#METABOLOMICS WORKBENCH borjalanzon_20241216_091130 DATATRACK_ID:5455 STUDY_ID:ST003738 ANALYSIS_ID:AN006138 PROJECT_ID:PR002324 VERSION 1 CREATED_ON February 18, 2025, 7:19 am #PROJECT PR:PROJECT_TITLE Impact of Trans-Fatty acids on renal health in mice with partial Tgfβ3 PR:PROJECT_TITLE deletion: a Metabolomic and Lipidomic analysis PR:PROJECT_TYPE Multiplatform non-targeted metabolomics PR:PROJECT_SUMMARY Trans-fatty acids, prevalent in Western diets, contribute to the onset and PR:PROJECT_SUMMARY progression of renal damage. This study investigates the effects of trans-fat PR:PROJECT_SUMMARY consumption on kidney health using a comprehensive approach encompassing PR:PROJECT_SUMMARY metabolomics, lipidomics, molecular, and cellular analyses. We analyzed both PR:PROJECT_SUMMARY wild-type mice and mice with partial transforming growth factor beta 3 (Tgfβ3) PR:PROJECT_SUMMARY deletion, a model for pre-existing renal damage. In control animals, the PR:PROJECT_SUMMARY trans-fat diet induced significant dysregulation in renal lipid metabolism. Omic PR:PROJECT_SUMMARY analyses revealed a reduction in triglycerides and an increase in plasmalogens, PR:PROJECT_SUMMARY which are associated with cellular protection and signaling. Furthermore, PR:PROJECT_SUMMARY upregulation of Tricarboxylic acid cycle (TCA) metabolites, malic and fumaric PR:PROJECT_SUMMARY acids, indicated mitochondrial dysfunction. In mice with partial Tgfβ3 PR:PROJECT_SUMMARY deletion, trans-fat consumption exacerbated renal fibrosis, and further depleted PR:PROJECT_SUMMARY triglycerides and plasmalogens. Despite similar lipid profiles in both groups, PR:PROJECT_SUMMARY Tgfβ3-deleted mice exhibited larger lipid droplets and worsened renal damage, PR:PROJECT_SUMMARY including a notable reduction in taurine levels. This study underscores the PR:PROJECT_SUMMARY value of detailed metabolic profiling to uncover mechanisms driving renal PR:PROJECT_SUMMARY dysfunction linked to harmful dietary habits and genetic mutations. PR:INSTITUTE Universidad Rey Juan Carlos PR:DEPARTMENT Basic Sciences of Health PR:LABORATORY Lipobeta PR:LAST_NAME Lanzon PR:FIRST_NAME Borja PR:ADDRESS Avenida de Atenas S/N PR:EMAIL borja.lanzon@urjc.es PR:PHONE 663692554 #STUDY ST:STUDY_TITLE Impact of Trans-Fatty acids on renal health in mice with partial Tgfβ3 ST:STUDY_TITLE deletion: a Metabolomic and Lipidomic analysis ST:STUDY_TYPE Multiplatform non-targeted metabolomics ST:STUDY_SUMMARY Trans-fatty acids, prevalent in Western diets, contribute to the onset and ST:STUDY_SUMMARY progression of renal damage. This study investigates the effects of trans-fat ST:STUDY_SUMMARY consumption on kidney health using a comprehensive approach encompassing ST:STUDY_SUMMARY metabolomics, lipidomics, molecular, and cellular analyses. We analyzed both ST:STUDY_SUMMARY wild-type mice and mice with partial Tgfβ3 deletion, a model for pre-existing ST:STUDY_SUMMARY renal damage. In control animals, the trans-fat diet induced significant ST:STUDY_SUMMARY dysregulation in renal lipid metabolism. Omic analyses revealed a reduction in ST:STUDY_SUMMARY triglycerides and an increase in plasmalogens, which are associated with ST:STUDY_SUMMARY cellular protection and signaling. Furthermore, upregulation of TCA cycle ST:STUDY_SUMMARY metabolites, malic and fumaric acids, indicated mitochondrial dysfunction. In ST:STUDY_SUMMARY mice with partial Tgfβ3 deletion, trans-fat consumption exacerbated renal ST:STUDY_SUMMARY