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

目的探究溶酶体Ca^(2+)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300μmol/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。

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 300μmol/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.