Application of TRPML1 agonist in preparation of medicine for promoting uranium discharge

Abstract

The invention relates to application of a TRPML1 agonist in preparation of a medicine for promoting uranium excretion, the TRPML1 agonist comprises rapamycin, the use dosage of the TRPML1 agonist is 1-8 mg / kg, the application realizes efficient uranium excretion by activating TRPML1 and promoting lysosome exocytosis, and the TRPML1 agonist is especially suitable for delayed treatment after uranium exposure. The invention discloses the process that the rapamycin promotes lysosomal exocytosis and biogenesis by activating the TRPML1, effectively reduces accumulation of uranium in the kidney and relieves renal toxicity for the first time.

Description

Technical Field

[0001] This invention relates to the field of uranium effluent, and more specifically to the application of a TRPML1 agonist in the preparation of a drug for promoting uranium effluent. Background Technology

[0002] Uranium, an actinide element with significant chemical toxicity and radioactivity, can enter the human body through the respiratory tract, digestive tract, and skin contact. It specifically accumulates in the lysosomes of proximal tubular epithelial cells (PTECs) of the kidney, forming insoluble phosphate complexes, leading to renal tubular damage and dysfunction. It is the main target organ of uranium toxicity.

[0003] Currently, the main clinical strategy for uranium poisoning is chelation therapy, such as using diethylenetriaminepentaacetic acid (DTPA) or sodium bicarbonate. These methods are based on coordination chemistry principles, accelerating the excretion of uranium ions in urine by forming soluble complexes with them. However, existing chelating agents have significant limitations, including low excretion efficiency (e.g., DTPA's 24-hour uranium excretion rate is only 12-18%), near ineffectiveness for delayed treatment (after 24 hours of exposure), and potential increased renal burden. While novel HOPO-type chelating agents have shown some improvement, they still face issues such as short plasma half-life and hepatotoxicity. Therefore, developing a new, highly efficient, low-toxicity uranium excretion strategy that is effective for delayed treatment is a critical technical challenge that urgently needs to be addressed in this field.

[0004] Recent studies have revealed that lysosomes are not only sites of uranium accumulation, but their inherent lysosomal exocytosis provides a novel pathway for the active removal of uranium. This process relies on the activation of the calcium ion channel TRPML1 on the lysosomal membrane. Rapamycin, a mature immunosuppressant, can directly activate TRPML1, independent of its traditional mTOR inhibitory target. Recent research suggests that lysosomal exocytosis may offer new insights into heavy metal detoxification, with the lysosomal membrane calcium ion channel TRPML1 being a key regulator of this process. Summary of the Invention

[0005] Based on the above problems, this invention overcomes the shortcomings of existing chelation uranium excretion therapies, such as low efficiency, delayed treatment, ineffectiveness, and potential toxic side effects. Based on the role of the TRPML1 channel in lysosomal excretion, this invention provides a novel treatment strategy that promotes uranium excretion by targeting and activating the TRPML1 channel and utilizing the lysosomal excretion mechanism.

[0006] This invention provides the use of a TRPML1 agonist in the preparation of a drug for promoting uranium expulsion.

[0007] Furthermore, the TRPML1 agonist includes rapamycin.

[0008] Furthermore, the TRPML1 agonist is used at a dose of 1-8 mg / kg.

[0009] Furthermore, the method for promoting uranium expulsion includes delayed administration of the TRPML1 agonist after uranium exposure; Preferably, the delayed administration time is 0-48 hours after uranium exposure.

[0010] Furthermore, the TRPML1 agonist is used to promote lysosomal exocytosis.

[0011] Furthermore, the TRPML1 agonist promotes uranium expulsion by activating the TRPML1-TFEB signaling pathway.

[0012] Furthermore, the promotion of uranium excretion includes promoting uranium excretion in urine and / or feces.

[0013] Furthermore, it also includes applications in the preparation of drugs for treating uranium poisoning.

[0014] Furthermore, the treatment of uranium poisoning includes reducing uranium-induced nephrotoxicity; Preferably, the reduction of uranium-induced nephrotoxicity includes one or more of the following effects: reducing uranium accumulation in the kidneys, improving renal tubular damage, reducing renal tissue apoptosis rate, downregulating the expression of the kidney-damaging molecule KIM-1, and improving renal function biochemical indicators.

[0015] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a TRPML1 agonist and a pharmaceutically acceptable carrier for promoting uranium excretion and / or treating uranium poisoning.

[0016] Compared with the prior art, the advantages of this invention are: This invention reveals for the first time the process by which rapamycin effectively reduces uranium accumulation in the kidneys and alleviates nephrotoxicity by activating TRPML1 to promote lysosomal exocytosis and biogenesis, and elucidates its mechanism of action. This not only provides a new target for the treatment of uranium poisoning but also offers insights into treatment strategies for other heavy metal toxicities. This research aims to provide a theoretical basis for developing novel uranium excretion strategies and to open new pathways for reducing uranium load in the body and mitigating toxic effects. From an application perspective, the development of TRPML1 agonists may become an important medical strategy for nuclear industry workers and those exposed to environmental pollution, with broad social and economic benefits. Attached Figure Description

