Relationship between Arterial Calcification and Fetuin-A Levels in Patients with Chronic Kidney Disease at the Onset of Hemodialysis

Relationship between Arterial Calcification and Fetuin-A Levels in Patients with Chronic Kidney Disease at the Onset of Hemodialysis

LI,Jiajia

Shandong Liuzhuang Hospital,Weihai Shandong 264200,China

【Abstract】Objective::Arterial calcification is a negative predictor of morbidity and mortality in end-stage renal disease.Traditional factors such as hypertension,dyslipidemia and non-traditional factors such as mineral disorders are involved in calcification.They are generally classified as promoters and inhibitors.Possi-ble changes in inhibiting factors,such as fetuin-A,are responsible for the path-ophysiology of vascular calcification.Methods:Arterial calcification was evalu-ated by withdrawing fragments of the artery to undergo the first arteriovenous fistula for hemodialysis.Four histological stains were used to identify the pres-ence of calcification while blood for fetuin determination was collected at the same time.Demographic,clinical and laboratory data were analyzed to test pos-sible relationships in the prevalence of vascular calcification.Results:Thir-ty-four patients were enrolled and vascular calcification was detected in eight.Statistical analysis revealed that calcification predictors were age and the pres-ence of diabetes.The correlation between arterial calcification and fetuin was 0.4(r=of 0.033).Conclusion:There is a tendency to an inverse correlation between arterial calcification and fetuin-A levels in patients with chronic kidney disease at the onset of hemodialysis.

【Keywords】Fetuin-A;alpha-2-HS-Glycoprotein;Vascular Calcification;Chronic Kidney Disease

1.Introduction

Patients with chronic kidney disease present with significantly higher morbidity and mortality than the general population.Cardiovascular diseases are the primary cause of death in this population,accounting for around 50%of all fatal cases[1].

The traditional risk factors for atherosclerotic cardiovascular disease do not explain the high cardiovascular mortality observed in patients with chronic kidney disease,who exhibit around 10 times the mortality rate of those without the disease[2].

One prevalent alteration in this population is vascular calcification.In patients with chronic kidney disease,histological and radiological evidence and vascular calcification are significantly greater than in the general popu-lation and have considerable influence on a number of indirect markers of mortality from heart disease[3].

It is known that changes in the metabolism of calcium,phosphorus and the parathormone are important factors in the severity and rapid progression of vascular calcification.However,these are not the only causes of arterial calcification;other factors,such as changes in fetuin-A serum levels,may play a significant role in physiopathol-ogy[4].

Non-traditional factors can be classified as promoters(calcium,phosphorus,PTH and other mediators)and in-hibitors such as fetuin-A.The serum is generally saturated with calcium while phosphorus and inhibition mecha-nisms,present in both the serum and the arterial wall,ensure that the formation of hydroxyapatite(a calcium phosphate salt)is limited to bone tissue[5].

Therefore,the loss of equilibrium between promoting and inhibiting factors is an essential condition for calci-um and phosphorus deposition on the arterial wall,leading to its hardening and consequent loss of elasticity,thereby complicating the cardiovascular situation of patients with chronic kidney disease[6].

In a study of patients with arterial calcification and chronic kidney disease undergoing hemodialysis Smith et al reported a sharper decrease in fetuin-A levels than in those with no arterial wall alterations[6].

Despite appearances,low fetuin-A levels are linked not only to patients who have been on hemodialysis for a considerable period of time,but also to those who have undergone it early in life,possibly exhibiting changes in calcium metabolism[7].

Given that studies on fetuin-A are quite recent,there is scarce evidence in the literature to definitively confirm the importance of this glycoprotein in the vascular calcification of patients with chronic kidney disease.However,results in a number of studies show that levels below 0.290 g/l of fetuin play a special role in the physiopathology of vascular calcification and that this may occur in the early stage of kidney disease[8].

Since literature studies report increased mortality with arterial calcification[1,9],it is important to confirm promoting or inhibiting factors that display alterations in the early stages of chronic kidney disease.Thus,the aim of the present study was to determine the relationship between arterial calcification and changes in the plasma fetuin levels of patients with chronic kidney disease at the onset of hemodialysis therapy.

2.Methods

The project was presented and approved by the research ethics committees of Federal University of Sao Paulo.The study was conducted at the nephrology facility of tertiary hospital.

This is a cross-sectional correlation study with a non-probability sample selected consecutively by convenience.All participants gave their informed consent.Included were patients aged eighteen years or older who would be submitted to arteriovenous fistula construction prior to the onset of hemodialysis.

Excluded were patients with chronic liver disease,pregnant women,prison inmates,indigenous people and pa-tients using oral anticoagulants.

