{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000878","ANALYSIS_ID":"AN001428","VERSION":"1","CREATED_ON":"October 24, 2017, 3:25 pm"},

"PROJECT":{"PROJECT_TITLE":"Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease","PROJECT_SUMMARY":"Autosomal Dominant Polycystic Kidney Disease (ADP.D  MIM ID's 173900, 601313, 613095) is estimated to affect almost 1/1000 and is the most common genetic cause of end stage renal disease (Torres et al., 2007). While advances have been made in slowing the progression of some other forms of chronic kidney disease, standard treatments have not reduced the need for renal replacement therapy in ADPKD (Spithoven et al., 2014). Unfortunately, several experimental interventions also have recently failed to show significant benefit in slowing the rate of functional decline (Serra et al., 2010; Walz et al., 2010; Schrier et al., 2014; Torres et al., 2014), and the only positive study reported very modest effects (Torres et al., 2012). These findings suggest new treatment strategies are required. A central dogma of molecular genetics is that discovery of the causative genes will lead to identification of key pathways and potential targets for intervention. In the case of ADPKD, the two genes mutated in the disorder, PKD1 and PKD2, were identified almost 20 years ago and yet their functions remain poorly understood. The PKD1 gene product, polycystin-1 (PC1), encodes a large membrane protein that requires the PKD2 gene product, polycystin-2 (PC2), for its trafficking to the primary cilium where the two are thought to form a receptor channel complex (Kim et al., 2014; Cai et al., 2014). What the complex senses and what it signals remains controversial. The primary cilium has emerged as a key player in the pathogenesis of PKD as mutations in dozens of different genes that encode either essential ciliary components or factors in ciliary signaling pathways result in PKD. A recent report suggests that the relationship between the polycystin complex and ciliary signaling is complicated, however.While ablation of primary cilia by mutation of core ciliary components results in cysts, these same perturbations done in the setting of Pkd1 or Pkd2 inactivation results in significant attenuation of cystic disease (Ma et al., 2013). These data suggest that the polycystin complex provides a suppressive signal for a novel, cilia-dependent growth-promoting pathway that is independent of MAPK/ERK, mTOR, or cAMP pathways, three effector pathways previously implicated as major drivers of cyst growth. The identities of the growth-promoting and growth-inhibiting pathways remain unknown. We have taken a systems-based approach to study Pkd1 gene function. Building on our previous work identifying markedly different outcomes in animals with induced Pkd1 inactivation before or after P12 and correlating this susceptibility with metabolic status (Piontek et al., 2007; Menezes et al., 2012), we now show that female sex is partially protective in adult-induced Pkd1 inactivation, that sex differences in metabolic status may account for this effect, and that cells lacking Pkd1 have abnormal fatty acid oxidation. Finally, manipulating diet in Pkd1 mouse models, we demonstrate a positive correlation between lipid content in mouse chow and cystic kidney disease severity. Our results therefore suggest that abnormal lipid metabolism is an intrinsic component of PKD and an important modifier of disease progression.","INSTITUTE":"National Institute of Diabetes and Digestive and Kidney Diseases","LABORATORY":"Polycystic Kidney Disease Laboratory","LAST_NAME":"Menezes","FIRST_NAME":"Luis","ADDRESS":"31 Center Dr, Bethesda, MD 20892","EMAIL":"menezeslf@mail.nih.gov","PHONE":"301-451-9614"},

