The Metabolism Core (Meta-Core) is a Biomedical Resource Core of the Washington University Kidney O’Brien Center for Chronic Kidney Disease Research.

Meta-Core Mission

Many proteins and modulators of chronic kidney disease (CKD) progression affect kidney metabolism. While there is increasing recognition of how metabolic alterations may affect kidney function and response to injury, there are several barriers to investigating how a given target impacts metabolism.

Some metabolic assays require expensive equipment that may not be accessible at every institution, but also the best approach to assess changes in metabolism and interpret the assays requires very specialized expertise.

Our goal is to facilitate the use of metabolic assays to further promote advances in CKD-related research.

How to get started using the Meta-Core:

1. User (investigator) has a research question related to metabolism and kidney injury research

2. Fill out the User Request Form

3. Core faculty will contact the User by email to clarify the goals/question and potentially further discuss virtually

4. A plan will be recommended which could involve one (or more) of the following:

  • Guidance in the design and/or interpretation of an assay that’s available at the User’s institution
  • Performance of appropriate metabolic assay by the Meta-Core for the User
  • Referral to a partner Core for the appropriate assay

Core Services

Bioflux analysis with Seahorse XF24/XF96

Through Washington University’s Diabetes Center, we have access to two Seahorse instruments, the 24 well and 96 well formats, for measurement of mitochondrial respiration and glycolysis. The Seahorse is ideally suited for adherent cells though in some cases can be adapted for use in tubules ex vivo. The Core can provide guidance for experimental design and proper controls to help the user best answer their specific question. There is also expertise at the Core for using the Seahorse on primary proximal tubules.

Substrate oxidation assays

These assays are designed to assess substrate oxidation using tissue ex vivo and radioactive substrates. As an example, we have used injured kidney tissue to measure fatty acid oxidation using 3H-labeled palmitate. If the palmitate is fully oxidized, then the 3H will be incorporated into 3H2O, which can be quantified using a scintillation counter. A similar technique can measure glucose/pyruvate oxidation as well.

High resolution respirometry (Oroboros)

Substrate-dependent respiration in kidney tissue can be measured using the Oroboros Oxygraph 2K through Washington University’s Nutrition Obesity Research Center (NORC). The Core has optimized methods for measuring respiration in permeabilized kidney tissue.

Analyses involve use of standardized reagents and an optimized sequential multiple substrate-uncoupler-inhibitor titration (SUIT) protocol for analysis of oxidative phosphorylation. Routine respiration is measured after addition of Complex I substrates (glutamate or malate), Complex II substrate (succinate) and ADP (state 3), followed by LEAK/State 4 respiration after addition of oligomycin and uncoupling measurements after added FCCP. Depending upon the project, palmitoylcarnitine or pyruvate may be used as well.

Untargeted Metabolomics

Global profiling of metabolites and lipids will be accomplished by using liquid chromatography/mass spectrometry (LC/MS). The platform can be applied to biofluids such as blood and urine or tissues such as those from the kidney.

This technique is useful to get a snapshot of compounds involved in glycolysis, the TCA cycle, the pentose-phosphate pathway, one-carbon metabolism, amino acid metabolism, fatty acid synthesis and oxidation, complex lipids, ceramides, sphingomyelins, and many more.

The metabolomics experiments will be performed in the Patti laboratory, which houses 20 mass spectrometers for metabolomics. We will use well-established protocols that have been developed by the Patti team over the past 15 years.  Analysis can also be performed by the Patti lab, which has pioneered interpretation of metabolomics data.  

Stable isotope flux studies

Whereas metabolomics provides information about the relative concentration of metabolites at a comprehensive scale, isotope tracer experiments provide information about the dynamics of a specific pathway of interest. The appropriate isotope tracer (e.g. glucose versus glutamine, uniformly labeled versus positionally labeled) and exact duration will depend upon each project’s goals.

In general, the isotope tracing capabilities will support three unique goals: (i) determining the metabolic flux of a pathway, (ii) mapping the fate of a particular nutrient, and (iii) quantitating how much of a given nutrient is metabolized by each pathway. The Patti laboratory regularly performs in vitro and in vivo isotope tracing using a variety of metabolic substrates.

Metabolism Core Personnel:

Leslie Gewin, MD, Director

Brian Finck, PhD

Gary Patti, PhD

Leah Shriver, PhD