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- Unpaywall
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- 1838-7640
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- 10.7150/thno.84655
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Abstract
Background: Cisplatin is a widely used anti-tumor agent but its use is frequently limited by nephrotoxicity. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel which is generally viewed as a sensor of oxidative stress, and increasing evidence supports its link with autophagy, a critical process for organelle homeostasis. Methods: Cisplatin-induced cell injury and mitochondrial damage were both assessed in WT and Trpm2-knockout mice and primary cells. RNA sequencing, immunofluorescence staining, immunoblotting and flowcytometry were applied to interpret the mechanism of TRPM2 in cisplatin nephrotoxicity. Results: Knockout of TRPM2 exacerbates renal dysfunction, tubular injury and cell apoptosis in a model of acute kidney injury (AKI) induced by treatment with cisplatin. Cisplatin-caused tubular mitochondrial damage is aggravated in TRPM2-deficient mice and cells and, conversely, alleviated by treatment with Mito-TEMPO, a 2+ mitochondrial ROS scavenger. TRPM2 deficiency hinders cisplatin-induced autophagy via blockage of Ca influx and subsequent up-regulation of AKT-mTOR signaling. Consistently, cisplatin-induced tubular mitochondrial damage, cell apoptosis and renal dysfunction in TRPM2-deficient mice are mitigated by treatment with a mTOR inhibitor. Conclusion: Our results suggest that the TRPM2 channel plays a protective role in cisplatin-induced AKI via 2+ modulating the Ca -AKT-mTOR signaling pathway and autophagy, providing novel insights into the pathogenesis of kidney injury. Keywords: TRPM2, autophagy, mitochondria, cisplatin, acute kidney injury Introduction Acute kidney injury (AKI) is a common clinical filtration function, mainly caused by hypotension, condition defined by a rapid decline of glomerular dehydration, sepsis, nephrotoxins and renal ischemia- https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4357 reperfusion (1-4). (4). The incidence of AKI during channel (18) and identified cADPR as an activator to hospitalization is estimated to be 10%-15% (5). TRPM2 channel via its specific interactions with the Cisplatin is a highly effective agent for treating ADPR Binding Pocket (19). A number of studies multiple solid tumors, but its dose-dependent support that TRPM2 plays a critical role in multiple nephrotoxicity frequently curtails its clinical use (6, 7). pathological conditions, such as post-ischemic brain It is estimated that approximately 30% of adult injury (20), diabetes (21) and liver damage (22). patients treated with cisplatin experienced AKI and a Nonetheless, some studies have highlighted the 2+ majority of patients who survived for at least 5 years physiological significance of TRPM2-mediated Ca suffered a permanent decline in glomerular filtration signaling in embryonic neurogenesis (23), radiation- function (8). To date, the mechanisms underlying induced DNA damage response (24), and bacterial cisplatin-induced nephrotoxicity remains poorly clearance in Escherichia coli sepsis (25), and plays a understood. Nonetheless, growing evidence supports protective role in cardiac ischemic injury (26) and that mitochondrial dysfunction plays a pivotal role in pneumonic bacterial infection (27). Moreover, TRPM2 the development of AKI caused by cisplatin (2, 9) and deficiency disturbed mitochondrial homeostasis and other conditions such as ischemia-reperfusion (1) and autophagic process in leukemia and gastric cancer sepsis (3). Mitochondria are the central cellular hub of cells, rendering them more susceptible to chemo- energy production and thus participate in a variety of therapy (28, 29). There is evidence that the TRPM2 physiological processes. Disruption of mitochondria channel mediates ischemic kidney injury (30). not only compromises production of energy but also However, the role of TRPM2 in AKI caused by other increases production of reactive oxygen species (ROS) etiologies such as treatment with cisplatin is still and induces release of cytochrome (Cyt)-c and unknown. mitochondrial DNA (mtDNA), triggering cell In this study, we investigated the role of TRPM2 apoptosis and inflammation (10-12). Therefore, in cisplatin-induced AKI pathogenesis, focusing on its mitochondria are a potential therapeutic target for role in mediating cisplatin-induced effects on treatment of AKI. mitochondrial function. Our results from in vivo and Autophagy is a lysosome-mediated degradation in vitro experiments consistently show that TRPM2 process that recycles cellular components including promotes cisplatin-induced autophagy to maintain proteins, lipids and organelles and thus is important mitochondrial homeostasis through inhibiting the in maintaining cellular homeostasis. Previous studies protein kinase B (AKT)-mammalian target of have shown that autophagy occurs at an early stage of rapamycin (mTOR) signaling pathway and protects cisplatin-induced AKI and plays a crucial role in against cisplatin-induced kidney injury. protecting against kidney injury (13), and that renal Methods proximal tubule-specific knockout of Atg7 or Atg5 expression impaired cisplatin-induced autophagy in Cell preparation and treatment tubules and aggravated cell apoptosis and kidney Primary mouse embryonic fibroblasts (MEFs) damage (14, 15). In addition, induction of transcrip- were obtained from embryos at E13.5 and cultured in tion factor EB-mediated autophagy by trehalose DMEM (Gibco, C11995500BT) supplemented with ameliorated cisplatin-induced mitochondrial damage 10% FBS and 1% penicillin/streptomycin at 37℃ in an and kidney injury (9). These findings support the incubator with 5% CO as previously described (31). importance of autophagy in AKI pathogenesis but the Primary culture of mouse renal tubular epithelial cells current understanding is still limited. (mRTECs) was performed as previously described Transient receptor potential melastatin 2 (32). Briefly, kidneys were harvested from 3- to (TRPM2) is the second member of the melastatin 5-week-old male mice, followed by cutting into pieces subfamily of transient receptor potential superfamily. and digesting with 2 mg/mL collagenase I 2+ -permeable none-selective cation channel, As a Ca (Worthington, LS004196) at 37 ℃ for 30 min. Cells TRPM2 is expressed in a variety of organs and tissues, were filtered through a 100-μm strainer and cultured including brain, heart, liver, kidney and pancreatic in RPMI 1640 (Sigma-Aldrich, R-8758) supplemented islet (16). Extracellular signals such as ROS and tumor with 10% FBS, 20 ng/mL epidermal growth factor necrosis factor-α can activate the TRPM2 channel, by (Peprotech, AF-100-15), ITS-X (Gibco, 51500-056) and promoting intracellular production of adenosine 1% penicillin/streptomycin. Staining with fibroblast diphosphate-ribose (ADPR) that specifically binds to marker Vimentin and tubular epithelial marker and activates the TRPM2 channel (17, 18), and AQP-1 was used for the verification of isolated MEFs contribute to oxidative damage and cell death (16). and mRTECs, respectively (Figure S1A-B). HK-2, Our previous studies revealed the structural and HCT116, SW480 and ACHN were purchased from functional basis of selectivity filter in human TRPM2 https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4358 American Type Culture Collection, and cultured in sacrificed. In some other experiments, mice were DMEM/F12 (Sigma-Aldrich, D8437) supplemented either injected i.p. with 1 mg/kg rapamycin or an with 10% FBS for HK-2 and RPMI 1640 supplemented equal volume of vehicle (5% DMSO+30% PEG400) 1 h with 10% FBS for other cell types. prior to and 1 day after cisplatin injection as To induce apoptosis, MEFs and HK-2 cells were previously described (15, 34). For inhibition of TRPM2 treated with 20 µM cisplatin (Selleck, S1166) and activity, mice were injected i.p. with 10 mg/kg 2-APB mRTECs with 5 μM cisplatin for 24 h, respectively. 1 h prior to cisplatin injection with minor modification Clotrimazole (CLT, 10 μM, Target Mol, T0506), 2-APB on the method from a previous study (35). For (20 μM, Sigma-Aldrich, D9754), ADPR (100 μM, inhibition of AKT activity, mice were injected i.p. with MedChemExpress, HY-100973A), chloroquine (CQ, 50 mg/kg VIII 1 h prior to cisplatin injection. 