Epidemiological Characteristics Of Calciphylaxis Based On Chinese Hemodialysis Population And The Mechanism Of Warfarin In Vascular Calcification

ObjectiveThe prevalence of vascular calcification in chronic kidney disease(CKD)is extremely high,which is the main reason for the decrease of survival rate of dialysis population.Calciphylaxis is a kind of vascular calcification syndrome commonly seen in CKD patients,which is characterized by systemic arteriolar medial calcification,intimal fibrosis and microthrombosis.The patient has persistent pain,ulcer or necrotic skin damage in multiple parts of the body.Early identification of calciphylaxis is a key measure to improve the cure rate and prognosis.The etiology and pathogenesis of the disease are still unclear.Hence,mastering its prevalence and clinical characteristics and identifying high-risk factors are helpful for early identification of clinical patients,providing important clues for exploring the pathogenesis and contributing to the development of diagnosis and treatment technology in the future.When calciphylaxis occurs,a large amount of hydroxyapatite accumulates in blood vessels and soft tissues,and matrix vesicles(MVs)are the center of hydroxyapatite crystal precipitation,which is considered as the starting site of calcification.Warfarin,as the strongest identifiable risk factor for calciphylaxis,may play an important role in regulating MVs.Therefore,this study is mainly carried out from two aspects:investigating the epidemiological characteristics of calciphylaxis and exploring the role of warfarin in promoting vascular calcification,with a view to providing data support and theoretical basis for early diagnosis schemes and potential therapeutic targets of severe vascular calcification such as calciphylaxis from clinical and pathogenesis aspects.This study includes three parts:Part Ⅰ:Analysis of risk factors of calciphylaxis in hemodialysis patientsMethods:A retrospective matched case-control study was conducted to analyze the risk factors of calciphylaxis.The case group involved 20 hemodialysis patients who were newly diagnosed with calciphylaxis from October 2017 to December 2018.The 40 non-calciphylaxis patients undergoing dialysis with the same age and duration of dialysis were randomly selected as controls.Results:Most of calciphylaxis patients were male and elderly,while overweight people were more susceptible to the disease.60%of the patients had intolerable pain.65%of the patients’condition deteriorated,in which 35%died during the one-year follow-up period.Although incidence of secondary hyperparathyroidism(SHPT)was higher in calciphylaxis patients,the differences in duration of elevated serum intact parathyroid hormone(iPTH)level and its highest value didn’t reach statistical significance compared with controls.No significant difference in warfarin therapy was discernible between two groups.It should be noticed that the case group used activated vitamin D and its analogues at a higher dose or for a longer period.The univariate regression analysis indicated that male,score of use of activated vitamin D and its analogues,corrected serum calcium level,serum phosphate,iPTH,albumin,alkaline phosphatase(ALP)and hypersensitive c-reactive protein level were significantly associated with calciphylaxis.Elevated levels of serum phosphate and ALP,decreased level of serum albumin were independent risk factors after multivariate analysis.Conclusions:Increased levels of serum phosphate and ALP,decreased level of serum albumin were vital high-risk factors for calciphylaxis in hemodialysis population.Unlike previous research from western countries,female and warfarin therapy didn’t show an increased risk in this study.Part Ⅱ:Epidemiological survey of calciphylaxis in hemodialysis patients in Jiangsu ProvinceMethods:In the cross-sectional survey during Oct.2018 to Oct.2019,a stratified sampling method was used to select 24 dialysis centers in Jiangsu Province.The participants were all adult patients in each center,who had been on hemodialysis for more than 6 months.This multicenter investigation was conducted in the form of questionnaires,which were filled in by doctors or nurses according to the actual situation of subjects.Calciphylaxis patients were uniformly diagnosed by the Calciphylaxis Study Group based on characteristic skin lesions and histopathological features.Results:A total of 3867 hemodialysis patients(average age of 55.33±13.89 years;2390[61.81%]of males)were included.48 cases were diagnosed with calciphylaxis,and the prevalence was 1.24%.In addition,394 patients had a variety of manifestations of skin lesions,and they did not meet current diagnostic criteria for calciphylaxis that were excluded from diagnosis.Among calciphylaxis patients,33 cases(68.75%)were male,and the average age and median dialysis duration were 53.85±15.17 years and 84.00(48.00,138.75)months,respectively.Older and overweight people were vulnerable to this disease.Skin biopsy was performed in 70.83%of calciphylaxis patients,and the positive rate was 64.71%.Meanwhile,the positive rate of bone scintigraphy in the diagnosis of calciphylaxis was 62.50%,which was mainly manifested by increased uptake of radiotracers in soft tissue.The prevalence of SHPT in the case group was as high as 72.92%with longer duration,and 42.86%of the patients had undergone parathyroidectomy.Warfarin significantly increased the risk of calciphylaxis,although only 4 patients had previously used this anticoagulant.Multivariate analysis indicated that increased BMI,prolonged dialysis duration,warfarin therapy,concomitant with SHPT,diabetes mellitus,low serum albumin and high serum alkaline phosphatase levels were high-risk factors for calciphylaxis.Among all the hemodialysis patients,the iPTH and serum calcium levels were well controlled,and the rate of reaching the standard was high.However serum phosphorus level was difficult to control,and more than half of the patients’ serum phosphorus was higher than the target value.Conclusions:The prevalence of calciphylaxis in hemodialysis patients in Jiangsu Province was 1.24%,but its actual prevalence would be presumably far beyond present data.Prolonged dialysis duration,increased BMI,concurrent SHPT,concomitant diabetes mellitus,warfarin therapy,decreased level of serum albumin and elevated level of ALP were independent high-risk factors for this disease.It’s urgent to improve clinical understanding of calciphylaxis,and multifaceted diagnostic methods should be applied for early screening.Part Ⅲ:Study on the mechanism of warfarin in vascular calcificationMethods:Vascular smooth muscle cells(VSMCs)were cultured in normal or osteogenic medium and stimulated with 10 μmol/L warfarin for 3-14 days.RT-PCR and Western Blot were used to detect the gene level and protein expression of its markers,the intracellular calcium content was measured,and alizarin red staining was performed.MVs were extracted by differential centrifugation and identified,and the expression and activity of transglutaminase 2(TG2)were detected.Results:Warfarin stimulation promoted the transdifferentiation of VSMCs,with increased expression of osteogenic markers Runx2,BMP2 and OPN,and decreased expression of SM22α.Although osteogenic medium alone could induce transdifferentiation of VSMCs,the induction effect was significantly enhanced by warfarin.In warfarin intervention group,intracellular calcium content was significantly increased and alizarin red staining was positive.After the same amount of VSMCs was cultured for 96 h,MVs were extracted from the supernatant of the same amount of culture medium.The quantitative results of total protein showed that warfarin promoted the secretion of MVs and increased the expression of related marker proteins TSG101,CD9 and CD81.The particle size of MVs was about 130 nm by nanoparticle tracking analysis,and transmission electron microscopy showed that MVs were bilayer vesicles.In addition,the formation of MVs was accompanied by calcium phosphorus crystal deposition.At the same time,warfarin intervention increased the expression and activity of TG2 in VSMCs,and gradually increased with the prolongation of intervention time.Inhibition of TG2 reduced the secretion of warfarin-stimulated MVs and the degree of calcification.Conclusions:Warfarin regulates the secretion of MVs and induces VSMCs calcification by upregulating the expression and activity of TG2.