Summary of Study ST002760

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 PR001719. The data can be accessed directly via it's Project DOI: 10.21228/M8HF01 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	ST002760
Study Title	Metabolic responses of normal rat kidneys to a high salt intake (Kidney outer medulla)
Study Type	Time-course metabolomics experiment
Study Summary	In this study, novel methods were developed which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic profiles before and following the switch from a 0.4% to 4.0% NaCl diet.
Institute	Medical College of Wisconsin
Department	Physiology
Laboratory	Dr. Allen W. Cowley
Last Name	Cowley
First Name	Allen
Address	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email	cowley@mcw.edu
Phone	4149558277
Submit Date	2023-06-26
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2023-07-02
Release Version	1

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Project:

Project ID:	PR001719
Project DOI:	doi: 10.21228/M8HF01
Project Title:	SD Rat Metabolomics in Response to Salt
Project Type:	Untargeted Four-Mode Metabolomics
Project Summary:	This study analyzed the effects of a high salt (HS; 4.0% NaCl) diet upon the kidney, arterial plasma, and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats.
Institute:	Medical College of Wisconsin
Department:	Physiology
Laboratory:	Dr. Allen W. Cowley
Last Name:	Cowley
First Name:	Allen
Address:	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email:	cowley@mcw.edu
Phone:	414-955-8277
Funding Source:	NHLBI
Contributors:	Satoshi Shimada, Brian R. Hoffmann, Chun Yang, Theresa Kurth, Andrew S. Greene, Mingyu Liang, Ranjan K. Dash, Allen W. Cowley Jr

Subject:

