Summary of Study ST002145

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001359. The data can be accessed directly via it's Project DOI: 10.21228/M8270X This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002145
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
Submit Date	2022-03-23
Num Groups	3
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-05-02
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001359
Project DOI:	doi: 10.21228/M8270X
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

Subject:

Subject ID:	SU002230
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype
SA205778	shchrebp HG-1	ChREBP knockdown
SA205779	shchrebp HG-4	ChREBP knockdown
SA205780	shchrebp HG-2	ChREBP knockdown
SA205781	shchrebp HG-3	ChREBP knockdown
SA205782	shctrl NG-3	Control
SA205783	shctrl NG-2	Control
SA205784	shctrl HG-2	Control
SA205785	shctrl HG-1	Control
SA205786	shctrl HG-3	Control
SA205787	shctrl NG-1	Control

Showing results 1 to 10 of 10

Showing results 1 to 10 of 10

Collection:

Collection ID:	CO002223
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 ID:	TR002242
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.

Treatment ID:

TR002242

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.

Sample Preparation:

Sampleprep ID:	SP002236
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.

Sampleprep ID:

SP002236

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.

Combined analysis:

Analysis ID	AN003511	AN003512
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Thermo Accucore C18 (100 x 2.1mm,2.6um)	Thermo Accucore C18 (100 x 2.1mm,2.6um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Fusion Orbitrap	Thermo Fusion Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	AUC/ngDNA	AUC/ngDNA

Chromatography:

Chromatography ID:	CH002592
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.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Accucore C18 (100 x 2.1mm,2.6um)
Column Temperature:	40
Flow Gradient:	0-3 min, 30% B; 3-13 min, 30-43% B; 13.1-33 min, 50-70% B; 33-48 min, 70-99% B; 48-55 min, 99% B; 55.1-60 min, 30% B
Flow Rate:	0.200 mL/min
Solvent A:	40% acetonitrile/60% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/9% acetonitrile/1% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003269
Analysis ID:	AN003511
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
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.
Ion Mode:	POSITIVE

MS ID:	MS003270
Analysis ID:	AN003512
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
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 negative 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.
Ion Mode:	NEGATIVE