{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002145","ANALYSIS_ID":"AN003511","VERSION":"1","CREATED_ON":"April 19, 2022, 3:54 pm"},

"PROJECT":{"PROJECT_TITLE":"The Carbohydrate Sensing Transcription Factor ChREBP Links Mitochondrial Lipidomes to Mitochondrial Dynamics and Progression of Diabetic Nephropathy","PROJECT_TYPE":"Biomarker under ChREBP controlled lipidome","PROJECT_SUMMARY":"Despite recent advances, diabetic nephropathy (DN) remains a major public health concern. The precise underlying molecular mechanisms of DN remain elusive. Accumulating recent evidence suggests that mitochondrial integrity and lipid metabolism in podocytes significantly contribute to the pathogenesis of DN. However, the interplay between these two key metabolic regulators of DN is not fully understood. This study examines the role of ChREBP (carbohydrate-response element-binding protein), a master regulator of lipogenesis, on mitochondrial morphology and progression of DN. Our findings suggest that diabetic mice with podocyte-specific deletion of ChREBP are protected against mitochondrial fragmentation and progression of DN. Using liquid chromatography coupled with mass spectrometry, we identified the central role of ChREBP-induced plasmalogen phospholipids in linking mitochondrial lipidomes with mitochondrial dynamics in DN.","INSTITUTE":"University of Texas MD Anderson Cancer Center","DEPARTMENT":"Nephrology","LABORATORY":"Danesh Laboratory","LAST_NAME":"Danesh","FIRST_NAME":"Farhad","ADDRESS":"1515 Holcombe Blvd, Houston, TX77030","EMAIL":"fdanesh@mdanderson.org","PHONE":"7135634498","CONTRIBUTORS":"Iqbal Mahmud, Philip L. Lorenzi"},

"STUDY":{"STUDY_TITLE":"The Carbohydrate Sensing Transcription Factor ChREBP Links Mitochondrial Lipidomes to Mitochondrial Dynamics and Progression of Diabetic Nephropathy","STUDY_TYPE":"Biomarker","STUDY_SUMMARY":"Despite recent advances, diabetic nephropathy (DN) remains a major public health concern. The precise underlying molecular mechanisms of DN remain elusive. Accumulating recent evidence suggests that mitochondrial integrity and lipid metabolism in podocytes significantly contribute to the pathogenesis of DN. However, the interplay between these two key metabolic regulators of DN is not fully understood. This study examines the role of ChREBP (carbohydrate-response element-binding protein), a master regulator of lipogenesis, on mitochondrial morphology and progression of DN. Our findings suggest that diabetic mice with podocyte-specific deletion of ChREBP are protected against mitochondrial fragmentation and progression of DN. Using liquid chromatography coupled with mass spectrometry, we identified the central role of ChREBP-induced plasmalogen phospholipids in linking mitochondrial lipidomes with mitochondrial dynamics in DN.","INSTITUTE":"University of Texas MD Anderson Cancer Center","LAST_NAME":"Danesh","FIRST_NAME":"Farhad","ADDRESS":"1515 Holcombe Blvd, Houston ,TX77030","EMAIL":"fdanesh@mdanderson.org","PHONE":"7135634498","NUM_GROUPS":"3"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"shchrebp HG-1",
"Factors":{"Genotype":"ChREBP knockdown"},
"Additional sample data":{"Treatment":"shchrebp HG-1","RAW_FILE_NAME":"210716_shchrebpHG-1_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shchrebp HG-2",
"Factors":{"Genotype":"ChREBP knockdown"},
"Additional sample data":{"Treatment":"shchrebp HG-2","RAW_FILE_NAME":"210716_shchrebpHG-2_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shchrebp HG-3",
"Factors":{"Genotype":"ChREBP knockdown"},
