Summary of Study ST003652

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 PR002262. The data can be accessed directly via it's Project DOI: 10.21228/M87N8N 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.

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Study ID	ST003652
Study Title	Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction Murine Model of CKD
Study Summary	In chronic kidney disease (CKD) research, animal models provide invaluable insights into the disease’s etiopathogenesis and progression, particularly through the evaluation of renal tissue. The unilateral ureteral obstruction (UUO) rodent model stands out for its widespread use in CKD studies, due to its advantages to generate renal fibrosis and accelerated mimicry of obstructive nephropathy in humans. Despite its extensive use, the molecular underpinnings driving kidney disease progression remain incompletely understood. Given the crucial interplay between metabolism and fibrosis in CKD, a thorough examination of the UUO renal tissue through metabolomics is required. Untargeted multiplatform analysis enables a comprehensive measurement of the sample metabolic profile, ensuring a maximum coverage of metabolite diversity to yield extensive insights into the metabolism of this renal injury model. Therefore, in this study, murine kidney tissue from the UUO model underwent analysis using three separation techniques—liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). The findings reveal metabolic changes associated with tubulointerstitial fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the renal tissue's lipidomic profile, characterized by a general decrease in most lipid classes and an increase in glycerophospholipids with ether substituents, hexosylceramides, and cholesterol esters compared to the control. These results underscore the relevance of the untargeted multiplatform approach to obtain a comprehensive overview of the alterations within the renal metabolic map, paving the way for further exploration of the molecular mechanisms underlying CKD.
Institute	Universidad CEU San Pablo
Last Name	Rupérez
First Name	Francisco
Address	Campus Montepríncipe. Ctra. M-501 km 0
Email	ruperez@ceu.es
Phone	913724753
Submit Date	2024-10-19
Raw Data Available	Yes
Raw Data File Type(s)	mzML, mzXML
Analysis Type Detail	GC-MS/LC-MS
Release Date	2025-04-21
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002262
Project DOI:	doi: 10.21228/M87N8N
Project Title:	Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction Murine Model of CKD
Project Summary:	In chronic kidney disease (CKD) research, animal models provide invaluable insights into the disease’s etiopathogenesis and progression, particularly through the evaluation of renal tissue. The unilateral ureteral obstruction (UUO) rodent model stands out for its widespread use in CKD studies, due to its advantages to generate renal fibrosis and accelerated mimicry of obstructive nephropathy in humans. Despite its extensive use, the molecular underpinnings driving kidney disease progression remain incompletely understood. Given the crucial interplay between metabolism and fibrosis in CKD, a thorough examination of the UUO renal tissue through metabolomics is required. Untargeted multiplatform analysis enables a comprehensive measurement of the sample metabolic profile, ensuring a maximum coverage of metabolite diversity to yield extensive insights into the metabolism of this renal injury model. Therefore, in this study, murine kidney tissue from the UUO model underwent analysis using three separation techniques—liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). The findings reveal metabolic changes associated with tubulointerstitial fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the renal tissue's lipidomic profile, characterized by a general decrease in most lipid classes and an increase in glycerophospholipids with ether substituents, hexosylceramides, and cholesterol esters compared to the control. These results underscore the relevance of the untargeted multiplatform approach to obtain a comprehensive overview of the alterations within the renal metabolic map, paving the way for further exploration of the molecular mechanisms underlying CKD.
Institute:	Universidad CEU San Pablo
Last Name:	Rupérez
First Name:	Francisco
Address:	Campus Montepríncipe. Ctra. M-501 km 0
Email:	ruperez@ceu.es
Phone:	913724753

Subject:

