#METABOLOMICS WORKBENCH tcavallo_20170120_073935 DATATRACK_ID:825 STUDY_ID:ST000540 ANALYSIS_ID:AN000821 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Kidney tissue metabolomic profiling of diabetic nephropathy in the steptozotocin PR:PROJECT_TITLE induced 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; NIH Center Grant # U24DK097193; NIH/NCATS award # PR:FUNDING_SOURCE UL1TR001111; NIH/NIGMS Grant # K01GM109320 PR:DOI http://dx.doi.org/10.21228/M80G60 #STUDY ST:STUDY_TITLE Kidney tissue metabolomic profiling of diabetic nephropathy in the steptozotocin ST:STUDY_TITLE induced type-1 diabetes mouse model. ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY This metabolomics study evaluated kidney tissue from wild-type and meprin β ST:STUDY_SUMMARY knockout mice after induction of diabetes with streptozotocin or treatment with ST:STUDY_SUMMARY sodium citrate control to understand how these factors influence the metabotype. ST:INSTITUTE RTI International ST:DEPARTMENT Discovery-Science-Technology 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 2017-01-20 #SUBJECT SU:SUBJECT_TYPE mice SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:GENDER Male 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 Treatment:- | Genotype:- Instrument Run Name (POS)=MO_KT_Equil6_POS.raw; Instrument Run Name (NEG)=MO_KT_Equil6_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS - Total Pool_1 Treatment:- | Genotype:- Instrument Run Name (POS)=MO_KT_Pool_1_POS.raw; Instrument Run Name (NEG)=MO_KT_Pool_1_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS - Total Pool_2 Treatment:- | Genotype:- Instrument Run Name (POS)=MO_KT_Pool_2_POS.raw; Instrument Run Name (NEG)=MO_KT_Pool_2_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS - Total Pool_3 Treatment:- | Genotype:- Instrument Run Name (POS)=MO_KT_Pool_3_POS.raw; Instrument Run Name (NEG)=MO_KT_Pool_3_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS - Total Pool_4 Treatment:- | Genotype:- Instrument Run Name (POS)=MO_KT_Pool_4_POS_rerun.raw; Instrument Run Name (NEG)=MO_KT_Pool_4_NEG.raw; Gender=- SUBJECT_SAMPLE_FACTORS - 383 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_383 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_NaC_383 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 387 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_387 _POS.raw; Instrument Run Name (NEG)=-; Gender=Male SUBJECT_SAMPLE_FACTORS - 390 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_390 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_NaC_390 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 391 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_391 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_NaC_391 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 398 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_398 _POS.raw; Instrument Run Name (NEG)=-; Gender=Male SUBJECT_SAMPLE_FACTORS - 399 Treatment:NaC | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_NaC_399 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_NaC_399 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 519 Treatment:NaC | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_NaC_519 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_NaC_519 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 520 Treatment:NaC | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_NaC_520 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_NaC_520 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 521 Treatment:NaC | Genotype:wild-type Instrument Run Name (POS)=-; Instrument Run Name (NEG)=MO_KT_WT_NaC_521 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 522 Treatment:NaC | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_NaC_522 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_NaC_522 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 523 Treatment:NaC | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_NaC_523 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_NaC_523 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 395 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_395 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_395 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 396 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_396 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_396 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 397 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_397 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_397 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 400 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_400 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_400 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 514 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_514 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_514 