[0017] Figure 1 CCK-8 assay for rapamycin cytotoxicity; Figure 2Effects of uranium exposure on HK-2 cell viability: A: CCK-8 assay of cytotoxicity of HK-2 cells after 12 h of uranium exposure; B: CCK-8 assay of cytotoxicity of HK-2 cells after 24 h of uranium exposure; C: CCK-8 assay of cytotoxicity of HK-2 cells after 48 h of uranium exposure. Figure 3 Effects of rapamycin pretreatment on the viability of uranium-treated HK-2 cells: A: Schematic diagram; B: CCK-8 assay of the effect of rapamycin on HK-2 cell viability after 24 h of exposure to different concentrations of uranium nitrate; C: CCK-8 assay of the effect of rapamycin on HK-2 cell viability after 48 h of exposure to different concentrations of uranium nitrate. Figure 4 Effects of rapamycin on the viability of uranium-exposed HK-2 cells: A: Schematic diagram of the time axis after rapamycin treatment at 2, 4, 6, and 24 h prior to U exposure and after U exposure at 2, 4, 6, and 24 h with delayed rapamycin treatment; B: Effect of CCK-8 assay on HK-2 cell viability after pretreatment with rapamycin (15 μM) at 2, 4, 6, and 24 h prior to U exposure, followed by 24 h exposure with uranyl nitrate (600 μM); C: Effect of CCK-8 assay on HK-2 cell viability after 2, 4, 6, and 24 h exposure with uranyl nitrate (600 μM) followed by 24 h treatment with rapamycin (15 μM). Figure 5 A. Grouping and timeline of uranium uptake and efflux experiments. B. ICP-MS detection of intracellular and extracellular uranium content in HK-2 cells after treatment with rapamycin (15 μM) for 30 min prior to and after delay of uranium exposure to low (50 μM), medium (100 μM), and high (600 μM) uranium concentrations. Figure 6 Image showing the release of β-hex in HK-2 cells after Rap treatment; Figure 7 Expression levels of TRPML-1 in HK-2 cells after Rap treatment. A, B: Western blotting analysis of TRPML1 in HK-2 cells after U exposure and Rap treatment. C, D: Representative images (red) of TRPML1 immunofluorescence staining in HK-2 cells after U exposure and Rap treatment. Figure 8 Western blotting analysis of TFEB in HK-2 cells after U exposure and Rap treatment; Figure 9 Expression levels of LAMP-1 in HK-2 cells after uranium exposure and Rap treatment. A, B: Western blotting analysis of LAMP-1 in HK-2 cells after U exposure and Rap treatment. C, D: Representative images (green) of LAMP-1 immunofluorescence staining in HK-2 cells after U exposure and Rap treatment. Figure 10 Representative images of HK-2 cells calcein-AM fluorescence staining after U exposure and Rap treatment (green). Figure 11 Representative images of HK-2 cells stained with caspase-3 fluorescence after U exposure and Rap treatment (red); Figure 12 The effect of rapamycin on mitochondrial membrane potential in uranium-exposed HK-2 cells was investigated by flow cytometry to detect the proportion of cells with positive mitochondrial membrane potential. Figure 13 Effects of rapamycin on uranium transport and excretion in mice. A: Schematic diagram of the experiment in BALB / c male mice after single-dose uranium exposure (2 mg / kg) followed by 2 mg / kg and 5 mg / kg Rap treatment. B: ICP-MS detection of uranium content in the kidney, bone, liver, spleen, heart and lung tissues, and urine and feces in the single-dose U exposure group and the single-dose U exposure followed by Rap treatment group, n=7; Figure 14 Representative H&E staining images of mouse kidneys after U exposure and Rap treatment; Figure 15 Detection of CER, BUN, TP, AST, ALT and AKP levels in plasma; Figure 16 Representative images of TUNEL staining of the S1, S2 and S3 segments of the proximal tubules of the renal cortex of mice after U exposure and Rap treatment; Figure 17 Immunohistochemical representations of the KIM segments S1, S2, and S3 of the proximal tubules of the mouse renal cortex after U exposure and Rap treatment.

[0018] Figure 18 Effects of immediate and delayed administration of sodium bicarbonate after a single uranium exposure in mice on uranium clearance and protection against uranium nephrotoxicity. A: Schematic diagram of the experiment in male BALB / c mice after immediate and delayed administration of sodium bicarbonate following a single uranium exposure. B: U content in kidney tissue of mice treated with immediate and delayed sodium bicarbonate after a single uranium exposure, n=3. C: U content in urine of mice treated with immediate and delayed sodium bicarbonate after a single uranium exposure, n=3. D: CRE levels in mice after immediate and delayed sodium bicarbonate treatment following a single uranium exposure, control group n=7, other groups n=3. E: BUN levels in mice after immediate and delayed sodium bicarbonate treatment following a single uranium exposure, control group n=7, other groups n=3. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0021] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0022] Example 1 This embodiment provides the application of a TRPML1 agonist in the preparation of a drug for promoting uranium expulsion.

[0023] As a further preferred embodiment, the TRPML1 agonist includes rapamycin.

[0024] Rapamycin (Rap) is an immunosuppressant primarily used clinically to combat rejection during organ transplantation. However, Rap also shows great potential in anticancer, neuroprotective, and anti-aging applications. By inhibiting the activity of the mammalian target of rapmycin (mTOR), Rap can activate and regulate TFEB, thereby upregulating lysosomal function. In this invention, Rap directly activates the lysosomal calcium channel TRPML1, an effect independent of mTOR. The opening of the TRPML1 channel releases lysosomal calcium ions. Calcineurin, acting as a receptor for this lysosomal-derived calcium signal, activates TFEB, thereby enhancing lysosomal function and promoting autophagy. Therefore, rapmycin accelerates uranium excretion by activating TRPML1, promoting lysosomal excretion. As a TRPML1-specific agonist, rapmycin activates lysosomal Ca²⁺ release, inducing lysosomal fusion with the plasma membrane, thus expelling uranium from the cell and reducing its accumulation in the kidneys.

[0025] As a further preferred embodiment, the TRPML1 agonist is used at a dose of 1-8 mg / kg.

[0026] Within a certain dosage range, the effect of rapamycin in activating TRPML1 is concentration-dependent. Too low a dose will have no effect, while too high a dose may affect the efficacy and safety due to the drug's own toxicity.

[0027] As a further preferred embodiment, the promotion of uranium excretion includes promoting uranium excretion in urine and / or uranium excretion in feces.

[0028] Uranium excreted into the renal tubules via lysosomal exocytosis is excreted in the urine; while uranium excreted into the interstitial space or via the hepatobiliary system may be excreted in the feces.

[0029] As a further preferred embodiment, the method for promoting uranium excretion includes delayed administration of the TRPML1 agonist after uranium exposure; preferably, the delayed administration time is 0-48 hours after uranium exposure.

[0030] Even if uranium has precipitated in lysosomes, activation of TRPML1 can still initiate the exocytosis clearance procedure of the precipitated uranium. This mechanism does not depend on immediate action before or during uranium entry into the cell, so delayed administration is still effective.

