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