中华放射医学与防护杂志

张洪静,王睿云,王毅斐,等.TRPML1激动剂ML-SA5对铀暴露人肾近端小管上皮细胞的促排解毒作用及机制[J].中华放射医学与防护杂志,2024,44(7):549-554.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].Chin J Radiol Med Prot,2024,44(7):549-554

TRPML1激动剂ML-SA5对铀暴露人肾近端小管上皮细胞的促排解毒作用及机制

Decorporation and detoxification effects of TRPML1 agonist ML-SA5 on human renal proximal tubular epithelial cells exposed to uranyl acetate

投稿时间：2023-11-20

DOI：10.3760/cma.j.cn112271-20231120-00179

中文关键词: 铀 TRPML1 ML-SA5 溶酶体胞吐 HK-2细胞

英文关键词:Uranium TRPML1 ML-SA5 Lysosomal exocytosis HK-2 cell

基金项目:国家自然科学基金面上项目(81972971)

作者	单位	E-mail
张洪静	复旦大学上海医学院放射医学研究所, 上海 200032
王睿云	复旦大学上海医学院放射医学研究所, 上海 200032
王毅斐	复旦大学上海医学院放射医学研究所, 上海 200032
张旭霞	复旦大学上海医学院放射医学研究所, 上海 200032
陈红红	复旦大学上海医学院放射医学研究所, 上海 200032	hhchen@shmu.edu.cn

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中文摘要:

目的探究溶酶体Ca²⁺通道瞬时受体电位粘脂蛋白1(TRPML1)激动剂ML-SA5通过促进溶酶体胞吐促进醋酸铀酰暴露的人肾近端小管上皮HK-2细胞内铀的排出及减轻铀致细胞损伤的作用及机制。方法将HK-2细胞分为空白对照组(Ctrl组)、ML-SA5组(M组)、Vacuolin-1组(V组)、ML-SA5+Vacuolin-1组(M+V组)、单纯铀染毒组(U组)、铀染毒+ML-SA5组(U+M组)、铀染毒+Vacuolin-1组(U+V组)和铀染毒+ML-SA5+Vacuolin-1组(U+M+V组),分别给予0和300 μmol/L醋酸铀酰暴露24 h后加入ML-SA5和/或溶酶体胞吐抑制剂Vacuolin-1作用0.5 h。采用免疫荧光法检测溶酶体相关膜蛋白-1(LAMP-1)在细胞质膜的定位情况,ICP-MS检测细胞内铀含量,免疫荧光法检测肾损伤分子1(KIM-1)蛋白表达,Calcein-AM/PI双染色法检测细胞死亡率,免疫荧光法检测转录因子EB(TFEB)的亚细胞定位、LAMP-1及TRPML1蛋白表达,采用溶酶体LysoTracker荧光探针检测溶酶体数量。结果与Ctrl组相比,U组细胞质膜定位的LAMP-1蛋白、细胞KIM-1蛋白表达、细胞死亡率明显增加(t = 12.86、18.86、38.53,P ＜ 0.05),TFEB核转位、TFEB下游靶基因LAMP-1和TRPML1蛋白表达及LysoTracker探针标记的溶酶体数量明显增加(t = 9.12、16.47、32.33、7.75,P ＜ 0.05);与U组相比,U+M组HK-2细胞质膜定位的LAMP-1明显增加(t = 3.33,P ＜ 0.05),细胞内铀含量明显减少(t = 5.01,P ＜ 0.05),细胞内KIM-1蛋白表达和细胞死亡率显著降低(t = 3.81、3.24,P ＜ 0.05),这些作用均能被溶酶体胞吐抑制剂Vacuolin-1所抵消;ML-SA5还可明显提高负载铀HK-2细胞的TFEB核转位(t = 9.20,P ＜ 0.05)、TFEB下游靶基因LAMP-1及TRPML1蛋白表达(t = 3.05、3.17,P ＜ 0.05)以及LysoTracker标记的溶酶体数量(t = 3.13,P ＜ 0.05)。结论 TRPML1激动剂ML-SA5通过刺激溶酶体胞吐,促进负载铀的HK-2细胞内铀排出及降低铀致细胞损伤/死亡,与其激活TFEB上调溶酶体生物发生和TRPML1蛋白表达有关。

英文摘要:

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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