Mercuric chloride induces sequential activation of ferroptosis and necroptosis in chicken embryo kidney cells by triggering ferritinophagy

Abstract

Mercuric chloride (HgCl₂) is an environmental pollutant with serious nephrotoxic effects, but the underlying mechanism of HgCl₂ nephrotoxicity is not well understood. Ferroptosis and necroptosis are two programmed cell death (PCD) modalities that have been reported singly in heavy metal-induced kidney injury. However, the interaction between ferroptosis and necroptosis in HgCl₂-induced kidney injury is unclear. Here, we established a model of HgCl₂-exposed chicken embryo kidney (CEK) cells to dissect the progresses and mechanisms of these two PCDs. We found that ferroptosis was initially activated in CEK cells after HgCl₂ exposure for 12 h, and necroptosis was activated subsequently at 24 h. Importantly, further study indicated that the shift from ferroptosis to necroptosis was driven by ROS, which was produced by iron-dependent Fenton reaction, and the iron chelation by DFO prevented the sequential activation of both ferroptosis and necroptosis. To investigate the source of intracellular iron, the regulation of iron homeostasis was first explored and demonstrated a tendency for intracellular iron overload in CEK cells. Interestingly, the cellular ferritin, a free iron depository, decreased in a time-dependent manner. Further studies revealed that the degradation of ferritin was attributed to the activation of selective cargo receptor nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, and the inhibition of ferritinophagy by CQ prevented the HgCl₂-induced cell death. In conclusion, our study demonstrated that HgCl₂ released excess free iron via ferritinophagy, led to a sustained accumulation of ROS and ultimately activated ferroptosis and necroptosis sequentially. These findings provide a new understanding for the nephrotoxic mechanism of HgCl₂.

Keywords: Ferritinophagy; Ferroptosis; Mercuric chloride; Necroptosis; Nuclear receptor coactivator 4.