Induction of lysosomal exocytosis and biogenesis via TRPML1 activation for the treatment of uranium-induced nephrotoxicity

Abstract

Uranium (U) is a well-known nephrotoxicant which forms precipitates in the lysosomes of renal proximal tubular epithelial cells (PTECs) after U-exposure at a cytotoxic dose. However, the roles of lysosomes in U decorporation and detoxification remain to be elucidated. Mucolipin transient receptor potential channel 1 (TRPML1) is a major lysosomal Ca²⁺ channel regulating lysosomal exocytosis. We herein demonstrate that the delayed administration of the specific TRPML1 agonist ML-SA1 significantly decreases U accumulation in the kidney, mitigates renal proximal tubular injury, increases apical exocytosis of lysosomes and reduces lysosomal membrane permeabilization (LMP) in renal PTECs of male mice with single-dose U poisoning or multiple-dose U exposure. Mechanistic studies reveal that ML-SA1 stimulates intracellular U removal and reduces U-induced LMP and cell death through activating the positive TRPML1-TFEB feedback loop and consequent lysosomal exocytosis and biogenesis in U-loaded PTECs in vitro. Together, our studies demonstrate that TRPML1 activation is an attractive therapeutic strategy for the treatment of U-induced nephrotoxicity.

Fig. 1. TRPML1 agonist ML-SA1 promotes renal U clearance and reduces the U-induced renal proximal tubule injury in mice after single- or multiple-dose U exposure.

a Schematic representation of the experiments on single- or multiple-dose U exposure followed by ML-SA1 treatment at 400 or 800 μg/kg in BALB/c male mice. n = 6 mice for control groups, ML-SA1 treatment alone groups, single dose U exposure alone groups and signal dose U exposure followed by ML-SA1 treatment groups, and n = 4 mice for multiple-dose U exposure alone group and multiple-dose U exposure followed by ML-SA1 treatment group. b U contents in kidney tissues and the last 24-h urine from mice with single- or multiple-dose U exposure and ML-SA1 treatment. c Representative immunohistochemical staining of KIM-1 in the S3 or S1 segment of proximal tubules in renal cortex of mice after U exposure and ML-SA1 treatment as indicated in the figure. Boxed areas are enlarged below. d Quantitative analysis of KIM-1 levels in the S1, S2, and S3 segments of proximal tubules as shown in c and Supplementary Fig. 4a. e The CRE and BUN levels in mice after single-dose (2.0 mg/kg) U exposure and treatment with ML-SA1 at 400 μg/kg or vehicle. f Representative H&E staining in the S3 segment of proximal tubules in renal cortex of mice after U exposure and ML-SA1 treatment as indicated in the figure. Boxed areas are enlarged below. g Quantitative analysis of pathological injury of proximal tubules with necrotic or exfoliated cells in the S1, S2, and S3 segments of proximal tubules as shown in f and Supplementary Fig. 4b. im: intramuscular injection; ip: intraperitoneal injection; ASI: a single injection; MDI: multiple dose injection; PT: proximal tubule. Data represent mean ± SD. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (b, d, e, g). Source data are provided as a Source Data file. All the images share the same scale bar (20 μm).

Fig. 2. TRPML1 agonist ML-SA1 increases the levels of lysosomal membrane proteins in the apical membrane of renal proximal tubules in mice after single- or multiple-dose U exposure.

The male mice with single- or multiple-dose U exposure and ML-SA1 treatment were described in Fig. 1a. n = 6 mice for single-dose U exposure alone groups, single-dose U exposure followed by ML-SA1 treatment groups, and corresponding control group and ML-SA1 treatment alone group. n = 7 mice for multiple-dose U exposure alone group and multiple-dose U exposure followed by ML-SA1 treatment group, and n = 9 mice for corresponding control group and ML-SA1 treatment alone group. a Representative immunohistochemical staining of LAMP-1 and TRPML1 in the S3 or S1 segment of the proximal tubules in renal cortex of mice after U exposure and ML-SA1 treatment as indicated in the figure. Boxed areas are enlarged below. Images share the same scale bar (20 μm). b Quantitative analysis of LAMP-1 and TRPML1 staining of the apical membrane in the S1, S2, and S3 segments of renal proximal tubules as shown in a and Supplementary Fig. 5. PT: proximal tubule. Data represent mean ± SD. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (b). Source data are provided as a Source Data file.

