| Zhong Dengqin,Li Qiang,Zhang Xuxia,Wang Mengmeng,Wang Ruiyun,Chen Honghong.Mechanism of lysosomal membrane permeabilization in uranyl acetate-induced death of renal proximal tubule epithelial cells[J].Chinese Journal of Radiological Medicine and Protection,2022,42(3):161-167 |
| Mechanism of lysosomal membrane permeabilization in uranyl acetate-induced death of renal proximal tubule epithelial cells |
| Received:November 05, 2021 |
| DOI：10.3760/cma.j.cn112271-20211105-00448 |
| KeyWords:Uranium Lysosomal membrane permeability Reactive oxygen species Lysosomal-dependent cell death HK-2 cells |
| FundProject:国家自然科学基金面上项目（81972971） |
| Author Name | Affiliation | E-mail | | Zhong Dengqin | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | | | Li Qiang | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | | | Zhang Xuxia | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | | | Wang Mengmeng | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | | | Wang Ruiyun | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | | | Chen Honghong | Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China | hhchen@shmu.edu.cn |
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| 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). 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. |
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