[0031] As a further preferred embodiment, the TRPML1 agonist is used to promote lysosomal exocytosis.

[0032] Lysosomes are important cell compartments that remove toxic heavy metals from the cytoplasm and isolate them within the vacuolar membrane in an insoluble detoxifying form, thus limiting their diffusion throughout the body. Uranium in the kidneys selectively concentrates in the lysosomes of the proximal tubular epithelial cells as insoluble phosphate. Other toxic heavy metals also accumulate and precipitate in lysosomes of various tissues / cells. Lysosomal exocytosis is a calcium-dependent process... 2+ The process involves the fusion of lysosomes with the plasma membrane, releasing their contents into the extracellular space. The lysosomal membrane contains the main Ca²⁺ channels, and their activation can induce lysosomal exocytosis. Activation of lysosomal Ca²⁺ channels... 2+ The TRPML1 channel can significantly improve the cytotoxicity of uranium.

[0033] As a further preferred embodiment, the TRPML1 agonist promotes uranium expulsion by activating the TRPML1-TFEB signaling pathway.

[0034] Upon activation of TRPML1, the released calcium ions activate calmophosphatase, leading to dephosphorylation and nuclear translocation of the transcription factor TFEB. After TFEB enters the nucleus, it upregulates the expression of lysosome-related genes, including TRPML1 itself, forming a positive feedback loop. This not only promotes exocytosis but also enhances lysosomal biogenesis, continuously improving the cell's detoxification capacity.

[0035] As a further preferred embodiment, it also includes use in the preparation of drugs for treating uranium poisoning.

[0036] The core danger of uranium poisoning lies in its accumulation in target organs, particularly the kidneys, and the resulting toxicity. Promoting uranium excretion is itself a fundamental treatment for poisoning.

[0037] The mechanism by which rapamycin reduces uranium-induced renal epithelial cell toxicity is that uranium forms a precipitate in the lysosomes of proximal tubular epithelial cells (PTECs) of the kidney. Rapamycin alleviates uranium-induced nephrotoxicity by clearing damaged lysosomes and reducing lysosome-dependent cell death.

[0038] As a further preferred embodiment, the treatment of uranium poisoning includes reducing uranium-induced nephrotoxicity.

[0039] By removing uranium from renal tubular epithelial cells, the chemical toxicity and radiation damage of uranium to cells are directly reduced, thereby reversing or alleviating a series of pathological manifestations of nephrotoxicity.

[0040] Preferably, the reduction of uranium-induced nephrotoxicity includes one or more of the following effects: reducing uranium accumulation in the kidneys, improving renal tubular damage, reducing renal tissue apoptosis rate, downregulating the expression of the kidney-damaging molecule KIM-1, and improving renal function biochemical indicators.

[0041] Example 2 A pharmaceutical composition comprising a therapeutically effective amount of a TRPML1 agonist and a pharmaceutically acceptable carrier for promoting uranium excretion and / or treating uranium poisoning.

[0042] Example 3 Cell Experiment HK-2 cells are a human renal proximal tubular epithelial cell line that can mimic the accumulation mechanism of uranium in the kidney, respond to the toxic effects of uranium exposure, and facilitate drug screening and mechanism studies. In this embodiment, HK-2 cells were chosen as an ideal cell model for studying uranium-induced nephrotoxicity.

[0043] Experimental Example 1 As a agonist of the calcium channel TRPML1, rapamycin's safety needs to be evaluated first to select a safe dosage for subsequent experiments. HK-2 cells were cultured at 5 × 10⁶ cells per well. 3个 The cells were seeded at a density of 100 μL / well in 96-well plates. After adhesion, a gradient concentration of rapamycin solution (125, 62.5, 31.25, 15.625, 7.8125, 3.9062, 0 μM) was added, with 6 replicates per group. Cytotoxicity was assessed by CCK-8 assay after 24 hours of continuous treatment. Results are as follows: Figure 1 The results showed that the half maximal inhibitory concentration (IC50) of rapamycin was 38.34 μM. To reduce the impact of drug toxicity on the experimental results, a dose with cell viability >75% was selected, i.e., the safe dose for cell experiments was 0-17 μM, preferably 15 μM. The dose of rapamycin administered in subsequent experiments was 15 μM.

[0044] Experimental Example 2: Effects of Uranium Poisoning on HK-2 Cell Viability HK-2 cells were exposed to different concentrations of uranium (0, 62.5, 125, 250, 500, 750, 1000 μM) to induce varying degrees of cell damage after uranium exposure. Treatment times were 12, 24, and 48 hours. Cell viability results showed that the IC50 values ​​for uranium nitrate exposure at 12, 24, and 48 h were 731.47, 519.85, and 498.83 μM, respectively, which are close to the IC50 values ​​reported in previous studies. (Results are as follows...) Figure 2 As shown, cell viability decreases in a dose-dependent manner with increasing uranium exposure time.

[0045] Experimental Example 3: Effect of Rapamycin Pretreatment on Viability of Uranium-Exposed HK-2 Cells HK-2 cells were seeded in 96-well plates and divided into the following groups: Rap pretreatment group 1 (Rap 1h + U 24h) was pretreated with rapamycin (15μM) for 1 hour, and then different concentrations (0, 62.5, 125, 250, 500, 750, 1000μM) of uranyl nitrate were added. After culturing for another 24 hours, cell viability was measured. Two Rap pretreatment groups (Rap 1h + U 24h) were pretreated with rapamycin (15 μM) for 1 hour, and then different concentrations (0, 62.5, 125, 250, 500, 750, 1000 μM) of uranyl nitrate were added. After culturing for 48 hours, cell viability was measured. U-treated group (U24h): Cell viability was measured after culturing with uranyl nitrate at different concentrations (0, 62.5, 125, 250, 500, 750, 1000 μM) for 24 hours; cell viability was measured after culturing for another 48 hours.

[0046] Cell viability was detected using the CCK-8 assay, such as... Figure 3 As shown, the experimental results indicate that rapamycin pretreatment can significantly improve the survival rate of HK-2 cells when the concentration of uranyl nitrate is below 750 μM.