Subject ID:	SU003782
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

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

mb_sample_id	local_sample_id	Group
SA398424	1WT-CT_LCMS	CT
SA398425	6WT-CT_GCMS	CT
SA398426	5WT-CT_GCMS	CT
SA398427	4WT-CT_GCMS	CT
SA398428	3WT-CT_GCMS	CT
SA398429	2WT-CT_GCMS	CT
SA398430	1WT-CT_GCMS	CT
SA398431	13WT-CT_LCMS	CT
SA398432	12WT-CT_LCMS	CT
SA398433	6WT-CT_LCMS	CT
SA398434	5WT-CT_LCMS	CT
SA398435	3WT-CT_LCMS	CT
SA398436	2WT-CT_LCMS	CT
SA398437	1WT-CT_CEMS	CT
SA398438	4WT-CT_LCMS	CT
SA398439	6WT-CT_CEMS	CT
SA398440	2WT-CT_CEMS	CT
SA398441	3WT-CT_CEMS	CT
SA398442	4WT-CT_CEMS	CT
SA398443	13WT-CT_CEMS	CT
SA398444	5WT-CT_CEMS	CT
SA398445	12WT-CT_CEMS	CT
SA398446	1WT-UUO_CEMS	UUO
SA398447	2WT-UUO_GCMS	UUO
SA398448	12WT-UUO_GCMS	UUO
SA398449	1WT-UUO_LCMS	UUO
SA398450	5WT-UUO_GCMS	UUO
SA398451	2WT-UUO_CEMS	UUO
SA398452	4WT-UUO_GCMS	UUO
SA398453	12WT-UUO_CEMS	UUO
SA398454	3WT-UUO_GCMS	UUO
SA398455	3WT-UUO_CEMS	UUO
SA398456	1WT-UUO_GCMS	UUO
SA398457	2WT-UUO_LCMS	UUO
SA398458	4WT-UUO_LCMS	UUO
SA398459	4WT-UUO_CEMS	UUO
SA398460	13WT-UUO_LCMS	UUO
SA398461	12WT-UUO_LCMS	UUO
SA398462	5WT-UUO_CEMS	UUO
SA398463	6WT-UUO_LCMS	UUO
SA398464	3WT-UUO_LCMS	UUO
SA398465	5WT-UUO_LCMS	UUO
SA398466	6WT-UUO_CEMS	UUO
SA398467	13WT-UUO_CEMS	UUO

Showing results 1 to 44 of 44

Showing results 1 to 44 of 44

Collection:

Collection ID:	CO003775
Collection Summary:	All animal-related procedures and sample collections were conducted at the Centro de Biología Molecular "Severo Ochoa" (CBMSO) in accordance with the guidelines outlined in Directive 2010/63/EU of the European Parliament, known as the Guide for the Care and Use of Laboratory Animals. The study (PROEX 098.0/22) received approval from the CBMSO Ethics Committee for Animal Experimentation, the ethics committee of the Consejo Superior de Investigaciones Científicas (CSIC) and the Regulatory Unit for Animal Experimental Procedures of the Comunidad de Madrid. Mice were housed in a specific pathogen-free animal facility at CBMSO, adhering to EU regulations.
Sample Type:	Kidney

Treatment:

Treatment ID:	TR003791
Treatment Summary:	The UUO murine model (UUO7d) was stablished using the surgical procedure described in previous studies. Briefly, mice were anesthetized with isoflurane and the left ureter was doubly ligated with subsequent cutting between the ligatures. Adequate analgesia was provided to these animals after surgery and sacrifice was carried out after seven days using CO2 overdose. Control and obstructed kidneys were harvested following perfusion with PBS. Kidney samples were categorized into two experimental groups: wild-type control (WTCT) and wild-type obstruction (WTOBS), each containing 8 samples.

Treatment ID:

TR003791

Treatment Summary:

The UUO murine model (UUO7d) was stablished using the surgical procedure described in previous studies. Briefly, mice were anesthetized with isoflurane and the left ureter was doubly ligated with subsequent cutting between the ligatures. Adequate analgesia was provided to these animals after surgery and sacrifice was carried out after seven days using CO2 overdose. Control and obstructed kidneys were harvested following perfusion with PBS. Kidney samples were categorized into two experimental groups: wild-type control (WTCT) and wild-type obstruction (WTOBS), each containing 8 samples.

Sample Preparation:

Sampleprep ID:	SP003789
Sampleprep Summary:	All analytical-grade organic solvents and chemicals utilized in this study were obtained from Merck/Sigma-Aldrich (Germany), VWR International/BHD Prolabo (Spain) and Agilent Technologies (USA). Sialylation-grade pyridine was procured from VWR International BHD Prolabo (Spain). Reference mass solutions for LC-MS and CE-MS were obtained from Agilent Technologies. Deionized water (Milli-Q) utilized throughout the study was obtained using a Milli-Q PLUS system from Millipore (Austria). Sample treatment Each kidney was dissected both lengthwise and crosswise to obtain four equal pieces. These sections were promptly frozen in liquid nitrogen and stored at -80ºC until further analysis. Sample treatment For tissue disruption and homogenization, a cold methanol solution of 50% was added to achieve a tissue-weight volume ratio of 1:10. Homogenization was conducted using a TissueLyser LT bead-mill homogenizer device (QIAGEN, Hilden, Germany) with 2.8 mm (mean diameter) steel beads 1. The homogenization process entailed vibrating at a maximum power of 50Hz for 5 minutes, carried out over 4 cycles, with a 1-minute interval on ice between each cycle. The weight range of the kidney tissue varied between 25 and 50 mg (WTCT, 32–52.8 mg; WTOBS, 27.4–50.5 mg). Once the homogenate was obtained, the metabolite extraction process followed different protocols depending on the analytical platform used 2,3. For CE-MS analysis, 100 µL of 0.2M formic acid was added to 100 µL of homogenate, followed by vortex-mixing and centrifugation (16,000×g for 10 minutes at 4 °C). The resulting supernatant was then transferred to a Centrifree ultracentrifugation device (Milipore Ireland Ltd., Cork, Ireland) equipped with a 30-kDa protein cutoff filter for deproteinization through centrifugation (2000×g for 70 minutes at 4 °C). The filtrate obtained was transferred to a chromacol vial and subsequently evaporated using a SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the residue was resuspended in 50 μL of 0.1 M formic acid containing 0.2 mM methionine sulfone as an internal standard (IS). In the case of LC-MS and GC-MS analysis, 320 μL of cold methanol was added to 100 μL of homogenate, followed by the addition of 80 μL of methyl tert-butyl ether. The mixture was vortex-mixed for 1 hour at room temperature. Subsequently, the samples were centrifuged at 4000 g at 20ºC for 20 minutes, and the resulting supernatants were divided for LC-MS and GC-MS analysis. For LC-MS analysis, 100 μL of the supernatant was directly injected into the UHPLC-MS system. Regarding GC-MS analysis, 300 μL of the supernatant was transferred to a chromacol vial and evaporated to dryness using a SpeedVac Concentrator system. The derivatization process commenced by reconstituting the samples with 20 μL of O-methoxyamine hydrochloride (15 ng/mL) in pyridine for the methoximation step. After vigorous ultrasonication and vortex-mixing, the vials were incubated in the dark at room temperature for 16 hours. Subsequently, for the silylation step, 20 μL of BSTFA:TMCS (99:1) was added, followed by vortex-mixing for 5 minutes and incubation at 70ºC in the oven for 1 hour. Finally, 100 μL of heptane containing 20 ppm of tricosane (IS) was added. It is worth mentioning that due to the limited availability of samples, the GC-MS analysis was performed with only 6 samples in each group. Quality control samples (QCs) were prepared for each analytical technique by pooling equal volumes of each homogenized kidney tissue. Blank samples, which lacked the analyte of interest but underwent the same extraction protocols, were also prepared alongside the experimental samples. These blank samples were analyzed at the beginning and at end of the analytical sequence. QC samples were injected at the beginning of the sequence to condition and stabilize the analysis system, and subsequently after every 5-6 experimental samples throughout the run to assess the stability and performance of the analysis, and finally, at the end of the analysis sequence. All experimental samples were randomized prior to sample preparation and analysis.

Sampleprep ID:

SP003789

Sampleprep Summary:

