#METABOLOMICS WORKBENCH bendovi_20230131_091306 DATATRACK_ID:3717 STUDY_ID:ST002458 ANALYSIS_ID:AN004010 PROJECT_ID:PR001585
VERSION             	1
CREATED_ON             	January 31, 2023, 11:25 am
#PROJECT
PR:PROJECT_TITLE                 	Mouse kidney metabolomic studies
PR:PROJECT_TYPE                  	MS quantitative analysis
PR:PROJECT_SUMMARY               	Chronic kidney disease secondary to cystic kidney disease is a leading cause of
PR:PROJECT_SUMMARY               	mortality in patients with tuberous sclerosis complex (TSC) disease. The
PR:PROJECT_SUMMARY               	mechanisms underlying TSC cystic kidney disease are obscure, with no
PR:PROJECT_SUMMARY               	interventions available to prevent cyst formation. Here, we reveal for the first
PR:PROJECT_SUMMARY               	time the misregulated metabolic pathways in TSC kidneys and their relevance to
PR:PROJECT_SUMMARY               	TSC-associated cytogenesis. To this end, we have analyzed the metabolic profile
PR:PROJECT_SUMMARY               	of the whole kidney as well as sorted proximal tubule cell (PTCs) extracts. The
PR:PROJECT_SUMMARY               	metabolomics data show that Tsc1 deletion in nephron progenitor cells changes
PR:PROJECT_SUMMARY               	the arginine biosynthesis pathway as well as a substantial number of metabolic
PR:PROJECT_SUMMARY               	pathways. These changes were associated with overexpression of argininosuccinate
PR:PROJECT_SUMMARY               	synthetase 1 (ASS1), a rate-limiting enzyme in the arginine biosynthetic pathway
PR:PROJECT_SUMMARY               	in TSC KO mice and human kidneys. The rise in ASS1 expression is dependent on
PR:PROJECT_SUMMARY               	mTORC1 activity. Arginine depletion in vitro and in vivo prevented the rise in
PR:PROJECT_SUMMARY               	mTORC1 activity and cell cycle progression and averted the overexpression of
PR:PROJECT_SUMMARY               	previously described cytogenetic signaling proteins, including c-Myc and P65.
PR:PROJECT_SUMMARY               	Accordingly, an arginine-depleted diet substantially reduced the TSC cystic
PR:PROJECT_SUMMARY               	load, indicating the potential therapeutic effects of arginine deprivation as a
PR:PROJECT_SUMMARY               	treatment of TSC-associated kidney disease.
PR:INSTITUTE                     	Hadassah Medical Center
PR:DEPARTMENT                    	Pediatric Nephrology
PR:LABORATORY                    	Wohl Institute for Translational Medicine
PR:LAST_NAME                     	Ben-Dov
PR:FIRST_NAME                    	Iddo
PR:ADDRESS                       	Ein Karem Campus, Jerusalem, NA, 9211001, Israel
PR:EMAIL                         	iddo@hadassah.org.il
PR:PHONE                         	+97226776881
PR:FUNDING_SOURCE                	This work was supported by grants from the Israel Science Foundation (MN 2030/21
PR:FUNDING_SOURCE                	and OV 2358/18), the TSC alliance research grant (OV), the research authority of
PR:FUNDING_SOURCE                	Hadassah Hebrew University Medical Center (OV). OV and MN are Wohl’s
PR:FUNDING_SOURCE                	Translation Research Institute research associates at Hadassah-Hebrew University
PR:FUNDING_SOURCE                	Medical Center
PR:CONTRIBUTORS                  	Athar Amleh, Lana Watad, Ifat Abramovich, Bella Agranovich, Eyal Gottlieb, Iddo
PR:CONTRIBUTORS                  	Z. Ben-Dov, Morris Nechama and Oded Volovelsky
#STUDY
ST:STUDY_TITLE                   	Mouse kidney metabolomics (Proximal tubular cells)
ST:STUDY_SUMMARY                 	Here, we reveal for the first time the misregulated metabolic pathways in TSC
ST:STUDY_SUMMARY                 	kidneys and their relevance to TSC-associated cytogenesis. To this end, we have
ST:STUDY_SUMMARY                 	analyzed the metabolic profile of the whole kidney as well as sorted proximal
ST:STUDY_SUMMARY                 	tubule cell (PTCs) extracts. The metabolomics data show that Tsc1 deletion in
ST:STUDY_SUMMARY                 	nephron progenitor cells changes the arginine biosynthesis pathway as well as a
ST:STUDY_SUMMARY                 	substantial number of metabolic pathways.
