MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14854/m_MTBLS14854_LC-MS_alternating_reversed-phase-chromatography_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14854/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14854/a_MTBLS14854_LC-MS_alternating_reversed-phase-chromatography.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14854/s_MTBLS14854.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14854MultiQuant 3.0.2 software was used to extract the original MRM data of MT1000 KIT metabolites and obtain the peak area of each metabolite for quantification from different samples.MetaboLightsPublicLiquid Chromatography MS - alternating - reversed-phase-chromatographyThe LC/MS portion of the platform was based on a Shimadzu Nexera X2 LC-30AD system equipped with an ACQUITY UPLC HSS T3 column (1.7 μm, 2.1 mm×100 mm, Wasters) and a triple quadruple mass spectrometer (5500 QTRAP, AB SCIEX). Metabolites were detected in electrospray negative-ionization and positive-ionization mode. The 2 μL samples were injected sequentially with LC autosampler. The ACQUITY UPLC HSS T3 column (1.7 μm, 2.1 mm×100 mm, Wasters) was heated to 40 °C under a flow rate of 200 μL/min. A gradient was used to separate the compounds consisted of 0.1% Formic acid aqueous solution (solvent A) and 100% acetonitrile (solvent B). The gradient started at 100% solvent A for 2.5 min and increasing linearly to 70% solvent A over 9 min, and then increasing linearly to 0% solvent A over 1 min following with a 5.4 min hold before returning the starting mixture during 0.1min and re-equilibrating for 2.5 min. QC samples were injected every several samples during acquisition to monitor the reproducibility of the test.Podophyllotoxin-induced nephrotoxicity via the microbiota-gut-kidney axis in SD rats based on the toxicological evidence chain (TEC) concept.The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and TechnologyXuejiao Lifecelmass spectrometry assayThe metabolites were extracted from cell residue with 1 mL precooled methanol/acetonitrile/water (v/v, 2:2:1) under sonication for 1 h in ice baths. The mixture was incubated at -20 °C for 1 h followed by centrifugation at 16,000 g, 4 °C for 20 min, and then transferred to the sampling vial for LC-MS analysis.Rattus norvegicushttps://www.ebi.ac.uk/metabolights/MTBLS14854Xuejiao Li. The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology. white_snow1987@163.com.Chuanxin Liu. The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology. 9906194@haust.edu.cn.The discriminating metabolites were obtained using a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model and two-tailed Student’s t test (p value) on peak area of each metabolite for two groups comparison. The p value was calculated by one-way analysis of variance (ANOVA) for multiple groups analysis. Metabolites with VIP> 1 and p value< 0.05 were considered to be statistically significant. The significantly differential metabolites were used to perform cluster analyses with R package.TreatmentDosewhite_snow1987@163.comEuthanasia was carried out after anesthesia induced with 1.25% tribromoethanol, and samples of blood, kidney, colon106tissues, and colonic feces were taken for further analysis, such as organ index assessment,107biochemical tests, histological inspection, and multi-omics researchMetabolomicsMetabolomicsmicrobiota-gut-kidney axistoxicological evidence