10 μM, MedChemExpress, HY-17589A), Mito-TEMPO Renal function, histopathology and (200 nM, MedChemExpress, HY-112879), BAPTA-AM immunohistochemistry analysis (5 μM, MedChemExpress, HY-100545), 3-methylade- Renal function was assessed by measuring nine (3-MA, 5 mM, Selleck, S2767), VIII (5 μM, serum creatinine using the FUJIDRI-CHEM 7000i Beyotime, SF2784) and rapamycin (50 nM, biochemistry analyzer (FUJIFILM, Tokyo, Japan). MedChemExpress, HY-10219) were added to cell Kidney tissues were fixed in 4% paraformaldehyde culture medium 1 h before incubation with cisplatin. (PFA), embedded in paraffin, and sliced into For induction of autophagy, cells were treated with 4-μm-thick sections for Periodic Acid-Schiff (PAS) H O (Sinopharm Chemical Reagent, 10011218) at 2 2 staining. Tubular injury was semi-quantitatively indicated concentrations for 3 h. To evaluate scored by the percentage of damaged tubules and autophagic flux, MEFs were transfected with histological injury: 0, no damage; 1, < 25%; 2, 25%– lentiviral vector carrying mRFP-GFP-LC3 (HANBIO, 50%; 3, 50%–75%; 4, > 75% (36). The assessment was HB-LP210 0001). To overexpress TRPM2 in mRTECs, performed by two pathologists blinded to the cells were infected with adenovirus encoding mouse experiments. TRPM2 (Adv-TRPM2, purchased from OBiO For immunohistochemistry, kidney sections Technology) for 24 h before subsequent interventions. from mice or human were immersed in EDTA/EGTA All experiments were performed in triplicate or in buffer (pH 9.0) and heated to boiling for antigen quadruplicate. retrieval. 8-OHdG (1:100, Santa Cruz, sc-66036) or Animals TRPM2 (1:100, ET1703-34, Huabio) antibody was C57BL/6 WT mice were purchased from incubated at 37 °C for 3 h. The images were captured -/- Shanghai SLAC Laboratory. Trpm2 transgenic mice using a Leica DM4000 microscope. were introduced from the University of Leeds and Immunofluorescence staining genetic validation was shown in Figure S1C as For immunofluorescence, PFA-fixed frozen previously described (33). These mice express a kidney sections or cells were permeabilized with 0.3% TRPM2 protein that is lack of transmembrane Triton X-100 for 20 min and blocked with 5% bovine domains 3 and 4 due to deletion of exons 17 and 18 serum albumin for 1 h. After washed with PBS, the and does not form functional channel. All mice were slides were incubated with primary antibodies housed in a specific-pathogen-free facility with a 12-h targeting Calnexin (1:100, Santa Cruz, sc-23954), light/dark cycle. All animal experiments were kidney injury marker-1 (Kim-1, 1:500, R&D Systems, approved by the Committees for Animal Experiments AF1817), LC3B (1:200, Sigma-Aldrich, L7543), of Zhejiang University (approval number, ZJU20 lysosomal associated membrane protein 1 (LAMP1, 220301) and performed following the policies of 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Protein- Zhejiang University. tech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; Cisplatin-induced AKI models 1:100, Bethyl Laboratories, A300-414A), TOM20 To induce AKI, 8- to 10-week-old WT and (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, -/- Trpm2 male mice were subjected to a single Huabio, HA500437) or AQP-1 (1:100, Huabio, intraperitoneal (i.p.) injection of 18 mg/kg cisplatin. ET1703-34), respectively or in combination at 4 ℃ The mice were euthanized at 72 h and the blood and overnight, followed by incubation with correspond- kidney tissues were harvested for further analysis. In ing secondary antibodies for 1 h. Brush borders of some experiments, mice were either injected i.p. with proximal tubules were labelled by Lotus tetragonolobus 7 mg/kg Mito-TEMPO or an equal volume of PBS lectin (LTL, 1:200, Vector Laboratories, B-1325), in once daily, starting 7 days before cisplatin injection combination with Cy3-conjugated streptavidin (1:500, and continuing until the day before the mice were Vector Laboratories, SA-1300). DAPI was used for https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4359 nuclear staining. All fluorescence images were 40704ES50) in Hank’s Balanced Salt Solution (HBSS) captured using a confocal microscope (Nikon A1). buffer in combination with 0.05% Pluronic F-127 at The numbers of positive tubules or cells from 5 37°C for 30 min. Cells were rinsed with HBSS three randomly selected fields were averaged for each times and detected using a confocal microscope kidney sample. (Nikon A1) at 488-nm excitation. 2+ Dihydroethidium (DHE) staining Measurement of mitochondrial Ca 2+ DHE staining was performed to determine the For the measurement of mitochondrial Ca , cells level of ROS in kidney tissues as previously described were loaded with 4 μM Rhod-2 AM (Yeasen, (37). Briefly, kidneys were rapidly harvested and 40776ES50) in HBSS buffer in combination with 0.05% freshly frozen in optimal cutting temperature (OCT) Pluronic F-127 at 37°C for 30 min. Cells were rinsed compound and sliced into 8-μm-thick slices. The with HBSS three times and detected under a confocal kidney slices were incubated with 20 μM DHE microscope (Nikon A1) at 550-nm excitation. (Beyotime, S0063) at room temperature in the dark for Mitochondrial isolation 30 min before counterstained with DAPI. Images were Isolation of mitochondria from kidney tissues captured using a confocal microscope (Nikon A1). was performed using Tissue Mitochondrial Extraction Transmission electron microscopy (TEM) Kit (Beyotime, C3606) following manufacturer’s Kidney cortex tissues in 1 mm and collected instructions. Isolation of mitochondria in cells was cells were firstly fixed with 2.5% glutaraldehyde and performed using Cell Mitochondrial Extraction Kit 1% osmic acid and then fixed and dyed with 2% (Beyotime, C3601) following manufacturer’s instruct- uranyl acetate, followed by dehydration in ethanol ions. The mitochondrial and cytoplasmic fractions and acetone. Tissues and cells were embedded and were isolated through differential centrifugation and polymerized at 37℃ overnight and sliced into lysed for further immunoblotting analysis. ultrathin sections, stained with uranyl acetate and RNA-seq profiling lead citrate. Images were captured using a RNA-sequencing was performed in Novogene transmission electron microscope (Philips). on Illumina Hiseq 2500 platform as previously Measurement of mitochondrial membrane described (38). Briefly, RNA was isolated from kidney potential (MMP) tissues using Trizol. Sequencing libraries were constructed using NEBNext Ultra II RNA Library The MMP was determined by JC-1 staining (MedChemExpress, HY-15534) following manufac- Prep Kit for Illumina (NEB, E7770). Principal turer’s instructions. Briefly, cells were incubated with Component Analysis (PCA) was conducted based on 2 μM JC-1 for 20 min at 37°C in the dark, washed with Fragments Per Kilobase Million from each sample. PBS, and analyzed by a flow cytometer with 488-nm Differentially expressed genes were defined as |log2(FoldChange)| > 1, P value < 0.05. Kyoto excitation. The relative MMP was calculated by the ratio of J-aggregate/monomer, i.e. red to green Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed by clusterProfiler fluorescence intensity emitted at 590 and 520 nm, respectively. software. Gene Set Enrichment Analysis (GSEA) was conducted based on the fold change of differentially Measurement of mitochondrial ROS (mtROS) expressed genes. The level of mtROS in living MEFs was detected Immunoblotting by MitoSOX (Invitrogen, M36008). Briefly, cells were incubated in 5 μM MitoSOX at 37 ℃ for 10 min before Kidney tissues and cells were lysed in RIPA harvested and detected by flow cytometry at 550-nm lysis buffer (Millipore, 20-188). Protein concentrations were determined using a Bradford assay (Beyotime, excitation. P0006C). 