ventilator free from start of first infusion of study drug through day 30, and days alive and KRT-free from start of first infusion of study drug through day 30; both will be defined as 0 if the patient dies before day 30. We will consider 2-sided p values <0.05 as statistically significant without adjustment for multiplicity. If the primary endpoint is not significantly different between groups, we will consider all analyses comparing secondary endpoints with p < 0.05 as nominally significant. We will conduct all analyses using SAS version 9.3 or a more recent version (SAS Institute Inc., Cary, NC, USA).

Safety data will be tabulated by the treatment group and presented for all treated patients. Exposure to the study drug, reasons for discontinuation, deaths and causes of deaths will be tabulated. Summaries will be provided for all AEs, AEs considered related to study drug, serious adverse events, and related serious adverse events, and these summaries will be based on the Medical Dictionary for Regulatory Activities. Laboratory test results will be summarized. The percentage of 30-day all-cause mortality will be calculated based on each patient’s date of death relative to start of first infusion of study drug, tabulated by treatment group.

CalciMedica monitoring and auditing procedures will be implemented and aligned with the ICH Harmonized Guideline for Good Clinical Practice, applicable local regulations, and the ethical principles listed in the Declaration of Helsinki. A CONSORT 2010 checklist was provided in online supplementary Table S6. Monitoring will be conducted either in person or remotely to ensure the study is conducted according to the protocol. An Independent Trial Steering Committee comprised of the authors of this publication, all experienced investigators in AKI and critical care, contributed to the study design and will provide ongoing oversight of the trial, including interfacing with site investigators and other personnel. An Independent Data Monitoring Committee (IDMC) will monitor patient safety and make recommendations concerning study conduct. Responsibilities of the IDMC will be governed according to a separate charter.

Patients with severe AKI who have associated AHRF have a high risk of mortality and morbidity. Currently, no disease-modifying treatment is approved for AKI. While KDIGO provides general AKI management guidelines for use in clinical practice, the current treatment paradigm has changed little over the past 4 decades. Treatment generally consists of the management of symptoms and complications of acutely decreased kidney function, identification of the cause, avoidance of aggravating insults, and if recovery is delayed, the provision of KRT. [1]. To date, clinical studies of therapeutics for AKI have predominantly focused on studies of prevention in the rare circumstances where the timing of AKI can be predicted (e.g., with administration of radiocontrast or potentially nephrotoxic chemotherapy, or following major cardiac or cardiovascular surgery). However, no therapy has been shown to effectively treat or prevent AKI, and mortality associated with severe AKI remains >50% for patients in the ICU.

Orai1 is a pore-forming plasma membrane protein that in combination with the calcium-sensing endoplasmic reticulum gating-protein stromal interaction molecule 1 (STIM1) constitutes a calcium release-activated calcium (CRAC) channel. Low levels of calcium within the endoplasmic reticulum cause the STIM1 protein to oligomerize and move to locations closely apposed to Orai1. When STIM1 binds to Orai1, the Orai1 Ca²⁺ pore opens, permitting entry of extracellular calcium into the cell through the CRAC channel. This process is referred to as store operated calcium entry (SOCE). Calcium entry in T-cells via CRAC channels is responsible for activation of multiple pro-inflammatory pathways, including the production of NFκB and the activation of nuclear translocation of nuclear factor of activated T-cells (NFAT). The latter results in the production of pro-inflammatory cytokines involved in the development of acute hypoxemic respiratory failure in the setting of AKI, including IL-6, TNFα, and IL-17. CRAC channel overactivation has also been suggested to increase endothelial cell permeability and translocation of fluid into the lung from the vascular space [12].

Orai1 inhibition in cultured human podocytes has been shown to ameliorate apoptosis and mitochondrial dysfunction seen in response to exposure to high levels of glucose and angiotensin 2 [13]. In addition, it reversed impaired mitochondria membrane potential (MMP), ATP production, and enhanced mitochondria superoxide generation induced by high glucose exposure [13]. Additionally, the knockdown, or blockade of the Orai1 Ca²⁺ channel in cultured human proximal tubular cells prevented epithelial-to-mesenchymal transition induced by TGF‐b1 [14]. These findings suggest that the inhibition of Orai1 may not only represent a therapeutic target in AKI but may also reduce the transition of AKI to chronic kidney disease.

Calcineurin inhibitors (CNIs) suppress the phosphorylation of NFAT and prevent the production of pro-inflammatory cytokines. In transplant recipients, however, they have been associated with acute nephrotoxicity due to renal vasoconstriction secondary to an increase in vasoconstrictor factors that include endothelin and thromboxane as well as activation of the renin-angiotensin system (RAS) [15]. CNIs have also been shown to reduce vasodilator factors like prostacyclin, prostaglandin E2, and nitric oxide (NO) [15]. These nephrotoxic effects likely counter the benefits of suppressing NFAT-mediated pro-inflammatory cytokine production in the setting of AKI, and no therapeutic trials of CNIs for AKI have been conducted. A single-center database analysis of CNI use in septic patients showed a significantly higher 28- and 365-day survival rate for CNI users compared to nonusers. There were no differences in the development of AKI or need for continuous renal replacement therapy between the 2 groups [16].

