Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells

Cadmium exposure is known to cause endoplasmic reticulum (ER) stress. In our current study, we examined the effects of salubrinal, a selective inhibitor of eukaryotic translation initiation factor 2 subunit α (eIF2α) dephosphorylation, on apoptotic cell death and ER stress-signaling events in HK-2 human renal proximal tubular cells exposed to cadmium chloride (CdCl2). Using phase-contrast microscopy and a cell viability assay, we observed that salubrinal suppressed CdCl2-induced cellular damage and cell death. Treatment with salubrinal reduced the number of TUNEL-positive cells and the cleavages of caspase-3 and poly(ADP-ribose) polymerase, but not the cleavage of light chain 3B, indicating protection from CdCl2-induced apoptosis but not autophagy. Although eIF2α remained phosphorylated after CdCl2 exposure to salubrinal-treated HK-2 cells, the expression of activating transcription factor 4 (ATF4) and the 78 kDa glucose-regulated protein (GRP78) was not increased. On the other hand, CdCl2-induced expression of C/EBP homologous protein (CHOP) was reduced by salubrinal treatment. Expression of ATF4, an upstream regulator of GRP78 and CHOP, appeared to be a prerequisite for full protection by salubrinal against cadmium cytotoxicity, because CdCl2-induced cellular damage was not fully suppressed in ATF4-deficient cells. Phosphorylated forms of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK), increased after CdCl2 exposure, whereas salubrinal suppressed the phosphorylation of JNK and p38 but not ERK. These results suggest that salubrinal protects CdCl2-exposed HK-2 cells from apoptosis by suppressing cell death signal transduction pathways.