ST:INSTITUTE                     	Hadassah Medical Center
ST:LAST_NAME                     	Ben-Dov
ST:FIRST_NAME                    	Iddo
ST:ADDRESS                       	Hadassah Medical Center, Jerusalem, Israel 91120
ST:EMAIL                         	iddo@hadassah.org.il
ST:NUM_GROUPS                    	3
ST:TOTAL_SUBJECTS                	26
ST:STUDY_TYPE                    	MS quantitative analysis
ST:PHONE                         	+97226776881
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENDER                        	Male and female
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	1	01_WT_1	Genotype:Wild-type | Treatment:none	RAW_FILE_NAME=01_WT_1.raw
SUBJECT_SAMPLE_FACTORS           	2	02_WT_2	Genotype:Wild-type | Treatment:none	RAW_FILE_NAME=02_WT_2.raw
SUBJECT_SAMPLE_FACTORS           	3	03_WT_3	Genotype:Wild-type | Treatment:none	RAW_FILE_NAME=03_WT_3.raw
SUBJECT_SAMPLE_FACTORS           	4	04_KO_1	Genotype:Tsc1-knockout | Treatment:none	RAW_FILE_NAME=04_KO_1.raw
SUBJECT_SAMPLE_FACTORS           	5	05_KO_2	Genotype:Tsc1-knockout | Treatment:none	RAW_FILE_NAME=05_KO_2.raw
SUBJECT_SAMPLE_FACTORS           	6	06_KO_3	Genotype:Tsc1-knockout | Treatment:none	RAW_FILE_NAME=06_KO_3.raw
SUBJECT_SAMPLE_FACTORS           	7	07_KO_Rapha1	Genotype:Tsc1-knockout | Treatment:rapamycin	RAW_FILE_NAME=07_KO_Rapha1.raw
SUBJECT_SAMPLE_FACTORS           	8	08_KO_Rapha2	Genotype:Tsc1-knockout | Treatment:rapamycin	RAW_FILE_NAME=08_KO_Rapha2.raw
SUBJECT_SAMPLE_FACTORS           	9	09_KO_Rapha3	Genotype:Tsc1-knockout | Treatment:rapamycin	RAW_FILE_NAME=09_KO_Rapha3.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	PTCs were isolated as previously described(PMID: 32484794). Briefly, kidneys
CO:COLLECTION_SUMMARY            	were excised in ice-cold HBSS buffer. The kidneys were sliced and chopped into
CO:COLLECTION_SUMMARY            	pieces (~0.5–1 mm) on ice using a surgical scalpel. The chopped kidneys were
CO:COLLECTION_SUMMARY            	transferred into an HBSS solution containing 1 μg/μL collagenase/dispase
CO:COLLECTION_SUMMARY            	(Sigma Aldrich, Cat. # 10269638001) and incubated for 25 minutes at 370 C. The
CO:COLLECTION_SUMMARY            	cells were filtered through a 40-μm nylon cell strainer (Corning, Cat. #
CO:COLLECTION_SUMMARY            	431750) and washed twice with cold HBSS. For PTC isolation, the cells were
CO:COLLECTION_SUMMARY            	stained with PE-conjugated antiCD133/prominin-1 antibody (Invitrogen, Cat. #
CO:COLLECTION_SUMMARY            	12-1331-82) according to the manufacturer's instructions. PE+ cells were
CO:COLLECTION_SUMMARY            	isolated by Aria III flow cytometry-based cell sorting.
CO:SAMPLE_TYPE                   	Proximal tubular cells
#TREATMENT
TR:TREATMENT_SUMMARY             	A mixture of six labeled internal standards was added to the extraction solution
TR:TREATMENT_SUMMARY             	for quality control (13C6-Glucose, 13C5-Glutamine, 13C5-Glutamate, 13C1-Alanine,
TR:TREATMENT_SUMMARY             	13C3-Pyruvate and 13C3-Lactate). The exact volume at each tube was adjusted
TR:TREATMENT_SUMMARY             	according to tissue weight (average volume of 200 µl). The samples were
TR:TREATMENT_SUMMARY             	homogenized using Precellys 24 tissue homogenizer (Bertin Technologies)
TR:TREATMENT_SUMMARY             	precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a 30 s gap
TR:TREATMENT_SUMMARY             	between each cycle) and later centrifuged at 18,000 g for 15 min at 4°C. The
TR:TREATMENT_SUMMARY             	supernatants were transferred into HPLC glass vials and kept at −80°C prior
TR:TREATMENT_SUMMARY             	to LC-MS analysis.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The samples were homogenized using Precellys 24 tissue homogenizer (Bertin
SP:SAMPLEPREP_SUMMARY            	Technologies) precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a