"STUDY":{"STUDY_TITLE":"Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease","STUDY_SUMMARY":"Autosomal Dominant Polycystic Kidney Disease (ADP.D  MIM ID's 173900, 601313, 613095) is estimated to affect almost 1/1000 and is the most common genetic cause of end stage renal disease (Torres et al., 2007). While advances have been made in slowing the progression of some other forms of chronic kidney disease, standard treatments have not reduced the need for renal replacement therapy in ADPKD (Spithoven et al., 2014). Unfortunately, several experimental interventions also have recently failed to show significant benefit in slowing the rate of functional decline (Serra et al., 2010; Walz et al., 2010; Schrier et al., 2014; Torres et al., 2014), and the only positive study reported very modest effects (Torres et al., 2012). These findings suggest new treatment strategies are required. A central dogma of molecular genetics is that discovery of the causative genes will lead to identification of key pathways and potential targets for intervention. In the case of ADPKD, the two genes mutated in the disorder, PKD1 and PKD2, were identified almost 20 years ago and yet their functions remain poorly understood. The PKD1 gene product, polycystin-1 (PC1), encodes a large membrane protein that requires the PKD2 gene product, polycystin-2 (PC2), for its trafficking to the primary cilium where the two are thought to form a receptor channel complex (Kim et al., 2014; Cai et al., 2014). What the complex senses and what it signals remains controversial. The primary cilium has emerged as a key player in the pathogenesis of PKD as mutations in dozens of different genes that encode either essential ciliary components or factors in ciliary signaling pathways result in PKD. A recent report suggests that the relationship between the polycystin complex and ciliary signaling is complicated, however.While ablation of primary cilia by mutation of core ciliary components results in cysts, these same perturbations done in the setting of Pkd1 or Pkd2 inactivation results in significant attenuation of cystic disease (Ma et al., 2013). These data suggest that the polycystin complex provides a suppressive signal for a novel, cilia-dependent growth-promoting pathway that is independent of MAPK/ERK, mTOR, or cAMP pathways, three effector pathways previously implicated as major drivers of cyst growth. The identities of the growth-promoting and growth-inhibiting pathways remain unknown. We have taken a systems-based approach to study Pkd1 gene function. Building on our previous work identifying markedly different outcomes in animals with induced Pkd1 inactivation before or after P12 and correlating this susceptibility with metabolic status (Piontek et al., 2007; Menezes et al., 2012), we now show that female sex is partially protective in adult-induced Pkd1 inactivation, that sex differences in metabolic status may account for this effect, and that cells lacking Pkd1 have abnormal fatty acid oxidation. Finally, manipulating diet in Pkd1 mouse models, we demonstrate a positive correlation between lipid content in mouse chow and cystic kidney disease severity. Our results therefore suggest that abnormal lipid metabolism is an intrinsic component of PKD and an important modifier of disease progression.","INSTITUTE":"University of California, Davis","DEPARTMENT":"Genome and Biomedical Sciences Facility","LABORATORY":"WCMC Metabolomics Core","LAST_NAME":"Fiehn","FIRST_NAME":"Oliver","ADDRESS":"1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616","EMAIL":"ofiehn@ucdavis.edu","PHONE":"(530) 754-8258"},

"SUBJECT":{"SUBJECT_TYPE":"Animal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"Fifth-generation C57/BL6 Pkd1tm2Ggg (Piontek et al., 2004) mice were crossed to the reporter mice C57/BL6 congenic B6.129S4-Gt(ROSA)26Sortm1Sor/J (stock 003474, Jackson Laboratories) and to C57/BL6 tamoxifen-Cre (B6.Cg-Tg(Cre/Esr1)5Amc/J mice (stock 004682), Jackson Laboratories) or Ksp-Cre B6.Cg-Tg(Cdh16-cre)91Igr/J(stock 012237), Jackson Laboratories).","GENDER":"Males","ANIMAL_ANIMAL_SUPPLIER":"Jackson Laboratories"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"83033_003",