chainPodophyllotoxinnephrotoxicityThermo Scientific Q ExactiveRattus norvegicusfecelexperimental blankselected reaction monitoringsemi-targeted analysisShimadzu Nexera UHPLC system (LC-30AD pump and CBM-20A controller)Metabolomicsmicrobiota-gut-kidney axistoxicological evidence chainPodophyllotoxinnephrotoxicityThermo Scientific Q ExactiveRattus norvegicusfecelexperimental blankselected reaction monitoringsemi-targeted analysisShimadzu Nexera UHPLC system (LC-30AD pump and CBM-20A controller)Widely targeted metabolites were quantified using multiple-reaction monitoring mode set up as previous report described (Cao, G., et al. 2020; Zheng, F., et al. 2020). All transitions from large-scale metabolites (MT1000 Kit developed by Shanghai BioProfile) were detected with optimized decluttering potential and collision energy. The MS conditions were set as follows: negative-ionization mode: Source Temperature 550℃, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF) :-4500V; positive-ionization mode: Source Temperature 550℃, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF) :5500V.DEOXYCARNITINEdGDPbeta-Hydroxymyristic acidCorticosteroneDL-2-Aminooctanoic acidLPE(18:2)PhenylacetylglutamineRiboflavinDocosatetraenoic acidCDPFolic acidN-Fructosyl isoleucineButyric acidDeoxyinosineEctoineDG(18:0/18:1)Carnitine-C12:1PG(18:2/16:1)KetoleucineCEAO-Succinyl-L-homoserineD-glucosamine 1-phosphateFlavoneO-Acetyl-L-serineOxoglutaric acidLPE(18:3)Cytidine diphosphate ethanolamineCitrullinealpha-Muricholic acidPC(16:0/18:3)1-MethylxanthineD-Glucuronic acidFlavin mononucleotideDimethyl-L-arginineOctadecanedioic acidHyocholic acidDG(18:0/18:2)PE(P-18:0/18:3)SM 40:4;33-Hydroxyanthranilic acidHexanoylcarnitineDeoxycytidine triphosphateAlpha-N-Phenylacetyl-L-glutamineDhCer d18:0/22:0DhCer d18:0/22:1Cinnamic acidHexose Pool5,6-trans-25-Hydroxyvitamin D3EstradiolLPE(18:0)HordenineMevalonic acidArginineDG(16:0/22:5)13S-hydroxyoctadecadienoic acidSM 42:0;2D-Glucose 1-phosphate2'-Deoxycytidine diphosphateTHPGlutathione12 Dehydrocholic Acid sulfateIsobutyryl-CoABeta-alanineN6-acetyl lysineFA(18:0)4-Methyl-2-oxovaleric acidPseudouridineLPE(18:1)N-Acetylcadaverine4-Ethyl-2-methoxyphenolCoenzyme Q(9)2-Ketoglutaric acid14,15-DHETPC(16:0/18:1)Oleamide3-phosphoglycerateTiglylcarnitine5alpha-CholestanolN-Acetylneuraminic acidThiazolidine-4-carboxylic acid4-Pyridoxic acidPantothenic acidGlycerol 3-phosphateDG(16:0/20:4)GuanosineSM 34:1;4CitrateFA(18:1)beta-D-GlucosaminePI(18:0/18:2)Xanthurenic acidD-Glucosamine 6-phosphateShikimic acidUrobilinogenCer d18:1/18:1Shikimate-3-phosphateAcetoacetic acidGlyceric acidcis-aconitic acidGamma-glutamyl-Alanine1alpha,25-Dihydroxyvitamin D3BiliverdinAlanylphenylalanineDG(16:0/20:5)FA(18:2)5-MethoxytryptaminePhenethylacetateCarnitine-C6DC1-CinnamoylpyrrolidineDG(18:1/20:4)NaringinUridine 5-monophosphate4-HydroxyquinolineL-ThyroninePyrophosphateUridineOrnithineD-Gluconic acidRetinalSM 40:2;3PS(18:0/18:2)Vitamin B13-Ureidopropanoate24-OH cholesterolSM 34:0;2Isovalerylcarnitine2-HydroxyadenineIndoleSerineArgpyrimidineProlineFA(18:3)6-Ketolithocholic acid5-Hydroxy-6,8,11,14,17-eicosapentaenoic acidDG(18:1/20:3)Lactamide3,7-Dimethyluric acidAsp-GluTridecanoic acid7-ketocholesterol2-Hydroxybenzoic acidValylphenylalanineInosine diphosphateCL(18:2/18:2/18:2/18:2)AdenosineL-Ascorbic acidPS(18:0/18:1)N-Acetyl-L-glutamic acidUric acidNADHomogentisic acidPI(18:2/18:3)11,12-DiHETrEPE(P-18:0/18:1)TMPD-Erythrose 