Fig. 3. TRPML1 agonist ML-SA1 reduces the U-induced LMP and LMP-related apoptosis of renal PTECs in mice after single- and multiple-dose U exposure.

The male mice with single- or multiple-dose U exposure and ML-SA1 treatment were described in Fig. 1a. n = 6 mice for control groups, ML-SA1 treatment alone groups, single dose U exposure alone groups and signal dose U exposure followed by ML-SA1 treatment groups. n = 4 mice for multiple-dose U exposure alone group and multiple-dose U exposure followed by ML-SA1 treatment group. a Representative immunohistochemical staining of galectin-1 in the S3 or S1 segment of the proximal tubules in renal cortex of mice after U exposure and ML-SA1 treatment as indicated in the figure. Boxed areas are enlarged below. b Quantitative analysis of galectin-1 staining in the S1, S2, and S3 segments of the proximal tubules as shown in a and Supplementary Fig. 6a. c Representative TUNEL staining in the S3 segment of the proximal tubules in renal cortex of mice after U exposure and ML-SA1 treatment as indicated in the figure. d Quantitative analysis of TUNEL staining in the S3 segment of the proximal tubules as shown in c. PT: proximal tubule; FI: fluorescence intensity. All the images share the same scale bar (20 μm). Data represent mean ± SD. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (b, d). Source data are provided as a Source Data file.

Fig. 4. TRPML1 activation with ML-SA1 triggers an efficient removal of intracellular U by lysosomal exocytosis in U-loaded renal epithelial HK-2 cells.

a The timeline of short-term U (50, 100, 600 μM) exposure for 24 h followed by ML-SA1 (10 μM) and Vacuolin-1 (2 μM) treatment for 30 min after washout of U. b Representative fluorescence images showing the LAMP-1 exposure on the PM in nonpermeabilized HK-2 cells and TRPML1 localization in permeabilized HK-2 cells after U exposure and ML-SA1 treatment. Images share the same scale bar (20 μm). c Comparison of β-hex release in HK-2 cells after U exposure and ML-SA1/Vacuolin-1 treatment. d Comparison of intracellular and extracellular U contents in HK-2 cells after U exposure and ML-SA1/Vacuolin-1 treatment. PM: plasma membrane. Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (c, d). Source data are provided as a Source Data file.

Fig. 5. TRPML1 activation with ML-SA1 reduces the U-induced LMP and LMP-related cell death via lysosomal exocytosis in U-loaded renal epithelial HK-2 cells.

The HK-2 cells were incubated with U at 0, 100, 600 μM for 24 h. After washout of U, the cells were treated with vehicle, ML-SA1 (10 μM), Vacuolin-1 (2 μM), and ML-SA1 (10 μM) plus Vacuolin-1 (2 μM) for 30 min and then analyzed. a Representative fluorescence images for colocalization of galectin-3 (red) and LAMP-1 (green) in HK-2 cells after U exposure and ML-SA1/Vacuolin-1 treatment as indicated in the figure. b Representative fluorescence images of Calcein-AM/PI staining in HK-2 cells after U exposure and ML-SA1/Vacuolin-1 treatment as indicated in the figure. The living cells were stained with Calcein-AM (green), and the nuclei of the dead cells were stained with PI (red). c Representative images of immunofluorescence staining of cleaved-caspase-3 (green) in HK-2 cells after U exposure and ML-SA1/Vacuolin-1 treatment as indicated in the figure. d Quantitative analysis of colocalization of galectin-3 with LAMP-1 under various treatment conditions shown in a. More than 30 cells were analyzed in each sample. e Quantitative analysis of the cell mortality under various treatment conditions shown in b. About 1000 cells were analyzed in each sample. f Quantitative analysis of cleaved-caspase-3 level under various treatment conditions shown in c. More than 500 cells were analyzed in each sample. FI: fluorescence intensity. All the images share the same scale bar (20 μm). Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (d–f). Source data are provided as a Source Data file.

Fig. 6. TRPML1 activation with ML-SA1 promotes lysosomal TRPML1-mediated Ca²⁺ release and nuclear translocation of TFEB and consequent lysosomal biogenesis in U-loaded renal epithelial HK-2 cells.

