Repurposing Zafirlukast Confers Renal Protection in Ischemia-Reperfusion Injury via Suppressing Macrophage METosis

Tacrolimus is widely used to prevent post-transplant acute kidney injury (AKI) but causes severe toxicities (e.g., nephrotoxicity, hyperglycemia). We repurposed zafirlukast (ZFK), an FDA-approved asthma drug, to address this limitation. In a murine kidney ischemia-reperfusion injury (KIRI) model, ZFK significantly attenuated renal dysfunction, reducing serum creatinine and blood urea nitrogen (BUN) to levels comparable to tacrolimus, without inducing metabolic adverse effects. RNA sequencing revealed that ZFK recapitulated tacrolimus' anti-inflammatory gene signatures while uniquely suppressing macrophage extracellular trap formation (METosis). Mechanistically, ZFK inhibited METosis by downregulating PAD4 and CitH3 expression, confirmed by immunofluorescence and flow cytometry. Single-cell transcriptomics (Tabula Muris and GEO database) identified macrophages as the primary target via CysLT1R antagonism. This study provides the first evidence that ZFK protects against KIRI by targeting METosis, a key driver of sterile inflammation. Given its established safety profile, ZFK could bypass Phase I trials, accelerating clinical translation as a safer alternative to calcineurin inhibitors. Our findings also highlight the broader potential of ZFK in METosis-related diseases (e.g., sepsis, atherosclerosis) and underscore drug repurposing as a strategic approach for rapid therapeutic development. Overall design: Male C57BL/6J mice were randomly assigned to two main experimental groups (6 mice per group) as follows: (i) I/R group: received daily oral administration of 0.9% saline for 1 consecutive days, followed by partial bilateral kidney ischemia for 30 minutes, and then a 24-hour reperfusion period; (ii) Zafirlukast + I/R group: received Zafirlukast (10 mg/kg/day, p.o.) or tacrolimus (1 mg/kg/day, p.o.) via oral gavage for 1 consecutive days, followed by laparotomy with bilateral kidneys occlusion for 30 minutes and then a 24-hour reperfusion period. Animals were anesthetized using an intraperitoneal injection of pentobarbital sodium (0.5%, 0.01 mL/g) At the conclusion of the experimental period, the animals were humanely sacrificed by decapitation. Blood was collected via cardiac puncture and centrifuged at 4,000 × g for 10 minutes. The resulting sera were isolated and stored at -80°C for biochemical assessments. The kidneys were excised and divided into three portions. The first portion was homogenized in phosphate-buffered saline, then centrifuged, and the supernatant was stored at -80°C for RNA sequencing assays. The second portion was rapidly frozen in liquid nitrogen and stored at -80°C for Western blot analysis and quantitative PCR. The third portion was immersed in 4% paraformaldehyde for histopathological and immunohistochemical investigations.