| BackgroundIn recent years,with the increasing incidence of population aging,diabetes and hypertension,the morbidity of chronic kidney disease(CKD)has been increasing,which has become a serious global health problem and caused adverse outcomes in developed and developing countries.Study showed that the morbidity of CKD in the general population worldwide was as high as 143%.The prevalence of CKD in Chinese people over 18 years old was 10.8%and it was higher in the elderly people over 60 years old.Moreover,the prevalence of CKD presents different economic geographical disparities.Recent studies have shown that the overall prevalence of CKD was 16.4%in a Chinese rural population.More CKD patients were associated with cardiovascular disease(CVD)before dialysis and a large number of CKD patients died from CVD even before initiation of dialysis.CKD patients were often diagnosed with the presence of vascular calcification(VC),which was associated with an increased risk for CVD.VC can occur in the early stage of CKD,and the incidence rate increased significantly with the decline of renal function,which was a high-risk factor for CKD combined with CVD and death.Studies have shown that the prevalence of VC in CKD patients with dialysis was as high as 80-90%,which was significantly higher than the incidence of 40%in CKD stage-3 patients.An epidemiological study reported that total prevalence of VC was 77.4%in Chinese dialysis population.A recent study of 151 patients with non-dialysis CKD stage 4-5 showed that the prevalence of abdominal aortic calcification(AAC)was 73%,including 47%with severe calcification.The incidence of cardiac valve calcification(CVC)in maintenance hemodialysis patients with non-diabetic patients reached 70%,which suggested that VC was prevalent in CKD patients.The extent of AAC was an independent risk factor for cardiovascular events in CKD non-dialysis patients.Therefore,early identification of VC and early intervention of factors affecting VC are important for improving the prognosis of patients with CKD.Studies have shown that VC was a pathological process of cell regulation,including many inhibiting factors and contributing factors.Experimental studies in vivo and in vitro have confirmed that klotho played a key role in reducing vascular calcification.Soluble klotho(sklotho)protein has been shown to protect endothelial cells from injury caused by high phosphate and oxidative stress and block the transformation of smooth muscle cells induced by high phosphate into osteoblasts.Other studies have confirmed that sklotho could directly inhibit the absorption of phosphate by vascular smooth muscle cells,thereby inhibited the occurrence of VC.The predictive value of serum sklotho for AAC and CVC in hemodialysis or peritoneal dialysis patients has been confirmed by several studies.There were still controversial researches on the prognosis of serum sklotho and CKD patients.Moreover,previous studies of correlation between VC and serum sklotho were mostly concentrated on dialysis patients or CKD stage 3-5 patients.There were few studies about the incidence of VC and its influencing factors in non-dialysis patients with CKD stage 1-5,especially CVC in non-dialysis patients with CKD.Relationship between serum sklotho level and VC in CKD stage 1-5 are not clear yet.It is also uncertain whether serum sklotho can be used as a biomarker for early diagnosis of VC and prognostic in non-dialysis patients with CKD.For this purpose,the prospective observational study was conducted in non-dialysis patients with CKD stage 1-5.Objective1.To investigate the prevalence of VC in non-dialysis patients with CKD stage 1-5;to explore the relationship between sklotho and VC in the occurrence and severity;to analyze the predictive value of serum sklotho for VC.2.To explore the prognostic impact of serum sklotho on adverse events such as acute non-fatal cardiovascular events,initiation of renal replacement therapy and aggravation of AAC in non-dialysis patients with CKD stage 1-5.Methods1.Inclusion and exclusion criter ia of objectsAccording to the ICD-10 system,non-dialysis patients with CKD stage 1-5 in outpatient and inpatient department of Linyi people' s hospital from January 2014 to December 2016 were recruited.The inclusion criteria:(1)The main diagnosis was in line with the CKD stage 1-5 diagnostic criteria of ICD-10;(2)The history of CKD?6 months.The exclusion criteria:(1)age<18 years old;(2)Kidney transplantation;(3)Serious cardiovascular and cerebrovascular events;(4)Acute infection;(5)Peripheral vascular disease or valvular heart disease.All study