#METABOLOMICS WORKBENCH ryant_20201211_121449 DATATRACK_ID:2351 STUDY_ID:ST001625 ANALYSIS_ID:AN002660 PROJECT_ID:000000 VERSION 1 CREATED_ON December 15, 2020, 11:24 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 Kidney (part-II) 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 - CKD1Kidney_Org Group:CKD RAW_FILE_NAME=CKD1Kidney_Org SUBJECT_SAMPLE_FACTORS - CKD2Kidney_Org Group:CKD RAW_FILE_NAME=CKD2Kidney_Org SUBJECT_SAMPLE_FACTORS - CKD4Kidney_Org Group:CKD RAW_FILE_NAME=CKD4Kidney_Org SUBJECT_SAMPLE_FACTORS - CKD5Kidney_Org Group:CKD RAW_FILE_NAME=CKD5Kidney_Org SUBJECT_SAMPLE_FACTORS - CKD6Kidney_Org Group:CKD RAW_FILE_NAME=CKD6Kidney_Org SUBJECT_SAMPLE_FACTORS - CKD7Kidney_Org Group:CKD RAW_FILE_NAME=CKD7Kidney_Org SUBJECT_SAMPLE_FACTORS - Con1Kidney_Org Group:Control RAW_FILE_NAME=Con1Kidney_Org SUBJECT_SAMPLE_FACTORS - Con2Kidney_Org Group:Control RAW_FILE_NAME=Con2Kidney_Org SUBJECT_SAMPLE_FACTORS - Con3Kidney_Org Group:Control RAW_FILE_NAME=Con3Kidney_Org SUBJECT_SAMPLE_FACTORS - Con4Kidney_Org Group:Control RAW_FILE_NAME=Con4Kidney_Org SUBJECT_SAMPLE_FACTORS - Con5Kidney_Org Group:Control RAW_FILE_NAME=Con5Kidney_Org SUBJECT_SAMPLE_FACTORS - Con6Kidney_Org Group:Control RAW_FILE_NAME=Con6Kidney_Org SUBJECT_SAMPLE_FACTORS - Con7Kidney_Org Group:Control RAW_FILE_NAME=Con7Kidney_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 Kidney 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 CKD1Kidney_Org CKD2Kidney_Org CKD4Kidney_Org CKD5Kidney_Org CKD6Kidney_Org CKD7Kidney_Org Con1Kidney_Org Con2Kidney_Org Con3Kidney_Org Con4Kidney_Org Con5Kidney_Org Con6Kidney_Org Con7Kidney_Org Factors 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 16.41582649 17.42026608 13.61593931 16.76655911 12.4863875 18.17918833 20.65412297 19.90621371 22.45758632 15.00164566 19.92642943 19.18525004 18.65785675 CH3-protons 244.1573002 242.3210022 184.9564173 321.3213512 200.7175933 276.0372407 373.4385258 433.6331534 396.4821548 303.2425276 405.2563369 330.6295335 253.6111102 C19-CH3 Chol 18.25296334 19.79628899 15.27728598 18.52842156 14.11961721 20.26084553 24.0998453 22.76010971 24.05192704 18.01993165 21.48764705 20.92659498 20.55739582 CH2n chol 362.3940848 357.2700567 283.5480583 581.2273242 313.0091808 484.1875256 696.1122492 782.2292041 817.3794508 446.2795499 783.9779597 683.5913018 571.6433928 CH2n aliphatic chains 347.1797339 338.004368 332.1224658 808.0201666 479.8437618 518.5803782 618.4088186 1026.77274 834.3864651 548.4211411 1072.523966 834.678048 432.6604844 Cholesterol2? 4.014712866 4.260999563 2.880106216 2.877166866 2.649390005 3.447528097 5.583295849 5.61609285 2.887065684 4.848315377 3.74257404 3.061170236 2.88408409 CH2-CH2-COO-beta 91.46507967 89.13470595 79.53918279 150.2046992 96.65045441 114.5318459 146.6462213 185.6168375 154.0135081 108.7888371 189.0105442 144.4699936 94.83868493 CH2-CH=CH-CH2-alpha 108.4282715 105.4413176 62.02553819 217.2751291 55.31006848 233.6929747 246.3539875 265.6959346 209.397066 130.3916308 245.5366747 195.5057964 123.4535771 CH2COO-alpha 22.38651247 17.54647532 12.19439457 169.1501231 11.29827946 66.77285794 65.12464479 143.3364771 102.0750153 85.20854355 134.9622536 98.94393758 46.90884461 CH-CH2-CH=CH 44.89601782 51.09860742 37.09675901 80.06679043 37.51888097 66.41382302 126.6489097 125.3064491 176.7085649 91.72985421 179.1670477 181.8181047 266.8385545 N+CH33 36.53637491 42.36680569 31.62807309 52.17267193 28.90840615 49.44988334 62.59576166 48.00652345 68.5745554 39.39541116 53.85000646 80.04383099 63.00947973 Cholesterol3? 5.194988827 5.005134997 5.681403635 7.684552832 5.549811623 8.161072781 10.76288578 10.31877624 11.03826976 6.690095292 13.77272046 12.97917592 12.90717784 3CH2 glycerophospholipids 19.46144554 21.47457178 16.3981836 32.09600329 15.29714601 30.05115654 49.29139675 39.05302201 49.41283821 24.88581957 43.2540413 46.64850602 41.82676212 CH2_TG1 8.003249571 5.593381681 8.433678015 27.76910852 15.86644387 14.26364151 15.22995384 33.08074584 27.17292889 18.3238535 39.15727899 27.64804739 12.33014674 CH2_TG2 6.905017096 8.016320851 11.39047208 32.76965298 15.38983604 18.63885258 10.97845995 35.09205822 35.43300839 22.1610167 46.14660713 35.71015828 19.17966998 1CH phospholipids+TG 7.114480465 7.749232578 5.313674604 11.32373801 2.484118419 10.19692496 9.969318763 14.3973278 17.2960257 8.895579875 15.00214773 16.10551409 13.53073763 CH phospholipid 6.559063533 7.179844087 5.079877167 11.45603838 3.73186186 9.960399773 16.34777594 15.08702698 19.13685135 9.645845454 16.00387008 16.22370386 14.46378533 CH TG 1.466895429 1.214647347 2.468628388 10.67371579 5.71303167 3.719363665 0.160893773 12.84157607 9.558672097 6.841586896 16.16005013 9.200783313 1.911740027 CH=CH 74.191989 80.79126578 68.75466503 160.9249396 85.80801663 116.3110012 190.203644 223.8475589 242.1908718 138.7093356 222.1901844 230.0223069 169.4830578 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name C18-CH3 chol 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