#3137 GASDERMIN D DRIVES THROMBOTIC ANGIOPATHY-RELATED AKI BY ACCELERATING IMMUNOTHROMBOSIS AS WELL AS NEUTROPHIL-MEDIATED NECROINFLAMMATION AND INFARCTION

Abstract Background and Aims Cholesterol crystal embolism (CCE) is a life-threatening complication of advanced atherosclerosis, in which CC can trigger clot formation, arterial occlusion, acute kidney injury (AKI), and ischemic infarction. Neutrophils play a role in this process by driving arterial crystalline immunothrombosis and undergoing regulated necrosis in necroinflammation surrounding ischemic kidney infarcts. In this study, we hypothesized that Gasdermin D (GSDMD) contributes to the pathogenesis of CCE by amplifying these processes. Method We injected CC (20 mg/kg) into the left kidney artery of wildtype (WT) or Gsdmd-knockout (KO) mice to induce CCE. The mice were sacrificed after 24 hours. Kidney injury was assessed through glomerular filtration rate (GFR), histological analysis using periodic acid-Schiff and TUNEL staining as well as assessment of infarct size and arterial obstruction via triphenyltetrazolium chloride and alpha-smooth muscle actin/fibrin staining, respectively. Neutrophils (Ly6G+) and mature neutrophils (Ly6G+ CD101+) were analyzed in several organs by flow cytometry. The presence of neutrophil extracellular traps (NETs) in kidney was evaluated by staining for citrullinated histone 3 (CitH3) and flow cytometry analysis of Ly6G+ CitH3+ neutrophils. Interventional studies using disulfiram, a GSDMD inhibitor, administered via intraperitoneal injection (50 mg/kg) to WT mice either 4 hours prior or 3 hours following CCE induction, followed by sacrifice and analysis after 24 hours. In vitro, healthy human neutrophils were stimulated with CC in the presence or absence of pretreatment with disulfiram, and the levels of IL-1β and lactate dehydrogenase (LDH) were evaluated in the culture supernatant. Results WT CCE kidneys showed increased expression of Gsdmd in peritubular cells as determined by immunohistochemical analysis, as well as an increased expression of cleaved Gsdmd by immunoblot analysis compared to WT control kidneys. Baseline levels of GFR and the number of both total and mature neutrophils were comparable in both groups under normal conditions. Compared to WT mice, Gsdmd-KO mice were partially protected from sudden GFR decline, tubular injury, kidney cell death, and kidney infarction following CCE. Arterial obstruction was significantly reduced in Gsdmd-KO mice following CCE. Gsdmd-deficiency reduced the number of mature neutrophils in bone marrow, spleen, and blood in comparison to WT mice. The amount of NETs in kidney exhibited a significant reduction in Gsdmd-KO mice. Consistently, preemptive and therapeutic treatment with disulfiram significantly reduced sudden GFR decline and kidney infarct size in WT mice compared to vehicle-treated controls. Blood and kidney neutrophils, as well as blood and kidney NETs were significantly reduced in WT CCE mice with disulfiram preemptive and therapeutic treatment in comparison to respective vehicle controls. In vitro, CC induced the release of IL-1β and LDH from healthy human neutrophils, which disulfiram suppressed in a dose-dependent manner. Conclusion The deficiency and pharmacological inhibition of GSDMD alleviate crystal clot formation, AKI, and ischemic infarct growth following CCE via suppressing neutrophil recruitment, maturation, and NET formation. Thus, neutrophils significantly contribute to immunothrombosis and ischemic kidney infarction.