participants provided a written informed consent by the hospital ethics committee.2.Clinical data collection and laboratory testsDemographic data including age,sex,primary disease,duration of CKD,smoking history,drinking history,medication history and other clinical data were collected.It was obtained by questionnaire and further confirmed by inquiring the outpatient and inpatient data and interviewing patients if necessary.After fasting for 8 to 10 hours overnight,blood and urine samples were collected in the morning.After the blood sample was centrifuged,the serum samples were stored in-20? low temperature refrigerator.Serum calcium(Ca),serum phosphorus(P),serum alkaline phosphatase(ALP),serum albumin,serum creatinine,blood urea nitrogen,total cholesterol,triglyceride,serum uric acid,c-reactive protein(CRP),homocysteine(Hcy)were detected by automatic biochemical analyzer.sklotho(American R&D),FGF23 and iPTH were detected by ELISA.The level of 1,25-dihydroxyvitamin D3 was detected by radioimmunoassay.10ml of urine in the middle section was cleaned in a urine cup,and urine microalbumin creatinine ratio(ACR)was tested within half an hour.3.Assessment of vascular calcification3.1 Abdominal aortic calcification(AAC)was detected and the degree of calcification was determinedAbdominal lateral X-ray were conducted to each patient.Two experienced physicians,who were blinded to the study,independently carried out the evaluations and the final results were presented as the average.A semi-quantitative scoring syst em was utilized to analyze abdominal AAC.Abdominal aorta in front of the first to t he fourth lumbar vertebra were involved.Calcific deposits in the abdomi nal aorta adjacent to each lumbar vertebra were assessed separately for the posterior and anterior wall of the aorta using the midpoint of the intervertebral space above and below the vertebrae as the boundaries.Lesions were graded as follows:0,no aortic calcific deposits;1,less than one-third of the corresponding aorta length;2,cal cification length was between one-third to two-thirds of the corresponding aorta length;3,two-thirds or more of the corresponding aorta length.The total score therefor varied from 0 to 24.AAC was divided into mild calcification group(0<AAC score<5),moderate calcification group(5?AAC score?15)and severe calci fication group(AAC score?16)3.2 Detection of cardiac valve calcification(CVC)and determination of calcification degreeEchocardiography was performed using a Philips EPIQ7 ultrasound system,probe frequency 2.5?3.5MHZ.Valve calcification was assessed by an experienced cardiac color ultrasound physician.The cardiologist confirmed all the echocardiographic results.Valvular calcifications were defined as a brillliant echo of>lmm in one or more cusps of the aortic or mitral valve or mitral ring.Calcification area is divided into 3 grades:mild valve calcification:only an isolated area of calcification Moderate calcificat ion:multiple areas of calcification;Severe calcification:calcification is extensive and diffuse.4.Grouping criteria4.1 According to estimated glomerular filtrat ion rate(eGFR)at baseline,the participants were divided into 5 groups Group I(CKD stage-1):eGFR?90ml/min/1.73m2;Group?(CKD stage-2):eGFR 60?89 ml/min/1.73m2;Group III(CKD stage-3):eGFR 30?59 ml/min/1.73m2;GroupIV(CKD stage-4):eGFR 15?29ml/min/1.73m2;Group V(CKD stage-5)eGFR<15 ml/min/1.73m2 non-dialysis patients.eGFR was calculated according to the formula of chronic kidney disease epidemiology collaboration(CKD-EPI2009).4.2 Four groups were divided according to the quarti le range of sklotho levels at baseline Group I:sklotho<Q1;Group?:Q1?sklotho<Q2;Group III:Q2?sklotho<Q3;Group ?:sklotho?Q3.5.Follow-upPatients were divided into 4 groups according to baseline serum sklotho quartile level,all the patients were followed up for 2 years(24 months).The patients were followed up once a month.Follow-up methods:telephone or enquiry of outpatient and inpatient medical records.Follow-up content:(1)Record adverse events during follow-up:non-fatal acute cardiovascular events(acute coronary syndrome,congestive heart failure,arrhythmia);initiation of renal replacement therapy;The time(month)of adverse events was detailed recorded,when the patient has multiple cardiovascular events,only the time of the first event was recorded.