Pretreatment of metanephric mesenchymal cells with catalpol mitigates acute kidney injury through VEGF-A secretion via multiple mechanisms.

Abstract

BACKGROUND: Metanephric mesenchymal cells (MMCs) hold therapeutic potential for acute kidney injury (AKI), but their efficacy is limited, and the mechanisms underlying their action remain unclear. This study aimed to investigate whether catalpol-pretreated MMCs (MMCs-cata) could enhance the efficacy of AKI treatment by regulating key signaling pathways. METHODS: An AKI model was established in C57BL6 mice via intraperitoneal injection of cisplatin, and the therapeutic effects of MMCs-cata were compared with those of untreated MMCs. RNA-Seq was performed to analyze differentially expressed genes, Western blotting and ELISA were used to measure VEGF-A levels in MMC-cata and the supernatants. An in vitro model of cisplatin-induced renal tubular epithelial cell injury was developed to investigate the signaling pathways upstream and downstream of VEGF-A. The role of VEGF-A/VEGFR2 was confirmed through experiments involving gene silencing, neutralizing antibodies, and VEGFR2 blockade. Additionally, molecular docking simulations and Western blotting were performed to explore the effects of catalpol on the Wnt signaling pathway. RESULTS: MMCs-cata significantly improved renal function in AKI model mice by suppressing inflammation, oxidative stress, and necroptosis. RNA-Seq, Western blotting and ELISA revealed the activation of VEGF-related genes in MMCs-cata along with elevated intracellular and supernatant VEGF-A levels. In both the in vivo and in vitro models, silencing VEGF-A in MMCs-cata, neutralizing VEGF-A in the supernatant, or blocking VEGFR2 in tubular epithelial cells abolished the protective effects of MMCs-cata, while exogenous VEGF-A supplementation alone exerted protective effects. Mechanistic studies indicated that MMCs-cata activated the p38 pathway and suppressed the STAT3 pathway. Molecular docking and Western blotting confirmed that catalpol binds to Wnt3A, activating the canonical Wnt pathway to drive VEGF-A secretion. CONCLUSION: Catalpol pretreatment enhances the therapeutic efficacy of MMCs by activating the canonical Wnt pathway to promote VEGF-A secretion. VEGF-A interacts with VEGFR2 on renal tubular epithelial cells, likely through both p38 pathway activation and STAT3 pathway inhibition, thereby suppressing inflammation, oxidative stress, and necroptosis to alleviate AKI. This study provides novel insights into the integration of traditional Chinese medicine components with stem cell therapy for AKI management.