Ubiquitin-specific protease 7 is associated with vascular calcification in chronic kidney disease by regulating RUNX2 stabilization

Background There is a severe problem of vascular calcification (VC) in chronic kidney disease (CKD). The purpose of this study was to explore the function and underlying mechanisms of ubiquitin-specific protease 7 (USP7) in VC-associated CKD. Methods Plasma USP7 was measured in 22 maintenance-hemodialysis patients with VC, 51 without VC, and 20 healthy controls. Calcification was modeled in MOVAS cells using 10 mM β-glycerophosphate and in mice using an adenine/high-phosphate diet; USP7 was manipulated by siRNA, overexpression, or HBX19818 treatment. Alizarin Red Staining and calcium content/alkaline phosphatase activity were used for calcification analysis. Protein interaction and stability analysis were performed by immunoprecipitation and MG132-rescue experiments. Results Plasma USP7 was significantly higher in hemodialysis patients with VC compared to those without VC and healthy subjects, and positively correlated with blood urea nitrogen. USP7, SMAD2, and RUNX2 were elevated; on the other hand, SM22α decreased in calcified VSMCs, calcified mouse arteries, and calcified radial arteries from dialysis patients. Knockdown of USP7 or application of HBX19818 significantly reduced high-phosphate-induced VSMC calcification, osteogenic differentiation and SMAD2/RUNX2 levels, and overexpression of USP7 increased calcification. HBX19818 treatment reduced arterial calcification in adenine-diet mice in vivo, decreased calcium deposition, restored SM22α levels. RUNX2 overexpression, but not SMAD2, abolished the anti-calcifying effect of USP7 silencing. MG132 rescue and co-immunoprecipitation were consistent with a calcification-dependent USP7-RUNX2 interaction and proteasome-related RUNX2 stabilization, but direct deubiquitination was not tested. Conclusions These findings reveal a calcification-dependent USP7-RUNX2 interaction and suggest that USP7 regulates RUNX2 protein stability via a proteasome-dependent mechanism, without establishing direct deubiquitination.