"Additional sample data":{"Treatment":"shchrebp HG-3","RAW_FILE_NAME":"210716_shchrebpHG-3_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shchrebp HG-4",
"Factors":{"Genotype":"ChREBP knockdown"},
"Additional sample data":{"Treatment":"shchrebp HG-4","RAW_FILE_NAME":"210716_shchrebpHG-4_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl HG-1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl HG-1","RAW_FILE_NAME":"210716_shctrlHG-1_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl HG-2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl HG-2","RAW_FILE_NAME":"210716_shctrlHG-2_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl HG-3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl HG-3","RAW_FILE_NAME":"210716_shctrlHG-3_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl NG-1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl NG-1","RAW_FILE_NAME":"210716_shctrlNG-1_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl NG-2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl NG-2","RAW_FILE_NAME":"210716_shctrlNG-2_Pos_high.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"shctrl NG-3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Treatment":"shctrl NG-3","RAW_FILE_NAME":"210716_shctrlNG-3_Pos_high.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Conditionally immortalized mouse podocytes were cultured as previously reported.25, 35 Podocytes were treated with 20μM of DL-α-palmitin (Sigma-Aldrich), 1-O-Hexadecyl-rac-glycerol (16:0-AG, Santa Cruz) ,1-O-Octadecyl-rac-glycerol (18:0-AG, Sigma-Aldrich) or 0.05% ethanol (vehicle) for 48 hours. An engineered miR-30 based ChREBP knockdown (KD) construct was assembled by PCR using previously validated ChREBP shRNA clone,34 followed by an optimized miR-E backbone. Stable podocyte cell line with ChREBP KD were generated by transfection into constructs together with PiggyBac transposase. After selection with 0.5 μg/ml of blasticidin alone or in combination with 1μg/ml puromycin, GFP-positive cells were sorted by FACS, and the top 30% population were collected.","SAMPLE_TYPE":"Epithelial cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"An engineered miR-30 based ChREBP knockdown (KD) construct was assembled by PCR using previously validated ChREBP shRNA clone,34 followed by an optimized miR-E backbone. Stable podocyte cell line with ChREBP KD were generated by transfection into constructs together with PiggyBac transposase. After selection with 0.5 μg/ml of blasticidin alone or in combination with 1μg/ml puromycin, GFP-positive cells were sorted by FACS, and the top 30% population were collected."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Mitochondria were isolated from mouse podocytes using Mitochondria Isolation Kit (Thermo Fisher). Mitochondria were pelleted by centrifugation at 3000x g for 15 minutes at 4°C, flash-frozen and stored at -80°C until use. 200 μL of ethanol containing 1% 10 mM butylated hydroxytoluene in methanol, and 2% Avanti SPLASH® LIPIDOMIX® Mass Spec Standards, pre-cooled to -80°C, was added to each mitochondria sample. The tubes were vortexed for 10 min, placed on ice for 10 min, then centrifuged at 4°C for 10 min at 17,000 x g. The supernatants were then collected for LC-MS analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Mobile phase A (MPA) was 40:60 acetonitrile:0.1% formic acid in 10 mM ammonium formate. Mobile phase B (MPB) was 90:9:1 isopropanol: acetonitrile: 0.1% formic acid in 10 mM ammonium formate. The chromatographic method included a Thermo Fisher Scientific Accucore C30 column (2.6 μm, 150 x 2.1 mm) maintained at 40°C, a mobile phase flowrate of 0.200 mL/min, and a gradient elution program as follows: 0-3 min, 30% MPB; 3-13 min, 30-43% MPB; 13.1-33 min, 50-70% MPB; 33-48 min, 70-99% MPB; 48-55 min, 99% MPB; 55.1-60 min, 30% MPB.