#METABOLOMICS WORKBENCH zxiaolu0802_20230321_013313 DATATRACK_ID:3797 STUDY_ID:ST002532 ANALYSIS_ID:AN004167 PROJECT_ID:PR001628 VERSION 1 CREATED_ON March 28, 2023, 6:46 pm #PROJECT PR:PROJECT_TITLE Nontargeted metabolomics studies PR:PROJECT_TYPE LC‒MS PR:PROJECT_SUMMARY Nontargeted metabolomics analysis was carried out using the kidney tissues of PR:PROJECT_SUMMARY mice killed at Day 3 after cisplatin treatment. PR:INSTITUTE Children's Hospital of Nanjing Medical University PR:LAST_NAME xiaolu PR:FIRST_NAME zhang PR:ADDRESS 72 Guangzhou Road PR:EMAIL zxiaolu0802@163.com PR:PHONE 18351976523 #STUDY ST:STUDY_TITLE Nontargeted metabolomics analysis on kidney tissue treated with cisplatin ST:STUDY_TYPE Nontargeted metabolomics analysis ST:STUDY_SUMMARY Cisplatin-induced acute kidney injury (AKI) is a severe clinical complication ST:STUDY_SUMMARY with no satisfactory therapies in the clinic. Tumor necrosis factor receptor ST:STUDY_SUMMARY (TNFR)-associated factor 1 (TRAF1) plays an important role in both inflammation ST:STUDY_SUMMARY and metabolism. However, the role of TRAF1 in cisplatin-induced AKI needs to be ST:STUDY_SUMMARY evaluated. In this study, TRAF1 expression was decreased in cisplatin-treated ST:STUDY_SUMMARY mice and mouse proximal tubular cells (mPTCs), suggesting a potential role of ST:STUDY_SUMMARY TRAF1 in cisplatin-associated kidney injury. Thus, TRAF1 plasmids were ST:STUDY_SUMMARY introduced into male C57BL/6J mice by a tail vein high-pressure injection method ST:STUDY_SUMMARY to overexpress TRAF1. Then, cisplatin was administrated by a single ST:STUDY_SUMMARY intraperitoneal (i.p.) injection (20 mg/kg). Mice were sacrificed 72 h after ST:STUDY_SUMMARY cisplatin administration. Serum and kidney tissues were collected for further ST:STUDY_SUMMARY analysis. In vitro, mPTCs were transfected with TRAF1 plasmids before treatment ST:STUDY_SUMMARY with cisplatin (5 µg/mL) for 24 h. Western blotting, Masson’s trichrome and ST:STUDY_SUMMARY hematoxylin-eosin (HE) staining and tandem mass spectrometry (LC‒MS/MS) ST:STUDY_SUMMARY analysis were employed to evaluate kidney injury. Following cisplatin treatment, ST:STUDY_SUMMARY we observed a marked downregulation of TRAF1 in mouse kidneys and mPTCs treated ST:STUDY_SUMMARY with cisplatin. In mice, TRAF1 overexpression attenuated cisplatin-induced AKI, ST:STUDY_SUMMARY as evidenced by decreased levels of serum creatinine (Scr) and blood urea ST:STUDY_SUMMARY nitrogen (BUN). Moreover, TRAF1 delivery obviously ameliorated cisplatin-induced ST:STUDY_SUMMARY renal tubular injury, as shown by the improved histological damage and blocked ST:STUDY_SUMMARY upregulation of NGAL and KIM-1. Moreover, the NF-κB activation and inflammatory ST:STUDY_SUMMARY cytokine production enhanced by cisplatin were significantly blunted by TRAF1. ST:STUDY_SUMMARY In line with the attenuated inflammatory response, the increased number of ST:STUDY_SUMMARY apoptotic cells (TUNEL staining) and enhanced expression of BAX and cleaved ST:STUDY_SUMMARY Caspase-3 were markedly decreased by TRAF1 overexpression. In vitro, TRAF1 also ST:STUDY_SUMMARY attenuated renal tubular cell inflammation and apoptosis induced by cisplatin. ST:STUDY_SUMMARY In addition, disordered cellular metabolism, which was reported as an important ST:STUDY_SUMMARY pathogenic factor of AKI, was examined by metabolomics analysis. Interestingly, ST:STUDY_SUMMARY a significant correction of the metabolic disturbance, including perturbations ST:STUDY_SUMMARY in energy generation and lipid and amino acid metabolism, was observed in the ST:STUDY_SUMMARY kidneys of cisplatin-treated mice. In conclusion, TRAF1 overexpression ST:STUDY_SUMMARY significantly attenuated cisplatin-induced nephrotoxicity, possibly by ST:STUDY_SUMMARY correcting the impaired metabolism, inhibiting inflammation, and blocking ST:STUDY_SUMMARY apoptosis in renal tubular cells. ST:INSTITUTE Children's Hospital of Nanjing Medical University ST:LAST_NAME xiaolu ST:FIRST_NAME zhang ST:ADDRESS 72 Guangzhou Road ST:EMAIL zxiaolu0802@163.com ST:PHONE 18351976523 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - T-A1 Genotype:control | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A1.mzML SUBJECT_SAMPLE_FACTORS - T-A2 Genotype:control | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A2.mzML SUBJECT_SAMPLE_FACTORS - T-A3 Genotype:control | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A3.mzML SUBJECT_SAMPLE_FACTORS - T-A4 Genotype:control | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A4.mzML SUBJECT_SAMPLE_FACTORS - T-A5 Genotype:control | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A5.mzML SUBJECT_SAMPLE_FACTORS - T-A6 Genotype:vehicle+cisplatin | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A6.mzML SUBJECT_SAMPLE_FACTORS - T-A7 Genotype:vehicle+cisplatin | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A7.mzML SUBJECT_SAMPLE_FACTORS - T-A8 Genotype:vehicle+cisplatin | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A8.mzML SUBJECT_SAMPLE_FACTORS - T-A9 Genotype:vehicle+cisplatin | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A9.mzML SUBJECT_SAMPLE_FACTORS - T-A10 Genotype:vehicle+cisplatin | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A10.mzML SUBJECT_SAMPLE_FACTORS - T-A11 Genotype:cisplatin+TRAF1 | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A11.mzML SUBJECT_SAMPLE_FACTORS - T-A12 Genotype:cisplatin+TRAF1 | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A12.mzML SUBJECT_SAMPLE_FACTORS - T-A13 Genotype:cisplatin+TRAF1 | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A13.mzML SUBJECT_SAMPLE_FACTORS - T-A14 Genotype:cisplatin+TRAF1 | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A14.mzML SUBJECT_SAMPLE_FACTORS - T-A15 Genotype:cisplatin+TRAF1 | Strain:WT Age=C57BL/6; Sex=8 weeks male; RAW_FILE_NAME=T-A15.mzML #COLLECTION CO:COLLECTION_SUMMARY 8 weeks old C57BL/6 male mice were randomly divided into three groups: control CO:COLLECTION_SUMMARY group (control; n=8), cisplatin-induced AKI group (cisplatin; n=8), and CO:COLLECTION_SUMMARY cisplatin plus TRAF1 plasmid injection group (cisplatin + TRAF1; n=8). Both CO:COLLECTION_SUMMARY cisplatin and cisplatin + TRAF1 groups were given 20 mg/kg cisplatin CO:COLLECTION_SUMMARY intraperitoneal (i.p.) injection, while the control group received the same CO:COLLECTION_SUMMARY amount of saline i.p. injection. The mice in cisplatin + TRAF1 group were CO:COLLECTION_SUMMARY pretreated with TRAF1 performed by a hydrodynamic-based gene delivery approach CO:COLLECTION_SUMMARY 36 h before cisplatin administration. Mice in the three groups were sacrificed CO:COLLECTION_SUMMARY after cisplatin treatment for 72 h. The kidney tissues were cut horizontally and CO:COLLECTION_SUMMARY collected. CO:COLLECTION_PROTOCOL_FILENAME protocol.pdf CO:SAMPLE_TYPE Kidney #TREATMENT TR:TREATMENT_SUMMARY 8 weeks old C57BL/6 male mice were randomly divided into three groups: control TR:TREATMENT_SUMMARY group (control; n=8), cisplatin-induced AKI group (cisplatin; n=8), and TR:TREATMENT_SUMMARY cisplatin plus TRAF1 plasmid injection group (cisplatin + TRAF1; n=8). Both TR:TREATMENT_SUMMARY cisplatin and cisplatin + TRAF1 groups were given 20 mg/kg cisplatin TR:TREATMENT_SUMMARY intraperitoneal (i.p.) injection, while the control group received the same TR:TREATMENT_SUMMARY amount of saline i.p. injection. The mice in cisplatin + TRAF1 group were TR:TREATMENT_SUMMARY pretreated with TRAF1 performed by a hydrodynamic-based gene delivery approach TR:TREATMENT_SUMMARY 36 h before cisplatin administration. Mice in the three groups were sacrificed TR:TREATMENT_SUMMARY after cisplatin treatment for 72 h. The kidney tissues were cut horizontally and TR:TREATMENT_SUMMARY collected. TR:TREATMENT_PROTOCOL_FILENAME protocol.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 1. Take all samples into a 2 mL EP tube, add 2 steel balls, put them into the SP:SAMPLEPREP_SUMMARY tissue grinder, grind at 50 Hz for 60 s, and then homogenize the samples; 2. SP:SAMPLEPREP_SUMMARY Accurately weigh 100 mg (±1%) of the homogenized sample into a 2 mL EP tube, SP:SAMPLEPREP_SUMMARY add accurately 0.6mL of methanol (including internal standard), and vortex for SP:SAMPLEPREP_SUMMARY 30 s 3. Add 2 steel balls, put them into the tissue grinder, and grind for 60 s SP:SAMPLEPREP_SUMMARY at 50 Hz; 4. Centrifuge at 4 ℃ for 10 min at 12000 rpm, and the supernatant SP:SAMPLEPREP_SUMMARY was filtered through 0.22 µm membrane to obtain the prepared samples for LC-MS; SP:SAMPLEPREP_SUMMARY 5. Take 20 µL from each sample to the quality control (QC) samples; (These QC SP:SAMPLEPREP_SUMMARY samples were used to monitor deviations of the analytical results from these SP:SAMPLEPREP_SUMMARY pool mixtures and compare them to the errors caused by the analytical instrument SP:SAMPLEPREP_SUMMARY itself) 6. Use the rest of the samples for LC-MS detection. SP:SAMPLEPREP_PROTOCOL_FILENAME protocol.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 0.1% formic acid in water and 0.1% formic acid in acetonitrile CH:SOLVENT_B 5 mM ammonium formate in water and acetonitrile CH:FLOW_GRADIENT 0~1 min, 2% B.D 1~9 min, 2%~50% B.D 9~12 min, 50%~98% B.D 12~13.5 min, 98% CH:FLOW_GRADIENT B.D 13.5~14 min, 98%~2% B.D 14~20 min, 2% D-positive model (14~17 min, 2% CH:FLOW_GRADIENT B-negative model) CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 CH:METHODS_FILENAME protocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE protocol.pdf #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS - MS:MS_RESULTS_FILE ST002532_AN004167_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END