CREB1/CRTC2 regulated tubular epithelial-derived exosomal miR-93-3p promotes kidney injury induced by calcium oxalate via activating M1 polarization and macrophage extracellular trap formation

Abstract

Background: Calcium oxalate (CaOx) crystals are known to cause renal injury and trigger inflammatory responses. However, the role of exosome-mediated epithelial-macrophage communication in CaOx-induced kidney injury remains unclear. Methods: To identify key molecules, miRNA sequencing was conducted on exosomes derived from CaOx-treated (CaOx-exo) and control (Ctrl-exo) epithelial cells, identifying miR-93-3p as significantly upregulated. A combination of dual-luciferase reporter assays, Western blot, RT-qPCR, immunofluorescence staining, flow cytometry, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation-qPCR (CHIP-qPCR) was used to explore the regulation of miR-93-3p by CREB1/CRTC2 and its downstream effects on NFAT5/Akt1/NIK/NF-κB2 signaling in macrophages. The functional roles of NFAT5 in macrophage polarization and macrophage extracellular traps (METs) formation were further evaluated both in vitro and in vivo. Results: Epithelial exosomes stimulated by CaOx crystals were found to promote kidney injury via macrophage polarization and METs formation. Treatment with NIK SMI1, a NIK inhibitor, or CI-amidine, a METs inhibitor, mitigated crystal deposition and CaOx-induced kidney damage. Overexpression of NFAT5 in a CaOx-induced mouse model reduced renal injury and crystal deposition, downregulated NIK and NF-κB2 levels, and decreased the number of M1-polarized macrophages. Mechanistic studies revealed that miR-93-3p directly targets NFAT5 mRNA, as confirmed by dual-luciferase assays, qRT-PCR, and Western blot. Additionally, we demonstrated that CREB1/CRTC2 acts as a transcriptional activator of miR-93-3p. Inhibition of miR-93-3p partially reversed NIK/NF-κB2 activation and alleviated kidney injury. Conclusions: CaOx crystals exacerbate renal interstitial injury by promoting M1 macrophage polarization and METs formation through the CREB1/CRTC2–exosomal miR-93-3p–NIK/NF-κB2 signaling pathway. Targeting this pathway may provide therapeutic avenues for mitigating crystal deposition-induced kidney damage.