UHPLC/Q-TOF-MS-based blood-component profiling and multi-omics analysis reveal potential protective mechanisms of...

Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, with no definitive treatment currently available for DKD-related kidney injury. Podocyte injury, apoptosis, and microcirculation dysfunction play critical roles in the pathogenesis and progression of DKD. Shenzhuo Formula (SZF), a traditional Chinese herbal formulation, has shown clinical potential in reducing proteinuria, but its chemical basis and protective mechanisms remain unclear. This study combined Ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC/Q-TOF-MS) analysis, network pharmacology, renal proteomics, metabolomics, single-nucleus RNA sequencing (snRNA-seq), and in vivo validation to explore the potential mechanisms of SZF against DKD-related renal injury. UHPLC/Q-TOF-MS analysis annotated 27 SZF-related serum constituents, including 19 putative prototype constituents and 8 putative metabolites. Network pharmacology predicted 87 overlapping targets between SZF-related constituents and DKD, mainly enriched in apoptosis, inflammatory responses, focal adhesion, and metabolic regulation. In db/db mice, SZF reduced urinary albumin-to-creatinine ratio and renal injury biomarkers, including NGAL, CysC, and KIM-1, increased estimated glomerular filtration rate, and alleviated glomerular basement membrane thickening, mesangial matrix expansion, inflammatory infiltration, and collagen deposition. Proteomic and metabolomic analyses suggested that SZF regulated pathways related to necroptosis, complement and coagulation cascades, platelet activation, amino acid metabolism, and the citrate cycle. snRNA-seq and histological validation indicated that SZF reduced apoptosis-related gene signatures and podocyte injury. Western blot analysis showed increased BCL-2 expression and decreased cleaved Caspase-3 and cleaved PARP1 expression after SZF treatment. In zebrafish models, SZF improved microcirculatory phenotypes by increasing vessel diameter, cardiac output, and blood-flow velocity, and by reducing thrombus incidence. These hypothesis-generating findings suggest that SZF warrants further investigation as a potential modulator of podocyte apoptosis and microcirculatory dysfunction in DKD.