Myoglobin-Induced Apoptosis: Two Pathways Related to Endoplasmic Reticulum Stress

Myoglobin plays an important role in rhabdomyolysis-induced acute kidney injury (AKI), but the underlying mechanisms are still unclear. The present study investigates myoglobin-induced apoptosis in HK-2 cells (human renal proximal tubule cells) to discover some of the mechanisms involved in rhabdomyolysis related AKI. Metmyoglobin is reduced to ferrous myoglobin by ascorbic acid, and then the HK-2 cells are incubated with ferrous myoglobin. Cell viability is measured by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and cell injury is tested by supernatant lactose dehydrogenase (LDH). Cell apoptosis is evaluated by fluorescent microscopy of Hoechst staining and by flow cytometry of Annexin V/PI double staining. The apoptosis related protein expression is determined by Western blot. HK-2 cells were incubated with 200 mu M ferrous myoglobin for 24 h, the cell viability decreased and supernatant LDH release increased. Hoechst staining indicated more apoptosis after incubation. Molecular chaperone glucose-related protein 78 (GRP78), cytochrome C, caspase-9 started to increase within 3 h after incubation while caspase-4, caspase-8 showed no significant change. (iii) When the inositol triphosphate receptor (IP3R) calcium channel was blocked by 2-aminoethoxydiphenyl-borinate (2-APB), caspase-9 was completely inhibited, GRP78 and caspase-4 increased dramatically, and caspase-3 expression was not affected. The apoptosis in HK-2 cells showed no significant change. Apoptosis in HK-2 cells incubated with ferrous myoglobin is an endoplasmic reticulum stress induced, IP3R calcium channel mediated, caspase-9 dependent intrinsic pathway. When the intrinsic pathway was inhibited using an IP3R calcium channel blocker, endoplasmic reticulum stress increased, resulting in the activation of caspase-4 that cleaved caspase-3 and generated a substitutive pathway of apoptosis.