| Zhang Hongjing,Wang Ruiyun,Wang Yifei,et al.Decorporation and detoxification effects of TRPML1 agonist ML-SA5 on human renal proximal tubular epithelial cells exposed to uranyl acetate[J].Chinese Journal of Radiological Medicine and Protection,2024,44(7):549-554 |
| Decorporation and detoxification effects of TRPML1 agonist ML-SA5 on human renal proximal tubular epithelial cells exposed to uranyl acetate |
| Received:November 20, 2023 |
| DOI：10.3760/cma.j.cn112271-20231120-00179 |
| KeyWords:Uranium TRPML1 ML-SA5 Lysosomal exocytosis HK-2 cell |
| FundProject:国家自然科学基金面上项目(81972971) |
| Author Name | Affiliation | E-mail | | Zhang Hongjing | 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 | | | Wang Yifei | 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 | | | 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 study the role of ML-SA5, an agonist of the lysosomal Ca2+ channel transient receptor potential mucolipin 1 (TRPML1), in promoting lysosomal exocytosis to facilitate intracellular uranium removal and alleviate uranium-induced cellular damage for human renal proximal tubule epithelial cells (HK-2) exposed to uranyl acetate. Methods HK-2 cells were divided into the following groups to be exposed to uranyl acetate at either 0 or 300 μmol/L for 24 h, followed by treatment with ML-SA5 and/or the lysosomal exocytosis inhibitor vacuolin-1 for 0.5 h: control group (Ctrl group), ML-SA5 group (M group), vacuolin-1 group (V group), ML-SA5 plus vacuolin-1 group (M+V group), uranium exposure group (U group), uranium exposure plus ML-SA5 group (U+M group), uranium exposure plus vacuolin-1 group (U+V group), and uranium exposure plus ML-SA5 plus vacuolin-1 group (U+M+V group). We localized lysosome-associated membrane protein-1 (LAMP-1) on the plasma membrane (surface LAMP-1) by immunofluorescence assay; measured intracellular uranium content by inductively coupled plasma mass spectrometry; measured the level of kidney injury molecule-1 (KIM-1) by immunofluorescence assay; measured the rate of cell death with Calcein-AM/PI double staining; determined the subcellular localization of transcription factor EB (TFEB) and the levels of LAMP-1 and TRPML1 proteins by immunofluorescence assay; and measured the number of lysosomes using LysoTracker probes. Results Compared with the Ctrl group, the U group showed significant increases in the surface LAMP-1 protein level (t = 12.86, P ＜ 0.05), KIM-1 protein level (t = 18.86, P ＜ 0.05), cell death rate (t = 38.53, P ＜ 0.05), TFEB nuclear translocation (t = 9.12, P ＜ 0.05), the protein expression levels of TFEB’s downstream target genes LAMP-1 (t = 16.47, P ＜ 0.05) and TRPML1 (t = 32.33, P ＜ 0.05), and the number of lysosomes labeled with LysoTracker probes (t = 7.75, P ＜ 0.05). Compared with the U group, the U+M group showed a significantly increased surface LAMP-1 level (t = 3.33, P ＜ 0.05) and significant decreases in the intracellular uranium level (t = 5.01, P ＜ 0.05), KIM-1 protein expression level (t = 3.81, P ＜ 0.05), and cell death rate (t = 3.24, P ＜ 0.05); all these effects in the U+M group could be neutralized by the lysosomal exocytosis inhibitor vacuolin-1; and in addition, ML-SA5 significantly increased TFEB nuclear translocation (t = 9.20, P ＜ 0.05), the protein expression levels of LAMP-1 (t = 3.05, P ＜ 0.05) and TRPML1 (t = 3.17, P ＜ 0.05), and the number of lysosomes labeled with LysoTracker probes (t = 3.13, P ＜ 0.05). Conclusions The TRPML1 agonist ML-SA5 can promote lysosomal exocytosis to enhance intracellular uranium clearance and reduce uranium-induced cellular damage/death in uranium-loaded HK-2 cells, through activating TFEB to up-regulate lysosome biogenesis and TRPML1 protein expression. |
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