The primary variable was the relationship between arterial calcification and fetuin-A levels and the secondary variables were fetuin-A levels,percentage arterial calcification,initial levels of calcium,phosphorus,calcium x phosphorus,and parathormone(PTH),age,systemic hypertension(SHT)and diabetes(DM).

Procedures

Demographic data were collected on an inpidual chart that included calcium,phosphorus,parathormone,fetuin-A and histopathological levels.To assess fetuin-A,blood was collected when the patient was being submit-ted to arteriovenous fistula construction.After collection,blood was centrifuged and the plasma was frozen in a freezer at-80ºC,until all samples were collected so that levels could be measured at the same time Fetuin-A was measured directly in the plasma using the ELISA human fetuin-A immunoassay kit(Quantikine®),manufactured and distributed by USA and Canada R&D Systems,Inc.614 McKinley Place NE,Minneapolis,MN 55413.

For the histological study of vascular calcification,fragments of patients’arteries were collected during arteriotomy.The fragments were fixed in 10%formaldehyde and processed for inclusion in paraffin.Histological cuts(4µm)were made,followed by Hematoxylin-eosin,Von Kossa,Masson and Verho eff-van Gienson(VVG)staining[10].Slides were read by an independent examiner.

Banff[11]criteria,adapted for upper limb arteries,and those used by Moe[12],were used to determine the de-gree of calcification of each sample.

Statistical Method:

Data analyses

The sample consisted of thirty-four patients,and data were tabulated and processed by Predictive Analytics

Software-PASW®Statistic version 21.0.Data analysis used tables and graphs of means,medians,standard devi-ations,confidence intervals and hypothesis tests.

After the data were characterized using descriptive statistics,the Kolmogorov-Smirnov test was applied to as-sess normality of variable distribution.

The correlation between percentage calcification and fetuin-A concentration was determined by the bivariate correction test and the degree of linear relationship was observed by Pearson’s coefficient.The Receiver Operat-ing Characteristic(ROC)curve was traced to show the cutoff point for the variables that exhibited relevant arterial calcification.The risks of arterial calcification in relation to the variables assessed by the ROC curve were then identified.

Mean fetuin-A concentration,calcium(mg/dl)and phosphorus(mg/dl)in the groups with and without calcifica-tion were compared using the Student’s t-test for independent samples,since their distributions follow the normal curve.The variables parathormone(pg/mL),time on dialysis,age,and Ca x Pwere compared using the Mann Whitney U test,given that they do not follow the normal distribution curve.

Finally,the importance of different variables(income greater than one minimum monthly salary of~US$240.00,older than 58.5 years,time on dialysis of more than 29 days,hypertension and diabetes mellitus)for the presence or absence of arterial calcification,considering the existence of all of them,was determined using the binary logistic regression test.The values were considered significant forp<0.05.

3.Results

The study sample was composed of thirty-four patients with mean age of 48.6 years±13.9 CI 43.9–53.1.Twenty-three of the patients were men.The etiologic agent of chronic kidney disease in the sample of patients with and without arterial calcification is described in Table 1.

Table 1.Etiologic agent of patients with and without calcification

Average time on dialysis was 23 days±23(CI 15.9–31.7).

The general mean fetuin-A level was 0.529 g/L±0.197(CI0.463–0.595).Mean fetuin-A level in men and women was 0.556 g/L±0.224(CI 0.459–0.653)and 0.474±0.110(CI 0.400–0.549),respectively.

Eight patients presented with arterial calcification,six of whom were men.

The mean value for calcium was 8.7 mg/dL±118(CI 8.3–9.1),for phosphorus 5.65 mg/dL±1.74(CI 5.07–6.23),for calcium x phosphorus 49.2;and for parathormone 358 pg/dL±320(CI 251–465).

Table 2 shows the clinical and laboratory aspects of patients with and without arterial calcification.

Table 2.Clinical and laboratory aspects of patients with and without calcification

The odds ratio test was applied for an age of 58 years or older,where there was a 1.4 greater likelihood of calci-fication than in younger inpiduals,with a 95%confidence interval between 0.89 and 2.45 fold;sensitivity of 62%and specificity of 76%.

The odds ratio test was carried out for fetuin-A≥0.482 g/L with a 1.6-fold greater likelihood of an inpidual not presenting with calcification than one with fetuin below this value and a 95%confidence level between 0.638 and 3.058 fold;with sensitivity of 62%and specificity of 66%.

Binary logistic regression was conducted for secondary variables that exhibited importance with respect to the presence of arterial calcification(Table 3).

Table 3.Binary logistic regression for secondary variables in relation to arterial calcification

*Significant bivariate correlation(Pearson’s correlation).**Levels near established significance.

