#METABOLOMICS WORKBENCH borjalanzon_20191219_092327 DATATRACK_ID:1879 STUDY_ID:ST001303 ANALYSIS_ID:AN002171 PROJECT_ID:PR000884 VERSION 1 CREATED_ON January 9, 2020, 1:15 pm #PROJECT PR:PROJECT_TITLE TGFβ3 heterozygous mice PR:PROJECT_TYPE Mice nephropathy in lipotoxic model PR:PROJECT_SUMMARY Transforming growth factor β (TGFβ) family comprises the main player in the PR:PROJECT_SUMMARY development of fibrosis including three isoforms: TGFβ1, TGFβ2 and TGFβ3. PR:PROJECT_SUMMARY TGFβ3 may play an antifibrotic role at the renal level, counteracting the role PR:PROJECT_SUMMARY of TGFβ1, using a mouse model heterozygous for the TGFβ3 gene (TGFβ3+/-). PR:PROJECT_SUMMARY Partial deletion of TGFβ3 causes in the mice albuminuria, loss of glomerular PR:PROJECT_SUMMARY filtration rate, accelerated fibrosis, epithelial-to-mesenchymal transition and PR:PROJECT_SUMMARY increment of glomerular basement membrane thickening. PR:INSTITUTE University Rey Juan Carlos PR:DEPARTMENT Basics Science of Health PR:LAST_NAME Lanzon PR:FIRST_NAME Borja PR:ADDRESS Avenida de Atenas S/N, Alcorcón, Madrid, 28922, Spain PR:EMAIL borja.lanzon@urjc.es PR:PHONE 663692554 #STUDY ST:STUDY_TITLE TGF-Beta 3 heterozygous mice ST:STUDY_TYPE Mice nephropathy in lipotoxic model ST:STUDY_SUMMARY Transforming growth factor β (TGFβ) family comprises the main player in the ST:STUDY_SUMMARY development of fibrosis including three isoforms: TGFβ1, TGFβ2 and TGFβ3. ST:STUDY_SUMMARY TGFβ3 may play an antifibrotic role at the renal level, counteracting the role ST:STUDY_SUMMARY of TGFβ1, using a mouse model heterozygous for the TGFβ3 gene (TGFβ3+/-). ST:STUDY_SUMMARY Partial deletion of TGFβ3 causes in the mice albuminuria, loss of glomerular ST:STUDY_SUMMARY filtration rate, accelerated fibrosis, epithelial-to-mesenchymal transition and ST:STUDY_SUMMARY increment of glomerular basement membrane thickening. ST:INSTITUTE University Rey Juan Carlos ST:DEPARTMENT Basics Science of Health ST:LAST_NAME Lanzon ST:FIRST_NAME Borja ST:ADDRESS Avenida de Atenas S/N ST:EMAIL borja.lanzon@urjc.es ST:PHONE 663692554 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 14 ST:NUM_MALES 14 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN c57bl6 SU:AGE_OR_AGE_RANGE 16 weeks SU:GENDER Male #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 - 269 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 269 SUBJECT_SAMPLE_FACTORS - 119 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 119 SUBJECT_SAMPLE_FACTORS - 267 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 267 SUBJECT_SAMPLE_FACTORS - 130 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 130 SUBJECT_SAMPLE_FACTORS - 127 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 127 SUBJECT_SAMPLE_FACTORS - 98 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 98 SUBJECT_SAMPLE_FACTORS - 25 HZCD Genotype:HZCD RAW_FILE_NAME=HZ-CD 25 SUBJECT_SAMPLE_FACTORS - 132 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 132 SUBJECT_SAMPLE_FACTORS - 251 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 251 SUBJECT_SAMPLE_FACTORS - 120 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 120 SUBJECT_SAMPLE_FACTORS - 79 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 79 SUBJECT_SAMPLE_FACTORS - 129 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 129 SUBJECT_SAMPLE_FACTORS - 128 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 128 SUBJECT_SAMPLE_FACTORS - 92 WTCD Genotype:WTCD RAW_FILE_NAME=WT-CD 92 SUBJECT_SAMPLE_FACTORS - QC1 Genotype:QC RAW_FILE_NAME=QC1 SUBJECT_SAMPLE_FACTORS - QC2 Genotype:QC RAW_FILE_NAME=QC2 SUBJECT_SAMPLE_FACTORS - QC3 Genotype:QC RAW_FILE_NAME=QC3 SUBJECT_SAMPLE_FACTORS - QC4 Genotype:QC RAW_FILE_NAME=QC4 SUBJECT_SAMPLE_FACTORS - QC5 Genotype:QC RAW_FILE_NAME=QC5 #COLLECTION CO:COLLECTION_SUMMARY Kidney samples were powdered with mortar and pestle. Method used for extraction CO:COLLECTION_SUMMARY was previously validated for tissue CO:SAMPLE_TYPE Kidney CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Kidney homogenate was prepared by adding cold (−20 °C) methanol/water (1:1, TR:TREATMENT_SUMMARY v/v), (1:10 tissue/solvent). Tissue disruption was achieved with Tissue- Lyser TR:TREATMENT_SUMMARY LT homogenizer (Qiagen, Germany) for metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 μL of kidney tissue homogenate was vortex-mixed with 320 μL of methanol SP:SAMPLEPREP_SUMMARY for 2 min, followed by the addition of 80 μL of MTBE for the extraction of SP:SAMPLEPREP_SUMMARY nonpolar compounds. Then, vials were rapidly capped and placed on a shaker for 1 SP:SAMPLEPREP_SUMMARY h at room temperature. The extracted samples were centrifuged at 4000g for 20 SP:SAMPLEPREP_SUMMARY min at 20 °C. For GC−MS analysis, 300 μL of supernatant was evaporated to SP:SAMPLEPREP_SUMMARY dryness (SpeedVac Concentrator System, Thermo Fisher Scientific, Waltham, MA). SP:SAMPLEPREP_SUMMARY Methoxymation was then performed with 20 μL of O-methoxyamine hydrochloride (15 SP:SAMPLEPREP_SUMMARY mg/mL in pyridine) and vigorously vortex-mixed for 5 min. Vials were then SP:SAMPLEPREP_SUMMARY incubated in darkness at room temperature for 16 h. For silylation, 20 μL of SP:SAMPLEPREP_SUMMARY BSTFA/TMCS (99:1) was added and vortex-mixed for 5 min, and capped vials were SP:SAMPLEPREP_SUMMARY placed in the oven at 70 °C for 1 h. Finally, 100 μL of heptane containing SP:SAMPLEPREP_SUMMARY tricosane (20 ppm) as internal standard (IS) was added to each vial prior to SP:SAMPLEPREP_SUMMARY injection. For LC−MS analysis, 90 μL of supernatant was transferred to an SP:SAMPLEPREP_SUMMARY ultra-high-performance liquid chromatography−mass spectrometry. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY GC-MS CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7890A MS:INSTRUMENT_TYPE GC QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS An Agilent GC instrument (7890A) coupled to an inert mass spectrometer with MS:MS_COMMENTS triple-Axis detector (5975C, Agilent Technologies) was used for kidney tissue MS:MS_COMMENTS fingerprinting. Briefly, 1 μL of derivatized samples were injected by an MS:MS_COMMENTS Agilent autosampler (7693). Samples were automatically injected in split mode MS:MS_COMMENTS (split ratio 1:12), into an Agilent ultra-inert deactivated glass wool split MS:MS_COMMENTS liner. Compound separation was achieved using a pre-column (10 m J&W integrated MS:MS_COMMENTS with Agilent 122-5532G) combined with a GC column DB5-MS (length, 30m; inner MS:MS_COMMENTS diameter, 0.25 mm; and 0.25 μm film of 95% dimethyl/5% diphenylpolysiloxane). MS:MS_COMMENTS The flow rate of helium carrier gas was constant at : 0.938 mL/min through the MS:MS_COMMENTS column. The lock of the retention time (RTL) relative to the internal standard MS:MS_COMMENTS (methyl stearate) peak at 19.66 minutes was performed. The column oven MS:MS_COMMENTS temperature was initially set at 60°C (maintained for 1 minute), then raised by MS:MS_COMMENTS 10°C/min until it reached 325°C, and then was held at this temperature for 10 MS:MS_COMMENTS minutes before cooling down. The injector and the transfer line temperatures MS:MS_COMMENTS were established at 250°C and 280°C, respectively. MS system: the electron MS:MS_COMMENTS impact ionization operating parameters were set as follows: filament source MS:MS_COMMENTS temperature, 230°C; electron ionization energy, 70 eV. Mass spectra were MS:MS_COMMENTS collected over a mass range of 50-600 m/z at a scan rate of 10 spectra/s. Data MS:MS_COMMENTS were acquired using the Agilent MSD ChemStation Software (Agilent Technologies). MS:MS_COMMENTS For retention index determination, a mixture of n-alkanes (C8-C28) dissolved in MS:MS_COMMENTS nhexane was run prior to the samples. Data were acquired using Agilent MSD MS:MS_COMMENTS ChemStation Software (Agilent Technologies). MS:MS_RESULTS_FILE ST001303_AN002171_Results.txt UNITS:Area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END