SP:SAMPLEPREP_SUMMARY            	30 s gap between each cycle) and later centrifuged at 18,000 g for 15 min at
SP:SAMPLEPREP_SUMMARY            	4°C. The supernatants were transferred into HPLC glass vials and kept at
SP:SAMPLEPREP_SUMMARY            	−80°C prior to LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	UPLC setup consisted a ZIC-pHILIC column (SeQuant; 150 mm × 2.1 mm,
CH:CHROMATOGRAPHY_SUMMARY        	5 μm; Merck). 5 µl of cells or kidney extracts were injected using an
CH:CHROMATOGRAPHY_SUMMARY        	autosampler. Compounds were separated using a 15 minutes gradient, starting at
CH:CHROMATOGRAPHY_SUMMARY        	20% aqueous (20 mM ammonium carbonate adjusted to pH 9.2 with 0.1% of 25%
CH:CHROMATOGRAPHY_SUMMARY        	ammonium hydroxide) and 80% organic (acetonitrile), terminated with 20%
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile. Flow rate and column temperature were kept at 0.2 ml/min and
CH:CHROMATOGRAPHY_SUMMARY        	45°C, respectively for a total run time of 27 min
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	20% water/80% acetonitrile; 20 mM ammonium carbonate; 0.1% of 25% ammonium
CH:SOLVENT_A                     	hydroxide (adjusted to pH 9.2)
CH:SOLVENT_B                     	80%water/20% acetonitrile
CH:FLOW_GRADIENT                 	15 min terminated with 20% acetonitrile
CH:FLOW_RATE                     	0.2 ml/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Technion Mass Spect
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	Other
MS:ION_MODE                      	UNSPECIFIED
MS:MS_COMMENTS                   	Briefly, Dionex Ultimate ultra-high-performance liquid chromatography (UPLC)
MS:MS_COMMENTS                   	system coupled to Orbitrap Q-Exactive mass spectrometer (Thermo Fisher
MS:MS_COMMENTS                   	Scientific) was used. The resolution was set to 35,000 at 200 mass/charge ratio
MS:MS_COMMENTS                   	(m/z) with electrospray ionization and polarity switching mode to enable both
MS:MS_COMMENTS                   	positive and negative ions across a mass range of 67–1000 m/z.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	concentration
MS_METABOLITE_DATA_START
Samples	04_KO_1	05_KO_2	06_KO_3	07_KO_Rapha1	08_KO_Rapha2	09_KO_Rapha3	01_WT_1	02_WT_2	03_WT_3
Factors	Genotype:Tsc1-knockout | Treatment:none	Genotype:Tsc1-knockout | Treatment:none	Genotype:Tsc1-knockout | Treatment:none	Genotype:Tsc1-knockout | Treatment:rapamycin	Genotype:Tsc1-knockout | Treatment:rapamycin	Genotype:Tsc1-knockout | Treatment:rapamycin	Genotype:Wild-type | Treatment:none	Genotype:Wild-type | Treatment:none	Genotype:Wild-type | Treatment:none
pyroglutamic acid	2120000	2440000	2570000	909000	1020000	647000	4750000	1640000	813000
acetyl-aspartate (N)	539000	464000	487000	118000	117000	43800	61400	173000	0
acetyl-carnitine	9830000	10500000	9320000	4560000	4720000	3290000	5270000	4460000	1590000
Adenine	831000	592000	750000	983000	876000	706000	903000	438000	250000
Adenosine	288000	288000	279000	1020000	521000	357000	870000	387000	67900
aKG	1540000	278000	442000	1100000	192000	107000	235000	0	0
Alanine	3430000	3750000	3570000	3180000	3410000	1830000	1610000	1950000	742000
aminoadipate	129000	140000	106000	247000	215000	128000	131000	98900	82300
Arachidonic acid	0	258000	0	792000	839000	0	281000	510000	0
Arginine	812000	829000	800000	635000	478000	667000	2960000	674000	259000
Asparagine	838000	1020000	802000	618000	633000	310000	342000	1080000	21000
Aspartate	6730000	6190000	5930000	5180000	5200000	3270000	4050000	3380000	1310000
betaine	19300000	16300000	16400000	9340000	7070000	6390000	14000000	16800000	1990000
Butyric acid	1400000	932000	797000	950000	712000	996000	1100000	731000	407000
Butyryl-carnitine	184000	209000	161000	121000	164000	44200	104000	78200	40900
carnitine	17500000	16400000	14000000	12700000	13100000	8170000	8000000	7750000	2420000
Citrulline	823000	834000	876000	337000	366000	257000	256000	132000	0
