Summary of Study ST002758

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	ST002758
Study Title	Metabolic responses of normal rat kidneys to a high salt intake (Plasma)
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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Sample Preparation:

Sampleprep ID:	SP002871
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:

SP002871

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.