ETHNOPHARMACOLOGICAL RELEVANCE:Diabetic kidney disease (DKD) is a prevalent microvascular complication of diabetes mellitus (DM) that significantly impairs patients' quality of life. Its early stage is characterized by glomerular filtration barrier damage and the occurrence of proteinuria. Without timely intervention, this condition progresses to renal fibrosis, and therapeutic options remain limited despite recent advances. Yitangkang decoction (YTK) has shown potential in ameliorating this barrier damage, thereby inhibiting proteinuria and ultimately preventing renal fibrosis; however, its precise mechanisms remain unclear.
AIM OF THE STUDY:This study aimed to elucidate the therapeutic mechanism by which YTK ameliorates glomerular filtration barrier damage in DKD.
MATERIALS AND METHODS:db/db mice were treated with high-, medium-, or low-dose YTK or with finerenone for 8 weeks. We employed a multi-omics approach-including data-independent acquisition (DIA) proteomics, transcriptomics, and palmitoyl-proteomics coupled with LC-MS/MS-to identify differentially expressed proteins, mRNAs, and palmitoylation sites related to YTK's renoprotective effects, as well as to characterize its absorbed serum components. In vitro validation was performed in D-glucose-injured MPC5 podocytes, mouse renal microvascular endothelial cells and mouse glomerular mesangial cells using YTK-medicated serum, finerenone-medicated serum, the active components puerarin and quercitrin, or pharmacological modulators of the AMPK and TGF-β1 pathways. YTK's efficacy was evaluated using biochemical, histopathological, and molecular biological indicators.
RESULTS:YTK treatment dose-dependently reduced urinary albumin-to-creatinine ratio (UACR), serum creatinine (Scr), urea, uric acid (UA), and lipid peroxide (LPO) levels, while restoring glutathione (GSH) levels and alleviating renal pathology in db/db mice. Multi-omics integration revealed enrichment in pathways involving extracellular matrix (ECM)-receptor interaction, AMPK signaling, and glutathione metabolism. YTK attenuated glomerular filtration barrier damage through several coordinated mechanisms: (1) Inhibition of the TGF-β/Smad pathway, downregulating TMEM2, TGF-β1, Smad2/3/4, and MMP-9; (2) Enhancement of SLC7A11 palmitoylation via the AMPKα1/ZDHHC8 axis, which stabilized SLC7A11 protein and inhibited ferroptosis-a role confirmed by site-directed mutagenesis of the critical Cys327 site; (3) Direct modulation of macrophage polarization, suppressing pro-inflammatory M1 markers and promoting anti-inflammatory M2 markers; and (4) Multi-target cytoprotection against renal cell injury. Surface plasmon resonance (SPR) analysis confirmed the direct, concentration-dependent binding of puerarin and quercitrin to GPX4 and TGF-β1. In vitro, YTK-medicated serum, puerarin, and quercitrin restored viability in injured MPC5 podocytes, renal microvascular endothelial cells and glomerular mesangial cells, induced consistent mRNA expression changes of key targets across both cell types, and inhibited apoptosis via modulation of the TGF-β/Smad and AMPK/SLC7A11 pathways. Specificity validation of the palmitoylation detection method reinforced the reliability of the omics findings.
CONCLUSIONS:This study comprehensively elucidates the multi-cellular protective mechanism of YTK on the glomerular filtration barrier. We demonstrate that YTK confers protection via a dual-pathway mechanism: (1) Suppression of the injury pathway by inhibiting TGF-β1/Smad overactivation via modulation of macrophage phenotype and ECM homeostasis, thereby mitigating inflammation and cellular damage; and (2) Activation of the protective pathway by enhancing SLC7A11 palmitoylation through the AMPKα1/ZDHHC8 axis, which stabilizes SLC7A11, restores glutathione metabolism, and inhibits ferroptosis. These two axes functionally interact to rebalance injury and protective signaling. Furthermore, puerarin and quercitrin were identified as key serum-absorbed constituents that directly bind to targets including TGF-β1 and GPX4, mediating the described dual-pathway regulation. In summary, YTK represents a promising multi-target therapeutic strategy that coordinately regulates injury and protective signaling networks to prevent and alleviate proteinuria in DKD.