fibrosis, and further depleted triglycerides and plasmalogens. Despite similar ST:STUDY_SUMMARY lipid profiles in both groups, Tgfβ3-deleted mice exhibited larger lipid ST:STUDY_SUMMARY droplets and worsened renal damage, including a notable reduction in taurine ST:STUDY_SUMMARY levels. This study underscores the value of detailed metabolic profiling to ST:STUDY_SUMMARY uncover mechanisms driving renal dysfunction linked to harmful dietary habits ST:STUDY_SUMMARY and genetic mutations. ST:INSTITUTE Universidad Rey Juan Carlos ST:DEPARTMENT Basic Sciences of Health ST:LABORATORY Lipobeta 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 4 ST:TOTAL_SUBJECTS 28 ST:NUM_MALES 28 ST:STUDY_COMMENTS Study is composed of 4 groups of mice: wild-type on control diet (WT-CD), ST:STUDY_COMMENTS wild-type on trans diet (WT-HFD), heterozygous TGFBeta 3 on control diet (HZ-CD) ST:STUDY_COMMENTS and heterozygous TGFBeta 3 on trans diet (HZ-HFD) #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 16 weeks SU:WEIGHT_OR_WEIGHT_RANGE 26 to 32 g SU:GENDER Male SU:ANIMAL_FEED Control diet: D12450B, Research Diets. Trans diet: TD.07011, ENVIGO SU:SPECIES_GROUP 4 groups: wild-type animals on control diet (WT-CD), wild-type on trans diet SU:SPECIES_GROUP (WT-HFD), Heterozygous TGFBeta 3 on control diet (HZ-CD) and Heterozygous SU:SPECIES_GROUP TGFBeta 3 on trans diet (HZ-HFD) #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 Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 269 SUBJECT_SAMPLE_FACTORS - 119 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 119 SUBJECT_SAMPLE_FACTORS - 267 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 267 SUBJECT_SAMPLE_FACTORS - 130 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 130 SUBJECT_SAMPLE_FACTORS - 127 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 127 SUBJECT_SAMPLE_FACTORS - 98 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 98 SUBJECT_SAMPLE_FACTORS - 25 HZCD Sample source:Kidney | Genotype:Heterozygous | Diet:Control RAW_FILE_NAME(Raw Data File)=HZ-CD 25 SUBJECT_SAMPLE_FACTORS - 132 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 132 SUBJECT_SAMPLE_FACTORS - 251 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 251 SUBJECT_SAMPLE_FACTORS - 120 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 120 SUBJECT_SAMPLE_FACTORS - 79 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 79 SUBJECT_SAMPLE_FACTORS - 129 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 129 SUBJECT_SAMPLE_FACTORS - 128 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 128 SUBJECT_SAMPLE_FACTORS - 92 WTCD Sample source:Kidney | Genotype:Wild-Type | Diet:Control RAW_FILE_NAME(Raw Data File)=WT-CD 92 SUBJECT_SAMPLE_FACTORS - 81 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=81 HZ-HFD SUBJECT_SAMPLE_FACTORS - 95 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=95 HZ-HFD SUBJECT_SAMPLE_FACTORS - 96 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=96 HZ-HFD SUBJECT_SAMPLE_FACTORS - 123 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=123 HZ-HFD SUBJECT_SAMPLE_FACTORS - 131 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=131 HZ-HFD SUBJECT_SAMPLE_FACTORS - 136 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=136 HZ-HFD SUBJECT_SAMPLE_FACTORS - 139 HZHFD Sample source:Kidney | Genotype:Heterozygous | Diet:Transfat RAW_FILE_NAME(Raw Data File)=139 HZ-HFD SUBJECT_SAMPLE_FACTORS - 29 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=29 