Single-cell transcriptomics and multi-omics integration identify necroptosis-driven core targets and tubular-immune...

Current pharmacotherapies for chronic kidney disease (CKD) are limited by adverse effects and a "one-target-one-drug" paradigm. Necroptosis is a key driver of renal deterioration, yet its regulatory network remains poorly defined. Modified Da-Huang-Fu-Zi Decoction (DHFZ) has shown clinical efficacy, although its underlying mechanism, particularly in relation to necroptosis, requires further clarification.

Bulk and single-cell RNA sequencing data from human kidneys were integrated to screen for necroptosis-related genes in CKD. Systems pharmacology was used to identify key DHFZ targets. SHAP interpretation, an independent GEO cohort, and the Nephroseq database were used to validate these targets. Molecular docking was performed to characterize binding modes between DHFZ bioactive constituents and candidate proteins. Pseudotime trajectory and cell-cell communication analyses were used to map intercellular signaling. The therapeutic effects of DHFZ were evaluated in a 5/6-nephrectomy rat model of CKD by assessing renal function, histopathology, and subcellular ultrastructure. Western blotting, qPCR, and ELISA were used to quantify core targets, necroptosis markers, and oxidative stress indices. Immunofluorescence and qPCR further verified modulation of the THBS1-CD47 axis.

Three necroptosis-related hub genes, TP53, SIRT1, and CASP1, were identified and independently validated. Aloe-emodin showed the highest binding affinity for all three proteins. Pseudotime analysis showed that VCAM1⁺ proximal tubular (PT) cells accumulated in inflamed branches with active CASP1, whereas SIRT1 was enriched in reparative CUBN⁺ PT subsets. In CKD rats, SIRT1 expression was downregulated, whereas TP53, CASP1, oxidative stress markers (4-HNE, MDA, and 8-OHdG), and necroptosis signatures (p-RIPK1, p-RIPK3, and p-MLKL) were markedly increased. DHFZ and the SIRT1 agonist SRT1720 restored SIRT1 levels and attenuated renal injury. VCAM1⁺ PT cells highly expressed THBS1, which engaged macrophage CD47 and promoted inflammation. DHFZ disrupted the THBS1-CD47 axis, thereby reducing macrophage infiltration and cytokine production.

Multi-omics profiling showed that DHFZ confers nephroprotection by restoring SIRT1-mediated suppression of necroptosis in VCAM1⁺ PT cells and disrupting maladaptive THBS1-CD47 tubular-macrophage crosstalk. These findings identify SIRT1 as a tractable therapeutic target in CKD.