Subject ID:	SU002867
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Source	CF
SA290817	20220110_Cowley3_OM_2_22-391_C18pos	HS14	OM	OM
SA290818	20220111_Cowley3_OM_13_24-402_C18neg_rep	HS14	OM	OM
SA290819	20220113_Cowley3_OM_4_28-406_HILICpos_rep	HS14	OM	OM
SA290820	20220113_Cowley3_OM_5_30-413_HILICpos_rep	HS14	OM	OM
SA290821	20220113_Cowley3_OM_11_24-402_HILICpos	HS14	OM	OM
SA290822	20220111_Cowley3_OM_13_24-402_C18neg	HS14	OM	OM
SA290823	20220113_Cowley3_OM_5_30-413_HILICpos	HS14	OM	OM
SA290824	20220111_Cowley3_OM_9_26-405_C18neg	HS14	OM	OM
SA290825	20220111_Cowley3_OM_6_22-391_C18neg	HS14	OM	OM
SA290826	20220111_Cowley3_OM_1_30-413_C18neg_rep	HS14	OM	OM
SA290827	20220111_Cowley3_OM_6_22-391_C18neg_rep	HS14	OM	OM
SA290828	20220111_Cowley3_OM_8_28-406_C18neg	HS14	OM	OM
SA290829	20220113_Cowley3_OM_11_24-402_HILICpos_rep	HS14	OM	OM
SA290830	20220111_Cowley3_OM_8_28-406_C18neg_rep	HS14	OM	OM
SA290831	20220111_Cowley3_OM_9_26-405_C18neg_rep	HS14	OM	OM
SA290832	20220113_Cowley3_OM_13_26-405_HILICpos	HS14	OM	OM
SA290833	20220119_Cowley3_OM_13_24-402_HILICneg_rep	HS14	OM	OM
SA290834	20220119_Cowley3_OM_13_24-402_HILICneg	HS14	OM	OM
SA290835	20220119_Cowley3_OM_14_28-406_HILICneg	HS14	OM	OM
SA290836	20220119_Cowley3_OM_14_28-406_HILICneg_rep	HS14	OM	OM
SA290837	20220119_Cowley3_OM_15_22-391_HILICneg_rep	HS14	OM	OM
SA290838	20220119_Cowley3_OM_15_22-391_HILICneg	HS14	OM	OM
SA290839	20220119_Cowley3_OM_10_26-405_HILICneg_rep	HS14	OM	OM
SA290840	20220119_Cowley3_OM_10_26-405_HILICneg	HS14	OM	OM
SA290841	20220113_Cowley3_OM_15_22-391_HILICpos	HS14	OM	OM
SA290842	20220113_Cowley3_OM_13_26-405_HILICpos_rep	HS14	OM	OM
SA290843	20220113_Cowley3_OM_15_22-391_HILICpos_rep	HS14	OM	OM
SA290844	20220119_Cowley3_OM_2_30-413_HILICneg	HS14	OM	OM
SA290845	20220119_Cowley3_OM_2_30-413_HILICneg_rep	HS14	OM	OM
SA290846	20220111_Cowley3_OM_1_30-413_C18neg	HS14	OM	OM
SA290847	20220113_Cowley3_OM_4_28-406_HILICpos	HS14	OM	OM
SA290848	20220110_Cowley3_OM_8_28-406_C18pos	HS14	OM	OM
SA290849	20220110_Cowley3_OM_6_24-402_C18pos_rep	HS14	OM	OM
SA290850	20220110_Cowley3_OM_8_28-406_C18pos_rep	HS14	OM	OM
SA290851	20220110_Cowley3_OM_9_30-413_C18pos	HS14	OM	OM
SA290852	20220110_Cowley3_OM_15_26-405_C18pos_rep	HS14	OM	OM
SA290853	20220110_Cowley3_OM_9_30-413_C18pos_rep	HS14	OM	OM
SA290854	20220110_Cowley3_OM_6_24-402_C18pos	HS14	OM	OM
SA290855	20220110_Cowley3_OM_15_26-405_C18pos	HS14	OM	OM
SA290856	20220110_Cowley3_OM_2_22-391_C18pos_rep	HS14	OM	OM
SA290857	20220113_Cowley3_OM_1_18-434_HILICpos	HS21	OM	OM
SA290858	20220113_Cowley3_OM_1_12-417_HILICpos_rep	HS21	OM	OM
SA290859	20220113_Cowley3_OM_1_18-434_HILICpos_rep	HS21	OM	OM
SA290860	20220113_Cowley3_OM_7_14-421_HILICpos_rep	HS21	OM	OM
SA290861	20220113_Cowley3_OM_7_14-421_HILICpos	HS21	OM	OM
SA290862	20220113_Cowley3_OM_1_12-417_HILICpos	HS21	OM	OM
SA290863	20220110_Cowley3_OM_4_18-434_C18pos	HS21	OM	OM
SA290864	20220110_Cowley3_OM_13_12-417_C18pos_rep	HS21	OM	OM
SA290865	20220110_Cowley3_OM_14_14-421_C18pos	HS21	OM	OM
SA290866	20220110_Cowley3_OM_13_12-417_C18pos	HS21	OM	OM
SA290867	20220110_Cowley3_OM_12_20-463_C18pos_rep	HS21	OM	OM
SA290868	20220110_Cowley3_OM_4_18-434_C18pos_rep	HS21	OM	OM
SA290869	20220110_Cowley3_OM_12_20-463_C18pos	HS21	OM	OM
SA290870	20220113_Cowley3_OM_8_20-463_HILICpos_rep	HS21	OM	OM
SA290871	20220113_Cowley3_OM_12_16-429_HILICpos_rep	HS21	OM	OM
SA290872	20220119_Cowley3_OM_7_12-417_HILICneg_rep	HS21	OM	OM
SA290873	20220119_Cowley3_OM_11_16-429_HILICneg	HS21	OM	OM
SA290874	20220119_Cowley3_OM_7_12-417_HILICneg	HS21	OM	OM
SA290875	20220119_Cowley3_OM_4_20-463_HILICneg	HS21	OM	OM
SA290876	20220119_Cowley3_OM_5_14-421_HILICneg	HS21	OM	OM
SA290877	20220119_Cowley3_OM_4_20-463_HILICneg_rep	HS21	OM	OM
SA290878	20220119_Cowley3_OM_11_16-429_HILICneg_rep	HS21	OM	OM
SA290879	20220119_Cowley3_OM_12_18-434_HILICneg	HS21	OM	OM
SA290880	20220110_Cowley3_OM_1_16-429_C18pos_rep	HS21	OM	OM
SA290881	20220119_Cowley3_OM_5_14-421_HILICneg_rep	HS21	OM	OM
SA290882	20220110_Cowley3_OM_1_16-429_C18pos	HS21	OM	OM
SA290883	20220110_Cowley3_OM_14_14-421_C18pos_rep	HS21	OM	OM
SA290884	20220119_Cowley3_OM_12_18-434_HILICneg_rep	HS21	OM	OM
SA290885	20220113_Cowley3_OM_12_16-429_HILICpos	HS21	OM	OM
SA290886	20220113_Cowley3_OM_8_20-463_HILICpos	HS21	OM	OM
SA290887	20220111_Cowley3_OM_4_20-463_C18neg_rep	HS21	OM	OM
SA290888	20220111_Cowley3_OM_4_20-463_C18neg	HS21	OM	OM
SA290889	20220111_Cowley3_OM_1_18-434_C18neg_rep	HS21	OM	OM
SA290890	20220111_Cowley3_OM_7_16-429_C18neg	HS21	OM	OM
SA290891	20220111_Cowley3_OM_14_12-417_C18neg_rep	HS21	OM	OM
SA290892	20220111_Cowley3_OM_11_14-421_C18neg	HS21	OM	OM
SA290893	20220111_Cowley3_OM_11_14-421_C18neg_rep	HS21	OM	OM
SA290894	20220111_Cowley3_OM_14_12-417_C18neg	HS21	OM	OM
SA290895	20220111_Cowley3_OM_1_18-434_C18neg	HS21	OM	OM
SA290896	20220111_Cowley3_OM_7_16-429_C18neg_rep	HS21	OM	OM
SA290897	20220110_Cowley3_OM_11_4-467_C18pos	LS	OM	OM
SA290898	20220110_Cowley3_OM_10_8-469_C18pos_rep	LS	OM	OM
SA290899	20220119_Cowley3_OM_6_2-466_HILICneg_rep	LS	OM	OM
SA290900	20220110_Cowley3_OM_11_4-467_C18pos_rep	LS	OM	OM
SA290901	20220113_Cowley3_OM_14_2-466_HILICpos_rep	LS	OM	OM
SA290902	20220113_Cowley3_OM_10_10-470_HILICpos	LS	OM	OM
SA290903	20220113_Cowley3_OM_10_10-470_HILICpos_rep	LS	OM	OM
SA290904	20220113_Cowley3_OM_14_2-466_HILICpos	LS	OM	OM
SA290905	20220110_Cowley3_OM_10_8-469_C18pos	LS	OM	OM
SA290906	20220119_Cowley3_OM_6_2-466_HILICneg	LS	OM	OM
SA290907	20220110_Cowley3_OM_7_6-468_C18pos_rep	LS	OM	OM
SA290908	20220110_Cowley3_OM_7_6-468_C18pos	LS	OM	OM
SA290909	20220110_Cowley3_OM_5_2-466_C18pos_rep	LS	OM	OM
SA290910	20220113_Cowley3_OM_9_4-467_HILICpos_rep	LS	OM	OM
SA290911	20220119_Cowley3_OM_1_10-470_HILICneg	LS	OM	OM
SA290912	20220119_Cowley3_OM_3_4-467_HILICneg_rep	LS	OM	OM
SA290913	20220119_Cowley3_OM_3_4-467_HILICneg	LS	OM	OM
SA290914	20220119_Cowley3_OM_1_10-470_HILICneg_rep	LS	OM	OM
SA290915	20220110_Cowley3_OM_5_2-466_C18pos	LS	OM	OM
SA290916	20220113_Cowley3_OM_6_6-468_HILICpos_rep	LS	OM	OM