15-20 µg proteins were separated by Mitochondrial morphology analysis SDS-PAGE and transferred to polyvinyl- Cells were incubated with 200 nM MitoTracker idene difluoride membranes. After blocked in 5% Red CMXRos (Invitrogen, M7512) at 37°C for 30 min. fat-free milk for 1 h, membranes were incubated in the Cells were rinsed with PBS twice and detected using a following primary antibodies at 4℃ overnight: confocal microscope (Nikon A1) at 550-nm excitation. anti-LC3B (1:1000, L7543) from Sigma-Aldrich; anti-Bcl2 (1:1000, 26593-1-AP); anti-caspase-3 (1:1000, 2+ Measurement of intracellular Ca 19677-1-AP), anti-Cyt-C (1:1000, 10993-1-AP), 2+ For the measurement of intracellular Ca , cells anti-GAPDH (1:5000, 60004-1-Ig) from Proteintech; were loaded with 4 μM Fluo-4 AM (Yeasen, anti-p62 (1:1000, A19700), anti-AMPKα1 (1:1000, https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4360 A1229), anti- adenovirus E1B 19-kDa-interacting Statistics protein 3 (BNIP3, 1:1000, A19593) from ABclonal; Quantitative data were expressed as mean ± anti-DRP1 (1:1000, #8570), anti-TOM20 (1:1000, standard error of the mean. Difference was examined #42406), anti-phospho-mTOR (Ser2448, 1:1000, using two-tailed unpaired Student’s t-test between #5536), anti-mTOR (1:1000, #2983), anti-phospho- two groups, or one-way ANOVA followed by Tukey’s AKT (Ser473, 1:1000, #9271), anti-AKT (1:1000, #4691), post-hoc test among three or more groups. All anti-phospho-p70S6K (Thr389, 1:1000, #9234) from statistical analysis was performed using GraphPad CST; anti-BAX (1:1000, D220073) from BBI; Prism 8 software. P < 0.05 was considered statistically anti-phospho-Beclin1 (Ser295, 1:1000, ab183313), significant. anti-VDAC (1:1000, ab14734) from Abcam; anti-TRPM2 (1:1000, PA1-46473) from Invitrogen; Results anti-phospho-AMPKα (Thr172, 1:1000, AA393) from TRPM2 is expressed in proximal tubules and Beyotime; anti-TRPM2 (1:1000, A300-414A) from mitochondria Bethyl Laboratories; anti-Beclin1 (1:1000, AP6020) The absorption and accumulation of cisplatin in from Bioworld; anti-PINK1 (1:1000, DF7742) from Affinity Biosciences. Image J software was used to kidneys, particularly in renal tubular epithelial cells, leads to the nephrotoxicity (6). To examine the role of quantify proteins. TRPM2 in cisplatin-induced kidney injury, we firstly Cell viability assay investigated the expression of TRPM2 in kidneys. As Cell viability was determined using the CCK-8 revealed by immunofluorescent imaging, TRPM2 was assay (MedChemExpress, HY-K0301) following expressed predominantly in the proximal tubules manufacturer’s instructions. Briefly, cells were seeded labelled by LTL and rarely in glomeruli or tubulo- onto 96-well plates at a density of 4×10 cells/well. interstitium in untreated mice (Figure 1A), as Cells were treated with cisplatin at indicated previously reported (30). Similarly, TRPM2 in human concentrations for 24 h and subsequently incubated kidneys had the similar distribution that was not with CCK-8 reagent at 37℃ for 3 h. The absorbance at noticeably changed due to a pathological manifesta- 450 nm was measured by a microplate reader tion of acute tubular necrosis (Figure S1D). Typically, (SpectraMax M5/M5e). TRPM2 was mainly localized to the cytoplasmic membranes. Intriguingly, TRPM2 also showed Quantification of mtDNA diffused intracellular distribution, including in the Total DNA was isolated from kidney tissues mitochondria indicated by the co-localization of using the Universal Genomic DNA Purification Mini TRPM2 and the mitochondrial marker TOM20 (Figure Spin Kit (Beyotime, D0063) following manufacturer’s 1B), but not in the lysosomes or endoplasmic instructions. The mtDNA level was indicated by the reticulum (Figure S1E-F). Consistently, TRPM2 was ratio of mtDNA to nuclear DNA. Quantitative PCR detected by immunoblotting in mitochondria isolated was performed to amplify Nd2 gene from from primary mRTECs but its mitochondrial mitochondrial genome and Gapdh gene from nuclear distribution was not altered by treatment with genome (39). Primer sequences used are as follows: cisplatin (Figure S1G). Finally, there seemed to be a Gapdh forward, 5’-CCTGCACCACCAACTGCT small and transient increase in the expression of TAG-3’; Gapdh reverse, 5’-GTGGATGCAGGGATG TRPM2 in kidneys after exposure to cisplatin (Figure TTC-3’; Nd2 forward, 5’-CCCATTCCACTTCTGATT S1H-I). ACC-3’; Nd2 reverse, 5’-ATGATAGTAGAGTTGAG TAGCG-3’. TRPM2 deficiency aggravates cisplatin-induced AKI in mice Analysis of apoptosis Next, we aimed to evaluate the role of TRPM2 in Apoptosis in kidney tissues was examined using cisplatin-induced AKI. The serum creatinine level in the In situ Cell Apoptosis Detection Kit (G1507, WT mice was significantly increased by treatment Servicebio) according to manufacturer’s instructions. with cisplatin, and such increase was aggravated by Here we used serial sections of paraffin-embedded TRPM2 knockout and by addition of TRPM2 inhibitor kidney tissues in both PAS staining and TUNEL 2-APB (Figure 1C-D). In addition, as shown by PAS assays. After staining, the images were captured using staining, treatment with cisplatin resulted in apparent a Leica DM4000 microscope and TUNEL-positive cells casts formation, necrosis of tubular epithelial cells and per 0.25 mm were calculated. Apoptosis in cultured loss of tubular brush border in the renal cortex in WT cells was analyzed by flow cytometry using Annexin mice, all of which became noticeably severer in V-FITC/PI apoptosis kit (70-AP101-100, Multi- -/- Trpm2 mice (Figure 1E-F). Moreover, the proportion Sciences). https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4361 of apoptotic cells induced by treatment with cisplatin closely associated with cisplatin-induced tubular cell -/- was significantly increased in Trpm2 mice compared death and kidney injury. TRPM2 in cardiomyocytes with that in WT mice, as shown by TUNEL assay was reported to be indispensable for maintaining (Figure 1G-H) and immunoblotting of cleaved mitochondrial function (26). Moreover, genetic caspase-3 and BAX (Figure 1I). Consistently, the depletion of TRPM2 in neuroblastoma impaired the expression of Kim-1, a specific marker of injured expression and activation of mitochondrial proteins tubules, shown by immunofluorescent imaging, was through downregulating cAMP-responsive element- -/- higher in cisplatin-treated Trpm2 mice than in WT binding protein and proline-rich tyrosine kinase 2 mice, indicating that TRPM2 deficiency resulted in (40). Hence, we sought to investigate the role of more severe tubular injury (Figure 1J-K). Collectively, TRPM2 on the integrity and function of mitochondria these data support that TRPM2 plays a protective role in kidneys. As shown using TEM imaging, treatment in cisplatin-induced AKI. with cisplatin led to mitochondrial damage in the tubular cells of renal cortex, which was characterized TRPM2 deficiency exacerbates cisplatin- by brightened matrix, mitochondrial swelling, loss of induced tubular mitochondrial injury and ROS cristae and even mitochondrial rupture (Figure 2A). production in kidneys These alterations in the renal tubules were Mitochondrial dysfunction is known to be substantially greater, albeit no spontaneous Figure 1. TRPM2 deficiency aggravates cisplatin-induced acute kidney injury in mice. (A) Representative confocal images showing TRPM2 expression in the renal cortex of WT mice without cisplatin treatment. LTL, Lotus Tetragonolobus Lectin; G, glomeruli. Scale bars, 100 μm. (B) Representative confocal images of tubular epithelial cells double-labeled TRPM2 and mitochondrial marker TOM20 (colocalization indicated by white arrows). Scale bars, 10 μm. (C) Kidney function assessed by the serum creatinine -/- level in Trpm2 and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h (n = 7). (D) Kidney function assessed by the serum creatinine level in WT mice pretreated with vehicle or 2-APB. (E, F) Representative PAS staining images and quantification of tubular injury score (n = 7). Scale bars, 100 μm. (G, H) Representative images of TUNEL staining and quantification of apoptotic cells (n = 7). Scale bars, 100 μm. (I) Immunoblotting analysis and quantification of cleaved and total caspase-3 (CASP3) and BAX. (J, K) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 7). Scale bars, 100 μm. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4362 mitochondrial abnormalities in morphology, in cisplatin, which was however not significantly -/- -/- Trpm2 mice (Figure 2A). Morphology analysis different between WT and Trpm2 mice (Figure 2H). revealed significant declines in mitochondrial area Meanwhile, cisplatin led to upregulation of and aspect ratio after exposure to cisplatin, with the mitochondrial fission marker DRP1 which was -/- latter being more apparent in Trpm2 kidneys (Figure exacerbated by TRPM2 deficiency and downregu- 2B-C). Treatment with cisplatin markedly increased lation of mitochondrial membrane marker TOM20 DHE-positive cells, indicating an elevated level of which was inhibited by TRPM2 deficiency (Figure 2I). ROS in kidneys, and the increase was significantly Moreover, treatment with cisplatin induced release of -/- higher in cells from Trpm2 mice (Figure 2D-E). In Cyt-c into the cytoplasm in kidneys, shown by -/- line with these observations, treatment with cisplatin immunoblotting, which was augmented in Trpm2 induced a higher level of oxidative damage to DNA, mice (Figure 2J). Taken together, these data suggest -/- demonstrated by 8-OHdG staining in Trpm2 mice that TRPM2 is critically engaged in maintaining than in WT mice (Figure 2F-G). There was also a tubular mitochondrial function in response to decline in the mtDNA level after treatment with treatment with cisplatin. Figure 2. TRPM2 deficiency exacerbates tubular mitochondrial fragmentation and oxidative damage in the kidneys of cisplatin-treated mice. (A-C) -/- Representative TEM images showing tubular mitochondrial morphology in the kidneys of Trpm2 and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h and related analysis on mitochondrial area and aspect ratio. Scale bars, 1 μm. (D, E) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 6). Scale bars, 100 μm. (F, G) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 6). Scale bars, 100 μm. (H) Relative mitochondrial DNA content calculated by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) (n = 4). (I) Immunoblotting analysis and quantification of DRP1 and TOM20 in kidneys. (J) Immunoblotting analysis and quantification of cytochrome-c (Cyt-c) in the fractions of mitochondria and cytoplasm deprived of mitochondria. Cyto, cytoplasm; Mito, mitochondria. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4363 Figure 3. TRPM2 deficiency increases cisplatin-induced cell apoptosis and mitochondrial dysfunction in tubular cells. (A) Comparison of cell viability between -/- Trpm2 and WT mRTECs exposed to indicated concentrations of cisplatin (CP) for 24 h (n = 4). (B, C) Cell apoptosis determined by flow cytometry in mRTECs treated with 5 μM CP or normal saline (NS) for 24 h (n = 3). (D) Cell viability of HK-2 cells incubated with TRPM2 inhibitor clotrimazole (CLT, 20 μM) or vehicle (Veh) for 1 h and then subjected to cisplatin (CP) at indicated concentrations for 24 h (n = 3). (E, F) Cell apoptosis determined by flow cytometry in HK-2 cells treated with 20 μM CP or normal saline (NS) for 24 h (n = 3). (G) Immunoblotting analysis and quantification of BAX, Bcl2, cleaved and total caspase-3 (CASP3) in mRTECs. (H) Mitochondrial membrane potential in mRTECs indicated by the ratio of red to green fluorescence intensity of JC-1 (n = 3). (I) Level of mitochondrial ROS in mRTECs determined by the mean fluorescence intensity (MFI) of Mito-SOX (n = 3). Data are presented as mean±SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test in (A, D) and one-way ANOVA with Tukey post-hoc test in (C, F, G, H and I). *p < 0.05, **p < 0.01, ***p < 0.001. against cisplatin-induced toxicity to renal tubular Inhibition of TRPM2 worsens cisplatin-induced cells. cell apoptosis and mitochondrial dysfunction in We also assessed the effects of treatment with vitro cisplatin on mitochondrial function. In both mRTECs We further explored the role of TRPM2 in and MEFs, treatment with cisplatin reduced the ratio cisplatin-triggered cell death in vitro. Treatment with of red to green fluorescence intensity of JC-1 (Figure cisplatin concentration-dependently reduced the 3H and S2D), suggesting loss of MMP, which was viability of primary mRTECs and MEFs isolated from exacerbated by TRPM2 deficiency. On the contrary, mice evaluated by the cell counting kit-8 (CCK-8) treatment with cisplatin markedly increased the assay (Figure 3A and S2A), and induced apoptotic cell fluorescence intensity of MitoSOX, an indicator of death determined by flow cytometry (Figure 3B-C and mitochondrial superoxide, in mRTECs and MEFs S2B). Cisplatin-induced cytotoxicity and apoptosis from WT mice and to a higher level in cells from -/- was worsened by TRPM2 deficiency. Similarly, Trpm2 mice (Figure 3I and S2E). Treatment with cisplatin-induced cytotoxicity and apoptosis was cisplatin elicited mitochondrial fission, detected by sensitized by treatment with CLT, a blocker of TRPM2 Mitotracker Red, which was also aggravated by channel in human proximal tubular epithelial cell line TRPM2 deficiency (Figure S2F). Of notice, TRPM2 HK-2 cells (Figure 3D-F). Treatment with cisplatin deficiency alone did not affect MMP, mtROS level and induced an increase in cleaved caspase-3 and a mitochondrial morphology, suggesting that TRPM2 decrease in the ratio of Bcl2 to BAX, demonstrated by causes no mitochondrial damage in the absence of immunoblotting, both of which became more stress. Collectively, the results support that TRPM2 prominent in mRTECs (Figure 3G) and MEFs (Figure plays a crucial role in protecting against -/- S2C) from Trpm2 mice. These data provide further cisplatin-induced cell apoptosis and mitochondrial evidence to reinforce the protective role of TRPM2 dysfunction. https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4364 Figure 4. Mitochondrial ROS scavenger Mito-TEMPO protects mice from cisplatin-induced kidney injury and mitochondrial damage. (A) Schematic diagram -/- of the experimental design. Briefly, Trpm2 and WT mice were injected daily with either Mito-TEMPO (Mito-T, 7 mg/kg) or vehicle one week before administration of cisplatin (CP, 18 mg/kg) until being sacrificed at 3 days. (B) Kidney function assessed by the serum creatinine level (n = 8). (C, D) Representative PAS staining images and quantification of tubular injury score (n = 8). Scale bars, 100 μm. (E, F) Representative images of TUNEL staining and quantification of apoptotic cells (n = 8). Scale bars, 100 μm. (G, H) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 8). Scale bars, 100 μm. (I) Immunoblotting analysis and quantification of BAX and cleaved and total caspase-3 (CASP3) in mice. (J) Representative TEM images showing mitochondrial morphology in the renal tubules. Scale bars, 1 μm. (K, L) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 8). Scale bars, 100 μm. (M, N) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 8). Scale bars, 100 μm. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. dysfunction and mtROS production were responsible Mito-TEMPO rescues increased tubular for the deterioration of kidney injury due to TRPM2 mitochondrial damage and cell apoptosis in silencing. Mito-TEMPO was shown to be effective in -/- Trpm2 mice during cisplatin-induced kidney reducing ischemic AKI (41) and renal fibrosis (42) injury through eliminating excessive mtROS. Therefore, we Based on the findings described above, we tested this hypothesis by determining the effects of hypothesized that increased mitochondrial pretreatment with Mito-TEMPO (Figure 4A). As https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4365 expected, treatment with Mito-TEMPO clearly after treatment with cisplatin, and such cisplatin- ameliorated renal dysfunction (Figure 4B) and tubular induced formation of mitolysosomes was inhibited by -/- injury (Figure 4C-D) in both WT and Trpm2 mice, TRPM2 deficiency (Figure 5B, D). but the WT mice experienced mild even insignificant In line with the above in vivo findings, treatment improvement on renal function and tubular injury with cisplatin increased the expression of LC3B II in a (Figure 4B-D). Treatment with Mito-TEMPO also concentration-dependent manner in MEFs from WT effectively reduced the number of apoptotic cells mice, but this effect was largely blunted in MEFs from -/- (Figure 4E-F) and Kim-1 positive tubular cells (Figure Trpm2 mice (Figure 5E). The accumulation of LC3B II 4G-H) and the expression of apoptotic biomarker and p62 was elevated further in MEFs treated with -/- (Figure 4I) in both cisplatin-treated WT and Trpm2 CQ, an inhibitor of lysosomal function. In the mice. In addition, the fragmentation of tubular presence of CQ, the expression of LC3B II was also mitochondria was considerably lessened in cisplatin- downregulated in TRPM2-deificent MEFs, indicating -/- treated Trpm2 mice following administration with suppressed autophagic flux (Figure 5F). In addition, Mito-TEMPO (Figure 4J). Moreover, the proportions the formation of autophagosomes and autolysosomes of DHE-positive cells and 8-OHdG-positive cells were was further assessed in cells that expressed mRFP- -/- substantially decreased in Trpm2 mice compared GFP-LC3 (Figure 5G). Consistently, the increased that in WT mice (Figure 4K-N). In summary, levels of both autophagosomes and autolysosomes elimination of mtROS by Mito-TEMPO preserved the after treatment with cisplatin were downregulated by integrity of mitochondria, reduced oxidative damage TRPM2 deficiency (Figure 5H). Moreover, treatment and alleviated cell apoptosis and renal dysfunction in with cisplatin led to a notable increase in the number -/- cisplatin-treated mice, particularly in Trpm2 mice. of mitolysosomes, i.e. co-localization of TOM20 and These data support the notion that TRPM2 activation LAMP2, which was markedly reduced by TRPM2 mediates the protection against cisplatin-induced silencing (Figure 5I-J), indicating a crucial role for mitochondrial dysfunction, apoptosis and loss of TRPM2 in eliminating damaged mitochondria. Taken renal function, likely through reducing mtROS together, these data suggest that TRPM2 is required production. for cisplatin-induced autophagy in vitro and in vivo. TRPM2 is required for cisplatin-induced TRPM2 protects against cisplatin-induced activation of autophagy kidney injury and mitochondrial damage by modulation of autophagy through inhibiting Autophagy is one of the most important the AKT-mTOR pathway mechanisms for degrading damaged organelles, including mitochondria. Increasing evidence from To elucidate the mechanism underlying the recent studies suggests strong connection of TRPM2 association between TRPM2 and cisplatin-induced with induction of autophagy (27-29, 43). We were kidney injury, we performed kidney RNA-sequencing interested in whether autophagy, particularly analysis. Distinct gene expression profile between WT -/- TRPM2-mediated autophagy is engaged in cisplatin- and Trpm2 kidneys was initially illustrated by PCA induced AKI. Treatment with cisplatin resulted in an analysis (Figure 6A). A total of 672 genes were increase in autophagic marker LC3B II in mouse significantly increased and 699 genes were -/- kidneys, revealed by immunoblotting analysis, which significantly decreased in Trpm2 mice (Figure 6B-C). was attenuated by TRPM2 deficiency (Figure 5A). The top downregulated pathways revealed by KEGG Treatment with cisplatin led to accumulation of the analysis were enriched in several metabolism selective autophagy receptor p62, which was higher in pathways, whereas the top upregulated pathways -/- Trpm2 mice, indicating compromised autophagic were mainly enriched in inflammatory responses clearance (Figure 5A). Autolysosomes form by the including TNF, IL-17 and NF-κB signaling pathways fusion of autophagosomes and lysosomes for the later and cell survival-related PI3K-AKT pathways (Figure substrate degradation. Treatment with cisplatin 6D). It is worth noting that the AKT-mTOR signaling induced co-localization of LC3 puncta and lysosomal pathway is well known for autophagy inhibition marker LAMP1 in renal cortex, suggesting formation (44-46) and is enriched by GSEA analysis in this study of autolysosomes, which was substantially reduced in (Figure 6E). Here, the phosphorylation levels of AKT -/- Trpm2 mice (Figure 5B-C). Mitophagy is a selective and downstream mTOR were both significantly -/- form of autophagy for degrading damaged increased in the kidneys of Trpm2 mice compared mitochondria (1). We further evaluated the formation with those in WT mice (Figure 6F). We subsequently of mitolysosomes, a late stage of mitophagy. The used rapamycin and VIII to block the activity of number of mitolysosomes in renal cortex, shown by mTOR and AKT, respectively, in cisplatin-treated -/- co-localization of TOM20 and LAMP1, was increased Trpm2 mice. Treatment with rapamycin and VIII https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4366 -/- both markedly elevated the LC3B II level in Trpm2 level that was approximate to that in WT mice. These mice by immunoblotting analysis (Figure 6G-H). In results suggest that the AKT-mTOR signaling addition, rapamycin markedly increased the number pathway is critically involved in TRPM2-mediated of autolysosomes (Figure 6I-J) and mitolysosomes autophagy activation in vivo. -/- (Figure 6I, K) in renal tubules of Trpm2 mice to a Figure 5. TRPM2 is required for cisplatin-evoked autophagy in vitro and in vivo. (A) Immunoblotting analysis and quantification of LC3B II and p62 in the kidneys of -/- Trpm2 and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h. (B) Formation of autolysosomes in the kidneys evaluated by immunofluorescence double-labeled LC3B and LAMP1. Scale bars, 25 μm. Formation of mitolysosomes in the kidneys evaluated by immunofluorescence double-labeled TOM20 and LAMP1. Scale bars, 20 μm. Quantification of (C) autolysosomes and (D) mitolysosomes in the renal tubules (n = 4). (E) Immunoblotting analysis and quantification of LC3B -/- -/- II in Trpm2 and WT MEFs treated with the indicated concentrations of CP for 24 h. (F) Immunoblotting analysis and quantification of LC3B II and p62 in Trpm2 and WT MEFs -/- incubated with 10 μM chloroquine (CQ) or vehicle for 1 h before treatment with 20 μM CP for 24 h. (G) Representative confocal images of LC3 puncta in Trpm2 and WT MEFs + + + - expressing mRFP-GFP-LC3. Scale bars, 20 μm. (H) Quantification of autophagosomes (yellow dots, RFP GFP ) and autolysosomes (red dots, RFP GFP ) (n = 4). (I) Formation of -/- mitolysosomes in Trpm2 and WT MEFs evaluated by immunofluorescence double-labeled TOM20 and LAMP2. Scale bars, 20 μm. (J) Quantification of mitolysosomes in MEFs (n = 4). Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4367 Figure 6. TRPM2 regulates cisplatin-induced autophagy in kidneys via suppressing the AKT-mTOR signaling pathway. (A) PCA analysis on the -/- RNA-sequencing data from cisplatin (CP)-treated Trpm2 and WT mice (n = 3). (B, C) Heatmap and volcano plot of differentially expressed genes (|log2(FoldChange)| > 1, P value < 0.05). (D) Top 10 upregulated and downregulated Kyoto Encyclopedia of Genes and Genomes pathway enrichments. ARVC, Arrhythmogenic Right Ventricular -/- Cardiomyopathy; TNF, tumor necrosis factor. (E) Gene Set Enrichment Analysis of PI3K-AKT-mTOR pathway generated from Trpm2 and WT mice subjected to cisplatin. (F) -/- Immunoblotting analysis and quantification of p-mTOR, mTOR, p-AKT, AKT in the kidneys of Trpm2 and WT mice. (G) Immunoblotting analysis and quantification of LC3B II in the kidneys of CP-treated mice. The mice were either injected i.p. with 1 mg/kg rapamycin (Rapa) or vehicle 1 hour prior to and 1 day after CP treatment. (H) Immunoblotting analysis and quantification of LC3B II in the kidneys of CP-treated mice. The mice were injected i.p. with either 50 mg/kg VIII or vehicle 1 h prior to cisplatin injection. (I) Formation of autolysosomes evaluated by immunofluorescence double-labeled LC3B and LAMP1. Scale bars, 25 μm. Formation of mitolysosomes evaluated by immunofluorescence double-labeled TOM20 and LAMP1. Scale bars, 20 μm. Quantification of (J) autolysosomes and (K) mitolysosomes in the renal tubules (n = 6). Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. We subsequently examined the role of (Figure 7H) confirmed an evident amelioration of TRPM2-mediated autophagy in cisplatin-induced apoptosis in kidneys following treatment with tubular mitochondrial dysfunction and kidney injury. rapamycin. Furthermore, mitochondrial damage was As expected, treatment with rapamycin resulted in a lessened in the renal tubules (Figure 7I), consistent marked decline of the serum creatinine level in with reduced oxidative damage, indicated by reduced -/- Trpm2 mice (Figure 7A), accompanied by reduced DHE positive (Figure 7J-K) and 8-OHdG positive degree of tubular damage (Figure 7B-C) and number (Figure 7L-M) cells. The content of mtDNA was of TUNEL positive cells (Figure 7D-E) and Kim-1 further decreased after treatment with rapamycin, positive cells in the renal cortex (Figure 7F-G). The indicating strengthened elimination of damaged alterations in the expression of cleaved caspase-3 mitochondria (Figure 7N). Consistently, the https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4368 -/- upregulation of DRP1 and TOM20 in Trpm2 mice Collectively, these data suggest that TRPM2-mediated was evidently inhibited by the addition of rapamycin inhibition of mTOR plays a key role in the regulation (Figure 7O). Furthermore, treatment with rapamycin of autophagy and the alleviation of mitochondrial increased localization of Cyt-c in the mitochondria damage and cell apoptosis associated with and reduced its release to the cytosol, suggesting cisplatin-induced AKI. improved mitochondrial integrity (Figure 7P). -/- Figure 7. mTOR inhibitor rapamycin alleviates cisplatin-induced kidney injury, cell apoptosis and mitochondrial dysfunction in Trpm2 mice. (A) Kidney -/- function assessed by serum creatinine level in cisplatin (CP, 18 mg/kg)-treated Trpm2 and WT mice (n = 6). The mice were either injected i.p. with 1 mg/kg rapamycin (Rapa) https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4369 or vehicle 1 hour prior to and 1 day after CP treatment. (B, C) Representative PAS staining images and quantification of tubular injury score (n = 6). Scale bars, 100 μm. (D, E) Representative images of TUNEL staining and quantification of apoptotic cells (n = 6). Scale bars, 100 μm. (F, G) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 6). Scale bars, 100 μm. (H) Immunoblotting analysis and quantification of cleaved and total caspase-3 (CASP3). (I) Representative TEM images showing the mitochondrial morphology in the renal tubules. Scale bars, 1 μm. (J, K) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 6). Scale bars, 100 μm. (L, M) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 6). Scale bars, 100 μm. (N) Relative mitochondrial DNA content calculated by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) (n = 4). (O) Immunoblotting analysis and quantification of DRP1 and TOM20. (P) Immunoblotting analysis and quantification of cytochrome-c (Cyt-c) in the fractions of mitochondria and cytoplasm deprived of mitochondria. Cyto, cytoplasm; Mito, mitochondria. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 8. TRPM2 protects against cisplatin-induced cell apoptosis and mitochondrial dysfunction in vitro by the regulation of autophagy via suppressing -/- the AKT-mTOR signaling pathway. Trpm2 MEFs or mRTECs were pre-incubated with AKT inhibitor VIII (5 μM) or mTOR inhibitor rapamycin (Rapa, 50 nM) for 1 h before -/- cisplatin (CP) intervention. (A) Immunoblotting analysis and quantification of LC3B II, p-AKT and p-p70S6K in Trpm2 and WT mRTECs following treatment with 5 μM CP for -/- 24 h. (B) Representative confocal images of LC3 puncta in Trpm2 and WT MEFs expressing mRFP-GFP-LC3 following treatment with 20 μM CP for 24 h. Scale bars, 20 μm. (C) + + + - Quantification of autophagosomes (yellow dots, RFP GFP ) and autolysosomes (red dots, RFP GFP ) (n = 4). (D, E) Representative confocal images and quantification of -/- mitolysosomes evaluated by immunofluorescence double-labeled TOM20 and LAMP2 in mRTECs (n = 4). Scale bars, 20 μm. Then, Trpm2 mRTECs and MEFs were pre-incubated with rapamycin (50 nM), Mito-TEMPO (Mito-T, 200 nM) or Rapamycin (50 nM) plus 3-MA (5 mM) for 1 h followed by intervention of 5 and 20 μM CP for 24 h, respectively. (F) CP-induced apoptosis in mRTECs determined by flow cytometry (n = 3). (G) Level of mitochondrial ROS in mRTECs determined by the mean fluorescent intensity (MFI) of Mito-SOX (n = 3). (H) Mitochondrial membrane potential of mRTECs indicated by the ratio of red to green fluorescence intensity of JC-1 (n = 3). (I) Mitochondrial morphology of MEFs detected by the fluorescence of MitoTracker Red. Scale bars, 20 μm. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. TRPM2 protects against cisplatin-induced cell phosphorylation level p70S6K, downstream of mTOR is considered a marker of the mTOR activity (47). apoptosis and mitochondrial damage in vitro by Treatment with cisplatin reduced the phosphorylation modulation of autophagy through inhibiting levels of AKT and p70S6K, both of which being higher the AKT-mTOR pathway in TRPM2-deficent mRTECs (Figure 8A) and MEFs We also investigated the role of the AKT-mTOR (Figure S3A). Administration of rapamycin or AKT signaling pathway in cisplatin-treated cells. The https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4370 inhibitor VIII dramatically inhibited the phospho- autophagy activation (Figure 9E-F). In addition, rylation level of p70S6K and increased the expression cisplatin-induced downregulation of phosphorylation of LC3B II in TRPM2 deficient cells (Figure 8A and levels of AKT and p70S6K was also evidently S3A). Further, autophagic flux was suppressed due to augmented after treatment with CLT or BAPTA-AM, TRPM2 silencing as shown by the declined number of suggesting that TRPM2 activity or, more specifically, 2+ autophagosome and autolysosome, both of which TRPM2-mediated Ca influx regulates the AKT- 2+ were restored by treatment with rapamycin and VIII mTOR signaling pathway (Figure 9G). Ca (Figure 8B-C). Consistently, the reduced number of homeostasis is critical for the activity of mitochondrial mitolysosomes in TRPM2 deficient mRTECs and respiratory chain, and we therefore investigated 2+ MEFs were also substantially increased by treatment whether TRPM2 regulated mitochondrial Ca . A 2+ with rapamycin and VIII (Figure 8D-E and S3B-C). marked increase in mitochondrial Ca level, These data further demonstrate that TRPM2 mediates indicated by an increase in the fluorescence of Rhod-2, cisplatin-induced autophagy via modulating the was detected in both WT and TRPM2 deficient AKT-mTOR signaling pathway. mRTECs, but there existed no significant difference Next, we examined the effects of TRPM2- (Figure 9H-I). Mitophagy is mediated mainly by mediated autophagy on mitochondrial homeostasis serine/threonine-protein kinase PINK1 and and cell apoptosis. Treatment with rapamycin not mitophagy receptor BCL2 and BNIP3 (49). The only improved the autophagic level but also led to a expression levels of both PINK1 and BNIP3 in marked increase in cell viability and a marked mRTECs was not affected by TRPM2 silencing (Figure decrease in apoptotic cells in TRPM2 deficient 9J). mRTECs and MEFs, similar with the effects of TRPM2 promotes the anti-tumor effect of Mito-TEMPO (Figure 8F and S3D-E). However, cisplatin concomitant use of 3-MA, a widely used inhibitor of TRPM2 was shown to facilitate tumor cell autophagy, blocked the anti-apoptotic effects of proliferation but also contribute to tumor rapamycin (Figure 8F and S3E). Furthermore, like susceptibility to neutrophil cytotoxicity (50). Thus, we treatment with Mito-TEMPO, treatment with further explored whether blocking or enhancing rapamycin effectively attenuated the increase of TRPM2 activity influenced the anti-tumor effect of mtROS production (Figure 8G and S3F), and rescued cisplatin. Two known TRPM2 inhibitors, 2-APB and the decline of MMP (Figure 8H and S3G) and the CLT, were tested in human colon cancer cell HCT116, fragmentation of mitochondria caused by cisplatin colorectal adenocarcinoma cell SW480 and renal (Figure 8I). However, all of these beneficial effects cancer cell ACHN. Treatment with 2-APB (Figure were blocked by treatment with 3-MA. These data S4A-C) and CLT (Figure S4D-F) markedly attenuated indicate that cisplatin-caused mitochondrial damage cisplatin-induced cytotoxicity in these tumor cell and cell apoptosis are worsened by TRPM2 deficiency lines, examined by the CCK-8 assays, Conversely, through perturbation of the AKT-mTOR incubation with ADPR, a TRPM2 activator, pathway-mediated autophagy. dramatically increased tumor cell death following 2+ TRPM2-mediated Ca influx modulates the treatment with cisplatin (Figure S4G-I). Such AKT-mTOR signaling pathway ADPR-induced reduction in cell viability was 2+ Accumulating studies show that Ca plays however not observed in mRTECs. multifaceted roles in the regulation of autophagy (48), Discussion and Conclusion raising the question of whether TRPM2-mediated 2+ Ca influx facilitates autophagy induction and, if it Cisplatin is among the most common drugs does, whether this effect depends on the AKT-mTOR causing AKI. To date, there is still unmet demand for signaling pathway. We firstly evaluated the effective preventive and therapeutic strategies for 2+ intracellular Ca level in MEFs and mRTECs using cisplatin-induced AKI. In this study, we provide the 2+ Fluo-4 AM, a fluorescent Ca indicator. Exposure to first line of evidence to suggest that TRPM2 deficiency cisplatin induced a significant increase in the in mice increases the susceptibility to cisplatin- fluorescence intensity, indicating an increase in induced nephrotoxicity through impairing activation 2+ intracellular Ca level, which was ablated by TRPM2 of autophagy and disturbing mitochondrial deficiency (Figure 9A-D). Consistently, pharmacolo- homeostasis. gical inhibition of TRPM2 by CLT or chelating TRPM2 has been linked to a diversity of 2+ intracellular Ca by treatment with BAPTA-AM oxidative stress-related disorders (16). Acetamino- impeded cisplatin-induced increase in the level of phen overdosing can induce oxidative stress and 2+ LC3B II in WT MEFs, indicating an inhibitory effect on TRPM2 activation, leading to Ca overload and https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4371 thereby causing hepatocellular death and liver neuroblastoma cells (53), but, to date, the exact role of toxicity (22). In ischemic AKI, TRPM2 has been shown TRPM2 in this intracellular organelle is not fully to interact with and promote the activation of RAC1, defined. Considering that only a small part of TRPM2 leading to increased NADPH activity and oxidative was presented in mitochondria and its expression was stress to cause damage to kidneys (30). The present not altered by treatment with cisplatin, it remains study shows that cisplatin-induced mitochondrial possible that TRPM2 regulates cisplatin-induced ROS production and oxidative damage in kidneys kidney injury via modulating various organelles, as were exacerbated in TRPM2-deficient mice, suggest- well as acting as an ion channel on cell surface. ing a context-dependent role of TRPM2 in kidneys. Mitochondria, while representing the major Notably, we observed substantial localization of source of intracellular ROS, are the intracellular TRPM2 in the cytoplasm of renal tubular cells, organelles that are highly vulnerable to damage by particularly in mitochondria, as shown in ROS. Due to a lack of protective histones, mtDNA is TRPM2-transfected HEK-293 cells (51). Mitochondria also more susceptible to cisplatin-induced damage expression of TRPM2 has also been reported in than nuclear DNA (54). Therefore, mitochondrial hippocampal neurons (52) and SH-SY5Y dysfunction is likely responsible for the deterioration 2+ 2+ Figure 9. TRPM2-mediated Ca influx is involved in the inhibition of the AKT-mTOR pathway in response to cisplatin. Intracellular Ca signals indicated by the -/- fluorescence of Fluo-4 AM in cisplatin (CP, 20 μM) or normal saline (NS) treated Trpm2 and WT MEFs (A, B) and mRTECs (C, D) (n = 7). Scale bars, 20 μm and 10μm, respectively. (E) Immunoblotting analysis and quantification of LC3B II in WT MEFs after incubation with clotrimazole (CLT, 10 μM) or vehicle (Veh) for 1 h followed by treatment with CP at the indicated concentration for 24 h. (F) Immunoblotting analysis and quantification of LC3B II in WT MEFs after incubation with 5 μM BAPTA-AM for 1 h followed by treatment with 20 μM CP for 24 h. (G) Immunoblotting analysis and quantification of p-AKT, AKT and p-p70S6K in WT MEFs after incubation with 5 μM 2+ BAPTA-AM or 10 μM clotrimazole for 1 h followed by treatment with 20 μM CP for 24 h. (H, I) Mitochondrial Ca signals indicated by the fluorescence of Rhod-2 in mRTECs treated by CP or NS (n = 7). Scale bars, 10 μm. (J) Immunoblotting analysis and quantification of BNIP3 and PINK1 in mRTECs treated by CP or NS. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4372 -/- -/- of kidney damage in Trpm2 mice. In support of this significant difference between WT and Trpm2 mice notion, we showed that cisplatin-induced mitochon- (Figure S5A). Intriguingly, several lines of evidence drial fragmentation, ROS production, oxidative suggest a positive role of TRPM2 in ROS-induced damage and release of Cyt-c in kidneys, all of which autophagy under various conditions, such as were aggravated by TRPM2 deficiency, were largely nano-ZnO-treated human perivascular cells (58) and prevented after treatment with the mtROS scavenger epidermal growth factor-treated human corneal Mito-TEMPO. Previous studies examining gastric epithelial cells (59). There is evidence that TRPM2 cancer cells and leukemia cells showed that TRPM2 activation can also inhibit H O -triggered autophagy 2 2 2+ depletion led to mitochondrial dysfunction through through a Ca -dependent feedback mechanism (43). impeding mitochondrial protein expression and We found that TRPM2 silencing increased the LC3 II 2+ mitochondrial Ca uptake (28, 29). Here, while we level in response to exposure to H O in a 2 2 found that TRPM2 was present in mitochondria, there concentration-dependent manner (Figure S5B). were no evident abnormalities in mitochondrial Therefore, further investigation is required to better -/- structure and mtDNA content in Trpm2 kidneys and understand the mechanisms underlying the no substantial differences in ROS production and regulation by TRPM2 of different stress-induced MMP between untreated WT and TRPM2 deficient autophagy. In this study, we showed that the mRTECs or MEFs. Cisplatin-induced mitochondrial AKT-mTOR signaling pathway was activated in vitro 2+ Ca uptake appeared to be unaffected by silencing and in vivo as a result of TRPM2 silencing. TRPM2. These discrepancies may be explained by the Pharmacological inhibition of AKT or mTOR led to different subcellular distribution and multiple substantial elevation of the LC3 II level and formation functions of TRPM2 in various tissues or cells, as well of autophagosomes and autolysosomes, supporting as the different ways of silencing TRPM2, as several that TRPM2 regulates autophagy via modulating the splice variants with distinct functions have been AKT-mTOR pathway. mTOR acts as a typical identified (55). Thus, we speculate that TRPM2 may negative regulator of autophagy via phosphorylating participate in certain mitochondria quality control autophagy-initiating kinase ULK1 at Ser 757 and mechanism instead of directly influencing thereby preventing its activation (60). Intensive mitochondrial function. studies have shown that targeting mTOR effectively It is known that the initiation of caspase- attenuated cisplatin-induced kidney injury through dependent apoptosis in response to cisplatin is improving autophagy (13, 15, 45). AKT could preceded by the increase in LC3 II and indirectly activate mTOR through inhibition of TSC2, autophagosome formation during AKI (56). Inhibiting a suppressor of mTOR (61). It has been reported that autophagy with CQ aggravates renal tubular cell AKT-mediated inhibition of autophagy could apoptosis induced by cisplatin (15), while promoting aggravate cisplatin-induced nephrotoxicity (45) and autophagy with metformin has an opposite effect (57). ototoxicity (62). In the present study, cisplatin-induced accumulation Our recent work (63) shows that TRPM2 of LC3 II and an increase in the number of deficiency attenuates cerebral ischemia-reperfusion autolysosomes were observed both in vitro and in vivo, injury through enhancing autophagy via promoting confirming that autophagy was activated by cisplatin. the activity of AMPK, an upstream suppressor of However, these alterations were markedly attenuated mTOR. Here, we observed a marked increase in the -/- in Trpm2 mice, suggesting a disturbance of phosphorylation level of AMPK after treatment with autophagy. Furthermore, administration of cisplatin, which however remained similar between -/- rapamycin not only restored the autophagy process Trpm2 and WT MEFs (Figure S5C), implying that but also abolished mitochondrial dysfunction, cell AMPK was not involved in the inhibition of apoptosis and kidney injury, implying a critical role of cisplatin-induced autophagy due to TRPM2 TRPM2-mediated autophagy in defending against deficiency. TRPM2 activation by oxidative stress can cisplatin-induced nephrotoxicity. In gastric cancer phosphorylate beclin1 at Ser295 through a 2+ cells, TRPM2 depletion impaired the expression of Ca -dependent mechanism, leading to the inhibition