Mechanistic elucidation of the therapeutic effects of Fushen Xiezhuo formula on chronic kidney disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Fushen Xiezhuo Formula (FSXZF) is a classical traditional Chinese medicine (TCM) prescription used to invigorate the spleen and reinforce the kidney, as well as to activate blood circulation and eliminate turbidity. Clinical practice has demonstrated its beneficial effects in chronic kidney disease (CKD). However, the specific bioactive constituents and molecular mechanisms responsible for its renoprotective actions remain insufficiently defined.

OBJECTIVE

The active chemical components and therapeutic mechanisms of FSXZF in an adenine-induced rat model of CKD were systematically investigated.

METHODS

A multidisciplinary strategy integrating serum pharmacochemistry, network pharmacology, molecular docking, and metabolomics was employed to comprehensively analyze the pharmacological basis of FSXZF against CKD. First, serum-absorbed constituents following oral administration of FSXZF were characterized using UHPLC-HRMS. CKD-related targets were subsequently predicted, and a component-target interaction network was constructed to identify potential hub targets. Molecular docking between the bioactive compounds and potential targets was conducted. A CKD model was further established by oral administration of adenine to rats (250 mg kg-1·day-1) for three consecutive weeks, a widely accepted protocol that recapitulates sustained renal dysfunction and pronounced tubulointerstitial fibrosis. Successfully modeled animals were randomly allocated to a Model group, a high-dose FSXZF group (FSXZF-H, 32.5 g kg-1·day-1), and a low-dose FSXZF group (FSXZF-L, 16.3 g kg-1·day-1), with treatments administered once daily for four weeks. A healthy Control group was maintained in parallel. At study termination, rats were euthanized for evaluation of body weight and renal functional parameters. Renal inflammatory cytokines (IL-1β, TNF-α, IL-17), oxidative stress markers (MDA, SOD, GSH-Px), and metabolic indicators (ATP, lactate, pyruvate) were quantified by ELISA. Western blotting was applied to assess protein changes related to the PI3K/AKT/mTOR-HIF-1α axis and glycolipid metabolism. Histopathological changes, fibrosis, and lipid accumulation were examined using H&E, Masson's trichrome, and Oil Red O staining, respectively. Mitochondrial ultrastructure was evaluated by transmission electron microscopy, and mitochondrial membrane potential and renal ROS levels were also assessed. Finally, untargeted UHPLC-HRMS-based metabolomics of kidney tissue was performed for the identification of differential metabolites and associated pathways, providing mechanistic insight into FSXZF-mediated metabolic regulation in CKD.

RESULTS

UHPLC-HRMS analysis identified 260 FSXZF-related circulating constituents, comprising 39 prototype compounds and 221 metabolites. Network pharmacology analysis based on the 39 parent compounds revealed significant enrichment of pathways related to PI3K/AKT signaling, HIF-1α signaling, and lipid metabolism and atherosclerosis. In vivo studies demonstrated that FSXZF exerted marked renoprotective effects in adenine-induced CKD rats. Treatment with FSXZF significantly increased body weight, reduced the kidney index, alleviated histopathological damage, and improved renal function, as evidenced by reductions in SCr, BUN, UA, and ACR (P < 0.05). In addition, FSXZF markedly suppressed renal overexpression of IL-1β, TNF-α, and IL-17, decreased MDA and ROS levels, enhanced SOD and GSH-Px activities, restored mitochondrial membrane potential, increased ATP production, reduced lactate and pyruvate accumulation, and was associated with lower phosphorylation levels of PI3K/AKT/mTOR and reduced HIF-1α expression (P < 0.05). Untargeted metabolomic analysis identified 32 core differential metabolites, primarily involved in energy and lipid metabolism pathways, including primary bile acid biosynthesis and one-carbon metabolism via the folate pool. These findings provide metabolic evidence supporting an association between FSXZF treatment and coordinated modulation of metabolic networks in CKD.

CONCLUSIONS

FSXZF ameliorated renal dysfunction and tubulointerstitial fibrosis in adenine-induced CKD rats. These benefits were associated with reduced activation of the PI3K/AKT/mTOR-HIF-1α axis, attenuated inflammation and oxidative stress, and partial restoration of disturbed energy metabolism. Integrated evidence from serum pharmacochemistry, network analysis, molecular docking, in vivo assays, and renal metabolomics supports FSXZF as a multi-target therapeutic candidate against CKD; however, pathway dependency remains to be validated.