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Thermo Accucore C18 (100 2.1mm, 2.6um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Fusion Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"A Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer with heated electrospray ionization source was operated in data- dependent acquisition mode, in positive ionization mode, with scan ranges of 150-827 and 825-1500 m/z. An Orbitrap resolution of 120,000 (FWHM) was used for MS1 acquisition and spray voltages of 3.6kV and -2.9kV were used for positive and negative ionization modes, respectively. For MS2 and MS3 fragmentation a hybridized HCD/CID approach was used. Each sample was analyzed using 4 x 10 µL injections making use of the two scan ranges, in both ionization modes. Data were analyzed using Thermo Scientific LipidSearch software (version 5.0.63) and R scripts written in house. The intensity of each peak was normalized to total lipid signal and to the internal standard."},

"MS_METABOLITE_DATA":{
"Units":"AUC/ngDNA",

"Data":[{"Metabolite":"PE(P-42:10)_3","shchrebp HG-1":"49134.7179","shchrebp HG-2":"56218.7115","shchrebp HG-3":"114022.3628","shchrebp HG-4":"54064.7601","shctrl HG-1":"33557.9512","shctrl HG-2":"97217.375","shctrl HG-3":"138077.1483","shctrl NG-1":"90109.1176","shctrl NG-2":"113783.9239","shctrl NG-3":"40650.8408"},{"Metabolite":"PS(O-18:1)_5","shchrebp HG-1":"25466.3693","shchrebp HG-2":"51708.2383","shchrebp HG-3":"21496.5387","shchrebp HG-4":"36271.9367","shctrl HG-1":"48274.8363","shctrl HG-2":"41567.8464","shctrl HG-3":"20076.652","shctrl NG-1":"34342.4586","shctrl NG-2":"15455.3382","shctrl NG-3":"24518.4022"},{"Metabolite":"PE(O-23:4_18:0)_6","shchrebp HG-1":"7099386.749","shchrebp HG-2":"3370190.699","shchrebp HG-3":"6153477.383","shchrebp HG-4":"7046640.994","shctrl HG-1":"5006141.935","shctrl HG-2":"630987.35","shctrl HG-3":"2520570.221","shctrl NG-1":"6034636.876","shctrl NG-2":"1247375.412","shctrl NG-3":"6244186.215"},{"Metabolite":"PS(P-18:0)_7","shchrebp HG-1":"25466.3693","shchrebp HG-2":"51708.2383","shchrebp HG-3":"21496.5387","shchrebp HG-4":"36271.9367","shctrl HG-1":"48274.8363","shctrl HG-2":"41567.8464","shctrl HG-3":"20076.652","shctrl NG-1":"34342.4586","shctrl NG-2":"15455.3382","shctrl NG-3":"24518.4022"},{"Metabolite":"PE(P-23:3_18:0)_8","shchrebp HG-1":"7099386.749","shchrebp HG-2":"3370190.699","shchrebp HG-3":"6153477.383","shchrebp HG-4":"7046640.994","shctrl HG-1":"5006141.935","shctrl HG-2":"630987.35","shctrl HG-3":"2520570.221","shctrl NG-1":"6034636.876","shctrl NG-2":"1247375.412","shctrl NG-3":"6244186.215"},{"Metabolite":"PC(19:0_20:4)_9","shchrebp HG-1":"355794.8275","shchrebp HG-2":"714890.0357","shchrebp HG-3":"631675.1671","shchrebp HG-4":"873498.149","shctrl HG-1":"597362.1505","shctrl HG-2":"672617.7442","shctrl HG-3":"638918.832","shctrl NG-1":"636470.2187","shctrl NG-2":"492715.3933","shctrl NG-3":"647959.9564"},{"Metabolite":"PC(19:0_20:4)_10","shchrebp HG-1":"613003.7814","shchrebp HG-2":"657520.3009","shchrebp HG-3":"504783.4603","shchrebp HG-4":"392596.6378","shctrl HG-1":"610662.0439","shctrl HG-2":"604524.8053","shctrl HG-3":"520799.2605","shctrl NG-1":"605899.4751","shctrl NG-2":"312146.5119","shctrl NG-3":"418699.4563"},{"Metabolite":"PC(39:4)_11","shchrebp HG-1":"36770.4242","shchrebp HG-2":"28740.7494","shchrebp HG-3":"38145.7942","shchrebp HG-4":"78864.1628","shctrl