4-phosphate17-OH-ProgesteroneN-Acetyl-D-glucosamine 1-phosphatetetranor-Prostaglandin E MetabolitS-Carboxymethyl-L-cysteineHypotaurinePE(P-18:1/22:5)SM 40:1;2DG(16:1/16:1)Cyclic adenosine monophosphate6-Phospho-D-gluconateBenzoic acid3-Hydroxybutanoic acidMelatoninAla-LeuMannosePI(16:0/16:0)GlucoseQuinolinic Acidgamma-GlutamyltyrosineGuanosine 5'-diphospho-beta-L-fucose2-Pyrocatechuic acidPE(18:0/14:0)CortexoloneCMASpermidineFA(14:0)SphingosineD-Glyceraldehyde 3-phosphateN-Acetyl-L-tyrosineD-Fructose 1,6-bisphosphateSM 42:3;2Carnitine-C16:1gamma-GlutamylleucineTaurocholic acidFA(24:4)S-Sulfo-L-cysteineCML2-Isopropylmalic acidThreonineTaurodeoxycholic acid Pool2-Ketobutyric acidIsodeoxycholic acid5-AminolevulinateCL(18:2/18:2/18:1/18:1)LPE(16:1)Carnitine-C4DCUrocanic acidValeric acidLysine4-Aminobenzoic acidMethylsuccinic AcidPE(18:1/22:6)Alpha-TOCHOPHERYL ACETATEN-Fructosyl tyrosineMetanephrineHydroxyprolineL-AbrineCarnitine-C125'-Methylthioadenosine4-Nitrophenol1-MethylnicotinamideNADHCarnitine-C16Carnitine-C141,5-anhydroglucitolAlpha-ketoisovaleric acidCarnitine-C18PC(18:2/16:1)CreatinineTaurolithocholic acidXanthosineAspartateN-Carbamoyl-L-aspartatePyridoxine3-Methyl-2-oxovaleric acidUridine diphosphate-n-acetyl-D-glucosamineN-Fructosyl phenylalanineRetinolChenodeoxycholic Acid sulfatePA(16:1/18:1)5-Aminopentanoic acidGlycolithocholic acid2-PhenylacetamideValineSuberic AcidCarnitine-C12OHArachidonyl carnitine2-ThiocytidineP-Toluenesulfonic acidHexaethylene glycolHydroxyisocaproic acidGlutathione disulfideN6-lactoylPhosphotyrosineL-Methionine sulfoxideAlloisolithocholic acidBilirubinGlycocholic acid5-MethyltetrahydrofolateTauro-alpha/beta/omega muricholic acidDG(18:1/18:2)BetaineTG58:6-FA22:57-DehydrocholesterolCarnitine-C14:1Indole-3-carboxaldehydeCarnitine-C10 OHSM 42:1;2Flavin adenine dinucleotide3-methoxytyramineDeoxyribose phosphateCarnitine-C14:2n-Capric acidPG(18:1/16:1)AMPPC(18:1/18:1)Cinnamoylglycine2-Hydroxy-3-methylbutyric acidGlutamateAsp-PheN6-formylTaurohyocholic acidDehydroascorbic acidDG(18:1/18:1)HexCer d18:1/12:0n-Caproic acidSM 36:0;23alpha-Hydroxy-12 Ketolithocholic Acid sulfatep-Aminohippuric acidPI(16:0/18:0)23-Nordeoxycholic acid1-MethylhistidinePC(18:0/18:1)Glyoxylic acidGamma-glutamyl-CysteineFA(16:0)SphinganineSM 42:2;2Taurolithocholic acid 3-sulfate7-Ketolithocholic acidL-LactateIndolelactatePE(O-18:0/18:1)ThymineGlycochenodeoxycholic acidFA(16:2)OleoylethanolamideCarnitine-C3DCDodecylbenzenesulfonic acid7-methylguanineFA(16:1)TG54:8-FA22:64-Hydroxyphenylacetic acidAlpha-D-Ribose 5-phosphateSarcosineSM 36:1;2Hexose Diphosphate PoolTaurineHexose Disaccharide PoolAllantoinPE(O-18:0/18:2)3,4-Dihydroxyphenylacetic acidGlucaric acidPS(18:0/22:6)O-PhosphoethanolamineLithocholic acidCholic acid sulfateL-HomoserineDephosphocoenzyme AL-Threonine4-Guanidinobutyric acidPhosphoenolpyruvateKynurenic acidN-FormylmethionineHexose Disaccharide Monophosphate PoolAgmatinePyridoxalFA(22:2)PalmitoylethanolamidePyrralineMethionine5-Hydroxyindole-3-acetic acidInosine triphosphateEstriol(E3)N-Acetyl-D-glucosamineApocholic acidAlpha-TocopherolDG(16:1/20:4)Oxaloacetic acidCarnitine-C5OHSM 44:3;3SM 44:3;2SM 36:2;3Thymidine 5-triphosphatePE(O-16:0/22:5)dTMP2-Naphthalenesulfonic acidPE(18:1/16:1)SM 30:1;4Tetranor-Prostaglandin F MetaboliteCystineN-AcetylglutamineLPE(22:6)FA(12:0)Glycolic acidLithocholenic acid sulfateDG(18:2/20:4)3-methylphenylacetic