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Collection:

Collection ID:	CO002860
Collection Summary:	Plasma was collected through an arterial and renal venous catheter throughout the study (200 µL of arterial and renal venous blood were sampled at the day 7, 14, and 21). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.
Sample Type:	Kidney outer medulla
Storage Conditions:	-80?

Treatment:

Treatment ID:	TR002876
Treatment Summary:	Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Treatment ID:

TR002876

Treatment Summary:

Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Sample Preparation:

Sampleprep ID:	SP002873
Sampleprep Summary:	Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Sampleprep ID:

SP002873

Sampleprep Summary:

Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Combined analysis:

Analysis ID	AN004483	AN004484	AN004485	AN004486
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Area	Area	Area	Area

Chromatography:

Chromatography ID:	CH003368
Chromatography Summary:	This chromatography method was utilized for all C18 positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 98% A1/2% B1, 1-13 minutes from 98% A1/2% B1 to 10% A1/90% B1, 13-15 minutes at 10% A1/90% B1, 15-16 minutes from 10% A1/90% B1 to 98% A1/2% B1, and was re-equilibrated from 16-25 minutes at 98% A1/2% B1
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003369
Chromatography Summary:	This chromatography method was utilized for all C18 negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003370
Chromatography Summary:	This chromatography method was utilized for all HILIC positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium formate in H2O with 0.1% formic acid (Solvent A2)
Solvent B:	90% ACN with 10 mM ammonium formate in H2O with 0.1% formic acid (Solvent B2)
Chromatography Type:	HILIC

Chromatography ID:	CH003371
Chromatography Summary:	This chromatography method was utilized for all HILIC negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium acetate in H2O, pH 9.0 with 0.1% AffinityLab Deactivator Inhibitor (Agilent, #5191-3940; Solvent A3)
Solvent B:	85% ACN with 10 mM ammonium acetate in H2O with 0.1% AffinityLab Deactivator Inhibitor (Solvent B3)
Chromatography Type:	HILIC

MS:

MS ID:	MS004230
Analysis ID:	AN004483
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 positive kidney outer medulla data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004231
Analysis ID:	AN004484
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 negative kidney outer medulla data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE

MS ID:	MS004232
Analysis ID:	AN004485
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC positive kidney outer medulla data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004233
Analysis ID:	AN004486
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC negative kidney outer medulla data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE