The Synergistic Action Of Phosphate And Interleukin-6 Enhances Senescence-associated Calcification Depending On P53 In Vascular Smooth Muscle Cells

Background and ObjectiveChronic kidney disease(CKD)is currently divided into 1-5 stages according to the guidelines of the National Kidney Foundation of American.The 5~thh stage CKD,which is named as uremia stage and required for kidney transplant,is usually defined the end-stage renal disease(ESRD)as the estimated glomerular filtration rate(eGFR)less than 15 ml/min.Cardiovascular calcification,including vascular calcification and valve calcification,is associated with cardiovascular mortality and morbidity of patients with ESRD.Hyperphosphatemia and many of the inflammatory makers and mediators,including interleukin-6(IL-6),are considered as the major risk factors of cardiovascular calcification.Though cellular senescence may be involved in cardiovascular calcification caused by phosphate overload and(or)IL-6 in patients with ESRD,it is less known about the underlying mechanisms for phosphate-and IL-6-induced senescence-associated calcification of vascular smooth muscle cells(VSMCs).In the present study,we tried to investigate correlation between cellular senescence and vascular calcification induced by loading phosphate and(or)IL-6 in VSMCs.Methods1.Organization studyingTo investigate the serum phosphate and IL-6 levels in patients with end-stage renal disease and non-dialysis-dependent chronic kidney disease and their relationship with vascular calcification.IL-6 and phosphates in the serum of patients were detected respectively,and vascular imaging data were collected.2.Cellular level studyingPrimary vascular smooth muscle cells derived from human umbilical arteries were used as cell models to investigatephosphate-or(and)IL-6-induced senescence-associated calcification..Western blot analysis was performed to detect alterations of SIRT1,STAT3 and phospho-STAT3,p53,acetylated-p53,p21 and other molecules,?-galactosidase dying analysis was used to detect cell aging.Both calcification dying and cellular calcium release experiments were used to detectmineral deposition in vascular smooth muscle cells.3.Molecular level researchIn order to explore the molecular mechanism of activated IL-6/STAT3/p53 signaling pathway regulating gene expression,luciferase reporter vector was constructed by cloning human p53 gene promoter,and the p-STAT3 specific binding sequence in the promoter region was deleted and mutated by using PCR,and the regulatory activities were detected with luciferase analysis.Results1.Serum deter minations of inorganic phosphate and IL-6 from patients with ESRD and non-dialysis-dependent chronic kidney disease(NDD-CKD)were collected in the present study.Data showed that serum inorganic phosphate was 1.63±0.58 mM,and serum IL-6 was 21.32±38.68 pg/ml in 159 cases of patients with ESRD.Serum inorganic phosphate and IL-6 was 1.28±0.31 mM and 12.37±31.00 pg/ml in 38 cases of patients with NDD-CKD,respectively.The non-parametric tests showed significant differences in the levels of serum inorganic phosphate(P=0.012)and IL-6(P<0.001)between the ESRD group and the NDD-CKD group.Among the 60 patients with ESRD combined with cardiovascular calcification,27 patients with high serum phosphate and high IL-6,the incidence was 45%.There were 9 patients only with high phosphate,and the incidence was 15%.15 patients were only accompanied by elevated serum IL-6level,with an incidence of 25%.There were 9 patients with low serum phosphate and IL-6 levels,with an incidence of 15%.Among the 84 patients without calcification,29patients with high phosphate and high serum IL-6,the incidence was 34.5%.There were15 patients only with high phosphate,and the incidence was 17.9%.20 patients were only accompanied by high serum IL-6 level,with an incidence of 23.8%.There were 20patients with low serum phosphate and IL-6 levels,with an incidence of 23.8%.2.By up-regulating the expression of p53,high phosphates promote the expression of senescence related marker molecules such as p21 and?-galactosidase in VSMCs,and aggravate the calcium salt deposition in VSMCs.Low concentration of resveratrol inhibits p53 by activating SIRT1,thus inhibiting senescence related calcification of VSMCs3.IL-6 induces age-related calcification of VSMCs by activating the IL-6/soluble IL6receptor(sIL6R)/STAT3/p53/p21 pathway.Low concentration of resveratrol inhibits p53 by activating SIRT1,thereby inhibiting senescence related calcification of VSMCs induced by IL-6.4.The synergistic effect of high phosphate and IL-6 enhances senescence related vascular calcification in a p53-dependent manner,and resveratrol inhibits the synergistic effect of the two factors in a dose-dependent manner.Conclusions1.Patients with end-stage renal disease are usually accompanied by high serum phosphate and high levels of IL-6,and have a high incidence of vascular calcification.2.High phosphate can induce the aging-associated calcification of vascular smooth muscle cells by up-regulating the expression of p53,3.IL-6 induces senescence of VSMC by activating IL-6/sIL-6R/STAT3/p53/p21signaling pathway and promotes the occurrence of calcium salt deposition.4.There is a synergistic effect between high phosphate and IL-6,which can induce the senescence of VSMC and promote its calcium salt deposition by upregulation of p53.The synergistic effect inhibited by resveratrol in a dose-dependent manner.