BNIP3, a key mitophagy receptor (29). However, in of autophagy (43). In contrast, the phosphorylation mRTECs, TRPM2 silencing resulted in no significant level of beclin1 was augmented in cisplatin-treated -/- decrease in the expression of BNIP3 or PINK1, Trpm2 MEFs (Figure S5D). The conflicting findings implying that TRPM2 does not regulate mitophagy can be in part explained by a direct interaction of AKT with beclin1 that promotes beclin1 phosphorylation directly by these two mechanisms. Our study revealed that TRPM2 was strongly (64). associated with cisplatin-induced autophagy, though Numerous studies have shown that intracellular 2+ GSEA analysis on autophagy pathway displayed no Ca homeostasis influences a variety of process, https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4373 including autophagy. Serval ion channels have the generation of ADPR was sufficient to provoke ability to modulate autophagy by controlling autophagy through activating TRPM2 channel, or intracellular ion concentrations. For example, TRPV2 perhaps that the protective effect of extra ADPR was and TRPV4 can promote autophagy through offset by a rise in oxidation caused by itself. Similarly, 2+ generating Ca signals that block the AKT activity overexpression of TRPM2 could neither facilitate the (46, 65). In this study, we sought to elucidate the induction of autophagy nor boost the formation of 2+ relationship of TRPM2-mediated Ca signal and autophagosomes and autolysosomes induced by autophagy activation. TRPM2 silencing blocked cisplatin (Figure S6D-F). It is possible that the basal 2+ cisplatin-induced Ca signal in the cytoplasm but not level of TRPM2 activity upon cisplatin treatment was in the mitochondria. Both blockage of the TRPM2 sufficient to initiate the downstream autophagic 2+ channel and chelation of intracellular Ca prevented response for the protection of tubular cells. In the the accumulation of LC3B II (Figure 9E-F) and, future, it is important to gain more insights into the moreover, significantly attenuated cisplatin-induced mechanisms by which TRPM2 determines cell fates in downregulation of the AKT-mTOR signaling different genetic background and such information is pathway (Figure 9G). Collectively, these findings essential for developing targeted and individualized indicate that TRPM2 mediates autophagy in a treatments. 2+ Ca -AKT-mTOR-dependent mechanism. Whether In summary, our study is the first to show that 2+ there is another mediator between Ca signal and TRPM2 is important in protecting cisplatin-induced AKT activity merits further investigation. AKI via the AKT-mTOR signaling pathway and It has been reported that cisplatin-induced cell autophagy. Our findings provide novel insights into death in human laryngeal squamous cancer cells and the mechanism of autophagy regulation and highlight brain tumor cells was enhanced by treatment with the multifaceted roles of TRPM2 under various curcumin (66) or eicosapentaenoic acid (67), pathogenic conditions. respectively, via activating TRPM2. However, cell Abbreviations death in normal kidney (MPK) cell was reduced by treatment with curcumin (66). In this study, the ADPR: adenosine diphosphate-ribose; AKI: anti-tumor effect of cisplatin was weakened by Acute kidney injury; AKT: protein kinase B; BNIP3: inhibiting TRPM2 with 2-APB and CLT and, adenovirus E1B 19-kDa-interacting protein 3; CLT: conversely, enhanced by activating TRPM2 with Clotrimazole; CQ: chloroquine; Cyt: cytochrome; ADPR, suggesting that TRPM2 is a potential target for DHE: Dihydroethidium; HBSS: Hank’s Balanced Salt cisplatin-based chemotherapy. It is generally accepted Solution; Kim-1: kidney injury marker-1; LAMP: that ADPR is generated by the consecutive actions of lysosomal associated membrane protein; LTL: Lotus poly (ADPR) polymerase and poly (ADPR) tetragonolobus lectin; MEFs: mouse embryonic glycohydrolase, two main enzymes critically engaged fibroblasts; MMP: mitochondrial membrane potential; in DNA repair after nuclear DNA being attacked by mRTECs: mouse renal tubular epithelial cells; ROS or DNA-damaging agents, such as cisplatin (16). mtDNA: mitochondrial DNA; mTOR: mammalian It is likely that TRPM2 activation augments oxidative target of rapamycin; mtROS: mitochondrial ROS; stress that sensitizes cancer cell to cisplatin-based OCT: optimal cutting temperature; PAF: paraformal- chemotherapies. Nonetheless, the clinical translation dehyde; PAS: Periodic Acid-Schiff; ROS: reactive of TRPM2 agonists is still limited due to several oxygen species; TEM: Transmission electron reasons. Firstly, TRPM2 activation has been involved microscopy; TRPM2: Transient receptor potential in a variety of diseases, such as ischemia brain injury melastatin 2; 3-MA: 3-methyladenine. (20) and liver damage (22), and approaches to avoid Supplementary Material these potential side effects are still lacking. Secondly, TRPM2 is critical for proliferation of tumor cells, and Supplementary figures. TRPM2 activation increases the risks of tumor https://www.thno.org/v13p4356s1.pdf progression (68). Thirdly, ADPR is implicated in ADP-ribosylation, a post-translational modification Acknowledgements that plays a pivotal role in a wide variety of cellular We thank Ping Yang in the Center of Cryo- biological events (69). How to overcome these Electron Microscopy (CCEM), Zhejiang University for disadvantages of TRPM2 agonists merits further her technical assistance on TEM. investigations. Here, we also found that addition of Funding ADPR failed to augment the level of autophagy and protect mRTECs against cisplatin-induced injury This study was supported by the funding from (Figure S6A-C). We surmised that cisplatin-induced Primary Research and Development Plan of Zhejiang https://www.thno.org Theranostics 2023, Vol. 13, Issue 13 4374 14. Takahashi A, Kimura T, Takabatake Y, Namba T, Kaimori J, Kitamura H, et al. Province (2020C03034) and National Natural Science Autophagy guards against cisplatin-induced acute kidney injury. Am J Pathol. Foundation of China (81770674) to Fei Han, National 2012;180(2):517-25. 15. Jiang M, Wei Q, Dong G, Komatsu M, Su Y, Dong Z. Autophagy in proximal Natural Science Foundation of China (82000637) to Xi tubules protects against acute kidney injury. Kidney Int. 2012;82(12):1271-83. Yao, Zhejiang Provincial Natural Science Foundation 16. Malko P, Jiang LH. TRPM2 channel-mediated cell death: An important mechanism linking oxidative stress-inducing pathological factors to associated of China (LZ22H050001), National Natural Science pathological conditions. Redox Biol. 2020;37:101755. 17. Wang L, Fu TM, Zhou Y, Xia S, Greka A, Wu H. Structures and gating Foundation of China (81970573) and Zhejiang mechanism of human TRPM2. Science. 2018;362(6421). provincial program for the Cultivation of High-level 18. Yu X, Xie Y, Zhang X, Ma C, Liu L, Zhen W, et al. Structural and functional basis of the selectivity filter as a gate in human TRPM2 channel. Cell Rep. Innovative Health talents to Weiqiang Lin. 2021;37(7):110025. 19. Yu P, Liu Z, Yu X, Ye P, Liu H, Xue X, et al. Direct Gating of the TRPM2 Author contributions Channel by cADPR via Specific Interactions with the ADPR Binding Pocket. Cell Rep. 2019;27(12):3684-95 e4. FH, WL and WY conceived and designed the 20. Ye M, Yang W, Ainscough JF, Hu XP, Li X, Sedo A, et al. TRPM2 channel study. BY, LJ, XY, YZ, GZ and FW performed the deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia. Cell Death Dis. 2014;5:e1541. experiments. BY, XS, QF, XY, YY and WL analyzed 21. Abuarab N, Munsey TS, Jiang LH, Li J, Sivaprasadarao A. High glucose-induced ROS activates TRPM2 to trigger lysosomal membrane the results. JC provided administrative support as the permeabilization and Zn(2+)-mediated mitochondrial fission. Sci Signal. director of the Kidney Disease Center. BY and LJ 2017;10(490). 22. Kheradpezhouh E, Ma L, Morphett A, Barritt GJ, Rychkov GY. TRPM2 wrote the manuscript. LX, LJ, WL, WY and FH channels mediate acetaminophen-induced liver damage. Proc Natl Acad Sci U supervised the study and revised the manuscript. S A. 2014;111(8):3176-81. 23. Li Y, Jiao J. Deficiency of TRPM2 leads to embryonic neurogenesis defects in L-HJ generated the TRPM2-KO mice used in the study hyperthermia. 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Published: Jan 1, 2023