INDUCTION OF FERROPTOTIC DEATH IN HUMAN PRIMARY PROXIMAL TUBULAR EPITHELIAL CELLS (PTEC) DURING ADENINE-INDUCED CHRONIC KIDNEY DISEASE

M KHAN1,2,3,4,  P NAG2,3,  K GIULIANI2,3, X ANNIE WANG2,3, A GRIVEI2,3, W HOY1,7,  H HEALY1,2,3,5,  V DIWAN1,7,  G GOBE1,2,6, A KASSIANOS2,3,5

1NHMRC CKD CRE (CKD.QLD) Univ Queensland, Brisbane, Australia, 2Faculty of Medicine Univ Queensland, Brisbane, Australia, 3Conjoint Internal Medicine Laboratory, Pathology Qld, Brisbane, Australia, 4Bangabandhu Sheikh Mujibur Rahman Science and Technology University, , Bangladesh, 5Kidney Health Service, RBWH, Herston, Australia, 6Translational Research Institute, Woolloongabba, Australia, 7Centre for Chronic Disease, UQ Faculty of Medicine, Herston, Australia

Aim: To investigate mitochondrial function and some newly-described modes of cell death in human primary proximal tubular epithelial cells (PTEC) in an in vitro model of chronic kidney disease (CKD).
Background: CKD is a global health burden, increasing in incidence in developed and developing countries. Cell loss of the high energy PTEC can predict long term outcomes in CKD. The mechanisms of such cell loss are poorly understood. A well-accepted pre-clinical model of adenine diet-induced CKD, a tubular crystalline nephropathy, was modified for in vitro study of how the PTEC die.
Methods: Human primary PTEC were cultured in presence or absence of adenine and assessed for mitochondrial function, mitochondrial superoxide expression and altered cell viability by flow cytometry. Expression levels of markers of cell death pathways of apoptosis, necroptosis, mitophagy and ferroptosis were analyzed by Western blotting.
Results: Cell death was significantly higher in adenine-treated PTEC compared to untreated controls. Their mitochondrial function was significantly down-regulated and expression of mitochondrial superoxides up-regulated. There was no significant difference in expression of the signals of apoptosis (activated caspase-3), necroptosis (pMLKL) and mitochondrial permeability transition-induced necrosis (PPIF) between adenine-treated and untreated PTEC. In contrast, the lipid repair enzyme GPX4, a biomarker for ferroptosis, was significantly reduced in adenine-treated PTEC, suggesting the pathway of ferroptototic cell death. Supporting this mechanism, the addition of the ferroptosis inhibitor baicalein, a plant-based flavonoid compound, significantly reduced levels of cell death in adenine-treated PTEC.
Conclusions: Our findings identify ferroptosis as a novel human PTEC cell death pathway in adenine-induced CKD. The anti-ferroptotic action of baicalein is now being explored for therapeutic efficacy in this model of CKD.


Biography:
I am a PhD student at the School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Australia. My current project is “Use of plant extracts for the treatment of chronic kidney disease”. I am very keen to identify potential alternative or complementary therapy for CKD patients, as this disease affects developed and developing nations worldwide. I completed Bachelor of Pharmacy and Master of Pharmacy (Thesis) at the Department of Pharmacy, University of Rajshahi, Bangladesh, and was an Assistant Professor at the Department of Pharmacy at BSMRSTU, Bangladesh before starting my PhD at UQ.

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