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Myoglobin‐Induced Apoptosis: Two Pathways Related to Endoplasmic Reticulum Stress
Author(s) -
Zhou Jianhui,
Kong Deyang,
Zhang Xu,
Wang Yuanda,
Feng Zhe,
Zhang Xueguang,
Zhang Li,
Wang Yan,
Xie Yuansheng,
Chen Xiangmei
Publication year - 2012
Publication title -
therapeutic apheresis and dialysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.415
H-Index - 53
eISSN - 1744-9987
pISSN - 1744-9979
DOI - 10.1111/j.1744-9987.2011.01057.x
Subject(s) - apoptosis , annexin , endoplasmic reticulum , viability assay , unfolded protein response , cytochrome c , myoglobin , microbiology and biotechnology , caspase 12 , caspase , medicine , biochemistry , programmed cell death , biology
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 µM 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.