4.Discussion

The present study involved a sample of thirty-four patients,similar to studies conducted by Schlieper[13]andWang[14].Both authors performed a histopathological analysis of patients in relation to arterial calcification,used a sample of 30 patients and found significant results.Losses were within expected values,since they corre-sponded to less that 10%of the sample,and resulted from potential participants’refusing to give their informed consent.

Sample characterization showed a predominance of systemic hypertension(SHT)as etiologic agent,different from the study by Wang[14]and Penkac[15].In these studies,glomerulonephritis was the predominant etiologic agent,and this may have occurred due to efficient public policies to prevent and control hypertension in areas where these investigations took place.

A total of 23.5%of patients exhibited arterial calcification,according to Banff criteria and Moe’s study[12],and no statistical difference between these classifications was identified.

The etiologic agent that predominated in chronic kidney disease in patients with arterial calcification was dia-betes mellitus,corroborating Cozzolino[16],who reported it as being the agent that most promotes changes in the arterial wall.Ford[17]found that fetuin-A is an independent risk factor that causes arterial wall hardening even in the absence of diabetes mellitus in patients with advanced chronic kidney disease.

The variables such calcium and phosphorus exhibit no statistically significant differences in the group with or without calcification.With respect to fetuin-A,we identified an important difference between the group with and without calcification,with lower mean values in patients exhibiting arterial calcification but not statistically sig-nificant,which does not invalidate our study since our objective was only to show the correlation between fetuin-A and arterial calcification.

Assessment of the means and standard deviations of parathormone,calcium x phosphorus and time on dialysis show no important numerical difference between the group with and without calcification or statistical signifi-cance.However,mean age was higher in the calcification group,albeit with no statistical difference.

Even though Cozzolino[16]conducted a revision showing that arterial calcification is directly linked to a rise in promoting factors such as increased calcium,phosphorus and age and a decline in inhibiting factors such as fetuin-A,our study did not find these changes likely because the objective was to analyze these factors at the onset of hemodialysis.

The ROC curve shows a cutoff point for age of 58 years in inpiduals with calcification,with an odds ratio in-dicating a 1.4-fold likelihood of presenting with calcification.When this variable is submitted to binary logistic regression(Table 3),it was not an important cause of arterial calcification and the only variable that had an influ-ence on the disease in the arterial wall was diabetes mellitus,but without much weight in physiopathology.

The ROC curve for the cutoff point of fetuin-A in relation to arterial calcification was constructed,but without reliable data,likely due to the number of patients with calcification.However,an ROC curve was drawn to iden-tify the cutoff point of fetuin-A,which would protect arterial calcification.This value was 0.482 g/L,whereby odds ratio showed a 1.6-fold greater likelihood of an inpidual not exhibiting calcification than one with fetuin-A below this value.

Hematoxylin-eosin,Von Kossa,Masson and Verhoeff-Van Gienson(VVG)staining were used to identify arte-rial calcification,but Masson and Verhoeff-Van Gienson were not sensitive.Hematoxylin-eosin(HE)and Von-Kossa exhibited good sensitivity to arterial calcification and Von-Kossa was able to identify the presence of calcification in two samples that HE was unable to,possibly owing to the reaction between silver ions and the phosphate[18].

The primary objective of the study was to determine if there was a correlation between fetuin-A and arterial calcification at the onset of hemodialysis using Pearson’s correlation.Pearson’s correlation used the data indicat-ing a moderate inverse correlation between fetuin-A and arterial calcification A.The fact that our correlation was moderate and not as strong as that observed by Zheng[19]does not invalidate the research,since this author stud-ied patients that had undergone a long period of hemodialysis.In addition,our main goal was to show the exist-ence of this relationship at the onset of dialysis and not the power of this relationship at that moment.

Our study did not aim to definitively establish the early change in fetuin-A or relate it to the primary cause of arterial calcification in patients with chronic kidney disease,but rather demonstrate that it exists and that further research is needed to determine the real importance of fetuin-A and other factors in arterial calcification,in an attempt at decreasing the morbidity and mortality of patients submitted to hemodialysis for the treatment of chronic kidney disease.

Many studies have tried to identify the relationship of fetuin and vascular calcification.One of them failed to demonstrate the clear protective relationship of fetuin[20].More recently,a study correlated fetuin levels with bone porosity in stone formers[21].In children with chronic kidney disease,a study observed the relationshipbetween fetuin levels and other risk factors for vascular calcification[22].

We conclude that there is a moderate inverse correlation between arterial calcification and fetuin-A in patients with chronic kidney disease at the onset of hemodialysis.

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