WT-HFD SUBJECT_SAMPLE_FACTORS - 83 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=83 WT-HFD SUBJECT_SAMPLE_FACTORS - 91 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=91 WT-HFD SUBJECT_SAMPLE_FACTORS - 124 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=124 WT-HFD SUBJECT_SAMPLE_FACTORS - 133 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=133 WT-HFD SUBJECT_SAMPLE_FACTORS - 134 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=134 WT-HFD SUBJECT_SAMPLE_FACTORS - 138 WTHFD Sample source:Kidney | Genotype:Wild-Type | Diet:Transfat RAW_FILE_NAME(Raw Data File)=138 WT-HFD SUBJECT_SAMPLE_FACTORS - QC1 Sample source:Kidney | Genotype:QC | Diet:QC RAW_FILE_NAME(Raw Data File)=QC1 SUBJECT_SAMPLE_FACTORS - QC2 Sample source:Kidney | Genotype:QC | Diet:QC RAW_FILE_NAME(Raw Data File)=QC2 SUBJECT_SAMPLE_FACTORS - QC3 Sample source:Kidney | Genotype:QC | Diet:QC RAW_FILE_NAME(Raw Data File)=QC3 SUBJECT_SAMPLE_FACTORS - QC4 Sample source:Kidney | Genotype:QC | Diet:QC RAW_FILE_NAME(Raw Data File)=QC4 SUBJECT_SAMPLE_FACTORS - QC5 Sample source:Kidney | Genotype:QC | Diet:QC RAW_FILE_NAME(Raw Data File)=QC5 #COLLECTION CO:COLLECTION_SUMMARY Kidney samples were powdered with mortar and pestle. Initially, approximately 60 CO:COLLECTION_SUMMARY mg of each pulverized kidney sample was collected, then 50% Methanol: H2O (1 mg CO:COLLECTION_SUMMARY tissue: 10 μL solvent) was added to each sample. After that, samples were CO:COLLECTION_SUMMARY homogenized using a Qiagen TissueLyser LT system (Hilden, Germany). Next, 100 CO:COLLECTION_SUMMARY μL of each kidney homogenate was vigorously mixed (vortex 2 min) with 320 μL CO:COLLECTION_SUMMARY of methanol. Subsequently, 80 μL of MTBE was added to each sample to extract CO:COLLECTION_SUMMARY the lipid fraction from the kidneys. The samples were vigorously mixed for one CO:COLLECTION_SUMMARY hour at room temperature, and then centrifuged (4000 g, 20 min, 20°C) to CO:COLLECTION_SUMMARY facilitate phase separation. CO:SAMPLE_TYPE Kidney CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The study focused on metabolic, histological, and metabolomic alterations TR:TREATMENT_SUMMARY induced by a trans-fat-rich diet using wild-type C57Bl/6J mice, starting at 4 TR:TREATMENT_SUMMARY weeks old to 16 weeks. Mice were split into groups with a control diet, CD, (10% TR:TREATMENT_SUMMARY fat, D12450B, Research Diets, USA) or a high trans-fat diet, TFD, (29% fat, TR:TREATMENT_SUMMARY 54.4% total kcal, TD.07011, ENVIGO, USA), containing 24% saturated, 61% TR:TREATMENT_SUMMARY monounsaturated, and 15% polyunsaturated fats. To further evaluate the renal TR:TREATMENT_SUMMARY effects of the TFD, Tgfβ3 heterozygous mice (Tgfβ3+/-) were also studied TR:TREATMENT_SUMMARY alongside wild-type mice (Tgfβ3+/+), with both groups fed either a CD or TFD TR:TREATMENT_SUMMARY from 4 to 16 weeks of age. Complete deletion of both Tgfβ3 alleles is lethal. TR:TREATMENT_SUMMARY This animal model were grouped with the control or the trans-fat diets. After 16 TR:TREATMENT_SUMMARY weeks of age, Kidneys were collected and stored at -80°C. Kidney homogenate was TR:TREATMENT_SUMMARY prepared by adding cold (−20 °C) methanol/water (1:1, v/v), (1:10 TR:TREATMENT_SUMMARY tissue/solvent). Tissue disruption was achieved with Tissue- Lyser LT TR:TREATMENT_SUMMARY homogenizer (Qiagen, Germany) for metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Depending on the specific metabolomic analysis, the samples were prepared as SP:SAMPLEPREP_SUMMARY follows: LC-MS Analysis: 90 μL of the supernatant from each sample was SP:SAMPLEPREP_SUMMARY transferred to chromatography vials. 