acidMyo-InositolPhenylalanineSerotonin(5-L-Glutamyl)-L-glutamineIsoleucylisoleucinePhenylacetylglycinePE(P-18:1/18:3)CholestenoneEpiandrosteroneThiamin pyrophosphateTestosteroneDeoxycytidine monophosphateFumarate3-(Methylthio)propionic acidLeucylprolinegamma-Glutamyl-L-methionineGamma-Aminobutyric acidEicosapentaenoic acidAconitateCarnitine-C16OHATPDydrogesteroneCarnitine-C18:2TryptamineL-KynureninePC(16:0/22:5)Dihydroxy bile acid glucuronideCreatineLPA(18:2)7-methylguanosineSphingosine 1-phosphatePyridoxal 5-phosphateSM 38:0;2ThreonylleucineOctulose-monophosphatePhytosphingosineN6-glycerinyl3-Methyluridine3-Hydroxykynurenine2-AcetylpyrazineGlycodeoxycholic acid sulfateCarnitine-C18:1D-Glucose 6-phosphatebeta-Muricholic acidBeta-CaroteneTyrosineFA(22:6)Tauro-Tetrahydroxy-5beta-cholanoic acidHomoarginine2'-Deoxyuridine 5'-triphosphateN6-oxalylN2,N2-Dimethylguanosine12-Ketolithocholic acidDG(14:0/18:3)5-Aminoimidazole-4-carboxamide1-Beta-D-ribofuranosideQuinolin-2,8-diolBMP(18:1/18:2)11-Hydroxyeicosatetraenoic acid2-keto-D-Gluconic acidLPE(22:5)3-Indolepropionic acidSpermineSepiapterinGABASuccinic acidHypoxanthineN6-methyladenosineL-MalatePhenyllactic acid2,4-DinitrophenolAdipic acidGluconolactoneNADPHPhenylalanylisoleucineGuanidinoacetic acid3-FORMYLINDOLEHippuric acidDesmosterolEnterolactoneN-Acetylaspartylglutamate(S)-4,5-DihydroorotateSedoheptulose monophosphateD-Ala-D-alaSM 38:1;2N,N-DimethylanilinePG(16:0/16:0)Homovanillic acidGlycocholic Acid sulfateCarnitine-C2Carnitine-C4Carnitine-C16:1OHCarnitine-C3Carnitine-C6Deoxyuridine monophosphateCarnitine-C85-Amino-4-carbamoylimidazoleDhCer d18:0/16:0Cyanidin-3-glucosidePyruvateDG(14:0/18:1)Deoxyadenosine triphosphatePE(18:2/20:1)BMP(18:1/18:0)FructoseCortisoneHeptadecanoic acidCitraconic acidInosineNorepinephrinePurineDimethylglycineSerylleucineGlutamineCarnitine-C14OHPE(18:1/18:1)N-Acetyl-L-phenylalaninePE(16:0/22:6)4-HydroxyphenylpyruvateFA(20:1)Dihydroxyacetone phosphateCholic acid2'-O-methyladenosine11,12-Epoxyeicosatrienoic acidHistidine20-Hydroxyeicosatetraenoic acidTetrahydrobiopterinAnserineNa-Methyl-L-ornithine monohydrochloride3-MethylhistidineMODICCaffeine4-Hydroxybenzoylcholine(S)-12-Hydroxyeicosatetraenoic acidPropionylglycineSM 40:0;23-HydroxybenzaldehydeDG(16:0/18:3)Allocholic acidN-acetylserineVanillylmandelic acid5-hydroxyeicosatatraeniocacidMalondialdehyde3, 5-TetradecadiencarnitineSebacic acid5,6-DHETbeta-SitosterolN-AcetylhistamineN4-AcetylcytidineNicotinamideSM 40:0;4GlycerophosphocholineDhCer d18:0/26:0FA(20:3)7-Ketodeoxycholic acidDG(16:0/18:2)Leucyl-leucineNG,NG-Dimethyl-L-argininePE(18:0/18:2)N-AcetylornithineTarginineGlycochenodeoxycholic Acid sulfateAspartylglycosaminePyroglutamic acidCoenzyme AN-Methylglutamic acidBetaine aldehydeLPE(20:4)Deoxyguanosine triphosphatePyridoxamineDG(16:0/18:1)Salicylic acidAlanineL-Argininosuccinic acidPI(18:0/18:1)FA(20:2)N-AcetylputrescineTryptophanAndrosteroneXanthinePE(18:0/18:3)S-AdenosylmethioninePentosidineTG46:3-FA18:3DeoxyguanosineQuinic acidCholinePC(16:0/20:4)S-D-Lactoylglutathione2-Deoxyinosine triphosphateAsparagineN-Acetyl-L-alanineAcetylcholinemethylglyoxal-hydroimidazolone3,7,12 Taurodehydrocholic acid13,14-Dihydro-15-keto PGF2a3-Aminoisobutyric acidLPI(18:3)2-Ketohexanoic acidPG(16:0/18:1)TG58:8-FA22:5ThymidineDiphenylamineAldosteroneD-Fructose 6-phosphateOrotic acidTiglylglycine4-HydroxyphenyllactateChenodeoxyglycocholic