POS-377 BAICALEIN ATTENUATES ADENINE-INDUCED FERROPTOSIS IN HUMAN KIDNEY PRIMARY PROXIMAL TUBULAR EPITHELIAL CELLS (PTEC) VIA MAINTENANCE OF HEME OXYGENASE-1 EXPRESSION

Abstract

Chronic kidney disease (CKD) is a global health burden, increasing in incidence worldwide. Adenine diet-induced CKD is a well-accepted pre-clinical model of CKD. In vitro, adenine-induced proximal tubular epithelial cell (PTEC) injury and loss are associated with increased oxidative stress, however little is known regarding the mechanisms of injury that cause PTEC loss. The aims of this study were (1) to investigate modes of cell death induced in human kidney primary PTEC by adenine, and (2) modulate any cell death with novel cell death inhibitors. Human kidney primary PTEC were cultured in the absence or presence of adenine and assessed for mitochondrial function, mitochondrial superoxides and altered cell viability by flow cytometry. Biomarkers of cell death pathways were analyzed by Western blotting. On identification of ferroptosis, rescue of PTEC by the plant-based anti-ferroptotic flavonoid baicalein was investigated. Adenine-treated PTEC displayed significantly reduced mitochondrial function and significantly increased mitochondrial superoxides and cell death compared with untreated PTEC. Further interrogation of cell death pathways revealed comparable expression of markers for apoptosis (activated caspase-3), necroptosis (pMLKL) and mitochondrial permeability transition-induced necrosis (PPIF) between untreated and adenine-treated PTEC. In contrast, the lipid repair enzyme GPX4, a biomarker for ferroptosis, was significantly reduced in adenine-treated PTEC, suggesting a pathway of ferroptototic cell death. Addition of the ferroptosis inhibitor baicalein restored expression of GPX4, maintained levels of antioxidant enzyme heme oxygenase-1 and reduced cell loss in adenine-treated PTEC. Our findings identify ferroptosis as a key human kidney PTEC cell death pathway in adenine-induced CKD. The anti-ferroptotic mechanisms of baicalein are now being explored for therapeutic translation in the clinical management of CKD.