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 515 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_515 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_515 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 516 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_516 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_516 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 518 Treatment:STZ | Genotype:Meprin bKO Instrument Run Name (POS)=MO_KT_KO_STZ_518 _POS.raw; Instrument Run Name (NEG)=MO_KT_KO_STZ_518 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 525 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_525 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_525 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 526 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_526 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_526 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 528 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_528 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_528 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 529 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_529 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_529 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 530 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_530 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_530 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 531 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_531 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_531 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 532 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_532 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_532 _NEG.raw; Gender=Male SUBJECT_SAMPLE_FACTORS - 533 Treatment:STZ | Genotype:wild-type Instrument Run Name (POS)=MO_KT_WT_STZ_533 _POS.raw; Instrument Run Name (NEG)=MO_KT_WT_STZ_533 _NEG.raw; Gender=Male #COLLECTION CO:COLLECTION_SUMMARY Kidneys were harvested at 8-weeks post STZ-injection. The mice were sacrificed CO:COLLECTION_SUMMARY by CO2 asphyxiation. The kidneys were excised, decapsulated and individually CO:COLLECTION_SUMMARY weighed, snap-frozen in liquid nitrogen, and stored at -80oC until used. CO:SAMPLE_TYPE Kidney Tissue CO:STORAGE_CONDITIONS -80C #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 Frozen kidney tissue samples on dry ice were transferred to pre-chilled, SP:SAMPLEPREP_SUMMARY pre-labeled tubes, and their weights recorded. For every 1 mg of tissue sample, SP:SAMPLEPREP_SUMMARY 2 µL of 50:50 Acetonitrile:Water was added to the tube. Ceramic beads (2.3 mm; SP:SAMPLEPREP_SUMMARY ~15-20 prewashed & dried) were added to the tubes, and the samples were SP:SAMPLEPREP_SUMMARY homogenized on the MagNA Lyser system using a 30 s pulse at 4,000 rpm. The SP:SAMPLEPREP_SUMMARY samples were centrifuged at room temperature at 16,000 rcf for 4 min. An aliquot SP:SAMPLEPREP_SUMMARY of the supernatant was transferred to pre-labeled 2.0 mL low protein-binding SP:SAMPLEPREP_SUMMARY microcentrifuge tube to make the individual study sample (40 µL). Another SP:SAMPLEPREP_SUMMARY aliquot of supernatant (18 µL) was combined with those from all other kidney SP:SAMPLEPREP_SUMMARY tissue samples in a 1.5 mL low protein-binding microcentrifuge tube to make the SP:SAMPLEPREP_SUMMARY total pool quality control sample. The total pool quality control sample was SP:SAMPLEPREP_SUMMARY aliquoted (40 µL) to make 4 Total Pool Samples and 4 Column Equilibration SP:SAMPLEPREP_SUMMARY samples. 132 µL of the total pool quality control sample was added to the total SP:SAMPLEPREP_SUMMARY study sample pool to make a pool of kidney, urine, and plasma samples. The total SP:SAMPLEPREP_SUMMARY study pool sample was used to prepare 9 Total Study Samples (40 µL) to aid in SP:SAMPLEPREP_SUMMARY alignment of the three studies. Of these, 3 total study samples were included in SP:SAMPLEPREP_SUMMARY the kidney tissue analysis. Acetonitrile containing the internal standard SP:SAMPLEPREP_SUMMARY Tryptophan-d5 (460 µL; 0.0125 mg/ml) was added to all samples, and samples were SP:SAMPLEPREP_SUMMARY vortexed on a multiple tube vortexer for 2 min at 5000 rpm followed by SP:SAMPLEPREP_SUMMARY centrifugation at 16,000 rcf for 4 min. The supernatants (420 µL) were SP:SAMPLEPREP_SUMMARY transferred into new, pre-labeled 2.0 mL low protein-binding microcentrifuge SP:SAMPLEPREP_SUMMARY tubes and capped with disposable rubber stoppers. Samples were placed at -80 °C SP:SAMPLEPREP_SUMMARY for 1.5 h and lyophilized for 18 h. Acetonitrile:Water (150 µL; 95:5) was added SP:SAMPLEPREP_SUMMARY to each tube and vortex mixed for 10 min at 5000 rpm, followed by centrifugation SP:SAMPLEPREP_SUMMARY at room temperature at 16,000 rcf for 4 min. The supernatants were transferred SP:SAMPLEPREP_SUMMARY to pre-labeled autosampler vials and 3 µL was 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