HG-1":"34063.1043","shctrl HG-2":"38557.1305","shctrl HG-3":"30762.5408","shctrl NG-1":"45701.5984","shctrl NG-2":"40720.7698","shctrl NG-3":"97237.9424"},{"Metabolite":"PC(16:1_23:3)_12","shchrebp HG-1":"293427.3315","shchrebp HG-2":"197234.3181","shchrebp HG-3":"256268.5626","shchrebp HG-4":"222207.4488","shctrl HG-1":"126935.7476","shctrl HG-2":"417386.5006","shctrl HG-3":"391624.8144","shctrl NG-1":"334561.3242","shctrl NG-2":"340214.8273","shctrl NG-3":"264789.0623"},{"Metabolite":"PC(39:4)_13","shchrebp HG-1":"260940.8569","shchrebp HG-2":"133224.0636","shchrebp HG-3":"181055.5288","shchrebp HG-4":"237758.843","shctrl HG-1":"176661.9718","shctrl HG-2":"178201.1817","shctrl HG-3":"214849.7246","shctrl NG-1":"181804.1796","shctrl NG-2":"192925.2566","shctrl NG-3":"278164.8208"},{"Metabolite":"PE(O-42:10)_15","shchrebp HG-1":"60275.3946","shchrebp HG-2":"94941.2867","shchrebp HG-3":"157496.3165","shchrebp HG-4":"52891.6441","shctrl HG-1":"58543.1364","shctrl HG-2":"154926.1708","shctrl HG-3":"189638.1421","shctrl NG-1":"154677.8758","shctrl NG-2":"142447.0297","shctrl NG-3":"80139.9014"},{"Metabolite":"PE(P-20:3_22:6)_16","shchrebp HG-1":"60275.3946","shchrebp HG-2":"94941.2867","shchrebp HG-3":"157496.3165","shchrebp HG-4":"52891.6441","shctrl HG-1":"58543.1364","shctrl HG-2":"154926.1708","shctrl HG-3":"189638.1421","shctrl NG-1":"154677.8758","shctrl NG-2":"142447.0297","shctrl NG-3":"80139.9014"},{"Metabolite":"PE(O-23:3_18:0)_17","shchrebp HG-1":"702681.2412","shchrebp HG-2":"834600.8234","shchrebp HG-3":"350082.9365","shchrebp HG-4":"864708.2305","shctrl HG-1":"637700.2147","shctrl HG-2":"753701.3869","shctrl HG-3":"598639.1026","shctrl NG-1":"521097.5846","shctrl NG-2":"757607.6526","shctrl NG-3":"418635.7622"},{"Metabolite":"PG(P-18:2_23:1)_20","shchrebp HG-1":"85339.3375","shchrebp HG-2":"3248992.144","shchrebp HG-3":"2334921.722","shchrebp HG-4":"92142.0108","shctrl HG-1":"91399.6733","shctrl HG-2":"2874554.061","shctrl HG-3":"2621710.563","shctrl NG-1":"2863820.9","shctrl NG-2":"2153878.043","shctrl NG-3":"2335662.204"},{"Metabolite":"PE(P-23:2_18:0)_21","shchrebp HG-1":"702681.2412","shchrebp HG-2":"834600.8234","shchrebp HG-3":"350082.9365","shchrebp HG-4":"864708.2305","shctrl HG-1":"637700.2147","shctrl HG-2":"753701.3869","shctrl HG-3":"598639.1026","shctrl NG-1":"521097.5846","shctrl NG-2":"757607.6526","shctrl NG-3":"418635.7622"},{"Metabolite":"PE(P-23:2_18:0)_22","shchrebp HG-1":"1637136.023","shchrebp HG-2":"1632188.915","shchrebp HG-3":"1124592.041","shchrebp HG-4":"1507048.427","shctrl HG-1":"1407684.461","shctrl HG-2":"1257572.295","shctrl HG-3":"1197087.097","shctrl NG-1":"1111234.188","shctrl NG-2":"1140023.646","shctrl NG-3":"1253970.987"},{"Metabolite":"PE(P-23:2_18:0)_23","shchrebp HG-1":"1637136.023","shchrebp HG-2":"1707375.563","shchrebp HG-3":"1208192.453","shchrebp HG-4":"810016.3435","shctrl HG-1":"1490775.073","shctrl HG-2":"1257572.295","shctrl HG-3":"1242394.442","shctrl NG-1":"1191926.357","shctrl NG-2":"850050.1823","shctrl NG-3":"1279741.614"},{"Metabolite":"PA(18:2_16:1)_24","shchrebp HG-1":"37977.7034","shchrebp HG-2":"24826.1087","shchrebp HG-3":"30426.9215","shchrebp HG-4":"42510.2433","shctrl HG-1":"35715.4745","shctrl HG-2":"11175.7341","shctrl HG-3":"25545.2868","shctrl NG-1":"23196.8527","shctrl NG-2":"15307.9565","shctrl NG-3":"39818.996"},{"Metabolite":"PC(O-20:2_20:2)_25","shchrebp HG-1":"382803.8314","shchrebp HG-2":"449211.1927","shchrebp HG-3":"277715.3982","shchrebp HG-4":"337693.4547","shctrl HG-1":"463823.6668","shctrl 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