#METABOLOMICS WORKBENCH ryant_20201211_131212 DATATRACK_ID:2357 STUDY_ID:ST001631 ANALYSIS_ID:AN002666 PROJECT_ID:000000 VERSION 1 CREATED_ON December 15, 2020, 11:30 am #PROJECT PR:PROJECT_TITLE Tissue-specific 1H-NMR metabolomic profiling in mice with adenine-induced PR:PROJECT_TITLE chronic kidney disease PR:PROJECT_TYPE Metabolomic profiling of the heart, liver, kidney, and skeletal muscles obtained PR:PROJECT_TYPE from mice with and without CKD via 1H NMR PR:PROJECT_SUMMARY This project is focused on a metabolomic analyses of the heart, liver, kidney, PR:PROJECT_SUMMARY and skeletal muscles obtained from mice with and without CKD. To accomplish this PR:PROJECT_SUMMARY objective, we extracted tissues from mice with CKD induced by long-term (24 PR:PROJECT_SUMMARY week) adenine-supplemented diet as well as their control-diet fed counterparts PR:PROJECT_SUMMARY with normal kidney function. Metabolites were extracted from tissues and 1H PR:PROJECT_SUMMARY nuclear magnetic resonance (NMR) was performed and coupled with multivariate PR:PROJECT_SUMMARY statistical analysis. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology and Kinesiology PR:LABORATORY Rm 42 and Rm 43 PR:LAST_NAME Ryan PR:FIRST_NAME Terence PR:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA PR:EMAIL ryant@ufl.edu PR:PHONE 352-294-1700 PR:FUNDING_SOURCE This research was funded by grants from the National Institutes of Health and PR:FUNDING_SOURCE the National Heart, Lung, and Blood, Institute numbers R01-HL149704 (to T.E.R.) PR:FUNDING_SOURCE and the American Heart Association grant number 18CDA34110044 (to T.E.R.). PR:PROJECT_COMMENTS CKD metabolomic study via NMR using mice model PR:PUBLICATIONS MDPI PR:CONTRIBUTORS Ram B. Khattri, Trace Thome, and Terence E. Ryan #STUDY ST:STUDY_TITLE Tissue-specific 1H-NMR metabolomic profiling in mice with adenine-induced ST:STUDY_TITLE chronic kidney disease - organic phase Quadricep (part-VIII) ST:STUDY_TYPE Metabolomic profiling of the heart, liver, kidney, and skeletal muscles obtained ST:STUDY_TYPE from mice with and without CKD via 1H NMR ST:STUDY_SUMMARY This project is focused on a metabolomic analyses of the heart, liver, kidney, ST:STUDY_SUMMARY and skeletal muscles obtained from mice with and without CKD. To accomplish this ST:STUDY_SUMMARY objective, we extracted tissues from mice with CKD induced by long-term (24 ST:STUDY_SUMMARY week) adenine-supplemented diet as well as their control-diet fed counterparts ST:STUDY_SUMMARY with normal kidney function. Metabolites were extracted from tissues and 1H ST:STUDY_SUMMARY nuclear magnetic resonance (NMR) was performed and coupled with multivariate ST:STUDY_SUMMARY statistical analysis. ST:INSTITUTE University of Florida ST:DEPARTMENT Applied Physiology and Kinesiology ST:LABORATORY Rm 42 and Rm 43 ST:LAST_NAME Ryan ST:FIRST_NAME Terence ST:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA ST:EMAIL ryant@ufl.edu ST:PHONE 352-294-1700 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 17 ST:NUM_MALES All ST:STUDY_COMMENTS CKD metabolomic study via NMR using mice model ST:PUBLICATIONS MDPI #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 13 months SU:WEIGHT_OR_WEIGHT_RANGE (32.86±1.21 g (control mice) vs 23.57±1.27 g (CKD mice), P < 0.0001, SU:WEIGHT_OR_WEIGHT_RANGE N=7/group). SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Stock # 000664) SU:ANIMAL_HOUSING Housed in a temperature of 22 oC SU:ANIMAL_LIGHT_CYCLE 12-hour light/12-hour dark