For experiments a, b, HK-2 cells were treated with U at 0, 100, and 600 μM for 24 h. After washout of U, cytosolic Ca²⁺ was measured after treatment with ionomycin (2 μM) followed ML-SA1 (10 μM) by Fluo-4 imaging. For experiments c–l, HK-2 cells or HK-2 cells transfected with either TRPML1 shRNA or PPP3CB siRNA or corresponding empty vector plasmid or scramble control siRNA were exposed to U at 0, 100 and/or 600 μM for 24 h. After washout of U, the cells were treated with vehicle or ML-SA1 (10 μM) for 30 min and then analyzed. a ML-SA1–induced lysosomal Ca²⁺ release in control and U-loaded HK-2 cells treated with 100 and 600 μM U for 24 h. b Quantification of cytosolic Ca²⁺ peak values shown in a. c, d Western blotting analysis of TFEB in HK-2 cells after U exposure and ML-SA1 treatment. e Representative images of immunofluorescence staining of TFEB (green) in HK-2 cells after U exposure and ML-SA1 treatment. Images share the same scale bar (20 μm). f Quantitative analysis of nuclear TFEB under various treatment conditions shown in e. g, h Western blotting analysis of LAMP-1 and TRPML1 in HK-2 cells after U exposure and ML-SA1 treatment. i, j Quantitative analysis of nuclear TFEB in TRPML1- or PPP3CB-knockdown HK-2 cells after U exposure and ML-SA1 treatment. Representative images of immunofluorescence staining of TFEB (green) are shown in Supplementary Fig. 8e, f. k, l Western blotting analysis of p-S6K and S6K in HK-2 cells after U exposure and ML-SA1 treatment. Iono: ionomycin. f, i, j More than 200 cells were analyzed in each sample. Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (b, d, f, h–j, l). Source data are provided as a Source Data file.

Fig. 7. Knockdown of TRPML1 and TFEB abolishes the ML-SA1 effects on the removal of intracellular U and reduction of the U-induced cell death in U-loaded renal epithelial HK-2 cells.

HK-2 cells transfected with either TRPML1 shRNA or TFEB shRNA or corresponding empty vector were exposed to U at 0, 50, 100, and 600 μM for 24 h. After washout of U, the cells were treated with either vehicle or ML-SA1 at 10 μM for 30 min and then analyzed. a Comparison of β-hex release from TRPML1-knockdown HK-2 cells or empty vector-transfected HK-2 cells after U exposure and ML-SA1 treatment. b Comparison of intracellular and extracellular U contents in TRPML1-knockdown HK-2 cells or empty vector-transfected HK-2 cells after U exposure and ML-SA1 treatment. c Comparison of β-hex release in TFEB-knockdown HK-2 cells or empty vector-transfected HK-2 cells after U exposure and ML-SA1 treatment. d Comparison of intracellular and extracellular U contents in TFEB-knockdown HK-2 cells or empty vector-transfected HK-2 cells after U exposure and ML-SA1 treatment. e, f TRPML1 or TFEB depletion enhances the cell death after U exposure, which was not reversed by ML-SA1 treatment. Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (a–f). Source data are provided as a Source Data file.

Fig. 8. TRPML1 activation with ML-SA1 promotes the removal of intracellular U in U-loaded renal epithelial HK-2 cells after long-term U exposure.

Comparison of intracellular U content in TRPML1-knockdown HK-2 cells or empty vector-transfected HK-2 cells after long-term U exposure (1, 5, 10 μM for 10, 20, 30 days) and the subsequent treatment of ML-SA1 at 10 μM for 30 min after washout of U. Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test. Source data are provided as a Source Data file.

Fig. 9. A schematic model for the mechanism by which TRPML1 activation with ML-SA1 promotes the intracellular U clearance and reduces the U-induced LMP and cell death in U-loaded renal PTECs after acute and chronic U exposure.

TRPML1 agonist ML-SA1 activates TRPML1 channels on the perimeter membranes of lysosomes, inducing lysosomal Ca²⁺ release and lysosomal exocytosis. Meanwhile, Ca²⁺-bound calcineurin dephosphorylates TFEB, which then translocates to the nucleus to activate the transcription of CLEAR genes to enhance the lysosomal biogenesis and function^,. In turn, TFEB activation may also further promotes the TRPML1-mediated release of lysosomal Ca²⁺ and induction of lysosomal exocytosis. Subsequently, lysosomal exocytosis is promoted, which facilitates the removal of intracellular U and clearance of damaged lysosomes with LMP under the cooperation of compensatory lysosomal biogenesis and consequently reduces the U-induced cell death.