Project Grant R21DK137147

INTEGRATED MULTI-TISSUE 13C FLUX ANALYSIS PLATFORM TO ASSESS RENAL METABOLISM IN VIVO - PROJECT SUMMARY/ABSTRACT PRIOR STUDIES HAVE ASSESSED DISEASE-ASSOCIATED CHANGES IN METABOLIC FLUXES WITHIN A SINGLE ORGAN BUT HAVE NOT ATTEMPTED TO SIMULTANEOUSLY EXAMINE FLUX ALTERATIONS WITHIN MULTIPLE ORGANS THAT TOGETHER CONTROL WHOLE-BODY NUTRIENT METABOLISM. FURTHERMORE, CURRENT ISOTOPE TRACER TECHNOLOGIES DO NOT HAVE THE ABILITY TO DISSECT METABOLIC FLUX DIFFERENCES BETWEEN SPATIAL LOCATIONS WITHIN A SINGLE ORGAN. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP A MULTI-TISSUE 13C METABOLIC FLUX ANALYSIS (MFA) PLATFORM TO ASSESS KIDNEY METABOLISM AND ITS INTERRELATIONSHIP WITH LIVER AND HEART METABOLISM. THE RATIONALE FOR THIS TECHNOLOGY IS THAT IT WILL ENABLE INVESTIGATORS TO ADDRESS IMPORTANT QUESTIONS ABOUT IN VIVO REGULATION OF RENAL METABOLISM THAT CANNOT BE ANSWERED THROUGH STUDIES OF SINGLE ORGANS OR ISOLATED CELLS/TISSUES. THE RESEARCH BUILDS FROM OUR RECENTLY PUBLISHED STUDY WHEREIN RENAL AND HEPATIC CONTRIBUTIONS TO GLUCONEOGENESIS (GNG) WERE SIMULTANEOUSLY ASSESSED IN FASTED MICE. TO OUR KNOWLEDGE, THIS WAS THE FIRST TIME 13C MFA HAD BEEN APPLIED TO ASSESS INTERACTIONS BETWEEN LIVER AND KIDNEY FLUXES IN A LIVE ANIMAL. THERE IS NOW A CRITICAL NEED TO, FIRST, EXPAND THIS FLUX MEASUREMENT TECHNOLOGY TO INCLUDE GLYCOLYTIC TISSUES AND, SECOND, TEST WHETHER REGIONAL DIFFERENCES IN METABOLIC FLUXES CAN BE DISTINGUISHED WITHIN THE KIDNEY. THE FIRST AIM WILL ESTABLISH A MULTI-ORGAN 13C MFA PLATFORM TO SIMULTANEOUSLY QUANTIFY IN VIVO METABOLISM OF GLUCONEOGENIC AND GLYCOLYTIC TISSUES OF THE KIDNEYS, LIVER, AND HEART. MICE WITH VEINOUS AND ARTERIAL CATHETERS WILL RECEIVE 13C-GLUCOSE INFUSIONS DURING HYPERINSULINEMIC-EUGLYCEMIC OR HYPERINSULINEMIC-HYPOGLYCEMIC CLAMPS. IN EACH CONDITION, THE ISOTOPES, MEASUREMENTS, AND MATHEMATICAL MODELING PROCEDURES WILL BE OPTIMIZED TO PRECISELY DETERMINE METABOLIC FLUXES IN EACH TISSUE. WE WILL THEN APPLY THIS NOVEL PLATFORM TO ASSESS METABOLIC FLUX ALTERATIONS IN A RELEVANT ANIMAL MODEL OF DIABETIC KIDNEY DISEASE: BKS DB/DB MICE WITH ENDOTHELIAL NITRIC OXIDE KNOCKOUT (ENOS-/-). THE SECOND AIM WILL EXPAND 13C-LABELED METABOLITE MEASUREMENTS TO ASSESS SPATIALLY RESOLVED METABOLISM IN THE KIDNEY. UNTARGETED HIGH-RESOLUTION LC-MS/MS PROFILING WILL BE APPLIED TO TISSUE EXTRACTS, AND METABOLITES ENRICHED BY 13C-GLUCOSE WILL BE IDENTIFIED USING NOVEL SOFTWARE THAT SCREENS FOR HITS AGAINST THE KEGG COMPOUND DATABASE. THEN, MALDI-BASED IMAGING MASS SPECTROMETRY (IMS) OF KIDNEY TISSUE SLICES WILL BE APPLIED TO LOCATE THESE LABELED METABOLITE FEATURES, DETERMINE THEIR SPATIAL PATTERNS OF 13C ENRICHMENT, AND PERFORM 13C MFA TO ASSESS METABOLIC FLUXES WITHIN DIFFERENT KIDNEY REGIONS. THE PROPOSED RESEARCH IS INNOVATIVE BECAUSE IT WILL ESTABLISH NEW TECHNOLOGIES FOR QUANTIFYING FLUX PHENOTYPES OF INTEGRATED MULTI-TISSUE METABOLIC NETWORKS IN LIVE ANIMALS. THE ANALYSIS PLATFORM WILL BE IMPLEMENTED AS A CORE SERVICE OF THE VANDERBILT MOUSE METABOLIC PHENOTYPING CENTER AND VANDERBILT O'BRIEN KIDNEY CENTER. THE RESEARCH IS SIGNIFICANT BECAUSE IT WILL ENABLE FUTURE STUDIES TO ASSESS HOW METABOLISM IS DYSREGULATED DURING PROGRESSION OF KIDNEY DISEASE AND HOW TREATMENTS THAT TARGET RENAL PATHWAYS IMPACT WHOLE-BODY METABOLIC HEALTH.