Creatinine	335000	352000	254000	404000	454000	173000	95500	137000	7160
Decanoic acid	3020000	4380000	1340000	1120000	1160000	672000	1160000	1620000	2130000
Dodecanoic acid/Lauric acid	7500000	8990000	1330000	943000	604000	1080000	1690000	3790000	5050000
Eicosapentaenoic acid	80800	1450000	0	1500000	1600000	0	989000	1840000	307000
Ethanolamine phosphate	3700000	3520000	3090000	1750000	1940000	1790000	1340000	1190000	0
Glucose	3.14E+08	3.07E+08	2.57E+08	6.88E+08	6.81E+08	4.56E+08	4.61E+08	4.66E+08	1.76E+08
Glutamate	14400000	15500000	13300000	12700000	12400000	7170000	9700000	7420000	2500000
Glutamine	3220000	3140000	3020000	3480000	3240000	2180000	1980000	2090000	469000
Glycine	2900000	3160000	3480000	1230000	1430000	640000	18700000	19400000	5810000
hypoxanthine	202000	161000	189000	110000	110000	30700	152000	148000	7500
Isocapric Acid	1300000	1340000	1010000	1040000	1020000	991000	1100000	1020000	682000
Lactate	13000000	14000000	2610000	4650000	3040000	3040000	5310000	4750000	9180000
Leucine	6860000	6840000	6740000	5480000	5510000	3740000	4960000	5220000	1790000
Linoleic acid	1530000	1140000	1010000	1130000	1080000	3260000	1840000	1640000	3150000
Lysine	434000	585000	311000	482000	567000	211000	381000	295000	201000
Malate	3000000	3100000	2820000	3830000	916000	811000	3060000	2160000	341000
Methionine	1580000	1780000	1340000	1140000	1370000	578000	1100000	884000	136000
Myristic acid	7690000	9670000	3590000	2790000	1900000	2510000	3640000	5710000	5880000
NAD+	191000	295000	120000	293000	285000	66200	72300	66700	0
nicotinamide	1330000	1440000	1200000	1160000	1180000	1160000	1400000	1280000	691000
Octanoic acid	2960000	4090000	1620000	1890000	1580000	1050000	1750000	2260000	1210000
Oleic acid	6890000	5280000	3820000	5650000	3430000	18500000	7770000	7740000	17700000
Palmitic acid	54800000	64900000	76100000	61200000	36400000	32100000	54900000	51800000	27100000
Palmitoleic acid	6320000	7890000	2550000	2270000	1580000	3160000	4110000	7130000	8370000
PEP	482000	292000	108000	72000	133000	218000	299000	83000	0
Phenylalanine	2720000	2720000	2500000	2160000	2040000	1610000	2240000	1460000	504000
Phosphocreatine	201000	344000	199000	10500	126000	222000	12300	30600	0
Proline	9650000	9400000	9570000	8340000	8230000	9220000	18100000	5980000	2000000
Propionyl-carnitine	1190000	1150000	957000	885000	917000	611000	566000	664000	388000
Pyruvate	1300000	948000	427000	1020000	519000	864000	1540000	551000	583000
Ribose	8570000	8400000	6750000	18900000	19200000	12800000	12700000	13100000	4980000
S-Adenosyl-L-methionine	162000	180000	132000	79500	332000	85200	11600	29000	0
Serine	622000	709000	674000	420000	385000	181000	212000	170000	7050
Stearic acid	33600000	40500000	54500000	37200000	26500000	21200000	36400000	31700000	15700000
Succinic Acid	1830000	335000	827000	1070000	209000	1560000	259000	0	1100000
Taurine	9850000	9370000	8220000	5370000	5500000	3770000	3310000	3370000	1310000
Threonine	2800000	3350000	3100000	2190000	2700000	2150000	1730000	2140000	1290000
Tryptophan	488000	507000	368000	210000	221000	143000	215000	196000	0
Tyrosine	307000	399000	287000	165000	145000	158000	141000	128000	39100
UDP-GlcNAc	153000	177000	44300	103000	137000	56600	22700	55900	8930
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
pyroglutamic acid
acetyl-aspartate (N)
acetyl-carnitine
Adenine
Adenosine
aKG
Alanine
aminoadipate
Arachidonic acid
Arginine
Asparagine
Aspartate
betaine
Butyric acid
Butyryl-carnitine
carnitine
Citrulline
Creatinine
Decanoic acid
Dodecanoic acid/Lauric acid
Eicosapentaenoic acid
Ethanolamine phosphate
Glucose
Glutamate
Glutamine
Glycine
hypoxanthine
Isocapric Acid
Lactate
Leucine
Linoleic acid
Lysine
Malate
Methionine
Myristic acid
NAD+
nicotinamide
Octanoic acid
Oleic acid
Palmitic acid
Palmitoleic acid
PEP
Phenylalanine
Phosphocreatine
Proline
Propionyl-carnitine
Pyruvate
Ribose
S-Adenosyl-L-methionine
Serine
Stearic acid
Succinic Acid
Taurine
Threonine
Tryptophan
Tyrosine
UDP-GlcNAc
METABOLITES_END
#END