(2)Record the general condition of the patient,given health guidance according to the condition such as correct anemia,correct the disorder of calcium and phosphorus metabolism and other treatments.The end point of follow-up was all cause death or initiation of renal replacement therapy.At the end of the 24 months follow-up period,the subjects were again examined lateral abdominal radiographs and AAC score was recorded again.The aggravation of AAC was defined as the occurrence of new calcification or increase of AAC score compared with pre-follow-up.6.Statistical analysesSPSS25.0 software was used for data analysis.All hypothesis tests used a bilateral test with a significance level of 0.05.The measurement data conformed to the normal distribution was presented as x±s the comparison between groups was performed by one-way ANOVA or independent sample t test.Measurement data of non-normal distribution were expressed by median and interquartile range M(Q1?Q3)and rank sum test was used for comparison between groups.The count data was expressed by rate or composition ratio and the comparison between the groups of the count data was performed by the chi-square(x2)test or the Fisher exact probabi lity method.Pearson correlation coefficient,Spearman correlation coefficient or point two series correlation analysis was used to analyze correlation between variables according to research purpose and data type.Binary logistic and ordinal logistic regression were used to screen for risk factors affecting the occurrence and severity of AAC and CVC.The receiver operating characteristic(ROC)was used to analyze the predictive value of sklotho for AAC.The Kaplan-Meier method was used to estimate the cumulative survival rate without adverse events,and the survival rate was compared using the Log-rank test.Risk factors for adverse events were analyzed using the Cox proport ional hazards regression model.The level of significance was set at P<0.05.Results1.Demographic and clinical characteristics of the candidates 980 patients met the inclusion criteria,475 patients were agreed by telephone or interview.23 patients were excluded from kidney transplantation,87 patients were acute infection(skin,respiratory tract,digestive tract,urinary tract),70 patients were severe cardiovascular and cerebrovascular diseases,and 24 patients were peripheral vascular diseases.271 patients entered the study,20 patients withdrew due to failure to the collection of relevant indicators.At the end,a total of 251 patients were completed.There were 38 in CKD stage-1,52 in CKD stage-2,64 in CKD stage-3,40 in CKD stage-4,and 57 in non-dialysis CKD stage-5.There were 158(62.95%)males and 93(37.05%)females.The age of the candi dates was 20?86 years,and the median age was 45(34?61)years.Duration of CKD:6(3.5? 11.5)years.There were 96(38.25%)with drinking history and 67(26.69%)with smoking history.Primary disease:there were 108(43.03%)patients with chronic glomerulonephritis,among them 44(40.74%)were confirmed by renal biopsy,including 16(36.36%)patients with focal segmental-sclerosing nephritis(FSGS),16(36.36%)patients with IgA nephropathy and 12(27.28%)patients with mesangial proliferative nephritis.There were 60(23.90%)patients with diabetic nephropathy,among of them 9(15%)patients were confirmed by renal biopsy.There were 36(14.34%)patients with nephrotic syndrome,30(83.33%)patients were confirmed by renal biopsy,including 17(56.67%)patients with membranous nephropathy,6(20%)patients with minimal change nephropathy,3(10%)patients with mesangial proliferative nephritis,and 4(13.33%)patients with focal segmental glomerulosclerosis(FSGS).There were 22(8.76%)patients with hypertension renal impairment,among of them 3(13.64%)patients were confirmed by renal biopsy.There were 8(3.19%)patients with interstitial nephritis,among of them 6(75%)patients were confirmed by renal biopsy.There were 7(2.79%)patients with polycystic kidney disease.Other 10(3.98%)patients was unknown causes.A total of 146(58.20%)patients took anti-hypertensive drugs,including 9(23.68%),14(26.92%),39(60.94%),37(92.50%)and 47(82.46%)patients according to CKD stagel-5 respectively.There were 118(47.01%)patients who took calcium-phosphorus binder,including 10(26.32%),12(23.08%),28(43.75%),27(67.50%)and 41(71.93%)patients respectively.There were 100(39.84%)patients who took active vitamin D,including 2(5.26%),4(7.69%),32(50%),27(67.50%)and 35(61.40%)patients respectively.There were 87(34.66%)patients who used erythropoietin(EPO),including 0(0%),2(3.85%),18(28.13%),18(45%),49(85.96%)patients according to CKD stage 1-5 respectively.The difference was statistically significant(P<0.001).There was a significant difference in serum sklotho levels between groups.Serum sklotho levels in group ?,?,?,and ? were significantly lower than those in group I after Bonferroni correction,the difference was statistically significant(P<0.001).There