Mass Spectrometry-based Multi-omics Profiling Identifies Bioactive Compound Astragaloside IV as a Therapeutic Agent Targeting Cltc K856 Acetylation to Restore Tyrosine Metabolic Zonation in Diabetic Nephropathy

• First visualization of kidney tyrosine metabolic zonation via MALDI-MSI in DN. • Astragaloside IV suppresses Cltc K856 acetylation to restore spatial tyrosine fluxes in DN. • ASIV binds Cltc via hydrogen/electrostatic interactions, validated by molecular dynamics simulations. • Cltc acetylation links tyrosine metabolites to renal dysfunction markers. • ASIV rescues podocyte injury by reprogramming acetylation-dependent tyrosine metabolism. Diabetic nephropathy (DN) progression is driven by spatial metabolic dysregulation. However, the role of acetylation-mediated tyrosine metabolism zonation in this process remains unexplored. Here, we integrate spatial metabolomics, acetyl-proteomics, and MALDI mass-spectrometry imaging (MSI) to unveil Clathrin heavy chain 1 (Cltc) K856 acetylation as a therapeutic nexus in DN. Multi-omics profiling of DN patients revealed tyrosine metabolism as the most dysregulated pathway, with L-tyrosine, dopaquinone, and 4-hydroxycinnamic acid exhibiting strong correlations with renal dysfunction markers. Spatial MSI of diabetic kidneys uncovered distinct zonation: L-tyrosine accumulation in the inner medulla, 4-hydroxycinnamic acid in the outer medulla, and dopaquinone enrichment in the cortex - patterns rectified by Astragaloside IV (ASIV), a bioactive phytochemical from Astragalus membranaceus . Astragaloside IV has pharmacological properties in the regulating metabolic disorders in DN, and acetylation modification impacting tyrosine metabolism need to be explored. Mechanistically, ASIV suppressed Cltc acetylation at K856, confirmed by acetyl-proteomics and MS/MS validation in db/db mice, thereby rebalancing tyrosine metabolic fluxes. Molecular dynamics simulations demonstrated ASIV’s stable binding to Cltc via hydrogen bonding and electrostatic complementarity, stabilizing low-energy conformations. In high glucose-stressed podocytes, ASIV temporally restored tyrosine homeostasis, with Cltc expression positively correlating with L-tyrosine/dopaquinone and inversely with 4-hydroxycinnamic acid. This study pioneers the visualization of kidney metabolic zonation and identifies ASIV as a first-in-class acetyl-Cltc inhibitor targeting spatial tyrosine reprogramming in DN. Our findings bridge functional food-derived therapeutics with precision modulation of spatiotemporal metabolic disorders, offering a paradigm for acetylation-targeted interventions in diabetic complications.