The anti-inflammatory and protective effects of zegocractin were evaluated in a mouse model of lipopolysaccharide-induced acute lung injury, where two intraperitoneal injections of zegocractin reduced levels of the pro-inflammatory cytokines IL-6 and TNFα in bronchoalveolar lung fluid and decreased peribronchiolar/perivascular edema width in lung tissue (unpublished data). The anti-inflammatory and protective effects of Auxora have also been demonstrated in an ischemia/reperfusion (I/R) model of AKI in rats [17]. Male rats subjected to 40 min of bilateral I/R injury and then randomized to receive Auxora or placebo starting 6 h afterward. Study drug was repeated at 24 and 48 h after the initial infusion and GFR was then evaluated at 72 h after I/R. There was a reduction of GFR by about 50% between 2 and 4 h after I/R versus baseline, while there was no difference in GFR prior to randomization (0.43 ± 0.02 versus 0.39 ± 0.05 mL/min/100 g in placebo versus Auxora, respectively). Recovery of GFR 72 h after I/R was significantly greater in Auxora-treated animals versus placebo-treated controls (0.81 ± 0.03 vs. 0.47 ± 0.04 mL/min/100 g in Auxora versus placebo, respectively; p < 0.001). In a subgroup of animals with a reduction in GFR by 66% or more, 3 of 3 placebo-treated animals did not survive 72 h, while 2 of 2 Auxora-treated animals survived 72 h, although GFR did not return to baseline with only three doses of Auxora, suggesting that additional doses may be necessary to receive the full benefit [17].

Auxora has been administered safely to critically ill subjects in multiple clinical studies. In patients with severe COVID-19 and others with acute pancreatitis and accompanying systemic inflammatory release syndrome (SIRS), Auxora treatment resulted in improved recovery and decreased mortality, along with decreased incidence of severe organ failure [18, 19]. In a post hoc analysis of the CARDEA trial (NCT04345614) [18], we examined results in patients with an estimated GFR below 60 mL/min/1.73 m², including 23 patients randomized to Auxora and 15 patients randomized to placebo. Four of 23 (17%) patients randomized to Auxora and 7 of 15 (47%) patients randomized to placebo died by day 60. Corresponding values for the number of days alive and ventilator-free was 23.7 ± 11.5 and 14.9 ± 14.2, respectively. Across studies, Auxora has been well tolerated and patients receiving Auxora (whether in open label or placebo-controlled settings) have demonstrated favorable clinical outcomes relative to standard of care. The AEs that have been more commonly seen in patients treated with Auxora compared to placebo were transient increases in blood triglycerides (13% vs. 6%) and liver transaminases (9% vs. 4%) [18].

In conclusion, severe AKI remains an elusive problem plaguing a broad range of critically ill patients, including those with acute hypoxemic respiratory failure. The mechanism of action of CRAC channel inhibition, along with extensive preclinical and adjacent clinical data in COVID-19 and acute pancreatitis, suggest that zegocractin (in the Auxora formulation) with action via multiple targets (Fig. 1) may prove to be a safe and efficacious agent for the management of severe AKI with AHRF.

Medical writing and editorial support were provided by Nicola Donelan, PhD. Statistical support provided by Jeffrey Zhang, PhD. of Princeton Pharmatech.

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and approved by WCG Clinical (study number 1378225, IRB tracking number 20240970). Written informed consent to participate in the study will be provided by each patient or legally authorized representative prior to screening.

L.S.C. is an employee of Exthera Medical and a shareholder of Silver Creek Pharma and MediBeacon. P.T.M. receives consulting fees from Novartis, AM-Pharma, and Alexion for serving on clinical trial steering committees. He receives consulting fees from Renibus Therapeutics, Calcimedica, Variant Bio, and Bioporto Diagnostics for serving on scientific advisory boards. S.L.G. reports receiving grant support and consulting fees from Baxter Healthcare, SeaStar Medical, BioPorto Diagnostics, Medtronic, Nuwellis, ExThera, AstraZeneca, Otsuka, Fresenius Medical Care; he is a Data Safety Monitoring Board (DSMB) Member for Clinical Trials at SeaStar Medical and Silver Creek Medical, and Director of Clinical Development with stock options at MediBeacon. A.C. and S.H. are employees of CalciMedica. A.C. has stock options and S.H. has stock and stock options in CalciMedica. R.G.W. is a consultant for Calcimedica and institutionally, Northwestern University has received clinical trial and investigator-initiated funding. G.M.C. has served as an advisor to CalciMedica. He has served as Chair or Co-chair of trial steering committees with Akebia, AstraZeneca, CSL Behring, and Vertex. He has served as an advisor to Avvio, Ardelyx, CloudCath, Durect, Eliaz Therapeutics, Miromatrix, Outset, Panoramic, Renibus, Toku, and Unicycive. He has served on DSMBs with Aethlon, Bayer, Mineralys, and ReCor. He has served on the Board of Directors of Satellite Healthcare, a non-profit dialysis provider. L.S.C., P.T.M., S.L.G., and G.M.C. have been granted stock options in CalciMedica. Dr Glenn Matthew Chertow (G.M.C.) was a member of the Journal’s Editorial Board at the time of submission.

The KOURAGE trial was funded by CalciMedica.

L.S.C., P.T.M., S.L.G., A.C., S.H., R.G.W., and G.M.C. contributed to the design of the trial. The initial draft was prepared by L.S.C., S.H., and G.M.C. P.T.M., S.L.G., A.C., S.H., R.G.W., and G.M.C. reviewed and edited prior versions of the manuscript and approved the final submitted version.

The data that support the findings of this study are not publicly available since the trial has not yet completed. When the trial has been completed, reasonable requests for data sharing will be granted by the trial sponsor. Data sharing requests should be sent to Sudarshan Hebbar, MD at [email protected].