acidLTB4LPE(20:2)UDPIsobutyrylglycineInositolLPI(18:2)FA(20:4)Hexose Monophosphate PoolIndoxyl sulfateIndole-3-acetaldehydeSM 32:1;2DG(16:1/20:0)PE(18:0/18:1)N1-Methyl-2-pyridone-5-carboxamideGlycyl-L-leucineS-AdenosylhomocysteineGlucosepaneIsocitrateL-Homocysteine5'-DeoxyadenosineLPE(20:3)DG(18:2/22:5)Tetrahydroxy-5beta-cholanoic acid2-PiperidoneIsovalerylglycineFA(22:1)NormetanephrineAllantoic acidPC(18:2/22:5)Thromboxane B2LeucineCitramalic acidAzelaic acidRiboseL-Carnosinep-Aminobenzoic acidBMP(18:1/20:4)Uridine triphosphateN-Acetyl-L-leucineL-2-Aminoadipic acidIsopentenyladenosinegamma-L-Glutamyl-L-phenylalanineImidazoleacetic acidFA(22:0)5-HydroxyindoleacetateCholic acid – 7-O-sulfateGALADeoxycholic acid2,3-Dihydroxybenzoic acidCarnitine-C18:1OHIndole-3-carboxylic acidThreonatePI(18:2/16:1)14,15-Epoxy-5,8,11-eicosatrienoic acidTaurolithocholic acid sulfateGlycodeoxycholic acid5-Hydroxy-L-tryptophan15-Hydroxyeicosatetraenoic acidLPE(20:1)falsePodophyllotoxin-induced nephrotoxicity via the microbiota-gut-kidney axis in SD rats based on the toxicological evidence chain (TEC) conceptPodophyllotoxin (PPT) exhibits limited clinical utility due to its nephrotoxicity, and its underlying mechanisms remain poorly understood. This study employs the toxicological evidence chain (TEC) framework and integrated multi-omics analyses to investigate the potential involvement of the microbiota-gut-kidney (MGK) axis in PPT- induced nephrotoxicity in SD rats. Toxicity was systematically evaluated through longitudinal monitoring of body weight, general behavior, biochemical markers, intestinal barrier function, and histopathological alterations. In parallel, multi-omics analyses, encompassing microbiome, metabolomics, and transcriptomics, were conducted to delineate the mechanistic underpinnings. The results showed that PPT exposure induced pronounced renal and intestinal damage, manifesting as significant weight loss, diarrhea, elevated renal injury biomarkers, increased lipopolysaccharide (LPS) levels, and diamine oxidase (DAO), along with histopathological lesions and enhanced apoptosis in renal and colonic tissues. PPT exposure perturbed gut microbiota homeostasis, characterized by depletion of beneficial taxa (e.g., Lactobacillus) and enrichment of potentially pathogenic genera (e.g., Bacteroides and Aggregatibacter), concomitant with diminished short-chain fatty acid (SCFA) production and altered metabolite profiles in fecal, serum, and renal samples. Integrated multi- omics analysis further revealed activation of the JAK1/2-STAT3 signaling pathway, upregulation of pro-inflammatory mediators (TNF-α, IL-6, IL-1β, LPS, and TMAO), and suppression of anti-inflammatory cytokines (IL-10 and IL-4). These in vivo molecular and inflammatory patterns were partially reproduced in HK-2 cells co-cultured with fecal microbiota supernatant from PPT-treated rats. In addition, the JAK1/2 inhibitor ruxolitinib attenuated PPT-induced JAK1/2-STAT3 phosphorylation and inflammatory cytokine secretion in HK-2 cells. Correlation network analysis further identified associations between gut dysbiosis, systemic inflammation, and metabolic perturbations. Collectively, these findings support a mechanistic hypothesis that MGK- axis disruption and JAK1/2-STAT3 signaling may contribute to PPT-associated nephrotoxicity. However, in vivo interventional studies are required to establish definitive causality2026-06-252026-06-25MTBLS14854