#METABOLOMICS WORKBENCH jgooding_20170217_113747 DATATRACK_ID:846 STUDY_ID:ST000559 ANALYSIS_ID:AN000859 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Urine metabolomic profiling of diabetic nephropathy in the steptozotocin induced PR:PROJECT_TITLE type-1 diabetes mouse model. PR:PROJECT_SUMMARY Diabetic nephropathy (DN) is the leading cause of end stage renal disease, and PR:PROJECT_SUMMARY is associated with high morbidity and mortality rates. The pathophysiology of DN PR:PROJECT_SUMMARY includes both glomerular and tubulointerstitial damage. Meprins are PR:PROJECT_SUMMARY metalloproteinases which are most abundantly expressed in the brush border PR:PROJECT_SUMMARY membranes of proximal kidney tubules. Meprins are also expressed in leukocytes PR:PROJECT_SUMMARY (monocytes and macrophages) and podocytes. Meprins have been implicated in the PR:PROJECT_SUMMARY pathology of acute and chronic kidney injury. Single nucleotide polymorphisms PR:PROJECT_SUMMARY (SNPs) in the meprin β gene were associated in human DN in the Pima Indians, PR:PROJECT_SUMMARY suggesting a role for meprins in the pathophysiology of DN. The current study PR:PROJECT_SUMMARY was done to determine the mechanisms by which meprins modulate the progression PR:PROJECT_SUMMARY of DN in mice. PR:INSTITUTE North Carolina A&T State University PR:DEPARTMENT Department of Biology PR:LAST_NAME Ongeri PR:FIRST_NAME Elimelda Moige PR:ADDRESS 1601 E Market Street, Greensboro, NC 27411 PR:EMAIL eongeri@ncat.edu PR:PHONE 336-285-2182 PR:FUNDING_SOURCE NIH/NIGMS Grant # SC3102049 To Elimelda Moige Ongeri; NIH Center Grant # PR:FUNDING_SOURCE U24DK097193 to Susan Sumner; NIH/NCATS award # UL1TR001111 to John Buse, UNC-CH, PR:FUNDING_SOURCE PI, Sumner- Director, Metabolomics core; and NIH/NIGMS Grant # K01GM109320 to PR:FUNDING_SOURCE Jessica Gooding. PR:DOI http://dx.doi.org/10.21228/M82889 #STUDY ST:STUDY_TITLE Urine metabolomic profiling of diabetic nephropathy in the steptozotocin induced ST:STUDY_TITLE type-1 diabetes mouse model. ST:STUDY_SUMMARY This metabolomics study evaluated urine from wild-type and meprin β knockout ST:STUDY_SUMMARY mice after induction of diabetes with streptozotocin or treatment with sodium ST:STUDY_SUMMARY citrate control to understand how these factors influence the metabotype. ST:INSTITUTE RTI International ST:LABORATORY NIH Eastern Regional Comphrehensive Metabolomics Resource Core at UNC Chapel ST:LABORATORY Hill (ERCMRC) ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 E. Cornwallis Road, Research Triangle Park, NC 27709 ST:EMAIL susan_sumner@unc.edu ST:PHONE 704-250-5000 ST:SUBMIT_DATE 2017-02-17 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER KO: bred and housed at the laboratory animal resource unit (LARU) of North SU:ANIMAL_ANIMAL_SUPPLIER Carolina A&T State University; WT: Charles River Laboratories SU:ANIMAL_HOUSING group cages of up to 5 mice per cage SU:ANIMAL_LIGHT_CYCLE 12:12h light:dark cycle SU:ANIMAL_FEED rat chow SU:ANIMAL_WATER water ad libitum SU:SPECIES_GROUP Mammal #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Equilibrium_6 Equilibrium_6 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Equil6.raw; "Subject Identifier Instrument Run Name (NEG)"=-; Gender=- SUBJECT_SAMPLE_FACTORS Total Pool_1 Total Pool_1 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Pool_1.raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U_Pool_1_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS Total Pool_2 Total Pool_2 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Pool_2.raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U_Pool_2_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS Total Pool_3 Total Pool_3 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Pool_3.raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U_Pool_3_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS Total Pool_4 Total Pool_4 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Pool_4.raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U_Pool_4_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS Total Pool_5 Total Pool_5 Treatment:- | genotype:- | Week:- "Subject Identifer Instrument Run Name (POS)"=MO_U_Pool_5.raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U_Pool_5_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS 383 383-4 Treatment:NaC | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_NaC_383 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_NaC_383 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 387 387-4 Treatment:NaC | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_NaC_387 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_NaC_387 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 391 391-4 Treatment:NaC | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_NaC_391 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_NaC_391 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 383 383-8 Treatment:NaC | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_NaC_383 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_NaC_383 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 387 387-8 Treatment:NaC | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_NaC_387 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_NaC_387 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 390 390-8 Treatment:NaC | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_NaC_390 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_NaC_390 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 391 391-8 Treatment:NaC | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_NaC_391 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_NaC_391 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 399 399-8 Treatment:NaC | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_NaC_399 