[0047] Conclusion: Rapamycin can alleviate uranium-induced cell damage by activating TRPML1 and has a significant protective effect on HK-2 cells, which is time- and concentration-dependent. This dose-dependent effect is consistent with the toxicity threshold characteristics of uranium in kidney cells, i.e., no significant cell damage is observed at low concentrations, while high concentrations (>100 μM) induce a significant decrease in survival.

[0048] Experiment 4: Effect of delayed rapamycin administration on the viability of uranium-exposed HK-2 cells HK-2 cells were seeded in 96-well plates and divided into the following groups: Pre-treatment with rapamycin (15μM Rap + 600μM U): After treatment with rapamycin (15μM) at 2, 4, 6 and 24 hours in advance, the patient was exposed to U (600μM) for 24 hours. Delayed treatment with rapamycin (600 μM U + 15 μM Rap): After exposure to U (600 μM) for 2, 4, 6, and 24 h, treatment with delayed rapamycin (15 μM) was performed for 24 h.

[0049] U exposure group (600 μM U): Only U (600 μM) exposure was performed, and the U exposure time was the same as that of the rapamycin treatment group.

[0050] After incubation in all three groups of experiments, CCK-8 was added to measure cell viability.

[0051] CCK-8 test results show that... Figure 4 As shown, both early and late administration of rapamycin significantly reduced uranium-induced cytotoxicity and improved HK-2 cell viability.

[0052] As a result, rapamycin can exert a protective effect at different stages of uranium exposure by activating the TRPML1 ion channel, effectively alleviating renal cell damage and providing a new potential strategy for the prevention and treatment of uranium-induced nephrotoxicity.

[0053] Experimental Example 5: Effect of rapamycin on uranium uptake and efflux in HK-2 cells HK-2 cells were seeded in 96-well plates. The effects of pre-treatment and delayed treatment with rapamycin (15 μM) for 30 min on the uptake and efflux of low-concentration (50 μM), medium-concentration (100 μM), and high-concentration (600 μM) uranium were investigated. ML-SA1 was used as a positive control, and cells were grouped according to time axis. Pretreatment group 1: Rap (15 μM) was administered 30 min in advance, and the patient was exposed to U (50 μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0054] Two pretreatment groups were pretreated: Rap (15 μM) was administered 30 min in advance, and the group was exposed to U (100 μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0055] Pretreatment group 3: Rap (15 μM) was administered 30 min in advance, and the samples were exposed to U (600 μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0056] Delayed administration group 1: Samples were taken 30 minutes after exposure to U (50 μM) for 24 hours, followed by administration of Rap (15 μM).

[0057] Two delayed-dose groups: 24 hours after exposure to U (100 μM), samples were taken 30 minutes after administration of Rap (15 μM).

[0058] Three delayed-dose groups: Samples were taken 30 minutes after 24 hours of exposure to U (600 μM) followed by administration of Rap (15 μM).

[0059] Positive control group 1: ML-SA1 (10μM) was administered 30 min in advance, and the patient was exposed to U (50μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0060] Two positive control groups: ML-SA1 (10 μM) was administered 30 min in advance, and the group was exposed to U (100 μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0061] Positive control group 3: ML-SA1 (10μM) was administered 30 min in advance, and the samples were exposed to U (600μM) for 24 hours. After discarding the supernatant, culture medium was added and samples were taken after 30 min.

[0062] Positive control group 4: After 24 hours of exposure to U (50 μM), samples were taken 30 minutes after administration of ML-SA1 (10 μM).

[0063] Positive control group 5: After 24 hours of exposure to U (100 μM), samples were taken 30 minutes after administration of ML-SA1 (10 μM).

[0064] Positive control group 6: After 24 hours of exposure to U (600 μM), samples were taken 30 minutes after administration of ML-SA1 (10 μM).

[0065] Group 1 exposed to U: After 24 hours of exposure to U (50 μM), the supernatant was discarded, culture medium was added, and samples were taken after 30 minutes.

[0066] Group 2 exposed to U: After 24 hours of exposure to U (100 μM), the supernatant was discarded, culture medium was added, and samples were taken after 30 minutes.

[0067] Group 3 exposed to U: After 24 hours of exposure to U (600 μM), the supernatant was discarded, culture medium was added, and samples were taken after 30 minutes.

[0068] Collect the supernatant and cells, count the cells, and measure the concentration of uranium in the supernatant and cells using ICP-MS. Figure 5As shown, compared with the uranium poisoning model group, pretreatment groups 1, 2, and 3, and delayed administration groups 1, 2, and 3, significantly reduced the intracellular uranium content and increased the extracellular uranium content in the uranium-loaded HK-2 cell culture medium. Therefore, pretreatment and delayed administration of rapamycin for 30 min can promote the expulsion of low, medium, and high concentrations of uranium.

[0069] Conclusion: Rap can reduce the cytotoxicity of uranium to HK-2 cells and improve cell viability. ICP-MS analysis confirmed that 30 min of Rap delayed treatment significantly enhanced intracellular uranium clearance efficiency and promoted uranium excretion.

[0070] Experiment 6: TRPML1 activation promotes the release of β-hexosaminoase (β-hex) from HK-2 cells. β-hexosaminidase (β-hex) is a lysosomal hydrolase. During lysosomal exocytosis, β-hex is released extracellularly. Therefore, measuring the β-hex activity in cell culture supernatant can indirectly reflect the extent of lysosomal exocytosis. This study assessed the promoting effect of rapamycin on lysosomal exocytosis and its mechanism of action in clearing intracellular uranium after uranium exposure by detecting the β-hex release level. HK-2 cells were exposed to different concentrations (0 μM (Control group), 100 μM, 300 μM, and 600 μM) of uranium for 24 hours, followed by treatment with rapamycin (15 μM) for 30 minutes. Cell culture medium was then collected, and the extent of lysosomal exocytosis was assessed by measuring β-hex activity using ELISA. Figure 6 As shown, Vehicle represents the solvent itself that dissolves the drug, but does not contain any active ingredients with therapeutic effects.

[0071] Control + Vehicle group: Normal HK-2 cells, without uranium or drug treatment, treated only with solvent (culture medium).

