#METABOLOMICS WORKBENCH staruschenkolab_20240731_081631 DATATRACK_ID:5064 STUDY_ID:ST003380 ANALYSIS_ID:AN005538 PROJECT_ID:PR002096 VERSION 1 CREATED_ON August 6, 2024, 3:31 pm #PROJECT PR:PROJECT_TITLE Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl PR:PROJECT_TITLE salt-sensitive rats PR:PROJECT_TYPE Untargeted metabolomic analysis PR:PROJECT_SUMMARY The objective of the project was to compare the metabolomic profiles in the PR:PROJECT_SUMMARY kidney, urine and plasma of SSWT and SSKcnj16-/- rats. Untargeted metabolomic PR:PROJECT_SUMMARY analysis revealed different metabolic profiles between SSWT and SSKcnj16-/- PR:PROJECT_SUMMARY rats, with 219, 790, and 31 differentially expressed metabolites in plasma, PR:PROJECT_SUMMARY urine, and kidney in SSKcnj16-/- rats compared to SSWT rats. PR:INSTITUTE University of South Florida PR:DEPARTMENT Molecular Pharmacology and Physiology PR:LABORATORY Alexander Staruschenko PR:LAST_NAME Xu PR:FIRST_NAME Biyang PR:ADDRESS 560 channelside drive, Tampa, FLORIDA, 33602, USA PR:EMAIL bxu@usf.edu PR:PHONE (332) 201-4356 #STUDY ST:STUDY_TITLE Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl ST:STUDY_TITLE salt-sensitive rats ST:STUDY_TYPE Untargeted metabolomis ST:STUDY_SUMMARY The inwardly rectifying K+ Channel Kir5.1 (Kcnj16) is essential in renal salt ST:STUDY_SUMMARY handling and blood pressure control. However, the underlying mechanisms are not ST:STUDY_SUMMARY fully understood. Here, we integrated transcriptomics and metabolomics to ST:STUDY_SUMMARY comprehensively profile the changes in genes and metabolites in the Dahl ST:STUDY_SUMMARY salt-sensitive (SS) rat lacking Kcnj16 to identify potential mechanisms. ST:STUDY_SUMMARY Consistent with the phenotype of knock-out (KO) rats, the transcriptomic profile ST:STUDY_SUMMARY predicted reduced blood pressure, kidney damage, and increased ion transport. ST:STUDY_SUMMARY Canonical pathway analysis suggested activation of metabolic-related while ST:STUDY_SUMMARY suppression of immune responses-related pathways in KO rats. Untargeted ST:STUDY_SUMMARY metabolomic analysis revealed different metabolic profiles between WT and KO ST:STUDY_SUMMARY rats. Integration of transcriptomic and metabolomic profiles suggested altered ST:STUDY_SUMMARY tricarboxylic acid (TCA) cycle, amino acid, and reactive oxygen species (ROS) ST:STUDY_SUMMARY metabolism that are related to SS hypertension. In conclusion, besides increased ST:STUDY_SUMMARY ion transport, our data suggest suppressed immune responses-related and altered ST:STUDY_SUMMARY metabolic-related pathways of SS rats lacking Kir5.1. ST:INSTITUTE University of South Florida ST:DEPARTMENT Molecular Pharmacology and Physiology ST:LABORATORY Alexander Staruschenko ST:LAST_NAME Xu ST:FIRST_NAME Biyang ST:ADDRESS 560 channelside drive Tampa FL 33602 ST:EMAIL bxu@usf.edu ST:PHONE 3322014356 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 11 ST:NUM_MALES 11 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENDER Male #FACTORS #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 - s.9052.01 Sample source:plasma | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-01.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-01.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-01.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-01.d SUBJECT_SAMPLE_FACTORS - s.9052.02 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-02.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-02.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-02.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-02.d SUBJECT_SAMPLE_FACTORS - s.9052.03 Sample source:plasma | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-03.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-03.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-03.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-03.d SUBJECT_SAMPLE_FACTORS - s.9052.04 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-04.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-04.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-04.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-04.d SUBJECT_SAMPLE_FACTORS - s.9052.05 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-05.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-05.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-05.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-05.d SUBJECT_SAMPLE_FACTORS - s.9052.06 Sample source:plasma | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-06.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-06.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-06.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-06.d SUBJECT_SAMPLE_FACTORS - s.9052.07 Sample source:plasma | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-07.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-07.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-07.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-07.d SUBJECT_SAMPLE_FACTORS - s.9052.08 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-08.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-08.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-08.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-08.d SUBJECT_SAMPLE_FACTORS - s.9052.09 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-09.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-09.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-09.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-09.d SUBJECT_SAMPLE_FACTORS - s.9052.10 Sample source:plasma | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-10.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-10.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-10.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-10.d SUBJECT_SAMPLE_FACTORS - s.9052.11 Sample source:plasma | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-28mar23-ms9052-11.d; RAW_FILE_NAME(nC18 filename)=nC18-31mar23-ms9052-11.d; RAW_FILE_NAME(philic filename)=philic-28mar23-ms9052-11.d; RAW_FILE_NAME(nhilic filename)=nhilic-29mar23-ms9052-11.d SUBJECT_SAMPLE_FACTORS - s.9052.12 Sample source:Urine | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-12.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-12.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-12.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-12.d SUBJECT_SAMPLE_FACTORS - s.9052.13 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-13.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-13.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-13.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-13.d SUBJECT_SAMPLE_FACTORS - s.9052.14 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-14.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-14.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-14.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-14.d SUBJECT_SAMPLE_FACTORS - s.9052.15 Sample source:Urine | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-15.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-15.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-15.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-15.d SUBJECT_SAMPLE_FACTORS - s.9052.16 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-16.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-16.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-16.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-16.d SUBJECT_SAMPLE_FACTORS - s.9052.17 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-17.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-17.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-17.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-17.d SUBJECT_SAMPLE_FACTORS - s.9052.18 Sample source:Urine | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-18.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-18.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-18.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-18.d SUBJECT_SAMPLE_FACTORS - s.9052.19 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-19.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-19.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-19.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-19.d SUBJECT_SAMPLE_FACTORS - s.9052.20 Sample source:Urine | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-20.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-20.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-20.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-20.d SUBJECT_SAMPLE_FACTORS - s.9052.21 Sample source:Urine | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-21.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-21.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-21.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-21.d SUBJECT_SAMPLE_FACTORS - s.9052.22 Sample source:Urine | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-03apr23-ms9052-22.d; RAW_FILE_NAME(nC18 filename)=nC18-05apr23-ms9052-22.d; RAW_FILE_NAME(philic filename)=philic-30mar23-ms9052-22.d; RAW_FILE_NAME(nhilic filename)=nhilic-31mar23-ms9052-22.d SUBJECT_SAMPLE_FACTORS - s.9052.23 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-23.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-23.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-23.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-23.d SUBJECT_SAMPLE_FACTORS - s.9052.24 Sample source:kidney cortex | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-24.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-24.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-24.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-24.d SUBJECT_SAMPLE_FACTORS - s.9052.25 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-25.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-25.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-25.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-25.d SUBJECT_SAMPLE_FACTORS - s.9052.26 Sample source:kidney cortex | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-26.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-26.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-26.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-26.d SUBJECT_SAMPLE_FACTORS - s.9052.27 Sample source:kidney cortex | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-27.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-27.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-27.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-27.d SUBJECT_SAMPLE_FACTORS - s.9052.28 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-28.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-28.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-28.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-28.d SUBJECT_SAMPLE_FACTORS - s.9052.29 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-29.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-29.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-29.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-29.d SUBJECT_SAMPLE_FACTORS - s.9052.30 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-30.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-30.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-30.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-30.d SUBJECT_SAMPLE_FACTORS - s.9052.31 Sample source:kidney cortex | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-31.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-31.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-31.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-31.d SUBJECT_SAMPLE_FACTORS - s.9052.32 Sample source:kidney cortex | Genotype:control RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-32.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-32.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-32.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-32.d SUBJECT_SAMPLE_FACTORS - s.9052.33 Sample source:kidney cortex | Genotype:KO RAW_FILE_NAME(pC18 filename)=pC18-06apr23-ms9052-33.d; RAW_FILE_NAME(nC18 filename)=nC18-07apr23-ms9052-33.d; RAW_FILE_NAME(philic filename)=philic-03apr23-ms9052-33.d; RAW_FILE_NAME(nhilic filename)=nhilic-04apr23-ms9052-33.d #COLLECTION CO:COLLECTION_SUMMARY Flash frozen kidney tissue, urine and plasma from arterial blood were collected CO:SAMPLE_TYPE urine, plasma, kidney cortex #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Kidney: 10 uL/mg 1X PBS was added to the pulverized kidney tissue followed by SP:SAMPLEPREP_SUMMARY vortexing to form homogenous mixture. 50 uL of homogenate was used. 500 ng Ring SP:SAMPLEPREP_SUMMARY 13C6-Phe IS (internal standard) were added to all samples and sonicated in ice SP:SAMPLEPREP_SUMMARY bath for 30 seconds x 4 times. 300 uL 50x50 MeOH: ACN mixture were then added SP:SAMPLEPREP_SUMMARY and incubated on ice for 30 min with occasional vortexing (every 10 min). The SP:SAMPLEPREP_SUMMARY mixture were then centrifuged at 18,000 g for 20 min at 4oC, and dry under N2 SP:SAMPLEPREP_SUMMARY stream on cold blocks. Urine: 50 uL urine sample was used. 500 ng Ring 13C6-Phe SP:SAMPLEPREP_SUMMARY IS (internal standard) were added to all samples and sonicated in ice bath for SP:SAMPLEPREP_SUMMARY 30 seconds x 4 times. 300 uL 50x50 MeOH: ACN mixture were then added and SP:SAMPLEPREP_SUMMARY incubated on ice for 30 min with occasional vortexing (every 10 min). The SP:SAMPLEPREP_SUMMARY mixture were then centrifuged at 18,000 g for 20 min at 4oC, and dry under N2 SP:SAMPLEPREP_SUMMARY stream on cold blocks. Plasma: 25 uL of plasma was used. 500 ng Ring 13C6-Phe IS SP:SAMPLEPREP_SUMMARY (internal standard) were added to all samples and sonicated in ice bath for 30 SP:SAMPLEPREP_SUMMARY seconds x 4 times. 150 uL 50x50 MeOH: ACN mixture were then added and incubated SP:SAMPLEPREP_SUMMARY on ice for 30 min with occasional vortexing (every 10 min). The mixture were SP:SAMPLEPREP_SUMMARY then centrifuged at 18,000 g for 20 min at 4oC, and dry under N2 stream on cold SP:SAMPLEPREP_SUMMARY blocks. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (150 x 2.1mm, 1.8um) CH:SOLVENT_A Water with 1% acetonitrile, 5mM ammonium acetate & 0.1% formic acid CH:SOLVENT_B Water with 95% acetonitrile, 5mM ammonium acetate & 0.1% formic acid CH:FLOW_GRADIENT 0 min, 0% B; 1 min, 0% B; 3 min, 5% B; 13.0 min, 100% B; 16 min, 100% B; 16.5 CH:FLOW_GRADIENT min, 0% B; and 20 min, 0% B CH:FLOW_RATE 400 uL/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For each column, the run time is 20 min using a flow rate of 400 μL/min. A MS:MS_COMMENTS total of four samples per run was performed to give maximum coverage of MS:MS_COMMENTS metabolites. Samples were injected in duplicate or triplicate, and a quality MS:MS_COMMENTS control sample, made up of a subset of samples from the study was injected MS:MS_COMMENTS several times during a run. All raw data files obtained were converted to MS:MS_COMMENTS compound exchange file format using Masshunter DA reprocessor software (Agilent, MS:MS_COMMENTS USA). Mass Profile Professional (Agilent, USA) was used for data alignment and MS:MS_COMMENTS to convert each metabolite feature (m/z x intensity x time) into a matrix of MS:MS_COMMENTS detected peaks for compound identification. Components that were matched were MS:MS_COMMENTS further examined by comparison to a purchased reference standard of the proposed MS:MS_COMMENTS compound. Mass accuracy of the Q-TOF method was <5ppm with retention time MS:MS_COMMENTS precision better than 0.2%. A 1.2x fold change can be detected with a precision MS:MS_COMMENTS of 4%. MS:MS_RESULTS_FILE ST003380_AN005538_Results.txt UNITS:peak area Has m/z:Yes Has RT:No RT units:No RT data #END