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_NaC_399 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 519 519-4 Treatment:NaC | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_NaC_519 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_NaC_519 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 520 520-4 Treatment:NaC | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_NaC_520 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_NaC_520 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 521 521-4 Treatment:NaC | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_NaC_521 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_NaC_521 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 523 523-4 Treatment:NaC | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_NaC_523 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_NaC_523 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 519 519-8 Treatment:NaC | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_NaC_519 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_NaC_519 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 520 520-8 Treatment:NaC | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_NaC_520 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_NaC_520 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 521 521-8 Treatment:NaC | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_NaC_521 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_NaC_521 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 522 522-8 Treatment:NaC | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_NaC_522 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_NaC_522 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 523 523-8 Treatment:NaC | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_NaC_523 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_NaC_523 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 395 395-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_395 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_395 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 396 396-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_396 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_396 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 397 397-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_397 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_397 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 400 400-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_400 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_400 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 514 514-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_514 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_514 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 515 515-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_515 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_515 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 516 516-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_516 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_516 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 518 518-4 Treatment:STZ | genotype:Meprin bKO | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_KO_STZ_518 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_KO_STZ_518 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 395 395-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_395 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_395 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 396 396-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_396 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_396 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 400 400-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_400 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_400 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 515 515-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_515 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_515 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 516 516-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_516 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_516 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 518 518-8 Treatment:STZ | genotype:Meprin bKO | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_KO_STZ_518 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_KO_STZ_518 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 525 525-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_525 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_525 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 526 526-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_526 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_526 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 527 527-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_527 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_527 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 528 528-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_528 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_528 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 529 529-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_529 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_529 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 530 530-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_530 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_530 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 531 531-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_531 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_531 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 532 