[0072] Control + Rap group: Normal HK-2 cells, without uranium addition, were treated with 15μM Rap for 30 min and then sampled. 100μM U + Vehicle group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0073] 100μM U + Rap group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with 15μM Rap solution, and samples were taken after 30 minutes.

[0074] 300μM U + Vehicle group: HK-2 cells were first treated with 300μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0075] 300 μM U + Rap group: HK-2 cells were first treated with 300 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 min.

[0076] 600μM U + Vehicle group: HK-2 cells were first treated with 600μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0077] 600 μM U + Rap group: HK-2 cells were first treated with 600 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 minutes.

[0078] Uranium exposure was observed to promote β-hex release, and this effect was enhanced by the TRPML1 activator Rap, indicating that TRPML1 accelerates uranium expulsion by promoting lysosomal exocytosis. This promoting effect may help mitigate uranium-induced cell damage.

[0079] Experimental Example 7: Rapamycin enhances lysosomal exocytosis and biogenesis by activating the TRPML1-TFEB pathway. After HK-2 cells were exposed to different concentrations of uranium (0 μM (Control group), 100 μM and 600 μM) for 24 hours, they were treated with Rap (15 μM) for 30 minutes. The expression levels of TRPML1 protein, transcription factor TFEB and LAMP-1 protein were quantitatively analyzed by Western blotting.

[0080] like Figure 7 As shown, Control + Vehicle group: Normal HK-2 cells, without uranium or drug treatment, treated only with solvent (culture medium).

[0081] Control + Rap group: Normal HK-2 cells, without uranium addition, were treated with 15μM Rap for 30 min and then sampled. 100μM U + Vehicle group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0082] 100μM U + Rap group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with 15μM Rap solution, and samples were taken after 30 minutes.

[0083] 600μM U + Vehicle group: HK-2 cells were first treated with 600μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0084] 600 μM U + Rap group: HK-2 cells were first treated with 600 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 minutes.

[0085] It was found that uranium exposure itself upregulated TRPML1 expression, and this upregulation effect was further enhanced after Rap treatment, indicating that Rap activated the TRPML1 channel. Fluorescence staining experiments provided direct evidence of the intracellular localization and expression level of TRPML1. By labeling TRPML1 with a specific antibody and observing it under a fluorescence microscope, it was found that TRPML1 is mainly located on the lysosomal membrane, and its fluorescence intensity increased significantly after uranium exposure and Rap treatment, further verifying the results of the Western blot experiment and confirming that TRPML1 is a key molecule in the regulation of lysosomal function.

[0086] like Figure 8 As shown, the specific grouping process is the same. Figure 7 After 24 hours of uranium exposure (0 μM (Control group), 100 μM and 600 μM), the expression level of TFEB in HK-2 cells was significantly increased, and Rap treatment further enhanced these effects, revealing the central role of TFEB in the uranium toxicity response and its molecular mechanism of regulating lysosomal function through a positive feedback loop.

[0087] LAMP-1 (lysosome-associated membrane protein 1) is one of the main proteins on the lysosomal membrane, participating in lysosomal biogenesis, fusion, and exocytosis. Changes in its expression level can reflect the function and state of the lysosome. Figure 9 As shown, the specific grouping process is the same. Figure 7 Rap treatment increased the expression level of LAMP-1 protein. Rap ​​promotes lysosomal biogenesis and exocytosis by activating the TRPML-1 channel, which can further promote the release of uranium from lysosomes to the extracellular space, thereby reducing intracellular uranium accumulation.

[0088] Experimental Example 8: Delayed treatment with rapamycin alleviates uranium exposure-induced apoptosis in HK-2 cells. In the experimental group, HK-2 cells were exposed to different concentrations of uranium (100 μM, 300 μM, and 600 μM) for 24 hours, and then treated with Rap (15 μM) for 30 minutes before sampling. ML-SA1 was used as a TRPML-1 agonist as a positive control group. HK-2 cells were exposed to different concentrations of uranium (100 μM, 300 μM, and 600 μM) for 24 hours, and then treated with ML-SA1 (10 μM) for 30 minutes before sampling. The control group served as a blank control group without any treatment. The U + Vehicle group was a uranium poisoning group with different concentrations of uranium added as a negative control. Calcein-AM staining experiments were performed by fluorescent staining.

[0089] like Figure 10 As shown, the Control + Vehicle group consists of normal HK-2 cells that have not been treated with uranium or drugs, but only with solvent (culture medium).

[0090] 100μM U + Vehicle group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0091] 100μM U + Rap group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with 15μM Rap solution, and samples were taken after 30 minutes.

[0092] 100μM U +ML-SA1 group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, and then replaced with 10μM ML-SA1 solution. Samples were taken after 30 minutes.

[0093] 300μM U + Vehicle group: HK-2 cells were first treated with 300μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0094] 300 μM U + Rap group: HK-2 cells were first treated with 300 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 min.

[0095] 300μM U +ML-SA1 group: HK-2 cells were first treated with 300μM uranium solution for 24 hours, and then replaced with 10μM ML-SA1 solution. Samples were taken after 30 minutes.

[0096] 600μM U + Vehicle group: HK-2 cells were first treated with 600μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0097] 600 μM U + Rap group: HK-2 cells were first treated with 600 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 minutes.

[0098] 600μM U +ML-SA1 group: HK-2 cells were first treated with 600μM uranium solution for 24 hours, and then replaced with 10μM ML-SA1 solution. Samples were taken after 30 minutes.

[0099] Compared to the uranium-exposed (U+Vehicle) group, treatment with rapamycin resulted in increased relative fluorescence intensity, increased number of viable cells, and improved uranium-induced apoptosis, further verifying that Rap can significantly reduce uranium-induced renal cell death by promoting lysosomal exocytosis.

[0100] like Figure 11 As shown, the Control + Vehicle group consists of normal HK-2 cells that have not been treated with uranium or drugs, but only with solvent (culture medium).

[0101] Control + Rap group: Normal HK-2 cells, without uranium addition, were treated with 15μM Rap for 30 min and then sampled. 100μM U + Vehicle group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0102] 100μM U + Rap group: HK-2 cells were first treated with 100μM uranium solution for 24 hours, then replaced with 15μM Rap solution, and samples were taken after 30 minutes.

