The Study On The Relationship Between Serum Bone-Specific Alkaline Phosphatases, Cardiovascular Calcification And Mortality In Maintenance Hemodialysis Patients

Objective:Serum bone-specific alkaline phosphatases(BAP)is a new biological marker of chronic kidney disease-mineral and bone disorder(CKD-BMD).The aim of this study was to analysis serum BAP level and associated factors,to examine the relationship of serum BAP activity withcardiovascular calcification and clinical outcomes in maintenance hemodialysis(MHD)patients.Further,we explored the mechanism of phosphate-induced vascular smooth muscle cells(VSMCs)calcification.Methods:(1)Uremia patients received hemodialysis treatment in Ruijin Hospital were recruited in July 1,2012.Inclusion criteria for this study were as follows: 18-80 years old,dialysis vintage of at least 3 months.All clinical data,biochemical data and medication were collected at baseline.Bood samples of MHD patients and were collected to detect serum BAP level by the MicroVue BAP immunoassay(Quidel,San Diego,CA).(2)Valvular and vascular calcification was evaluated by the echocardiogram and lateral abdominal radiograph.Followed these patients for 4 years until July 1,2016,and record the date of outcome(death,kidney transplantation,or transfer to other dialysis center).Survival analyses were performed to assess associations of BAP levels with all-cause and cardiovascular mortality.(3)Rat VSMCs was cultured in vitro,the multiplication-stimulating activityof VSMCstreated with different concentrations of phosphate was detected by cell counting kit-8(CCK-8).VSMCstreated with high phosphate(3 mmol/L)for 3 days or 6 days,the evaluation of calcium precipitation was detected by alizarin red staining.The activity of alkaline phosphatase(ALP)and calcium content in the VSMCs was detected by ALP assay kit and calcium colorimetric assay kit respectively.The expression of mRNA and protein level of bone morphogenetic protein 2(BMP-2),runt related transcription factor 2(Runx2)and Klotho was detected by quantitative real-time polymerase chain reaction(RT-PCR)and western blot(WB).Results:(1)Two hundreds and nineteenMHD patientswere involved in this study with a median age of 58.00(interquartile range,48.00-67.00)years old,hemodialysis duration of 41.00(interquartile range,21.00-76.00)months,and 10.96% with diabetes.Median levels of BAP were 19.67(14.83-26.98)U/L.No significant differences were found in serum BAP levels of MHD female patients versus female controls,but the BAP levels of MHD male patients were significantly lower than male controls.In MHD patients,serum BAP levels were significantly higher in female than in male.There were positive correlations between ALP,parathyroid hormone(PTH),BAP/ALP,hemodialysis duration,single-pool Kt/V(spKt/V),total iron binding capacity(TIBC)and BAP.Blood bicarbonate level and post-dialysis systolic blood pressure were negatived associated with BAP.Multivariate logistic regression showed that female,using β-blocker,and higherPTH level were independently correlated with BAP.(2)In this part of study,179 MHD patients underwent echocardiogram,and 191 finished lateral abdominal radiograph.20(11.17%)patients had mitral valve calcification,55(30.73%)aortic valve calcification,and 123(64.40%)abdominal aortic calcification.Compared to patients withoutmitral valve calcification calcification,BAP was significantly higher in patients withcalcification.BAP/ALP ratio was significantly lower in patients with abdominal aortic calcification.After 4 years follow-up,a total of 60(26.67%)patients died,of whom 28 patients died of cardiovascular causes.Kaplan–Meier survival analysis showed higher all-cause and cardiovascular mortality in the first tertile of BAP/ALP ratio than in the third tertile.Comparing patients in the thirdBAP tertile,patients in the first tertile had a 2.52 times for all-cause mortality and 2.89 times for cardiovascular mortality.After adjusted by gender,age,dialysis duration and diabetes mellitus,BAP/ALP ratio is no longer the independent risk factor for mortality.(3)VSMCscultured in Higherphosphate media could suppress the proliferation of cells.Calcification was induced by incubation in high-phosphate media for 6 days and calcium content was significantly increased.RT-PCR showed that BMP-2 and Runx2 mRNA was higher in high-phosphate media compared with normal phosphate media.Consistent with the mRNA data,BMP-2 and Runx2 protein levels were also increased.On the other hand,high-phosphate media significantly inhibited the expression of Klotho in both mRNA and protein level compared with normal media.Conclusion:(1)MHD patients who are female,with treatment of β-blocker and higher PTH level may have a higher serum BAP levels.(2)The serum levels of BAP were associated withthe presence of cardiovascular calcification in MHD patients.BAP/ALP ratio can predict all-cause and cardiovascular mortality rather than single BAP level.(3)High-phosphate medium increased the expression of BMP-2 and Runx2 by inhibiting Klotho expression in VSMCs.This mechanism may contribute,in part,to hyperphosphatemia-related VSMCs differentiation and calcification.