Mechanism of lysosomal membrane permeabilization in uranyl acetate-induced death of renal proximal tubule epithelial cells
10.3760/cma.j.cn112271-20211105-00448
- VernacularTitle:溶酶体膜通透化在铀诱导肾近端小管上皮细胞死亡中的作用及机制
- Author:
Dengqin ZHONG
1
;
Qiang LI
;
Xuxia ZHANG
;
Mengmeng WANG
;
Ruiyun WANG
;
Honghong CHEN
Author Information
1. 复旦大学上海医学院放射医学研究所，上海 200032
- Keywords:
Uranium;
Lysosomal membrane permeability;
Reactive oxygen species;
Lysosomal-dependent cell death;
HK-2 cells
- From:
Chinese Journal of Radiological Medicine and Protection
2022;42(3):161-167
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the mechanism of lysosomal membrane permeabilization(LMP)inuranyl acetate-induced death of human kidney proximal tubular epithelial HK-2 cells.Methods:HK-2 cells were exposed to uranyl acetate at concentrations of 100, 300 and 600 μmol/L for 24 h, then in tracellular reactive oxygen species (ROS)and mitochondrial superoxide were measured by DCFH-DA and MitoSOX probe, respectively. HK-2 cells were divided into four groups: blank control group, NAC or CA-074 Me group, uranyl acetate exposure group and uranyl acetate exposure plus NAC or CA-074 Me group. Two-color immune of luorescence staining was used to detect the co-localization of galectin-1 and lysosomal associated membrane protein-1 (LAMP-1) to measure the extent of LMP, and to detect the non- co-localization of cathepsin B and LAMP-1 to reflect the release of cathepsin B in lysosomes. Calcein-AM/PI double staining method was used to detect cell death. One-color immune of luorescence staining of cleaved-caspase-3 expression was used to detect apoptosis. Results:Intracellular ROS and mitochondrial superoxide levels were significantly increased in HK-2 cells after exposure with 100, 300 and 600 μmol/L uranyl acetate for 24 h, about 1.1-2.5 times or 4.0-28 times, respectively( tROS=17.98, 11.84, 11.75, P< 0.05; tmitochondrial superoxide=6.14, 16.02, 13.06, P< 0.05), and they also increased with uranyl acetate concentrations ( tROS=10.10, 10.37, 5.59, P< 0.05; tmitochondrial superoxide=21.50, 15.16, 5.93, P< 0.05). The percentage of co-localization of galectin-1 and LAMP-1 and the percentage of non- co-localization of cathepsin B and LAMP-1 were markedly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, 5.4-6.7 times or 1.5-2.1 times, respectively ( tGalectin-1=15.85, 12.70, P< 0.05; tCathepsin B=5.95, 6.69, P< 0.05), but these increases were inhibited by NAC ( tGalectin-1=4.74, P<0.05; tCathepsin B=4.51, P< 0.05). Moreover, the cell death rate and the cleaved-caspase-3 expression level were also significantly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, about 28-47 times or 2.4-6.0 times, respectively( tPI=30.40, 10.34, P<0.05; tCleaved-caspase-3=18.49, 9.52, P<0.05), and these increases were obviously diminished by CA-074 Me ( tPI= 6.76, P<0.05; tCleaved-caspase-3=13.47, P<0.05). Conclusions:Exposure to uranyl acetate induces a burst of intracellular ROSthat leads to LMP and consequently causes leakage of cathepsin B from lysosomes to cytoplasm, in turn triggering the lysosomal-dependent cell death and mitochondrial-regulated apoptosis of HK-2 cells.
