Emp3 promotes the progression of renal fibrosis in mice with unilateral ureteral obstruction by activating TGF-β/Smad3 signaling pathway.

BACKGROUND

Epithelial membrane protein 3 (Emp3), an N-glycosylated tetraspanin, is known to regulate tumor progression; however, its role in renal fibrosis (RF) remains unclear. This study aims to elucidate the function of Emp3 in RF.

METHODS

To evaluate Emp3's role, we utilized electroporation to deliver the Emp3-targeting RfxCas13d knockdown plasmid into the kidney cells of C57BL/6 mice, followed by the well-established unilateral ureteral obstruction (UUO) model. Renal pathology and injury were subsequently assessed. Additionally, immunohistochemistry, quantitative real-time PCR, and Western blot analyses were performed to evaluate fibrosis severity. In vitro, knockdown and overexpression of Emp3 were achieved via transfection in renal tubular epithelial cells (TCMK1) to further investigate Emp3's role. Co-immunoprecipitation (Co-IP) assays were employed to determine the interaction between TGF-β receptor 2 (TGFBR2) and Emp3.

RESULTS

Emp3 was found to be significantly upregulated in chronic kidney disease (CKD) patients and animal models. Knockdown of Emp3 using the RfxCas13d system in the UUO rodent model resulted in notable improvements in renal pathology and reduced the progression of renal fibrosis. Furthermore, Emp3 inhibition markedly suppressed TGF-β/Smad3 signaling activation. In TCMK1 cells, Emp3 knockdown produced similar results, while Emp3 overexpression accelerated pro-fibrotic cellular response. Mechanistically, Co-IP assays revealed a direct interaction between Emp3 and TGFBR2, which activates the TGF-β/Smad3 signaling pathway.

CONCLUSION

Our research demonstrates that Emp3 plays a critical role in the development of renal fibrosis by facilitating the activation of the TGF-β/Smad3 signaling pathway. Targeting Emp3 with RfxCas13d-mediated inhibition presents a promising therapeutic strategy for addressing RF in CKD.