Verapamil Reduces Collagen Synthesis to Alleviate Ureteral Scar Formation by Inhibiting KDM1A-Mediated Epigenetic Silencing of SESN2.

INTRODUCTION

Ureteral scar formation is a fibrotic process linked with collagen synthesis. Although autophagy prevents collagen synthesis, its role in ureteral scarring remains unclear. Therefore, this study investigated the modulation of SESN2-mediated autophagy by verapamil during ureteral scar formation.

METHODS

An in vitro fibrosis model was constructed using transforming growth factor-β1 (TGF-β1)-stimulated primary fibroblasts. We established a rat model of ureteral stricture (US), and KN-93 was used to inhibit the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMK II). Histopathological alterations and collagen accumulation in rat ureteral tissues were examined using hematoxylin-eosin and Masson staining. The levels of vimentin, α-SMA, and LC3 were determined using immunofluorescence. Autophagic flux was assessed using an mCherry-GFP-LC3 assay. mRNA and protein levels were assessed by quantitative real-time PCR and Western blot, respectively. Additionally, co-immunoprecipitation and GST pull-down assays were used to detect the binding relationship between CaMK II and lysine-specific histone demethylase 1A (KDM1A). The interaction of H3K4me2 and KDM1A with the SESN2 promoter in fibroblasts was verified using chromatin immunoprecipitation.

RESULTS

Verapamil increased autophagy in TGF-β1-stimulated fibroblasts and alleviated ureteral fibrosis in US rats by inhibiting CaMK II phosphorylation (p-CaMK II) to reduce collagen synthesis. Moreover, p-CaMK II promoted the nuclear translocation of KDM1A by phosphorylating KDM1A. Additionally, the effects of KDM1A knockdown on the autophagy and collagen synthesis in TGF-induced fibroblasts were reversed by SESN2 knockdown. Finally, KDM1A knockdown further enhanced the effect of verapamil in promoting SESN2-mediated fibroblast autophagy and suppressed collagen synthesis.

CONCLUSION

Verapamil reduced collagen synthesis during ureteral scar formation by inhibiting the phosphorylation of KDM1A by p-CaMK II, thus, decreasing the demethylation of H3K4me2 on the SESN2 promoter to enhance SESN2-mediated autophagy.