E3 Ubiquitin Ligase RNF10 Negatively Regulates Rbpjk Expression During Vascular Calcification in Chronic Kidney Disease

BACKGROUND: Vascular calcification is frequent among patients with chronic kidney disease, in which vascular smooth muscle cells (VSMCs) play a key pathological role. Our previous research revealed that the E3 ubiquitin ligase RNF10 (ring finger protein 10) regulates VSMC hyperproliferation and apoptosis; however, the function of RNF10 in vascular calcification remains unknown. METHODS: Serum RNF10 levels were assessed in 2 independent cross-sectional chronic kidney disease patient cohorts with vascular calcification. RNF10 expression was assessed in calcified rat aortas and VSMCs. The effects of RNF10 on vascular calcification were evaluated in Rnf10 knock-in rats and RNF10 overexpressed VSMCs. To investigate the underlying mechanism, RNA-seq, ChIP-seq, ChIP-qPCR, and luciferase reporter assays were performed. Gain- and loss-of-function experiments targeting Rbpjk were conducted in vivo and in vitro. RESULTS: Circulating RNF10 levels were significantly reduced in chronic kidney disease patients with vascular calcification, and RNF10 protein expression was reduced in calcified rat aortas and VSMCs. Rnf10 knock-in alleviated vascular calcification without obvious adverse effects on other major organs. In vitro, RNF10 overexpression attenuated calcification and reduced the expression of osteogenic markers. Notably, pharmacological inhibition of the ubiquitin-proteasome system did not impair RNF10's anticalcific activity. Nuclear RNF10 expression was increased in calcified VSMCs, supporting a noncanonical and transcriptional regulatory role. Integrated analyses identified that RNF10 negatively regulated Rbpjk expression during vascular calcification, rather than acting through the ubiquitin-proteasome system. Moreover, viral Rbpjk overexpression partially reversed the protective effects of RNF10 in vivo and in vitro and increased osteogenic marker expression, whereas siRNA-mediated Rbpjk knockdown reduced the marker expression. CONCLUSIONS: RNF10 depletion serves as the initiating factor that triggers Rbpjk-driven osteogenic differentiation during chronic kidney disease-associated vascular calcification. The protective role of RNF10 involves Rbpjk negative expression regulation rather than E3 ubiquitin ligase activity. This RNF10-Rbpjk regulatory axis provides a new perspective for developing prevention and treatment strategies for vascular calcification.