Spatial Metabolomics Identifies Riboflavin Metabolism as a Therapeutic Target of Huangqi Guizhi Wuwu Decoction in Diabetic Nephropathy

Understanding kidney metabolic heterogeneity is critical for unraveling diabetic nephropathy (DN) pathogenesis. While Huangqi Guizhi Wuwu decoction (HGD) shows efficacy against DN, its spatial metabolic effects remain unknown. We integrated liquid chromatography-mass spectrometry (LC-MS) metabolomics, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and tandem mass tag (TMT) proteomics to map systemic and microregional metabolic landscapes in HGD-treated db/db mice. Spatial metabolomics identified seven compartmentalized metabolites: citrulline, dibutyl phthalate, and hydroxykynurenine showed pan-renal distribution, whereas riboflavin, γ-glutamylcysteine, 7-methylguanine, and L-4-hydroxyphenylglycine localized specifically to the cortical outer layer. HGD restored glomerular architecture, reduced hyperglycemia, and normalized glycated hemoglobin, with MALDI-MSI visualizing cortical metabolite redistribution. Proteomics revealed 10 differentially expressed proteins, and multi-omics integration linked HGD's effects to riboflavin metabolism, arginine biosynthesis, and pancreatic secretion pathways. Crucially, spatial correlation analysis uncovered a cortex-specific metabolic axis between riboflavin and citrulline, indicating compartmentalized cross-talk between vitamin metabolism and urea cycle regulation. This establishes HGD as a spatial metabolic modulator rectifying microregional imbalance in DN and demonstrates how MALDI-MSI-guided multi-omics deciphers tissue-specific herbal pharmacodynamics, providing a paradigm for investigating traditional medicine through spatially resolved molecular mapping.