SU:ANIMAL_FEED Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) SU:ANIMAL_WATER free access to food and water (3-5 animals per cage). SU:ANIMAL_INCLUSION_CRITERIA (3-5 animals per cage). #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 - CKD1Quad_Org Group:CKD RAW_FILE_NAME=CKD1Quad_Org SUBJECT_SAMPLE_FACTORS - CKD2Quad_Org Group:CKD RAW_FILE_NAME=CKD2Quad_Org SUBJECT_SAMPLE_FACTORS - CKD3Quad_Org Group:CKD RAW_FILE_NAME=CKD3Quad_Org SUBJECT_SAMPLE_FACTORS - CKD4Quad_Org Group:CKD RAW_FILE_NAME=CKD4Quad_Org SUBJECT_SAMPLE_FACTORS - CKD5Quad_Org Group:CKD RAW_FILE_NAME=CKD5Quad_Org SUBJECT_SAMPLE_FACTORS - CKD6Quad_Org Group:CKD RAW_FILE_NAME=CKD6Quad_Org SUBJECT_SAMPLE_FACTORS - CKD7Quad_Org Group:CKD RAW_FILE_NAME=CKD7Quad_Org SUBJECT_SAMPLE_FACTORS - Con1Quad_Org Group:Control RAW_FILE_NAME=Con1Quad_Org SUBJECT_SAMPLE_FACTORS - Con2Quad_Org Group:Control RAW_FILE_NAME=Con2Quad_Org SUBJECT_SAMPLE_FACTORS - Con3Quad_Org Group:Control RAW_FILE_NAME=Con3Quad_Org SUBJECT_SAMPLE_FACTORS - Con4Quad_Org Group:Control RAW_FILE_NAME=Con4Quad_Org SUBJECT_SAMPLE_FACTORS - Con5Quad_Org Group:Control RAW_FILE_NAME=Con5Quad_Org SUBJECT_SAMPLE_FACTORS - Con6Quad_Org Group:Control RAW_FILE_NAME=Con6Quad_Org SUBJECT_SAMPLE_FACTORS - Con7Quad_Org Group:Control RAW_FILE_NAME=Con7Quad_Org #COLLECTION CO:COLLECTION_SUMMARY While under isoflurane anesthesia, tissues were rapidly dissected and snap CO:COLLECTION_SUMMARY frozen in liquid nitrogen and stored at -80°C until metabolite extraction. The CO:COLLECTION_SUMMARY following tissues were used in this study: kidney, liver, heart (left CO:COLLECTION_SUMMARY ventricle), skeletal muscle (quadriceps). Euthanasia was carried out by CO:COLLECTION_SUMMARY thoracotomy followed by cervical dislocation. CO:SAMPLE_TYPE Muscle CO:COLLECTION_METHOD While under isoflurane anesthesia, tissues were rapidly dissected and snap CO:COLLECTION_METHOD frozen in liquid nitrogen and stored at -80°C until metabolite extraction CO:COLLECTION_LOCATION University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, CO:COLLECTION_LOCATION Gainesville, FL 32611 CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY To induce CKD, we utilized an established adenine-diet model. Briefly, mice were TR:TREATMENT_SUMMARY assigned to a casein-base chow for 7-days, followed by induction of renal TR:TREATMENT_SUMMARY tubular injury by supplementing the diet with 0.2% adenine for 7-days, and TR:TREATMENT_SUMMARY subsequently maintained on a 0.15% adenine diet for 5 months and two weeks. CKD TR:TREATMENT_SUMMARY mice were then placed back on casein control diet for 2-weeks prior to TR:TREATMENT_SUMMARY euthanasia and terminal experiments, allowing for a washout period of adenine TR:TREATMENT_SUMMARY based chow. Control mice received casein diet for the duration of the study. TR:TREATMENT_SUMMARY Total duration of CKD encompassed 6-months. TR:ANIMAL_VET_TREATMENTS none TR:ANIMAL_ANESTHESIA isoflurane TR:ANIMAL_FASTING non-fasted TR:ANIMAL_ENDP_EUTHANASIA Euthanasia was carried out by thoracotomy followed by cervical dislocation. TR:ANIMAL_ENDP_TISSUE_COLL_LIST kidney, liver, heart (left ventricle), skeletal muscle (quadriceps) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A modified form of FOLCH extraction protocol was used to extract metabolites SP:SAMPLEPREP_SUMMARY from the tissues. Wet weights of all tissue samples were recorded prior to SP:SAMPLEPREP_SUMMARY extraction. Tissue samples were immediately homogenized to prevent any possible SP:SAMPLEPREP_SUMMARY enzymatic action using 1 mL of ice-cold methanol in a PowerLyzer 24 Homogenizer SP:SAMPLEPREP_SUMMARY (QIAGEN Group, Hilden, Germany). The mixture was centrifuged using 13,200 rpm at SP:SAMPLEPREP_SUMMARY 4oC for 30 minutes and the resulting supernatant was transferred to a new glass SP:SAMPLEPREP_SUMMARY vial consisting 3 mL of ice cold chloroform:methanol (2:1, v/v) mixture. The SP:SAMPLEPREP_SUMMARY homogenate was vortexed and left in an ice bath for 15 minutes to allow for SP:SAMPLEPREP_SUMMARY phase separation. Next, 1 mL of 0.9% of saline was added, vortexed it for couple SP:SAMPLEPREP_SUMMARY of minutes followed by a second incubation in an ice bath for 30-45 min for SP:SAMPLEPREP_SUMMARY complete phase separation. The upper aqueous layer was transferred to a new SP:SAMPLEPREP_SUMMARY falcon tube. To the remaining organic phase sample, 1 mL of 0.9% of saline was SP:SAMPLEPREP_SUMMARY added again followed by vigorous mixing and letting it stand in ice bath (15 SP:SAMPLEPREP_SUMMARY minutes) for a second phase separation. This second aqueous phase was combined SP:SAMPLEPREP_SUMMARY with the first. The resulting aqueous and organic layers were dried separately. SP:SAMPLEPREP_SUMMARY The aqueous layer was dried overnight with a Labconco freezer dryer (Labconco SP:SAMPLEPREP_SUMMARY Corporation, MO, USA) and the organic layer was dried via inert nitrogen gas. SP:SAMPLEPREP_SUMMARY These two dried powders (aqueous and organic phases) were stored at -80oC until SP:SAMPLEPREP_SUMMARY performing NMR experiments. SP:PROCESSING_METHOD Lyophilization and Homogenization SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Modified FOLCH extraction SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Deuterated chloroform (80 microliter) with 10 mM pyrazine was used to re-suspend SP:SAMPLE_RESUSPENSION organic phase samples. SP:SAMPLE_SPIKING 10 mM of pyrazine for organic phase samples. #ANALYSIS AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker Avance Neo 600 MHz/54mm console NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterated chloroform NM:STANDARD_CONCENTRATION 10mM pyrazine NM:SPECTROMETER_FREQUENCY 600.2328273 MHz NM:NMR_PROBE 1.7 mm TXI CryoProbe NM:NMR_SOLVENT Deuterated chloroform NM:NMR_TUBE_SIZE 1.7 mm O.D. NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION none NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 101 NM:OFFSET_FREQUENCY 2827.31 Hz NM:CHEMICAL_SHIFT_REF_CPD CDCl3 at 7.26 ppm and pyrazine at 8.61 ppm NM:TEMPERATURE 25 o C NM:NUMBER_OF_SCANS 128 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 4 s NM:RELAXATION_DELAY 1 s NM:SPECTRAL_WIDTH 7142.9 Hz NM:NUM_DATA_POINTS_ACQUIRED 28571 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.22 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Spline NM:CHEMICAL_SHIFT_REF_STD 7.26ppm for CDCl3 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS A.U. NMR_METABOLITE_DATA_START Samples CKD1Quad_Org CKD2Quad_Org CKD3Quad_Org CKD4Quad_Org CKD5Quad_Org CKD6Quad_Org CKD7Quad_Org Con1Quad_Org Con2Quad_Org Con3Quad_Org Con4Quad_Org Con5Quad_Org Con6Quad_Org Con7Quad_Org Factors Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control C18-CH3 chol+V26C1C1:W25 1.352512305 6.79055712 5.214212162 6.217660115 6.882135128 7.530424993 5.067882268 5.134854796 6.947460058 6.055182393 7.293510265 7.816577315 5.405729082 6.463383225 CH3-protons 60.81737246 167.243107 182.4154585 173.5529004 272.1220385 251.372669 208.998134 169.0404786 300.5869788 218.6860695 283.6749383 369.0786487 276.635628 260.9057101 C19-CH3 Chol 2.439786453 9.180012688 7.80413926 8.538126365 9.99220021 11.34442875 7.953732793 7.106837343 10.81982311 8.934834505 10.58942823 12.20924083 9.506546369 9.91723251 CH2n chol 124.4930337 422.7877341 461.7124188 422.2863917 749.2143397 579.9001199 508.3263059 473.5535217 687.8175599 533.6129085 751.1959604 892.2297346 604.9207559 614.5152418 CH2n aliphatic chains 66.7749811 219.8749928 275.3909396 212.2657652 570.4056019 326.1702674 337.5291036 276.6803831 400.7305127 341.9663828 563.6956124 684.2560665 405.0361546 401.2008459 Cholesterol2? 1.246445112 1.986560372 3.028305622 2.118593347 4.680194731 4.547926848 4.689323459 1.217765849 3.55286684 3.755979225 4.57649127 3.98102331 5.519117357 3.074594751 CH2-CH2-COO-beta 24.2376162 59.08729978 74.0979317 57.41924701 135.1708429 97.45051294 87.90550606 66.8824953 98.45664934 82.47397156 131.2234314 155.3387058 106.3109079 93.89611142 CH2-CH=CH-CH2-alpha 37.96248194 63.68956387 80.31511821 61.32291643 152.3780321 120.4131159 92.42614577 81.16287868 107.2501732 88.91763563 155.467666 194.7807475 152.4840202 115.8052909 CH2COO-alpha 81.50013056 75.86729463 115.9875219 74.10562297 125.0400115 181.8604495 239.3533937 61.82792463 102.7856772 71.09976677 118.0990094 222.1498938 307.768222 360.5408118 CH-CH2-CH=CH 23.32967578 89.32506436 91.50111983 97.53978909 203.7659981 131.875225 92.9063062 98.67858485 227.0031807 205.0603704 344.6794684 136.3389715 99.52301771 111.3493427 N+CH33 19.35186441 73.69070166 71.42118944 69.31264238 97.60056736 76.62789517 68.37926096 130.2974356 109.0196691 99.34599385 116.3360328 98.22226103 73.11210914 82.70472584 Cholesterol3? 0.370733594 3.241827041 3.600959896 3.128334495 8.566628019 11.81391289 1.684225961 91.16554838 14.31481506 16.7612169 11.04433463 3.937794676 2.268840817 2.78632335 3CH2 glycerophospholipids 5.477607394 27.30160963 29.16222283 26.72238432 40.23873477 36.65264861 25.26508319 53.28156829 47.29778909 41.06138445 50.45301038 44.13053528 31.25525771 36.91607056 CH2_TG1 3.661985976 15.53507231 18.61895714 14.90089687 37.43322624 24.4636771 13.34234147 28.63103624 29.74030149 26.23515341 37.09688436 40.43752692 21.72711639 21.54131845 CH2_TG2 6.375800914 22.32777889 23.32261467 20.63907052 40.68264624 25.93117711 10.86821902 24.7324318 32.07464373 26.74091563 40.55253328 44.18512217 18.30726625 23.68617106 1CH phospholipids+TG 4.786412804 17.08417062 16.61103544 15.36178637 24.21740229 19.43162831 5.368748192 15.05194201 25.03044523 22.05319052 27.06168684 25.21493629 7.626488596 16.37504849 CH phospholipid 2.945031848 12.75342605 13.39554673 12.53830406 17.76754883 13.49457303 12.58968053 14.90360089 19.47702072 15.75458238 22.76097826 21.54487006 14.14654813 17.88295207 CH TG 0.210702118 1.256641434 1.890722584 0.987930057 7.940967244 1.593358264 1.147448804 2.693209227 2.75866352 1.785135398 6.24739573 9.710194444 2.987634624 2.827942137 CH=CH 26.57651751 116.7375018 127.0385734 110.050205 198.9381347 143.3098323 134.8679586 135.2018031 174.5582375 156.8994219 223.9405158 231.2882921 150.2000141 169.4725923 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name C18-CH3 chol+V26C1C1:W25 CH3-protons C19-CH3 Chol CH2n chol CH2n aliphatic chains Cholesterol2? CH2-CH2-COO-beta CH2-CH=CH-CH2-alpha CH2COO-alpha CH-CH2-CH=CH N+CH33 Cholesterol3? 3CH2 glycerophospholipids CH2_TG1 CH2_TG2 1CH phospholipids+TG CH phospholipid CH TG CH=CH METABOLITES_END #END