All analytical-grade organic solvents and chemicals utilized in this study were obtained from Merck/Sigma-Aldrich (Germany), VWR International/BHD Prolabo (Spain) and Agilent Technologies (USA). Sialylation-grade pyridine was procured from VWR International BHD Prolabo (Spain). Reference mass solutions for LC-MS and CE-MS were obtained from Agilent Technologies. Deionized water (Milli-Q) utilized throughout the study was obtained using a Milli-Q PLUS system from Millipore (Austria). Sample treatment Each kidney was dissected both lengthwise and crosswise to obtain four equal pieces. These sections were promptly frozen in liquid nitrogen and stored at -80ºC until further analysis. Sample treatment For tissue disruption and homogenization, a cold methanol solution of 50% was added to achieve a tissue-weight volume ratio of 1:10. Homogenization was conducted using a TissueLyser LT bead-mill homogenizer device (QIAGEN, Hilden, Germany) with 2.8 mm (mean diameter) steel beads 1. The homogenization process entailed vibrating at a maximum power of 50Hz for 5 minutes, carried out over 4 cycles, with a 1-minute interval on ice between each cycle. The weight range of the kidney tissue varied between 25 and 50 mg (WTCT, 32–52.8 mg; WTOBS, 27.4–50.5 mg). Once the homogenate was obtained, the metabolite extraction process followed different protocols depending on the analytical platform used 2,3. For CE-MS analysis, 100 µL of 0.2M formic acid was added to 100 µL of homogenate, followed by vortex-mixing and centrifugation (16,000×g for 10 minutes at 4 °C). The resulting supernatant was then transferred to a Centrifree ultracentrifugation device (Milipore Ireland Ltd., Cork, Ireland) equipped with a 30-kDa protein cutoff filter for deproteinization through centrifugation (2000×g for 70 minutes at 4 °C). The filtrate obtained was transferred to a chromacol vial and subsequently evaporated using a SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the residue was resuspended in 50 μL of 0.1 M formic acid containing 0.2 mM methionine sulfone as an internal standard (IS). In the case of LC-MS and GC-MS analysis, 320 μL of cold methanol was added to 100 μL of homogenate, followed by the addition of 80 μL of methyl tert-butyl ether. The mixture was vortex-mixed for 1 hour at room temperature. Subsequently, the samples were centrifuged at 4000 g at 20ºC for 20 minutes, and the resulting supernatants were divided for LC-MS and GC-MS analysis. For LC-MS analysis, 100 μL of the supernatant was directly injected into the UHPLC-MS system. Regarding GC-MS analysis, 300 μL of the supernatant was transferred to a chromacol vial and evaporated to dryness using a SpeedVac Concentrator system. The derivatization process commenced by reconstituting the samples with 20 μL of O-methoxyamine hydrochloride (15 ng/mL) in pyridine for the methoximation step. After vigorous ultrasonication and vortex-mixing, the vials were incubated in the dark at room temperature for 16 hours. Subsequently, for the silylation step, 20 μL of BSTFA:TMCS (99:1) was added, followed by vortex-mixing for 5 minutes and incubation at 70ºC in the oven for 1 hour. Finally, 100 μL of heptane containing 20 ppm of tricosane (IS) was added. It is worth mentioning that due to the limited availability of samples, the GC-MS analysis was performed with only 6 samples in each group. Quality control samples (QCs) were prepared for each analytical technique by pooling equal volumes of each homogenized kidney tissue. Blank samples, which lacked the analyte of interest but underwent the same extraction protocols, were also prepared alongside the experimental samples. These blank samples were analyzed at the beginning and at end of the analytical sequence. QC samples were injected at the beginning of the sequence to condition and stabilize the analysis system, and subsequently after every 5-6 experimental samples throughout the run to assess the stability and performance of the analysis, and finally, at the end of the analysis sequence. All experimental samples were randomized prior to sample preparation and analysis.

Chromatography:

Chromatography ID:	CH004557
Chromatography Summary:	The GC-MS analysis was carried out utilizing an Agilent 7890B GC instrument coupled with a 7250 QTOF mass spectrometer system (Agilent Technologies). The analysis conditions were consistent with those described earlier . In summary, 1 μL of the derivatized sample was injected through an Agilent DB5-MS GC Capillary Column (30 m length, 0.25 mm, 0.25 µm film 95% dimethylpolysiloxane/5% diphenylpolysiloxane) using an Agilent autosampler (7693A). The samples were injected in a split ratio of 1:10 into a Restek 20782 deactivated glass-wool split liner. The injector port was established at 250ºC, the flow rate of helium carrier gas was set at 1 mL/min through the column. The temperature gradient was programmed at 60ºC as initial oven temperature (maintained for 1 min), increasing to 325ºC at a rate of 10ºC per minute. This temperature was kept for 10 min before cooling down. The total time of the analysis run was 37.5 minutes.
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:	Programmed Temperature Gradient
Flow Gradient:	-
Flow Rate:	1 mL/min
Solvent A:	-
Solvent B:	-
Chromatography Type:	GC