[0103] 600μM U + Vehicle group: HK-2 cells were first treated with 600μM uranium solution for 24 hours, then replaced with fresh culture medium, and samples were taken after 30 minutes.

[0104] 600 μM U + Rap group: HK-2 cells were first treated with 600 μM uranium solution for 24 hours, then replaced with 15 μM Rap solution, and samples were taken after 30 minutes.

[0105] Immunofluorescence staining revealed that cleaved caspase-3 expression levels were significantly enhanced in HK-2 cells after 24 hours of uranium exposure (100 μM and 600 μM), indicating that uranium induced significant apoptosis. Treatment with Rap significantly reduced the fluorescence intensity of cleaved caspase-3, suggesting that Rap effectively inhibited uranium-induced apoptosis. This further validated that Rap significantly reduced uranium-induced renal cell death by promoting lysosomal exocytosis, demonstrating a clear molecular link between lysosomal function regulation and apoptosis.

[0106] Example 9: Delayed rapamycin treatment alleviates uranium exposure-induced mitochondrial damage in HK-2 cells. Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime Biotechnology, China, #305818), following the instructions. A brief summary of the procedure is as follows: HK-2 cells were cultured at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in complete medium for 24 h. After cell adhesion, cells were exposed to 100 or 600 μM uranium (U) solution for 24 h, followed by treatment with 15 μM rapamycin for 30 min. After treatment, cells were collected by trypsin digestion, washed twice with pre-chilled PBS, and resuspended in 1 mL of JC-1 staining working solution, and incubated at 37°C in the dark for 30 min. Subsequently, cells were washed twice with JC-1 staining buffer, and finally resuspended in 500 μL of the buffer and analyzed immediately using flow cytometry (BD FACS Celesta, USA). JC-1 dye accumulates in the matrix in response to the mitochondrial membrane potential gradient. Under high membrane potential, JC-1 forms aggregates and produces red fluorescence (excitation / emission wavelength: 585 / 590 nm); while under decreased membrane potential, JC-1 exists as monomers and emits green fluorescence (excitation / emission wavelength: 514 / 529 nm). Changes in mitochondrial membrane potential can be quantitatively assessed by calculating the ratio of red to green fluorescence intensity.

[0107] Grouping: Blank control group (control) - normally cultured HK-2 cells; Uranium exposure group (U 100μM): HK-2 cells were exposed to 100μM uranium for 24 hours; Uranium exposure group (U 600μM): HK-2 cells were exposed to 600μM uranium for 24 hours; Uranium exposure + Rap treatment group (U 100μM + Rap): HK-2 cells were exposed to 100μM uranium for 24 hours and then treated with 15μM Rap for 30 minutes.

[0108] Uranium exposure + Rap treatment group (U 600μM + Rap): HK-2 cells were exposed to 600μM uranium for 24 hours and then treated with 15μM Rap for 30 minutes.

[0109] Dynamic changes in mitochondrial membrane potential were detected using the JC-1 fluorescent probe. For example... Figure 12 As shown, JC-1 aggregates in the control group cells exhibited typical orange-red fluorescence. After 24 hours of exposure to 100 and 600 μM uranium, the mitochondrial membrane depolarization rate significantly increased, and the green fluorescence intensity was significantly enhanced, indicating that uranium exposure led to the disruption of mitochondrial membrane integrity. Treatment with Rap for 30 minutes after uranium exposure significantly reduced the mitochondrial membrane depolarization rate and restored the red-green fluorescence ratio, indicating that rapamycin can effectively antagonize uranium-induced mitochondrial damage and maintain normal cellular physiological functions.

[0110] Conclusion: This invention reveals the mechanism by which rapamycin (Rap) promotes lysosomal biogenesis and exocytosis, thereby facilitating uranium expulsion, by activating the TRPML1-TFEB signaling pathway. Delayed Rap treatment not only upregulates TRPML1 expression and promotes TFEB nuclear translocation but also increases the release of lysosomal markers (β-hex and LAMP-1), while reducing the expression of the apoptosis-related protein caspase-3 and improving mitochondrial function, thus effectively alleviating uranium-induced cytotoxicity. These findings provide an important molecular mechanism basis and therapeutic target for developing novel therapeutic strategies against uranium poisoning.

[0111] Experiment Example 4: Animal Experiment 1. Effects of rapamycin on uranium transport and excretion in mice Grouping: (6 BALB / c mice per group) Experimental Group 1 (2 mg / kg U + 2 mg / kg Rap): BALB / c mice were acutely exposed to uranium (2 mg / kg) via a single intravenous injection via the tail vein. Twenty-four hours later, they were treated with the TRPML1-specific agonist Rap (2 mg / kg) via gavage. Feces and urine were collected over 24 hours, and blood, heart, liver, spleen, lungs, kidneys, and femurs were harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis. The remaining tissues were digested with 70% HNO3, and the uranium content was determined by ICP-MS. Experimental Group 2 (2 mg / kg U + 5 mg / kg Rap): BALB / c mice were acutely exposed to uranium (2 mg / kg) via a single tail vein injection. After a 24-hour delay, they were treated with the TRPML1-specific agonist Rap (5 mg / kg) via gavage. Feces and urine were collected over 24 hours, and blood, heart, liver, spleen, lungs, kidneys, and femurs were harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis. The remaining tissues were digested with 70% HNO3, and the uranium content was determined by ICP-MS. Uranium exposure group (2 mg / kg U + Vehicle): BALB / c mice were acutely exposed to uranium (2 mg / kg) for 24 hours via a single tail vein injection. After 24 hours, feces and urine were collected, and blood, heart, liver, spleen, lungs, kidneys, and femurs were harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis. The remaining tissues were digested with 70% HNO3, and the uranium content was determined by ICP-MS.

[0112] Control group 1 (control + vehicle): BALB / c mice received no uranium exposure or drug treatment. Twenty-four hours after a single intravenous injection of 0.9% saline via tail vein, they were administered 0.9% saline via gavage. Feces and urine were collected over 24 hours. Blood, heart, liver, spleen, lungs, kidneys, and femur were then harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis.

[0113] Control group 2 (control + 2 mg / kg Rap): BALB / c mice were treated with the TRPML1-specific agonist Rap (2 mg / kg) via gavage 24 hours after a single injection of 0.9% saline into the tail vein without uranium exposure. Feces and urine were collected over 24 hours, and blood, heart, liver, spleen, lungs, kidneys, and femurs were harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis.

[0114] Three control groups (control + 5 mg / kg Rap): BALB / c mice were treated with the TRPML1-specific agonist Rap (5 mg / kg) via gavage 24 hours after a single injection of 0.9% saline into the tail vein without uranium exposure. Feces and urine were collected over 24 hours, and blood, heart, liver, spleen, lungs, kidneys, and femurs were harvested. One kidney was fixed with 4% paraformaldehyde. Blood was centrifuged, and plasma was collected for biochemical analysis.

[0115] Positive control group 1 (sodium bicarbonate (NaHCO3immedidte): uranyl nitrate exposure (uranyl nitrate 2mg / kg, tail vein injection) + immediate gavage administration of 1.0g / kg sodium bicarbonate can induce urine alkalinization (pH 8-9).

[0116] Positive control group 2: (Sodium bicarbonate (Na HCO3 24h-delay): Uranyl nitrate exposure (uranyl nitrate 2mg / kg, tail vein injection) + 1.0g / kg sodium bicarbonate administered by gavage after a 24-hour delay can induce urine alkalinization (pH 8-9).

[0117] The physiological process of uranium excretion via the kidneys leads to its specific accumulation in mouse kidney tissue. Quantitative analysis by ICP-MS, such as... Figure 13 As shown, over 80% of uranium is accumulated in the kidneys and bones, which is consistent with the physiological function of the kidneys as the main organ for uranium excretion. After Rap delayed treatment, the uranium content in the heart, liver, spleen, lungs, kidneys, and bone tissues decreased significantly, while the uranium content in feces and urine increased significantly, indicating that Rap promotes uranium excretion in mice by activating TRPML-1.

[0118] 2. Effects of rapamycin on uranium-induced kidney damage - He pathological analysis HE staining of one kidney from mice in the experimental group, control group, and uranium-treated group was performed for pathological analysis, such as... Figure 14 As shown, the renal tubular epithelial cells in the control group have intact tissue structure, with no pathological changes such as inflammation or necrosis observed. In the uranium-treated group, the renal tubular epithelial cells have lost their nuclei, the lumen morphology is severely damaged, and a large amount of proteinaceous mucus exudates from the tubules (red arrows), with disordered tissue structure (blue arrows). The cells in the experimental group show mild edema, and regularly shaped round vacuoles are visible within the cells (green arrows), indicating reduced lesion damage.

[0119] 3. Effects of rapamycin on uranium-induced liver and kidney function damage - liver and kidney function biochemical indicators In existing technologies, when the amount of uranium ingested by the human body exceeds 2 mg / kg, or when the amount of uranium deposited in the kidneys reaches 3 μg / g, significant kidney damage occurs, and a significant increase in serum creatinine and blood urea nitrogen levels can be observed. Uranium induces oxidative stress in hepatocytes and mitochondrial dysfunction, leading to a significant increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), resulting in acute liver injury and significant pathological changes in liver tissue, including hepatocyte necrosis, inflammatory cell infiltration, and fibrosis, severely affecting the liver's detoxification and metabolic functions. Therefore, to evaluate the protective effect of rapamycin against uranium-induced liver and kidney injury in mice, blood was collected from the eyes of mice 48 hours after uranium exposure (using heparin sodium anticoagulant tubes). The mice were divided into four groups: experimental group 1, group 2, control group, and uranium-exposed group (2 mg / kg U + Vehicle). After standing at 4 ℃ for 2 hours, the blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. Creatinine (Cr), blood urea nitrogen (BUN), total protein (TP), alkaline phosphatase (AKP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured. Figure 15 As shown, plasma biochemical indicators indicate that uranium poisoning can cause liver and kidney damage, manifested as elevated levels of CRE, BUN, AST, ALT, and AKP, and decreased levels of TP. Rap ​​treatment can effectively alleviate liver and kidney damage.

[0120] 4. Effects of rapamycin on kidney damage induced by uranium poisoning in mice - TUNEL apoptosis analysis TUNEL staining allows for a direct observation of the number and distribution of apoptotic cells in kidney tissue, thereby assessing the extent of kidney damage caused by uranium poisoning and the therapeutic effect of Rap.

[0121] Cells with the same DAPI blue label were selected as the total cells. Each photograph was analyzed to determine the number of positive cells and the total number of cells. The percentage of positive cells (number of positive cells / total number of cells * 100) was calculated as the apoptosis rate (%). TUNEL apoptosis analysis was performed, with groups as described above: experimental group 1, group 2, uranium poisoning group, and control group. Figure 16 As shown, no significant apoptosis was observed in the control group. Compared with the control group, the number of TUNEL-positive cells and the apoptosis rate were significantly increased in the S1, S2, and S3 segments of the kidneys of mice exposed to uranium, indicating that uranium exposure induced significant renal cell apoptosis, with the most significant apoptosis occurring in the S3 segment of the proximal tubule. Rap ​​treatment effectively alleviated uranium-induced apoptosis in various parts of the kidneys. In mice treated with rapamycin, the number of TUNEL-positive cells and the apoptosis rate in the kidneys after uranium exposure were significantly lower than those in the uranium-exposed group alone.

[0122] 5. Effects of rapamycin on KIM-1 expression in the kidney tissue of uranium-poisoned mice KIM-1 is a sensitive biomarker for acute kidney injury (AKI). Its immunohistochemical detection can accurately locate the site of injury (such as the proximal convoluted tubule) and quantify the degree of injury. Within each group, at least three 200x fields of view were randomly selected from each slide for imaging. During imaging, the tissue should fill the entire field of view as much as possible to ensure consistent background lighting in each image. ImageJ software was used to select the same brownish-yellow color as a unified standard for judging the positivity of all images, and the cumulative optical density (IOD) of each positive image was analyzed. Figure 17 As shown, compared with the control group, the IOD values ​​of the S1, S2, and S3 segments of the kidney in the uranium exposure group were significantly increased, indicating that uranium exposure significantly induced KIM-1 expression, causing significant acute kidney injury affecting different sites. After rapamycin administration, the IOD value was lower than that in the uranium exposure group. Rapamycin activated the TRPML1 pathway, promoted uranium excretion, reduced kidney damage, and decreased KIM-1 expression. In addition, different parts of the kidney showed different responses to uranium exposure and intervention, with the S3 segment being more sensitive to uranium exposure.

[0123] Animal studies showed that in a BALB / c mouse model induced by a single high-dose uranium exposure (2.0 mg / kg), delayed administration of the TRPML1-specific agonist Rap (2.0 and 5.0 mg / kg) for 24 hours significantly reduced uranium accumulation in the heart, liver, spleen, lungs, kidneys, and bone tissues and promoted uranium excretion in feces and urine. Uranium poisoning causes liver and kidney damage, manifested by elevated levels of CRE, BUN, AST, ALT, and AKP, and decreased TP; Rap effectively alleviated this damage. Immunohistochemical analysis revealed that Rap treatment significantly reduced the expression of the proximal tubule injury marker KIM-1 and the degree of pathological damage, particularly in the S3 segment of the renal cortex. Furthermore, TUNEL apoptosis analysis further demonstrated that uranium exposure significantly induced acute kidney injury and apoptosis, and that Rap could alleviate both damage and apoptosis. These findings provide reliable preclinical evidence for TRPML1-targeted therapy against uranium nephrotoxicity.

[0124] Currently, drugs used clinically to promote uranium excretion, such as sodium bicarbonate, rely on urine alkalization as their mechanism of action. Figure 18As shown, immediate gavage administration of sodium bicarbonate (1.0 g / kg) after a single high-dose uranium exposure (2.0 mg / kg) effectively promoted urinary alkalization (pH 8–9), significantly reduced renal uranium accumulation, and increased 24-hour uranium excretion. However, under delayed administration, the excretion-promoting effect of sodium bicarbonate was significantly limited. Simultaneously, immediate administration of sodium bicarbonate also improved renal function impairment, manifested by a decrease in CRE and BUN levels. However, delayed administration of sodium bicarbonate for 24 hours failed to alleviate renal tubular damage. This result is consistent with its ineffective promotion of uranium excretion under delayed administration, indicating that sodium bicarbonate is only effective when administered immediately after uranium exposure and is essentially ineffective for delayed treatment or chronic uranium exposure, representing a significant unmet clinical need. Compared to existing drugs like sodium bicarbonate, the rapamycin of this invention, administered as a single dose 24 hours after uranium exposure, still significantly promotes uranium excretion via urine and feces and effectively alleviates uranium-induced renal tissue pathological damage, functional abnormalities, and apoptosis, demonstrating superior advantages in delayed treatment.

[0125] in conclusion: Cellular-level experiments revealed that Rap can reduce uranium-induced cytotoxicity in HK-2 cells and improve cell viability. ICP-MS analysis confirmed that Rap delayed treatment for more than 30 minutes significantly enhanced intracellular uranium clearance efficiency and promoted uranium excretion. Its mechanism of action involves activating the TRPML1-TFEB signaling pathway, promoting lysosomal biogenesis and exocytosis, thereby accelerating uranium clearance. Rap ​​delayed treatment not only upregulates TRPML1 expression and promotes TFEB nuclear translocation but also increases the release of lysosomal markers (β-hex and LAMP-1), while reducing the expression of the apoptosis-related protein caspase-3 and improving mitochondrial function, thus effectively alleviating uranium-induced cytotoxicity.

[0126] At the animal level, a mouse model of single high-dose uranium exposure was established. Animal experimental results showed that delayed administration of Rap could significantly reduce the uranium content in animal tissues, promote uranium excretion in feces and urine, improve uranium-induced kidney and liver damage, KIM-1 expression and pathological changes in proximal tubule damage markers caused by uranium poisoning, and effectively reduce uranium-induced apoptosis in various parts of kidney cells.

[0127] This invention elucidates for the first time that rapamycin effectively alleviates uranium-induced nephrotoxicity by targeting and activating TRPML1, inducing lysosomal exocytosis and biogenesis, promoting the clearance of uranium accumulated in proximal renal tubular epithelial cells, and reducing uranium-induced cell death. This research not only reveals the core role of the TRPML1-TFEB pathway in heavy metal detoxification but also provides important theoretical support for developing novel therapeutic strategies targeting uranium and other heavy metal nephrotoxicity.

[0128] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of a TRPML1 agonist in the manufacture of a medicament for promoting uranium excretion.

2. Use according to claim 1, characterized in that, The TRPML1 agonist comprises rapamycin.

3. Use according to claim 2, characterized in that, The TRPML1 agonist is used at a dose of 1-8 mg / kg.

4. Use according to claim 3, characterized in that, The method for promoting uranium excretion comprises delayed administration of the TRPML1 agonist after uranium exposure.

5. Use according to claim 4, characterized in that, The TRPML1 agonist is used for promoting lysosomal exocytosis.

6. Use according to claim 5, characterized in that, The TRPML1 agonist promotes uranium excretion by activating the TRPML1-TFEB signaling pathway.

7. The use according to claim 1, characterized in that, The promotion of uranium excretion comprises promotion of urinary uranium excretion and / or fecal uranium excretion.

8. The use according to claim 1, characterized in that, Also included is the use in the manufacture of a medicament for treating uranium poisoning.

9. Use according to claim 8, characterized in that, The treatment of uranium poisoning comprises alleviating uranium-induced nephrotoxicity. Preferably, the alleviating of uranium-induced nephrotoxicity comprises one or more of the following effects: reducing kidney uranium accumulation, improving renal tubular injury, reducing kidney tissue apoptosis rate, down-regulating the expression of kidney injury molecule KIM-1, improving kidney function biochemical indicators.

10. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising a therapeutically effective amount of a TRPML1 agonist and a pharmaceutically acceptable carrier, for promoting uranium excretion and / or treating uranium poisoning in a subject.

Images