"Sample ID":"141215bctsa20_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"101241_004",
"Sample ID":"141215bctsa21_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"95833_008",
"Sample ID":"141215bctsa22_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"83031_001",
"Sample ID":"141215bctsa24_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"95834_009",
"Sample ID":"141215bctsa27_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"101243_005",
"Sample ID":"141215bctsa28_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"83032_002",
"Sample ID":"141215bctsa30_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"95868_007",
"Sample ID":"141215bctsa33_1",
"Factors":{"Treatment*":"mutant"}
},
{
"Subject ID":"95868_015",
"Sample ID":"141215bctsa19_1",
"Factors":{"Treatment*":"control"}
},
{
"Subject ID":"83073_012",
"Sample ID":"141215bctsa23_1",
"Factors":{"Treatment*":"control"}
},
{
"Subject ID":"101242_013",
"Sample ID":"141215bctsa25_1",
"Factors":{"Treatment*":"control"}
},
{
"Subject ID":"450_011",
"Sample ID":"141215bctsa26_1",
"Factors":{"Treatment*":"control"}
},
{
"Subject ID":"449_010",
"Sample ID":"141215bctsa29_1",
"Factors":{"Treatment*":"control"}
},
{
"Subject ID":"103431_014",
"Sample ID":"141215bctsa31_1",
"Factors":{"Treatment*":"control"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Kidneys from two Pkd1cko/cko animals (121112-C: male, bP12; and 94414: male, P463) were harvested, minced and digested using a collagenase/hyaluronidase solution (Stemcell technologies, cat. no. 07912) followed by proximal or collecting/distal tubule cell enrichment using, respectively, biotinylated Lotus tetragonolobus Lectin (LTL) (Vector Laboratories, cat. no. B-1325) or biotinylated Dolichos biflorus Agglutinin (DBA) (Vector Laboratories, ca. no. B-1035) and Cellection Biotin Binder kit (ThermoFischer, cat. no. 11533D). Cells were immortalized using the large T antigen (Addgene plasmid no. 22298). Pkd1 was conditionally inactivated using cre recombinase (121112C-LTL cells; Excellgen, cat. no. EG-1001) or viral transduction (121112-C DBA and 94414-LTL/DBA cells) using LV-Cre (Addgene plasmid no. 12106). At the time of inactivation, a corresponding control was generated using viral transductionwith plasmid LV-Lac (Addgene plasmid no. 12108). Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1). mCCDcl1 (mCCD) cells were a kind gift from the Rossier lab (Gaeggeler et al., 2005). mCCD Pkd1 knock-down cells were generated using viral transduction with the shRNA clone TRCN0000072085 and the corresponding pLKO.1 TRC21 control (Addgene plasmid 10,879). Cells were grown in DMEM/F12 media (Life cat. no. 21041–025) with 2% FBS (GEMINI Bio-Products cat. no. 100–106), 1X Insulin-Transferrin-Selenium (Thermo Fisher Scientific, cat. no. 41400–045), 5 uM dexamethasone (SIGMA, cat. no. D1756), 10 ng/ml EGF (SIGMA, cat. no. SRP3196), 1 nM 3,3′,5-Triiodo-L-thyronine (SIGMA, cat. no. T6397) and 10 mM HEPES (CORNING, cat. no. 25-060-CI). Mouse embryonic fibroblasts (MEFs) were obtained from E12.5 and E13.5 Pkd1 knockout (Pkd1tm1GGG (Bhunia et al., 2002)) and control mice. Briefly, whole embryos were minced, washed in PBS and cultured in six-well tissue culture plate in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). A total of 6 MEF lines was used for this study: three from E13.5 mouse littermates (2 Pkd1ko/ko and 1 Pkd1wt/wt) immortalized using large T antigen (Addgene plasmid no. 22,298) and an additional set of three primary (passage 2, non-immortalized) E12.5 embryos (1 Pkd1ko/ko and 2 Pkd1wt/wt littermate controls).","SAMPLE_TYPE":"Tissue"},

"TREATMENT":{"TREATMENT_SUMMARY":"Metabolomics analysis of 14 male kidneys, 8 ADPKD mutants and 6 controls."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"1. Weigh 50 mg tissue sample in to a 25 ml conical polypropylene centrifuge tube. 2. Add 2.5mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent. 3. Centrifuge the samples at 2500 rpm. for 5 minutes. Aliquot 2 X 500μl supernatant, one for analysis and one for a backup sample. Store backup aliquot in the -20°C freezer. 4. Evaporate one 500μl aliquot of the sample in the Labconco Centrivap cold trap concentrator to complete dryness 5. The dried aliquot is then re-suspended with 500l 50% acetonitrile (degassed as given) 6. Centrifuge for 2 min at 14000 rcf using the centrifuge Eppendorf 5415. 7. Remove supernatant to a new Eppendorff tube. 8. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. 9. Submit to derivatization.","SAMPLEPREP_PROTOCOL_FILENAME":"SP_SOP_Extraction_of_Mammalian_Tissue.pdf"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 6890N","COLUMN_NAME":"Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um) 0.25 μm film made of 95% dimethyl/5%diphenylpolysiloxane)","FLOW_RATE":"1 ml/min","COLUMN_TEMPERATURE":"50 - 330°C","COLUMN_PRESSURE":"7.7 PSI (initial condition)","INJECTION_TEMPERATURE":"50°C ramped to 250°C by 12°C/s","SAMPLE_INJECTION":"0.5 uL","OVEN_TEMPERATURE":"50°C for 1 min, then ramped at 20°C/min to 330°C, held constant for 5 min","TRANSFERLINE_TEMPERATURE":"230°C","WASHING_BUFFER":"Ethyl Acetate","SAMPLE_LOOP_SIZE":"30 m length x 0.25 mm internal diameter","RANDOMIZATION_ORDER":"Excel generated"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","DETECTOR_TYPE":"TOF","SOFTWARE_VERSION":"Chromatof","DATA_FORMAT":".netcdf"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Leco Pegasus III GC TOF","INSTRUMENT_TYPE":"GC-TOF","MS_TYPE":"EI","ION_MODE":"POSITIVE","ION_SOURCE_TEMPERATURE":"250°C","IONIZATION_ENERGY":"70eV","MASS_ACCURACY":"Nominal","SCAN_RANGE_MOVERZ":"85-500","SCANNING_CYCLE":"17 Hz","SCANNING_RANGE":"80-500 Da","SKIMMER_VOLTAGE":"1850","MS_RESULTS_FILE":"ST000878_AN001428_Results.txt UNITS:Counts"},

"MS_METABOLITE_DATA":{
"Units":"Counts",

"Data":[{"Metabolite":"xylulose NIST","141215bctsa20_1":"856","141215bctsa21_1":"154","141215bctsa22_1":"280","141215bctsa24_1":"401","141215bctsa27_1":"351","141215bctsa28_1":"168","141215bctsa30_1":"882","141215bctsa33_1":"158","141215bctsa19_1":"173","141215bctsa23_1":"162","141215bctsa25_1":"386","141215bctsa26_1":"247","141215bctsa29_1":"226","141215bctsa31_1":"280"},{"Metabolite":"xylose","141215bctsa20_1":"4674","141215bctsa21_1":"1656","141215bctsa22_1":"1076","141215bctsa24_1":"3531","141215bctsa27_1":"1325","141215bctsa28_1":"2398","141215bctsa30_1":"6069","141215bctsa33_1":"1155","141215bctsa19_1":"1227","141215bctsa23_1":"1285","141215bctsa25_1":"1562","141215bctsa26_1":"1095","141215bctsa29_1":"1094","141215bctsa31_1":"1241"},{"Metabolite":"xylitol","141215bctsa20_1":"3345","141215bctsa21_1":"3149","141215bctsa22_1":"8868","141215bctsa24_1":"5521","141215bctsa27_1":"7919","141215bctsa28_1":"5153","141215bctsa30_1":"4828","141215bctsa33_1":"9426","141215bctsa19_1":"7068","141215bctsa23_1":"3143","141215bctsa25_1":"11743","141215bctsa26_1":"12699","141215bctsa29_1":"7941","141215bctsa31_1":"9430"},{"Metabolite":"xanthurenic acid","141215bctsa20_1":"1296","141215bctsa21_1":"191","141215bctsa22_1":"57","141215bctsa24_1":"899","141215bctsa27_1":"66","141215bctsa28_1":"222","141215bctsa30_1":"1210","141215bctsa33_1":"56","141215bctsa19_1":"66","141215bctsa23_1":"91","141215bctsa25_1":"37","141215bctsa26_1":"62","141215bctsa29_1":"43","141215bctsa31_1":"80"},{"Metabolite":"xanthine","141215bctsa20_1":"3068","141215bctsa21_1":"2323","141215bctsa22_1":"5179","141215bctsa24_1":"3260","141215bctsa27_1":"5578","141215bctsa28_1":"3994","141215bctsa30_1":"2726","141215bctsa33_1":"5342","141215bctsa19_1":"4438","141215bctsa23_1":"2986","141215bctsa25_1":"6582","141215bctsa26_1":"4983","141215bctsa29_1":"6537","141215bctsa31_1":"6004"},{"Metabolite":"valine","141215bctsa20_1":"89924","141215bctsa21_1":"35413","141215bctsa22_1":"48648","141215bctsa24_1":"73167","141215bctsa27_1":"54944","141215bctsa28_1":"40710","141215bctsa30_1":"76800","141215bctsa33_1":"47161","141215bctsa19_1":"52971","141215bctsa23_1":"39657","141215bctsa25_1":"62077","141215bctsa26_1":"62471","141215bctsa29_1":"62170","141215bctsa31_1":"55058"},{"Metabolite":"uridine-5'-monophosphate","141215bctsa20_1":"2028","141215bctsa21_1":"684","141215bctsa22_1":"131","141215bctsa24_1":"962","141215bctsa27_1":"227","141215bctsa28_1":"456","141215bctsa30_1":"1056","141215bctsa33_1":"229","141215bctsa19_1":"177","141215bctsa23_1":"656","141215bctsa25_1":"133","141215bctsa26_1":"491","141215bctsa29_1":"236","141215bctsa31_1":"183"},{"Metabolite":"uridine","141215bctsa20_1":"3329","141215bctsa21_1":"3135","141215bctsa22_1":"8177","141215bctsa24_1":"4490","141215bctsa27_1":"6274","141215bctsa28_1":"5088","141215bctsa30_1":"3144","141215bctsa33_1":"5110","141215bctsa19_1":"3087","141215bctsa23_1":"4210","141215bctsa25_1":"7488","141215bctsa26_1":"5515","141215bctsa29_1":"3294","141215bctsa31_1":"4150"},{"Metabolite":"uric acid","141215bctsa20_1":"2177","141215bctsa21_1":"1113","141215bctsa22_1":"817","141215bctsa24_1":"2416","141215bctsa27_1":"889","141215bctsa28_1":"1148","141215bctsa30_1":"2251","141215bctsa33_1":"622","141215bctsa19_1":"673","141215bctsa23_1":"689","141215bctsa25_1":"763","141215bctsa26_1":"505","141215bctsa29_1":"651","141215bctsa31_1":"916"},{"Metabolite":"urea","141215bctsa20_1":"401966","141215bctsa21_1":"596235","141215bctsa22_1":"288535","141215bctsa24_1":"686152","141215bctsa27_1":"360015","141215bctsa28_1":"440771","141215bctsa30_1":"713488","141215bctsa33_1":"207091","141215bctsa19_1":"250247","141215bctsa23_1":"296645","141215bctsa25_1":"272596","141215bctsa26_1":"237995","141215bctsa29_1":"264314","141215bctsa31_1":"267974"},{"Metabolite":"uracil","141215bctsa20_1":"8228","141215bctsa21_1":"7802","141215bctsa22_1":"11657","141215bctsa24_1":"7811","141215bctsa27_1":"11746","141215bctsa28_1":"9478","141215bctsa30_1":"7755","141215bctsa33_1":"10825","141215bctsa19_1":"8019","141215bctsa23_1":"9425","141215bctsa25_1":"11074","141215bctsa26_1":"10093","141215bctsa29_1":"13595","141215bctsa31_1":"9758"},{"Metabolite":"UDP-N-acetylglucosamine","141215bctsa20_1":"959","141215bctsa21_1":"461","141215bctsa22_1":"680","141215bctsa24_1":"811","141215bctsa27_1":"609","141215bctsa28_1":"939","141215bctsa30_1":"809","141215bctsa33_1":"637","141215bctsa19_1":"668","141215bctsa23_1":"1008","141215bctsa25_1":"622","141215bctsa26_1":"698","141215bctsa29_1":"409","141215bctsa31_1":"918"},{"Metabolite":"UDP-glucuronic acid","141215bctsa20_1":"1239","141215bctsa21_1":"3732","141215bctsa22_1":"2153","141215bctsa24_1":"1694","141215bctsa27_1":"1817","141215bctsa28_1":"2666","141215bctsa30_1":"1727","141215bctsa33_1":"2421","141215bctsa19_1":"5050","141215bctsa23_1":"2286","141215bctsa25_1":"2493","141215bctsa26_1":"2318","141215bctsa29_1":"2671","141215bctsa31_1":"2038"},{"Metabolite":"tyrosine","141215bctsa20_1":"77017","141215bctsa21_1":"19497","141215bctsa22_1":"34848","141215bctsa24_1":"52838","141215bctsa27_1":"37415","141215bctsa28_1":"34002","141215bctsa30_1":"73809","141215bctsa33_1":"36874","141215bctsa19_1":"35984","141215bctsa23_1":"28394","141215bctsa25_1":"44524","141215bctsa26_1":"44910","141215bctsa29_1":"51021","141215bctsa31_1":"40523"},{"Metabolite":"tryptophan","141215bctsa20_1":"22326","141215bctsa21_1":"5300","141215bctsa22_1":"7248","141215bctsa24_1":"12040","141215bctsa27_1":"9640","141215bctsa28_1":"7887","141215bctsa30_1":"22350","141215bctsa33_1":"9105","141215bctsa19_1":"7749","141215bctsa23_1":"7012","141215bctsa25_1":"9666","141215bctsa26_1":"9218","141215bctsa29_1":"10293","141215bctsa31_1":"9187"},{"Metabolite":"tocopherol 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