#METABOLOMICS WORKBENCH ryant_20201211_123721 DATATRACK_ID:2353 STUDY_ID:ST001627 ANALYSIS_ID:AN002662 PROJECT_ID:000000 VERSION 1 CREATED_ON December 15, 2020, 11:27 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 Heart (part-IV) 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 - CKD1Heart_Org Group:CKD RAW_FILE_NAME=CKD1Heart_Org SUBJECT_SAMPLE_FACTORS - CKD2Heart_Org Group:CKD RAW_FILE_NAME=CKD2Heart_Org SUBJECT_SAMPLE_FACTORS - CKD3Heart_Org Group:CKD RAW_FILE_NAME=CKD3Heart_Org SUBJECT_SAMPLE_FACTORS - CKD4Heart_Org Group:CKD RAW_FILE_NAME=CKD4Heart_Org SUBJECT_SAMPLE_FACTORS - CKD5Heart_Org Group:CKD RAW_FILE_NAME=CKD5Heart_Org SUBJECT_SAMPLE_FACTORS - CKD6Heart_Org Group:CKD RAW_FILE_NAME=CKD6Heart_Org SUBJECT_SAMPLE_FACTORS - CKD7Heart_Org Group:CKD RAW_FILE_NAME=CKD7Heart_Org SUBJECT_SAMPLE_FACTORS - Con1Heart_Org Group:Control RAW_FILE_NAME=Con1Heart_Org SUBJECT_SAMPLE_FACTORS - Con2Heart_Org Group:Control RAW_FILE_NAME=Con2Heart_Org SUBJECT_SAMPLE_FACTORS - Con3Heart_Org Group:Control RAW_FILE_NAME=Con3Heart_Org SUBJECT_SAMPLE_FACTORS - Con4Heart_Org Group:Control RAW_FILE_NAME=Con4Heart_Org SUBJECT_SAMPLE_FACTORS - Con5Heart_Org Group:Control RAW_FILE_NAME=Con5Heart_Org SUBJECT_SAMPLE_FACTORS - Con6Heart_Org Group:Control RAW_FILE_NAME=Con6Heart_Org SUBJECT_SAMPLE_FACTORS - Con7Heart_Org Group:Control RAW_FILE_NAME=Con7Heart_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 Heart 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 CKD1Heart_Org CKD2Heart_Org CKD3Heart_Org CKD4Heart_Org CKD5Heart_Org CKD6Heart_Org CKD7Heart_Org Con1Heart_Org Con2Heart_Org Con3Heart_Org Con4Heart_Org Con5Heart_Org Con6Heart_Org Con7Heart_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 1.715362437 3.446219673 4.240249353 2.72567459 3.187529436 3.121843956 3.000172 3.183130399 4.009918095 4.383724097 3.894144512 3.825026828 5.769907871 3.936838171 CH3-protons 91.78128667 190.2183547 268.8769938 164.9110301 149.2940384 144.5166038 197.4852482 167.7287932 261.5612584 208.0801012 257.5203476 179.4070507 242.888475 167.9307077 C19-CH3 Chol 3.069515412 6.013150892 7.952671201 5.11037618 4.915859604 4.7695979 5.795169559 4.725637214 7.689481204 7.42793361 7.469772988 5.743314883 8.84680047 5.886012067 CH2n chol 164.4205246 347.0998106 452.4659213 284.7526636 312.1499804 299.2202147 351.2439843 376.8276568 449.0964235 427.3562294 451.0021185 351.3779875 478.0779584 360.6079347 CH2n aliphatic chains 109.4802813 241.9731243 297.1131522 175.4759101 227.8106969 210.1796941 246.2429867 200.3058341 344.8450717 284.9107154 322.1985164 258.5974058 310.3417132 232.4233941 Cholesterol2? 1.265308572 2.428127047 3.977492767 2.437893767 1.752484809 1.268981512 2.525371607 1.548115804 3.452161654 2.081295312 3.573655439 1.74237967 2.018431127 1.613832766 CH2-CH2-COO-beta 26.16857155 54.89296839 70.99020143 46.92781426 49.84016971 45.0611107 55.5263637 50.83051234 77.31275283 67.85456306 76.71609779 53.92033972 73.97353713 52.85511642 CH2-CH=CH-CH2-alpha 72.65886808 69.37209886 41.12970036 23.07038386 47.06401925 51.36559949 34.20894269 15.39491954 53.95076165 83.12568154 47.39376419 67.90225442 122.728719 84.84613065 CH2COO-alpha 32.55121339 66.64534721 69.25294968 40.47733025 63.20091472 91.0403968 56.45861662 73.16545993 85.82162833 90.11636806 81.87619861 72.82674027 90.73277231 85.98038126 CH-CH2-CH=CH 31.1828628 61.9598806 66.63942297 43.49662914 56.07356936 58.22798203 55.27308562 44.27572139 82.49473149 81.63795085 80.61453159 69.37379068 69.25943961 86.75090555 N+CH33 17.97134192 32.74170089 34.92195944 17.18729654 34.86571952 37.21719427 25.33182688 33.68178625 31.01839204 47.61383243 33.1795887 38.68908694 42.68129885 44.57182099 Cholesterol3? 0.367536841 0.672930746 0.242467893 0.191432253 0.628362416 0.726844516 0.304003258 0.501675944 0.686434681 2.432726215 0.842856198 0.947796983 0.890396693 0.63440771 3CH2 glycerophospholipids 10.14637985 19.80641546 20.54745566 11.84744429 19.11615555 22.00085932 16.65230866 17.00264215 17.10793881 26.92821187 19.72539248 23.29658634 22.75850238 23.62982364 CH2_TG1 4.538931326 9.34395049 9.99852014 6.293905584 9.435912068 9.802816704 9.616290719 11.97548025 14.58262335 16.1281471 12.66181188 12.80004686 15.68002209 10.98294124 CH2_TG2 1.372863658 2.353884972 1.741457948 0.638380601 2.579067425 4.372497162 1.893831427 8.405253137 4.047965426 3.012184915 2.910733823 4.558941032 7.819385953 3.138340866 1CH phospholipids+TG 4.481605861 9.526153969 10.98241572 7.180938814 8.70897821 8.825913098 9.497690831 6.042699418 11.84221263 6.995765636 12.23789404 9.545177505 9.635058991 10.78102762 CH phospholipid 4.420871409 8.38644974 9.611318156 5.436857256 8.674417244 9.113652831 7.656967271 6.110465246 9.424748384 10.44490042 9.937878882 9.478522972 9.888574892 11.12771885 CH TG 0.343805713 0.416695014 0.889561742 0.212637421 0.956038058 0.834501268 0.887829458 2.079377371 1.958144748 1.150320008 1.519086436 1.885745607 2.425277103 1.111464142 CH=CH 48.17762602 98.75850247 105.4651768 67.2730979 92.64926222 95.49977885 90.63989014 34.30806856 133.7191446 121.5466946 125.6433861 110.1518207 109.6449525 126.4845592 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