Research On The Mechanism Of Osteogenic Transdifferentiation Of Vascular Smooth Muscle Cells In Chronic Kidney Disease-associated Vascular Calcification

Background and objective:Chronic kidney disease(CKD)is a major factor that affects global health.Vascular calcification is a common complication of CKD and a critical component of chronic kidney disease-mineral and bone disorder(CKD-MBD).Substantial clinical evidence confirms that CKD-associated vascular calcification increases the incidence and mortality of cardiovascular events in patients with CKD.Vascular calcification has become a research hotspot in CKD owing to its high prevalence,multiple risk factors,and severe prognostic impact;but its mechanism remains unclear,and its prevention and treatment are difficult.Recent studies have shown that osteogenic transdifferentiation of vascular smooth muscle cells(VSMCs)is a key processe in the occurrence of vascular calcification in patients with CKD.Therefore,this study aimed to clarify the specific mechanism of vascular calcification in CKD,observe the abnormal phenotype,discuss the mechanism of CKD-MBD,elucidate the mechanism of osteogenic transdifferentiation of VSMCs in CKD-associated vascular calcification,identify potential therapeutic targets and elucidate their role,so as to effectively prevent and treat vascular calcification and improve the quality of life and survival rate of patients with CKD.Methods:First,a vascular calcification model of 5/6 nephrectomy with a highphosphorus diet was established in rats.Kidney function and aortic calcification were assessed using biochemical analyses,routine pathological analysis and alizarin red S staining.The microstructures of the femur and mandibular alveolar bone were evaluated using micro-CT,and serum metabolic parameters were evaluated using ELISA.Differential gene profiles were detected by full-length transcriptome sequencing of mRNA in the aortic tissue of CKD rats and were analyzed using bioinformatics.The levels of USP47,SOST and WNT5A/WNT5B in the aortas of CKD rats were detected using immunohistochemical staining,and the levels of ITPR2 in the serum of CKD rats were detected using ELISA.Meanwhile,rat VSMCs were induced by high phosphorus levels,and alizarin red S and Von kossa staining were used to observe calcium salt deposition.Label-free quantification was performed to detect VSMCs using nano liquid chromatography-mass spectrometry.Differentially expressed proteins were enriched and analyzed.VSMCs were transfected with USP47 siRNA.Real-time PCR or western blotting were used to verify USP47,ITPR2,SOST,WNT5A,and WNT5B expression in rat VSMCs,MiRNAs targeting WNT5 A or WNT5B were predicted using TargetScan and verified by real-time PCR in rat VSMCs and aortic tissue.Additionally,clinical samples were collected from patients with CKD,and USP47 levels in calcified vascular tissues were determined using immunohistochemistry.Serum USP47 and ITPR2 levels in patients with CKD-associated vascular calcification were measured using ELISA,and clinical correlations were analyzed.Results:1)Serum creatinine,blood urea nitrogen,and the urinary protein/creatinine ratio were significantly increased,kidney injury and vascular calcification were evident,and the CKD-MBD phenotype was observed in 5/6 nephrectomy rats with a highphosphorus diet.The bone phenotype was characterized by deterioration of the trabecular microstructure in the femur and aggravated alveolar bone loss in CKD rats.The abnormal metabolic phenotype showed disordered bone resorption and formation in CKD-MBD.After high-phosphorus treatment,RUNX2 increased and TAGLN decreased in rat VSMCs,suggesting that the cell contractile phenotype switched to the synthetic phenotype,showing the phenotypic characteristics of osteogenic transdifferentiation.2)Label-free quantification analysis showed that the expression of USP47 significantly increased after high-phosphorus treatment,which was confirmed by western blotting.The expression of USP47 increased in patients and rat vascular tissues after CKD-associated vascular calcification.Serum USP47 levels were slightly increased in patients with CKDassociated vascular calcification,and USP47 expression was positively correlated with blood urea nitrogen levels and negatively correlated with dialysis vintage.USP47 gene silencing can alleviate calcium deposition induced by high phosphorus levels in rat VSMCs and significantly reduced BTRC/AKT1 expression,affecting the mRNA levels of FGF23 and MGP.3)Gene ontology enrichment analysis of full-length transcriptome sequencing data showed that important biological processes were related to bone remodeling and smooth muscle cell differentiation.ITPR2 expression was significantly downregulated in the aortas of CKD rats and VSMCs after high-phosphorus treatment.Serum ITPR2 levels in CKD rats first decreased at 4 weeks,followed by a slight increase without a statistical difference at 16 weeks.Serum ITPR2 levels were significantly increased in patients with CKD-associated vascular calcification,positively correlated with TRACP-5B,and negatively correlated with blood urea nitrogen.4)Kyoto Encyclopedia of Genes and Genomes analysis showed that the potential key pathways were related to the process of osteogenic transdifferentiation in VSMCs,including the Wnt signaling pathway.Aortic sequencing showed that the expression of SOST was upregulated,and that of WNT5A/WNT5B was downregulated in CKD rats.Interaction analysis showed that SOST and WNT5A are closely linked to RUNX2 through RUNX2 to TAGLN.SOST,WNT5A,and WNT5B were closely linked to each other.Sclerostin increased,WNT5A and WNT5B decreased,RUNX2 increased,and TAGLN decreased in the arterial medial layer of CKD rats.Similarly,sclerostin increased and WNT5A and WNT5B levels decreased in rat VSMCs after high-phosphorus treatment.Among the miRNAs targeting WNT5A or WNT5B,the expression levels of miR-542-3p,miR-2983p,miR-376b-5p,and miR-3568 were significantly reduced,whereas that of miR-742-3p was significantly increased in rat VSMCs after high-phosphorus treatment.The expression levels of miR-542-3p,miR-298-3p,miR-376b-5p,miR-3568,miR-742-3p,and miR-22-5p were significantly reduced in CKD rat aortas.With CKD progression,the expression levels of miR-542-3p,miR-376b-5p,miR-3568,and miR-22-5p were greatly decreased in CKD rat aortas.Conclusion:Based on the rat model of vascular calcification in 5/6 nephrectomy with a high-phosphorus diet and the rat VSMCs model induced by high phosphorus,our study revealed that:1)USP47 may be associated with vascular calcification in CKD by regulating the osteogenic transdifferentiation of VSMCs through the BTRC/AKT1 pathway.2)ITPR2 may be involved in the osteogenic transdifferentiation of VSMCs in CKD-associated vascular calcification and play an important role in CKD-MBD.3)Sclerostin increased in CKD-associated vascular calcification following a reduction of the non-canonical Wnt signaling WNT5A/WNT5B.They may become potential biological targets for diagnosis,prognosis assessment,and treatment,and provide more reference for the clinical prevention and treatment of CKD-associated vascular calcification.