Chromatography ID:	CH004558
Chromatography Summary:	To perform LC-MS metabolomics analysis, an Agilent Technologies 1290 Infinity II UHPLC system was employed, coupled with a 6545-quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies). The analysis encompassed both ESI+ (positive) and ESI- (negative) modes, enabling the detection of a wide range of metabolite ions. The analysis conditions were consistent with those previously described, 1.5 μL of samples were injected with a multiwash option using the Agilent 1290 Infinity II Multisampler system with a sample temperature of 15ºC. Reversed-phase chromatography was used with an InfinityLab Poroshell 120 EC-C8 (2.1 x 150 mm, 2.7 µm) column and a suitable guard column (Agilent Technologies) at 60ºC. Mobile phases for the positive ionization mode were composed by, for aqueous phase (solvent A), 10 mM ammonium formate in Milli-Q water and for organic phase (solvent B), 10mM ammonium formate in methanol/isopropanol (85/15, v/v) with a flow rate of 0.5 mL/min. The mobile phases gradient started at 75% of solvent B, increasing to 96% B at minute 23 and kept for 8 min. The gradient then increased to 100% of solvent B by minute 31.5 and was maintained until minute 32.5. At minute 33, the initial condition was returned, followed by a 7 min-re-equilibration time, with a total run time of 40 min.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC-C8 (150 x 2.1mm,2.7um)
Column Temperature:	60
Flow Gradient:	The mobile phases gradient started at 75% of solvent B, increasing to 96% B at minute 23 and kept for 8 min. The gradient then increased to 100% of solvent B by minute 31.5 and was maintained until minute 32.5. At minute 33, the initial condition was returned, followed by a 7 min-re-equilibration time, with a total run time of 40 min.
Flow Rate:	0.5 mL/min
Solvent A:	100% water; 10 mM ammonium formate
Solvent B:	85% methanol/15% isopropanol; 10mM ammonium formate
Chromatography Type:	Reversed phase

Chromatography ID:	CH004559
Chromatography Summary:	To conduct the CE-MS analysis, an Agilent Technologies 7100 capillary electrophoresis system coupled with a 6224 TOF Mass Spectrometer (Agilent Technologies) was utilized. The system was equipped with an electrospray ionization source (ESI). The analysis conditions have been previously documented. In brief, a fused-silica capillary (Agilent Technologies; total length 96 cm; i.d.; 50 μm), previously flushed for 5 min with background electrolyte (BGE) (1 M formic acid in 10% methanol solution), was used for the separation analysis. Samples were injected over 50s at 50mbar, and BGE was co-injected for 20s at 100mbar after each sample injection to improve the reproducibility of the analysis. The composition of the sheath liquid included methanol/water (1/1, v/v), formic acid (1 mM), and two reference masses (purine, m/z 121.050873; HP-0921, m/z 922.009798). The total time of the analytical run was 23 min. The separation was carried out at a pressure of 25 mbar and a voltage of +30kV, in positive ionization mode, with a flow rate of 0.6 mL/min and a split set to 1/100.
Instrument Name:	Agilent 7100 CE
Column Name:	Agilent fused-silica (96cm x 50um)
Column Temperature:	25
Flow Gradient:	Samples were injected over 50s at 50mbar, and BGE was co-injected for 20s at 100mbar after each sample injection to improve the reproducibility of the analysis.
Flow Rate:	0.6 mL/min
Solvent A:	90% water/10% methanol; 1 M formic acid
Solvent B:	10% methanol/90% water; 1 M formic acid
Chromatography Type:	CE

Analysis:

Analysis ID:	AN005998
Analysis Type:	MS
Chromatography ID:	CH004557
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003652_AN005998_Results.txt
Units:	Peak area

Analysis ID:	AN005999
Analysis Type:	MS
Chromatography ID:	CH004558
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003652_AN005999_Results.txt
Units:	Peak area

Analysis ID:	AN006000
Analysis Type:	MS
Chromatography ID:	CH004559
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003652_AN006000_Results.txt
Units:	Peak area