532-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_532 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_532 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 533 533-4 Treatment:STZ | genotype:wild-type | Week:4 "Subject Identifer Instrument Run Name (POS)"=MO_U4_WT_STZ_533 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U4_WT_STZ_533 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 525 525-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_525 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_525 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 526 526-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_526 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_526 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 528 528-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_528 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_528 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 529 529-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_529 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_529 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 530 530-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_530 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_530 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 531 531-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_531 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_531 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 532 532-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_532 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_532 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS 533 533-8 Treatment:STZ | genotype:wild-type | Week:8 "Subject Identifer Instrument Run Name (POS)"=MO_U8_WT_STZ_533 .raw; "Subject Identifier Instrument Run Name (NEG)"=MO_U8_WT_STZ_533 _NEG.raw; Gender=Male #COLLECTION CO:COLLECTION_SUMMARY Urine samples were collected at 4 and 8 weeks post-STZ injection. Urine samples CO:COLLECTION_SUMMARY were obtained by bladder massage unto sterile petri dishes and stored at -80oC CO:COLLECTION_SUMMARY until used. CO:SAMPLE_TYPE Urine CO:STORAGE_CONDITIONS -80 C #TREATMENT TR:TREATMENT_SUMMARY Type 1 diabetes was induced at age 8 weeks. The mice were fasted for 6 hours TR:TREATMENT_SUMMARY prior to being injected with low dose STZ (50 mg/kg body weight) in sodium TR:TREATMENT_SUMMARY citrate buffer (10 mmol/L, pH 4.5) daily for 5 days following the protocols TR:TREATMENT_SUMMARY described by Tesch and Allen and recommended by the Animal Models of Diabetic TR:TREATMENT_SUMMARY Complications Consortium (AMDCC; amdcc.org)1. Control mice were injected with TR:TREATMENT_SUMMARY equivalent volumes of the sodium citrate buffer. Diabetes was confirmed by TR:TREATMENT_SUMMARY measuring fasting blood glucose levels using a standard glucose meter at 10 days TR:TREATMENT_SUMMARY post-STZ injection. Mice with a fasting glucose level ≥280 mg/dL were TR:TREATMENT_SUMMARY considered diabetic. STZ-injected mice with a fasting blood glucose of <280 TR:TREATMENT_SUMMARY mg/dL (15mmol/L) were culled and eliminated from the study. TR:TREATMENT_COMPOUND streptozotocin TR:TREATMENT_DOSE 50 mg/kg body weight TR:TREATMENT_DOSEDURATION daily; 5 days TR:TREATMENT_VEHICLE sodium citrate TR:ANIMAL_ENDP_TISSUE_COLL_LIST plasma, urine, kidney tissue #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Urine samples were thawed on ice for 30–60 min and vortexed on a multi-tube SP:SAMPLEPREP_SUMMARY vortexer for 2 mins at 5,000 rpm, followed by centrifugation at room temperature SP:SAMPLEPREP_SUMMARY at 16,000 rcf for 4 min. An aliquot of the supernatant was transferred to SP:SAMPLEPREP_SUMMARY pre-labeled 1.5 mL low protein-binding microcentrifuge tube to make the SP:SAMPLEPREP_SUMMARY individual study sample (40 µL). Another aliquot of supernatant (11 µL) was SP:SAMPLEPREP_SUMMARY combined with those from all other urine samples in a 1.5 mL low protein-binding SP:SAMPLEPREP_SUMMARY microcentrifuge tube to make the total pool quality control sample. The total SP:SAMPLEPREP_SUMMARY pool quality control sample was aliquoted (40 µL) to make 5 Total Pool Samples SP:SAMPLEPREP_SUMMARY and 4 Column Equilibration samples. 132 µL of the total pool quality control SP:SAMPLEPREP_SUMMARY sample was added to the total study sample pool to make a pool of kidney, urine, SP:SAMPLEPREP_SUMMARY and plasma samples. The total study pool sample was used to prepare 9 Total SP:SAMPLEPREP_SUMMARY Study Samples (40 µL) to aid in alignment of the three studies. Of these, 3 SP:SAMPLEPREP_SUMMARY total study samples were included in the urine analysis. Acetonitrile containing SP:SAMPLEPREP_SUMMARY the internal standard Tryptophan-d5 (120 µL; 0.0167 mg/ml) was added to all SP:SAMPLEPREP_SUMMARY samples, and samples were vortexed on a multiple tube vortexer for 2 min at 5000 SP:SAMPLEPREP_SUMMARY rpm followed by centrifugation at 16,000 rcf for 4 min. The supernatants (420 SP:SAMPLEPREP_SUMMARY µL) were transferred into new, pre-labeled 2.0 mL low protein-binding SP:SAMPLEPREP_SUMMARY microcentrifuge tubes and capped with disposable rubber stoppers. Samples were SP:SAMPLEPREP_SUMMARY placed at -80 °C for 1.5 h and lyophilized for 18 h. Acetonitrile:Water (150 SP:SAMPLEPREP_SUMMARY µL; 95:5) was added to each tube and vortex mixed for 10 min at 5000 rpm, SP:SAMPLEPREP_SUMMARY followed by centrifugation at room temperature at 16,000 rcf for 4 min. The SP:SAMPLEPREP_SUMMARY supernatants were transferred to new, pre-labeled autosampler vials and 3 uL was SP:SAMPLEPREP_SUMMARY injected into SYNAPT G2-Si. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Hydrophilic Interaction Liquid Chromatography (HILIC) Gradient Seperation CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm,1.7um) CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #END