Metabolite-proteome interactions are pleiotropic disease drivers in kidney disease

Study objective The objective of this study was to identify toxic gut-derived metabolites that accumulate in kidney tissue in acute kidney injury (AKI) and chronic kidney disease (CKD), characterise their molecular targets, and assess their potential pathogenic roles to understand the mechanisms of kidney disease. Hypothesis Metabolites are trapped in kidney tissue and bind to proteins, altering their expression and stability and thereby disrupting functional pathways in disease. Methodology Integrated multi-omics strategy combining metabolomics, proteomics, phosphoproteomics, kinome profiling (kinobeads), and thermal proteome profiling (TPP) across cisplatin, adenine, and ischemia-reperfusion injury (IRI) mouse models of AKI and CKD. Data Metabolomics analysis of kidney cortex tissue identified 25 upregulated metabolites in AKI related to tryptophan, indole, and amino acid metabolism (AAM). Three metabolites - 3-indoxyl sulfate (3IS), nudifloramide (2PY), and indole-3-lactic acid (ILA) were elevated across both AKI and CKD models and are known gut-derived toxic metabolites. Proteomics results supported metabolite accumulation, revealing that 45 transporters were downregulated across all kidney injury models, including Slc22a8, Slc22a6, and Abcc2, which mediate renal excretion of toxic metabolites. TPP of AKI kidneys identified 197 destabilised and 243 stabilised proteins, which were regulated independently at the expression level. Phosphoproteomics revealed that a cyclin-dependent kinase (CDK) motif is overrepresented in AKI, although this is not driven by toxic metabolites, as confirmed by kinome profiling. TPP of control kidney tissue treated with 3IS, 2PY and ILA revealed that they bound the same proteins observed in TPP of AKI kidneys, among them their known transporters - Slc22a8, Slc22a2, Slc22a6, Slc47a1, Abcc4, Abcc2, putative - Slc16a1, Slc3a2, Slc13a3, but also mitochondrial proteins of the complex I of the respiratory chain (Ndufv3, Ndufb6). Microphysiological coupled metabolomics of 3IS-treated native tubules showed changes in tricarboxylic acid cycle (TCA) intermediates. Complex I- and II-linked substrates malate, citrate, and lactate were decreased, while riboflavin and adenosine, as readouts of mitochondrial function, increased, and biotin, as an energy-generating pathway's cofactor, decreased. Summary of results In kidney injury, metabolites accumulate in kidney tissue, altering both protein abundance and stability. Although CDKs change in AKI, these shifts are not driven by 3IS, ILA, or 2PY. Instead, 3IS, elevated across all injury models, perturbs mitochondria, transport, and core metabolic pathways, such as the TCA, fatty acid oxidation, and AAM, by altering the stability and expression of key enzymes, ultimately contributing to the progression of kidney dysfunction. Conclusions Our study shows that toxic gut-derived metabolites accumulating in injured kidneys interact with metabolic enzymes and transporters, disrupting key pathways and contributing to kidney dysfunction. 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.