MP244
TWEAK/FN14 MEDIATES SECONDARY CELL DEATH DURING AKI Free

INTRODUCTION AND AIMS: Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily that induces renal tubular cell death in a proinflammatory environment. TWEAK, TNFα and IFNγ (TTI)-induced cell death has features of apoptosis and is associated to activation of caspases and mitochondria stress. However, inhibition of caspases with zVAD in vitro and in vivo was not protective. Recently, it was reported that ferroptosis plays a key role in the initial wave of tubular cell death in folic acid-induced acute kidney injury (AKI), and beyond cell death, it mediates upregulation of inflammatory proteins, including Fn14, the TWEAK receptor. We hypothesized that the TWEAK/Fn14 axis could contribute to a secondary wave of inflammation-related cell death during AKI.

METHODS: Fn14 knockout (Fn14-KO) and wild type (WT) received a single i.p. injection of folic acid and were killed 72 h later. Cultured proximal tubular MCT cells were also studied. Protein studies were performed by western blot. Cell death was analyzed by annexin V/7AAD and TMRM staining by flow cytometry. Protein expression was knocked down using siRNA for RIPK1, RIPK3 and MLKL.

RESULTS: Fn14-KO mice were protected from AKI, as assessed by serum BUN and creatinine levels, and reduced cell death, assessed by TUNEL, at 72h.This suggests that TWEAK may mediates secondary cell death in AKI. In cultured cells, TTI induces caspase activation, but zVAD changed the mode of cell death from apoptosis to necrosis. However, the RIPK1 inhibitor Nec-1 prevented both cell death induced by TTI and by TTI+zVAD (TTI/Z), suggesting that RIPK1 is implicated in both apoptosis and necrosis. Specifically, Nec-1 prevented caspase activation and loss of mitochondrial membrane potential induced by TTI. By contrast, RIPK3 and MLKL siRNA only prevented TTI/Z-induced cell death suggesting that this process is mediated by necroptosis.

CONCLUSIONS: The TWEAK/Fn14 axis plays a key role in secondary, inflammation-related cell death in AKI. In vitro experiment showed that TTI-induced cell death has features of apoptosis but is dependent on the kinase activity of RIPK1. By contrast, caspase inhibition changed the mode of cell death to necroptosis. These results open a new therapeutic window to treatment of AKI once established.