Taxifolin alleviates podocyte injury and macrophage chemotaxis associated with diabetic nephropathy by directly targeting RBP1 identified via multi-omics and machine learning

Abstract

Podocyte injury contributes to diabetic nephropathy (DN) progression. This study aimed to screen and validate the key functional proteins related to podocyte injury in DN, and to explore the protective effect of bioactive ingredients derived from traditional Chinese medicine Linggui Zhugan (LGZG) formula against DN. Proteomics, weighted gene co-expression network analysis, machine learning, and repeated out-of-fold exact SHapley Additive exPlanations (TreeSHAP) analysis were integrated to screen the top 15 core differentially expressed proteins in kidney tissues of DN patients. Single-cell transcriptomics and cell-cell communication analysis were further used to identify the podocyte protein retinol-binding protein 1 (RBP1). In vitro , a palmitic acid (PA)-induced podocyte injury model and gene knockdown experiments were performed to verify the regulatory function of RBP1. Molecular docking and microscale thermophoresis (MST) were applied to screen and confirm the binding interaction between bioactive ingredients of LGZG formula and RBP1. The renoprotective effect of taxifolin was further evaluated in this cell model. Integrative multi-omics and machine learning identified RBP1 as a key pathogenic molecule in DN patients. In vitro , PA stimulation increased RBP1 protein expression in podocyte injury, with the downregulation of podocyte-specific slit diaphragm proteins nephrin and synaptopodin, and the upregulation of pro-inflammatory mediators including C-C motif chemokine ligand 2 ( CCL2 ), interleukin-6 ( IL-6 ), and tumor necrosis factor-α ( TNF-α ), as well as enhanced macrophage chemotaxis. Notably, RBP1 knockdown reversed these PA-induced alterations to alleviate podocyte injury. Taxifolin from LGZG formula directly bound RBP1, as confirmed by molecular docking and MST. Furthermore, taxifolin was found to bind to RBP1. And then it suppressed nuclear factor κB (NF-κB) signaling pathway, reduced CCL2 , IL-6 and TNF-α mRNA expression, inhibited macrophage chemotaxis, and upregulated nephrin and synaptopodin expression, thereby ameliorating PA-induced podocyte injury. This study identified RBP1 as a novel driver of podocyte injury and macrophage chemotaxis associated with DN via multi-omics and machine learning. Taxifolin, a bioactive compound from the LGZG formula, directly bound to RBP1 and alleviated podocyte injury. These findings highlight RBP1 as a promising therapeutic target and support the potential of taxifolin as a targeted intervention for DN progression.