Effect Of EPO On Vascular Calcification And The Related Mechanism In CKD Rats

Background and Objective Cardiovascular disease is the leading cause of death in patients with chronic kidney disease(CKD).Vascular calcification is very common in CKD patients and is an important cause of high incidence of cardiovascular disease in CKD patients.Erythropoietin(EPO)is currently the first choice for the treatment of renal anemia,but studies have found that the dose of EPO is closely related to the incidence and mortality of cardiovascular diseases in patients with CKD.On the other hand,recent studies have revealed that EPO can promote the maturation of osteoblasts and chondrocytes,enhance bone healing,and this process is very similar to the pathological process of CKD vascular calcification.The aim of this study was to investigate whether EPO could affect the process of vascular calcification in CKD rats and to explore the related mechanism.Methods 1.Experiments in vivo(1)CKD rat model was established by 5/6 nephrectomy(5/6 Nx) and high phosphorus diet was given to promote the occurrence of vascular calcification.(2)Twenty-four SD rats were randomly divided into four groups(6rats in each group): sham-operated control group,sham-operated+EPO group,CKD group and CKD+EPO group.Eight weeks after the intervention of EPO(6000 IU/kg/week,subcutaneous injection),rats in each group were sacrificed.Blood samples were taken to detect hemoglobin,creatinine,urea nitrogen,calcium and phosphorus,and abdominal aorta was taken for Von Kossa staining and calcium quantitative detection.2.Experiments in vitro(1)Rat thoracic aorta was isolated and primary vascular smooth muscle cells(VSMCs)were cultured by tissue explants adherent method.(2)Primary VSMCs were treated with EPO and the effects of EPO on extracellular matrix calcium deposition and osteogenic markers were detected by alizarin red staining and Western Blot respectively.(3)Western Blot was used to detect whether EPO could activate JAK2/STAT3/BMP-2 axis and specific STAT3 inhibitor(Cryptotanshinone)was used to detect whether JAK2/STAT3/BMP-2 axis was involved in EPO-induced VSMCs calcification.(4)Western Blot and immunofluorescence were used to detect whether EPO could activate the NF-?B pathway,and BAY11-7082 was used to detect whether the NF-?B pathway was involved in EPO-induced VSMCs calcification.Results 1.Results in vivo1.1 Serum biochemical index(1)The serum creatinine level in the CKD group and the CKD+EPO group was significantly higher than that in the sham operation group,and the urea nitrogen was slightly increased,indicating that the surgical modeling was successful.(2)The serum phosphorus levels in the CKD group and the CKD+EPO group were significantly higher than those in the sham-operated group,and there was no significant difference in blood calcium levels between the groups.1.2 Rat abdominal aorta test results(1)The results of Von Kossa staining showed that there was no obvious black calcium salt deposition in the aortic wall of the sham operation group,and there was obvious scattered black calcium salt deposition in the CKD group.Calcium deposition in CKD+EPO group was significantly higher than that in CKD group.(2)The results of tissue calcium quantitative test were consistent with Von Kossa staining.The calcium content of arterial wall in CKD group was significantly higher than that in non-CKD group,and EPO could significantly increase calcium deposition on the arterial wall of CKD group.2.Results in vitro 2.1 Effects of EPO on extracellular matrix calcium deposition and the expression of osteogenic markers(1)EPO promoted extracellular matrix calcium deposition in a dose-dependent manner.(2)EPO could upregulate osteogenic markers: Runx2 and alkaline phosphatase.(3)EPO up-regulated the expression of VSMCs contractile markers SM22? and SM-?-actin.2.2 After applying the specific STAT3 inhibitor(Cryptotanshinone),the pro-calcification effect of EPO was partly inhibited.2.3 NF-?B pathway inhibitor(BAY 11-7082)partially inhibited the pro-calcification effect of EPO.Conclusions 1.EPO can significantly accelerate the progression of vascular calcification in CKD rats.2.EPO can up-regulate osteogenic transcription factors and promote VSMCs calcification in vitro.3.The JAK2/STAT3/BMP-2 axis and the NF-?B pathway are involved in the pro-calcification effect of EPO.