Time-resolved metabolomes integrate kidney tubule nutrient exposure and stress dynamics

Background and Hypothesis: Measuring renal metabolism at the organ level is challenging, as metabolite signals in tissues and urine are transient and can reflect transport, metabolism, interorgan communication, and incorporation into macromolecules - making it difficult to disentangle the kidney’s specific role. A system enabling controlled manipulation and time-resolved read-outs in living kidney tissues would overcome these limitations. We engineered a microfluidic platform and workflow that, for the first time, allows precise modulation of metabolic inputs and continuous monitoring of metabolic outputs from native kidney tissues. Here, we apply microfluidic-coupled metabolomics to tubules and glomeruli to resolve their time-dependent metabolic responses under basal, nutrient-defined, and stressor conditions. Methods: Tubules and glomeruli were isolated from mice (C57BL/6NTac) and rats (SD/Janvier) using established protocols. Tissues were exposed to basic buffer (electrolytes, hydrogen phosphates, α-ketoglutarate, gluconate, acetate, and glycine) or amino acid- and/or hydrogen peroxide - containing buffers. Outputs were collected at 4°C, extracted, and analyzed using high-sensitivity targeted metabolomics, yielding more than 45,000 time-resolved metabolite measurements. Results: Fluidic-coupled metabolomics revealed distinct metabolic signatures of renal tubules versus glomeruli. Tubules secreted tricarboxylic acid cycle intermediates and acetylated amino acids, whereas glomeruli released purine-related metabolites. Amino acid application rapidly modulated tubular metabolism. Pronounced sex differences in tubular metabolism were uncovered under baseline, amino acid, and hydrogen peroxide application. Oxidative stress triggered rapid lactate release, followed by fumarate and malate during prolonged exposure, while impairing glucose release, putatively from gluconeogenesis. These changes were partially rescued by amino acid application, which moreover ameliorated tubular damage. Lysine oxidation to hydroxylysine emerged as a stress-response pathway. Hydroxylysine formation was markedly increased under oxidative stress, correlated with AKI severity in humans and pigs, indicating translational relevance as a potential metabolic injury marker, leaving a footprint in the tubular proteome of pigs. Conclusion: Microfluidic-coupled metabolomics of native kidney tubules and glomeruli enable precise, time-resolved interrogation of renal metabolism, i.e., core physiological functions as nutrient processing and stress responses. This approach provides mechanistic insight into acute kidney injury. Further efforts will be made to apply the system to a wide range of translational questions in health and disease. Disclosure of funding sources: MMR received commercial (Novo Nordisk), public (DFG), and private (Carlsberg Foundation) grants. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.