#METABOLOMICS WORKBENCH ryant_20201211_125408 DATATRACK_ID:2355 STUDY_ID:ST001629 ANALYSIS_ID:AN002664 PROJECT_ID:000000 VERSION 1 CREATED_ON December 15, 2020, 11:28 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 Liver (part-VI) 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 - CKD1Liver_Org Group:CKD RAW_FILE_NAME=CKD1Liver_Org SUBJECT_SAMPLE_FACTORS - CKD2Liver_Org Group:CKD RAW_FILE_NAME=CKD2Liver_Org SUBJECT_SAMPLE_FACTORS - CKD3Liver_Org Group:CKD RAW_FILE_NAME=CKD3Liver_Org SUBJECT_SAMPLE_FACTORS - CKD4Liver_Org Group:CKD RAW_FILE_NAME=CKD4Liver_Org SUBJECT_SAMPLE_FACTORS - CKD5Liver_Org Group:CKD RAW_FILE_NAME=CKD5Liver_Org SUBJECT_SAMPLE_FACTORS - CKD6Liver_Org Group:CKD RAW_FILE_NAME=CKD6Liver_Org SUBJECT_SAMPLE_FACTORS - CKD7Liver_Org Group:CKD RAW_FILE_NAME=CKD7Liver_Org SUBJECT_SAMPLE_FACTORS - Con1Liver_Org Group:Control RAW_FILE_NAME=Con1Liver_Org SUBJECT_SAMPLE_FACTORS - Con2Liver_Org Group:Control RAW_FILE_NAME=Con2Liver_Org SUBJECT_SAMPLE_FACTORS - Con3Liver_Org Group:Control RAW_FILE_NAME=Con3Liver_Org SUBJECT_SAMPLE_FACTORS - Con4Liver_Org Group:Control RAW_FILE_NAME=Con4Liver_Org SUBJECT_SAMPLE_FACTORS - Con5Liver_Org Group:Control RAW_FILE_NAME=Con5Liver_Org SUBJECT_SAMPLE_FACTORS - Con6Liver_Org Group:Control RAW_FILE_NAME=Con6Liver_Org SUBJECT_SAMPLE_FACTORS - Con7Liver_Org Group:Control RAW_FILE_NAME=Con7Liver_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 Liver 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 DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) 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 CKD1Liver_Org CKD2Liver_Org CKD3Liver_Org CKD4Liver_Org CKD5Liver_Org CKD6Liver_Org CKD7Liver_Org Con1Liver_Org Con2Liver_Org Con3Liver_Org Con4Liver_Org Con5Liver_Org Con6Liver_Org Con7Liver_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 8.90038123 12.35616233 13.28316415 12.78085323 12.19640729 12.69537932 14.14442041 12.79518073 11.93463847 13.08714193 12.68703691 12.70700412 11.85417207 16.53813246 CH3-protons 222.1247435 337.9814288 451.9984312 400.7833301 387.5587623 290.3474821 286.0560385 322.4512762 357.1315084 498.605293 347.0821748 414.115702 307.8967914 403.4248255 C19-CH3 Chol 11.49533173 16.44547102 18.42099619 18.4576512 14.35474776 14.03529134 13.9149493 14.36947408 13.31093594 19.49088954 14.02745314 13.88165706 13.18337175 17.78345995 CH2n chol 324.9067621 508.4106207 476.8886544 515.8002078 577.0966543 447.6294652 448.3906883 553.7899122 625.7864973 616.5298359 626.7538771 686.6998098 545.8362345 731.4203089 CH2n aliphatic chains 400.9194403 586.3896271 671.5592965 588.2983408 838.6786337 643.5867777 660.5994183 861.437899 992.6286481 939.7676663 882.2282357 1223.461459 835.6709888 1148.043696 Cholesterol2? 3.44480047 5.176937852 8.707172774 7.345379107 4.134785644 2.863734764 2.078243383 2.297809787 2.441298575 9.881985713 2.438108986 3.070293549 2.785347698 2.276111352 CH2-CH2-COO-beta 76.41941328 113.6800956 126.6346214 121.0685382 139.5655892 111.2778543 109.2757421 135.9184684 155.6581156 173.6757124 145.787432 188.3106471 134.2121315 183.0862045 CH2-CH=CH-CH2-alpha 75.86083572 110.0098959 138.7709869 111.2200408 292.6254589 131.2556136 137.0886244 168.6871618 183.0127606 179.300511 170.5780307 315.0166483 164.634896 236.160702 CH2COO-alpha 68.88652068 128.6821348 97.30449706 253.7177694 126.1870263 258.7564073 325.8914734 156.834585 307.3638668 317.8600418 387.3707131 189.3706907 133.6721153 191.8052287 CH-CH2-CH=CH 101.7134834 113.0883695 84.85680641 92.94209337 92.84397245 89.44338178 93.53884707 150.0143766 109.143974 140.7490316 112.4410594 106.070655 368.9847912 204.563217 N+CH33 63.61998009 82.91623686 20.31518525 82.68181036 40.09450543 65.98241931 70.17580736 76.58664409 76.03660769 67.46458637 91.00241172 67.93062255 118.6551496 93.67082674 Cholesterol3? 4.236673879 4.963109873 3.342419269 4.926524233 3.495152531 4.415903174 4.532718056 6.745507172 5.307629671 6.262387913 6.015676745 4.105264184 15.36212564 6.193583417 3CH2 glycerophospholipids 25.34670158 34.17777785 14.16251775 33.53332943 20.91740785 28.08175151 30.96023207 35.85266483 34.27746037 33.49531728 37.36923436 31.54924011 39.9649379 44.47198954 CH2_TG1 10.0431903 13.7089281 12.58964025 12.64885424 20.22776584 15.16532561 15.90038513 25.53189707 28.41605776 28.95051098 26.41805682 37.64950621 28.06549657 38.89479916 CH2_TG2 15.41977932 18.92697861 9.307901704 17.58041771 14.95545679 13.91077389 18.29960104 27.89535167 30.23907809 27.36065949 32.8571117 38.26862866 26.183014 42.71063507 1CH phospholipids+TG 11.8588853 15.99764106 9.043933756 15.99860843 13.06864208 7.983969488 15.12626471 17.37793504 16.78865202 15.90860678 17.43291624 15.28639151 16.57467272 21.40119584 CH phospholipid 12.79837424 17.46210795 9.776339002 16.82077108 13.07946694 14.24309795 16.70667644 18.87657726 18.24045841 16.86196308 19.34939658 16.70943147 17.44907314 23.82416242 CH TG 1.909084072 2.619569619 4.527497796 1.732882754 6.89257014 3.149743846 4.542234748 8.894273731 10.53895721 10.64993935 9.016134534 15.35453655 8.06404135 14.65727181 CH=CH 108.8612618 155.5770794 143.70299 139.7206265 162.669693 155.7564101 159.8510837 219.6357702 191.5984853 220.8499269 193.5294914 213.7239732 194.7643847 282.8425716 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