#METABOLOMICS WORKBENCH borjalanzon_20241216_091130 DATATRACK_ID:5455 STUDY_ID:ST003738 ANALYSIS_ID:AN006136 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 LC-MS (+) A UHPLC system (1290 Infinity UHPLC system, Agilent Technologies, CH:CHROMATOGRAPHY_SUMMARY Waldbronn, Germany), consisting of two degassers, two binary pumps, and a CH:CHROMATOGRAPHY_SUMMARY thermostated autosampler (maintained at 4°C) coupled with 6545 QTOF MS CH:CHROMATOGRAPHY_SUMMARY detector, was used in positive and negative ESI modes. In brief, 1 μL of each CH:CHROMATOGRAPHY_SUMMARY sample was injected into a reverse-phase Poroshell 120 Infinity Lab EC-C8 (2.1 x CH:CHROMATOGRAPHY_SUMMARY 150 mm, 2.7 µm) (Agilent Technologies) thermostated at 60°C. The gradient used CH:CHROMATOGRAPHY_SUMMARY for the analysis consisted of a mobile phase A (10 mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY Milli-Q water) and mobile phase B (10 mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY methanol:isopropanol, 85:15) pumped at 0.5 mL/min. The chromatography gradient CH:CHROMATOGRAPHY_SUMMARY started at 82% phase B, increasing to 90% B in 17 min. The gradient then CH:CHROMATOGRAPHY_SUMMARY increased to 100% B by minute 18 and was maintained for 2 minutes until 20 min. CH:CHROMATOGRAPHY_SUMMARY The starting condition was returned to by 21.5 min, followed by an 8.5 min CH:CHROMATOGRAPHY_SUMMARY reequilibration time, taking the total run time to 30 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C8 (150 x 2.1mm,2.7um) CH:SOLVENT_A 100% water; 10 mM ammonium formate CH:SOLVENT_B 85% methanol/15% isopropanol; 10 mM ammonium formate CH:FLOW_GRADIENT gradient started at 82% phase B, increasing to 90% B in 17 min. The gradient CH:FLOW_GRADIENT then increased to 100% B by minute 18 and was maintained for 2 minutes until 20 CH:FLOW_GRADIENT min. The starting condition was returned to by 21.5 min, followed by an 8.5 min CH:FLOW_GRADIENT reequilibration time, taking the total run time to 30 min CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometry signals were processed following the procedure described MS:MS_COMMENTS by Armitage and colleagues (DOI:10.1128/AAC.02095-17). Data files generated from MS:MS_COMMENTS the instrumental analysis were processed using Agilent's Mass Hunter Profinder MS:MS_COMMENTS (B.08.00), a software employed for deconvolution, background noise removal, and MS:MS_COMMENTS integration of features obtained in the kidney samples of mice.The significant MS:MS_COMMENTS compounds obtained in the LC-MS analysis were annotated by combining information MS:MS_COMMENTS from spectral libraries (databases) and the data generated during LC-MS/MS MS:MS_COMMENTS (tandem mass spectrometry) experiments in both positive and negative ionization MS:MS_COMMENTS modes. The mass acquisition range was set in the interval of 50 to 3000 m/z for MS:MS_COMMENTS both ionizations. The identities were fragmented at 20 and 40 eV, and 1.5 µL of MS:MS_COMMENTS quality control samples were injected for each method developed in positive and MS:MS_COMMENTS negative ionization modes. Various spectral libraries were used for the MS:MS_COMMENTS annotation of unknown identities. Data were collected in full scan mode from 100 MS:MS_COMMENTS to 1200 m/z, with a scan rate of 1.02 scans/s. The capillary voltage was set to MS:MS_COMMENTS 3500 V; the drying gas flow rate was 12 L/min at 290°C and gas nebulizer 45 MS:MS_COMMENTS psi, fragmentor voltage 175 V, and octopole radio frequency voltage (OCT RF Vpp) MS:MS_COMMENTS 750 V. Two reference masses were used over the course of the whole analysis: m/z MS:MS_COMMENTS 121.0509 (protonated purine) and m/z 922.0098 (protonated hexakis, MS:MS_COMMENTS (1H,1H,3H-tetrafluoropropoxy)phosphazine (HP-921)). These masses were MS:MS_COMMENTS continuously infused into the system to provide constant mass correction. MS:MS_COMMENTS Samples were randomly analyzed throughout the run. MS:MS_RESULTS_FILE ST003738_AN006136_Results.txt UNITS:arbitrary units, a.u. Has m/z:Yes Has RT:Yes RT units:Minutes #END