45 μL was used for analysis in positive SP:SAMPLEPREP_SUMMARY ionization mode (+), and another 45 μL was used for analysis in negative SP:SAMPLEPREP_SUMMARY ionization mode. The supernatants were directly injected into the system for SP:SAMPLEPREP_SUMMARY analysis. For GC−MS analysis, 300 μL of supernatant was evaporated to dryness SP:SAMPLEPREP_SUMMARY (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 (10 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 An Agilent GC instrument (7890A) coupled to an inert mass spectrometer CH:CHROMATOGRAPHY_SUMMARY with triple-Axis detector (5975C, Agilent Technologies) was used for kidney CH:CHROMATOGRAPHY_SUMMARY tissue fingerprinting. Briefly, 1 μL of derivatized samples were injected by an CH:CHROMATOGRAPHY_SUMMARY Agilent autosampler (7693). Samples were automatically injected in split mode CH:CHROMATOGRAPHY_SUMMARY (split ratio 1:12), into an Agilent ultra-inert deactivated glass wool split CH:CHROMATOGRAPHY_SUMMARY liner. Compound separation was achieved using a pre-column (10 m J&W integrated CH:CHROMATOGRAPHY_SUMMARY with Agilent 122-5532G) combined with a GC column DB5-MS (length, 30m; inner CH:CHROMATOGRAPHY_SUMMARY diameter, 0.25 mm; and 0.25 μm film of 95% dimethyl/5% diphenylpolysiloxane). CH:CHROMATOGRAPHY_SUMMARY The flow rate of helium carrier gas was constant at 0.938 mL/min through the CH:CHROMATOGRAPHY_SUMMARY column. The lock of the retention time (RTL) relative to the internal standard CH:CHROMATOGRAPHY_SUMMARY (methyl stearate) peak at 19.66 minutes was performed. The column oven CH:CHROMATOGRAPHY_SUMMARY temperature was initially set at 60°C (maintained for 1 minute), then raised by CH:CHROMATOGRAPHY_SUMMARY 10°C/min until it reached 325°C, and then was held at this temperature for 10 CH:CHROMATOGRAPHY_SUMMARY minutes before cooling down. The injector and the transfer line temperatures CH:CHROMATOGRAPHY_SUMMARY were established at 250°C and 280°C, respectively. The injector and the CH:CHROMATOGRAPHY_SUMMARY transfer line temperatures were established at 250°C and 280°C, respectively. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE - CH:COLUMN_TEMPERATURE Programmed temperature gradient: The column oven temperature was initially set CH:COLUMN_TEMPERATURE at 60°C (maintained for 1 minute), then raised by 10°C/min until it reached CH:COLUMN_TEMPERATURE 325°C, and then was held at this temperature for 10 minutes before cooling CH:COLUMN_TEMPERATURE down. #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE 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. MS system: the electron impact ionization operating parameters MS:MS_COMMENTS were set as follows: filament source temperature, 230°C; electron ionization MS:MS_COMMENTS energy, 70 eV. Mass spectra were collected over a mass range of 50-600 m/z at a MS:MS_COMMENTS scan rate of 10 spectra/s. Data were acquired using the Agilent MSD ChemStation MS:MS_COMMENTS Software (Agilent Technologies). For retention index determination, a mixture of MS:MS_COMMENTS n-alkanes (C8-C28) dissolved in nhexane was run prior to the samples. Data were MS:MS_COMMENTS acquired using Agilent MSD ChemStation Software (Agilent Technologies). MS:MS_RESULTS_FILE ST003738_AN006138_Results.txt UNITS:arbitrary units, a.u. Has m/z:Yes Has RT:Yes RT units:Minutes #END