were no significant differences in serum sklotho levels between groups IV and V(P>0.05).2.The incidence of AAC and CVC at baseline levelThe total incidence of AAC was 21.51%.The incidence of AAC in CKD stagel-5 was 5.26%,7.69%,20.31%,27.50%and 42.11%respectively.Comparison between groups,the incidence of AAC in CKD stage-5 was higher than CKD stage-1,2(P<0.05)after Bonferroni correction.The independent sample Kruskal-Wallis test showed that the AAC score in CKD stage-5 was significantly higher than that of CKD stage 1-3 and the difference was statistically significant(P<0.001).The total incidence of CVC was 20.72%and the incidence of CVC in patients with CKD stage 1-5 was 2.63%,11.54%,16.63%,30.00%and 40.35%respectively.The incidence of CVC in CKD stage-5 was significantly higher than that in CKD stage 1-3 after Bonferroni correction,the incidence of CVC in CKD stage-4 was higher than that in CKD stage-1(P<0.05).3.Correlation analysis between serum sKlotho and other cl ini cal indicators3.1 Correlation analysis results between continuous variablesSerum sklotho was negatively correlated with age(r=-0.562,P<0.001),duration of CKD(r=-0.497,P<0.001),BMI(r=-0.128,P=0.042),FGF23(r=-0.898,P<0.001),serum uric acid(r=-0.557,P<0.001),serum phosphorus(r=-0.669,P<0.001),ALP(r=-0.221,P<0.001),Hey(r=-0.264,P<0.001),iPTH(r=-0.777,P<0.001),triglyceride(r=-0.135,P=0.032)and CRP(r=-0.243,P<0.001).It was positively correlated with eGFR(r=0.824,P<0.001),HCT(r=0.621,P<0.001),Hb(r=0.699,P<0.001),serum calcium(r=0.607,P<0.001),1,25(OH)2-VitD3(r=0.598,P<0.001).There was no correlation with urinary ACR(r=0.031,P=0.628),albumin(r=-0.112,P=0.077)and total cholesterol(r=-0.026,P=0.681).3.2 Pomit-biserial correlation analysis of serum sklotho with binary variablesThere was no correlation between sklotho and gender(r=-0.043,P=0.502).There was a significant positive correlation between sklotho and history of drinking(r=0.269,P<0.001)and smoking(r=0.269,P<0.001);There was a significant negative correlation between sklotho and history of drug use(P<0.001).sklotho was negatively correlated with AAC(r=-0.303,P<0.001)and CVC(r=-0.301,P<0.001).3.3 Rank correlation analysis results sklotho was negatively correlated with AAC(r=-0.330,P<0.001)and CVC(r=-0.301,P<0.001)severity.4.Binary logistic regression analysis of the occurrence of AAC and CVC at baseline levelTaking the AAC as a dependent variable,after single factor screening,factors with P<0.05 were included in multivariate analysis,corrected the effects of confounding factors,multivariate binary logistic regression analysis showed that age {OR:1.077,95%CI:1.029?1.126,P=0.001),diabetes nephropathy(OR:127.639,95%CI:27.603?590.128,P<0.001),FGF23(OR:2.045,95%CI:1.988?7.999,P=0.015),serum phosphorus(OR:3.615,95%CI:1.227?10.649,P=0.020)were independent risk factors for the AAC.sklotho(OR:0.856,95%CI:0.761?0.962,P=0.009)was a protective factor for the AAC.Taking the CVC as a dependent variable,after single factor screening,factors with P<0.05 were included in multivariate analysis,corrected the effects of confounding factors,multivariate binary logistic regression analysis showed that sklotho(OR:1.003,95%CI:0.998?1.008,P=0.282)had no statistically effect on CVC.Age {OR:1.123,95%CI:1.081?1.666,P<0.001),diabetes nephropat hy(OR:9.580,95%CI:3.979?23.063,P<0.001),CRP(OR:1.032,95%CI:1.001?1.085,P=0.021)were independent risk factors for the CVC.5.Ordinal logistic regression analysis of the severity of AAC and CVC at baseline LevelThe severity of AAC and CVC(mild,moderate,severe)was used as the dependent variable.After single factor screening,the factors with P<0.05 were included in the multivariate analysis.Adjusted for the influence of confounding factors,ordinal logistic regression analysis showed that that only diabetic nephropathy was an independent risk factor for the severity of AAC.The probability of moderate and severe AAC in non-diabetic nephropathy patients was 17.5%of patients with diabetic nephropathy(OR:0.175,95%CI:0.005?0.806,P=0.033).It was also an independent risk factor for the severity of CVC.The probability of moderate and severe CVC in non-diabetic nephropathy patients was 18.5%of patients with diabetic nephropathy(OR:0.185,95%CI:0.013?0.241,P=0.002).6.ROC curve of the diagnostic value of sklotho for AACArea under ROC curve(AUC)showed that the AUC of sklotho in the diagnosis of AAC was 0.712(95%CI:0.638?0.787,P<0.001)and the cut-off point was 588pg/ml,the sensitivity was 83.33%and the specificity was 59.42%.7.Grouped by serum sklotho quartile and its follow-up results7.1 Groups by sklotho quarti le Group ?:sklotho<416pg/ml,62;Group?:416pg/ml?sklotho<568pg/ml,61;Group ?:568pg/ml?sklotho<746 pg/ml,65;Group IV:sklotho?746 pg/ml 63.End time of follow-up was January 2019.A total of 15(5.97%)patients were excluded from follow-up because of the need for renal replacement therapy(10 patients with hemodialysis,5 patients with peritoneal dialysis).Non-fatal acute cardiovascular events occurred in 16(6.37%)patients and continued to follow up until 24 months.6 patients were lost during the follow-up and no all-cause deaths occurred.A second abdominal plain film examination was completed and scored within 1 week after the end of follow-up.There were 32(12.75%)patients(including 9 patients with new calcification and 23 patients with increased calcification score)which AAC score increased after the follow-up.The number of acute nonfatal cardiovascular events in group ?,?,?,and ? were 9(14.52%),4(6.56%),2(3.08%)and 1(1.59%),Fisher' s exact probability test showed that the difference between the groups was statistically significant(P<0.05).The number of renal replacement therapy in group ?,?,? and IV was 14(22.58%),1(1.64%),0(0%)and 0(0%).Group I was significantly higher than group II(P<0.05).The number of aggravation of AAC scores in group I,II,III,and IV was 20(32.26%),7(11.48%),4(6.15%)and 1(1.59%),the difference was statistically significant(P<0.001).Comparison between groups,the number of AAC scores in group I was significantly higher than that in group II,III,and IV after Bonferroni correction(P<0.05).7.2 Binary logistic regression analysis of increased AAC score at the end of 2 years fol low-upAt the end of follow-up,6 patients who were lost to foll ow-up and 15 patients with renal replacement therapy were excluded.The remained 230 patients were subjected to AAC score on abdominal X-ray examination again.The patients were divided into 2 groups according to the increase of AAC score:group with no increase of AAC score(n=198)and group with increase of AAC score(n=32).After single factor screening at baseline,the factors with P<0.05 were included in the multivariate binary logistic regression analysis.Adjusted for the confounding factors,sklotho(OR:0.892,95%Cl:0.886?0.999,P=0.023)was an independent influence factor for the increase of AAC score in non-dialysis patients with CKD within 2 years.7.3 ROC curve of serum sklotho for predicting increase of AAC scoreThe area under the ROC curve(AUC)showed that the AUC of the serum sklotho diagnosis aggravation of AAC scores was 0.778(95%CI:0.697?0.859,P<0.001)and the cut-off value was 471.5 pg/ml.The sensitivity was 81.25%and the specificity was 72.15%.7.4 Analysis of the survival of adverse events by Kaplan-Meier methodAcute non-fatal cardiovascular event and initiation of renal replacement therapy during the follow-up period were considered as composite adverse event,the mean survival time of no adverse events in group ?(sklotho<416pg/ml)was 21.25±0.63 months,which was significantly lower than group ? 23.73±0.14 months,group ?(24 months)and ?(23.93±0.09)months.The difference was statistically significant(Log-rank test ?2=41.716,P<0.001).7.5 Analysis of the risk factor for adverse events by COX proportional hazard regression modelAfter single factor screening,factors with P<0.05 were included in multivariate analysis.Adjusted the confounding factors,multivariate Cox proportional hazard regression analysis showed that age(HR:1.024,95%CI 1.002?1.046,P=0.032)and serum sklotho(HR:0.993,95%CI 0.987?0.999,P=0.023)were influencing factors for acute non-fatal cardiovascular events and initiation of renal replacement therapy in non-dialysis patients with CKD.Gonclusion1.VC is occurred in the early stage in non-dialysis patients with CKD,and the incidence and severity of VC in CKD stage-5 are significantly higher than CKD stage 1-3;Serum sklotho is positively correlated with eGFR and negatively correlated with the occurrence and severity of AAC and CVC.2.Serum sklotho is the protective factor for AAC in CKD patients without dialysis,which has certain predictive and diagnostic value for the occurrence of AAC.It is also an independent predictor of AAC progress in 2 years,but has no influence on the severity of AAC.Serum sklotho has no effect on the occurrence and severity of CVC.3.Low-level serum sklotho is associated with a higher risk of adverse events.Serum sklotho is a predictor of acute non-fatal cardiovascular events and initiation of renal replacement therapy in non-dialysis patients with CKD.Maintaining high levels of serum sklotho may improve prognosis of non-dialysis patients with CKD. |
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