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.

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.