A Multivariate Analysis-Factors Related To Vascular Calcification On Regular Hemodialysis Patient; First In Indonesia

Introduction: Cardiovascular disease is one of the main causes of death in patients with chronic kidney disease (CKD) undergoing hemodialysis or peritoneal dialysis therapy. This is related to calcification in large blood vessels such as the aorta. Although some known coronary risk factors, such as age, dyslipidemia, diabetes and smoking, play an important role in cardiovascular disease in patients undergoing hemodialysis, but several other risk factorsy such as anemia, uremic toxins, oxidative stress, and disorders of mineral and bone metabolism also associated with CKD.Method: This study was an analytical cross-sectional study with subjects were from CKD patients who underwent regular hemodialysis (for at least 3 months) in hemodialysis unit of RSKG Rasyida Medan from May 2018 and aged > 18 years. Interviews were conducted regarding age, duration of hemodialysis. Laboratory examination of calcium, phosphate and fetuin-A levels and lateral abdominal X-ray examination were conducted, then a statistical analysis was performed to determine the relationship of these factors with blood vessel calcification in regular hemodialysis patients.Results: Of 113 subjects, duration of hemodialysis in no calcifications group was 52.09 ± 21.725, while mild grade calcification underwent HD for 51.81 ± 29.115, and severe calcification group underwent HD for 69.06 ± 36.030. A total of 10 patients without calcification had a history of DM, which were total 14 patients with calcification (mild and severe group) had history of DM. The mean serum calcium level in the group without calcification was 9,467 ± 0,843 mg / dl, 9,638 ± 0,708 mg / dl in mild group, while the average calcium level was 5,412 ± 0,645 mg / dl in severe group.Conclusion: Duration of hemodialysis, diabetes mellitus, calcium levels and fetuin-A significantly influence the occurrence of calcification in the abdominal blood vessels. (p = 0.004, p = 0.049, p = 0.005 and p = 0.004)

3 Background Cardiovascular disease is one of the main causes of death in patients with chronic kidney disease (CKD) undergoing hemodialysis or peritoneal dialysis therapy. Increased mortality caused by cardiovascular disease, associated with calcification in large blood vessels, such as the aorta, where this can cause stiffness in the arteries and increase pulsation pressure and decreased myocardial perfusion at diastole. 1 Therefore, prevention of cardiovascular disease is important to reduce the incidence of morbidity and mortality. Although some known coronary risk factors, such as age, dyslipidemia, diabetes and smoking, play an important role in cardiovascular disease in patients undergoing hemodialysis, but several other risk factors, which are associated with chronic kidney disease are also involved, such as anemia, uremic toxins, oxidative stress, and mineral and bone metabolic disorders. 2 For this reason, many researchers are now finding out the relationship between a disorder of mineral metabolism and bone and vascular calcification.
The mechanism of ectopic calcification is very diverse and is not clearly known. The most studied is the pathology of vascular calcification as a major risk factor for cardiovascular mortality. Basically, regulation of calcification is regulated by maintaining extracellular calcium and phosphate concentration. 3 There are many methods that can be use to diagnose a vascular calcification such as ultrasound mostly for superficial vascular, multi-slice computed tomography (MSCT) but the downside is it cannot differentiate a intima or media tunica calcification, and lateral lumbal X-ray. There are no any gold standard to diagnose a vascular calcification.
Although it is still a controversy, The Kidney Disease Improving Global Outcome (KDIGO) suggest a lateral abdominal radiography can be use for detection of vascular calcification. 4

Study Samples
Samples were from populations of CKD patients who underwent regular hemodialysis in the hemodialysis unit of RSKG Rasyida Medan from May 2018 (for at least 3 months) and aged> 18 years. The subject had received information and gave consent to participate in informed and voluntary research. Patients who underwent irregular hemodialysis or were unstable were excluded from this study.

Study Design
This study is an analytical cross-sectional study. The proportion formula is used to determine the number of samples. After obtaining approval from the ethics committee, the subjects who met the inclusion and exclusion criteria were given an explanation and asked to provide informed consent to take part in the study. Interviews were then conducted to determine the age, history of diabetes mellitus and the duration of the patient's hemodialysis. Then blood sampling is performed to determine serum calcium levels and serum phosphorus, as well as lateral abdomen x-ray. After the data collected, data processing and data analysis were then conducted.

Statistical Analysis
Univariate analysis was conducted to obtain an overview of each variable studied.
Bivariate analysis is used to state the analysis of two variables, which are the dependent variable and the independent variable. To see the strength of the relationship between the dependent and independent variables, the value of the Prevalence Ratio (RP) is used. In bivariate analysis anova test is used because the independent variable is numerical data and the dependent variable is ordinal data.
Multivariate analysis is used to see the relationship of several independent variables with the dependent variable by connecting independent variables with the dependent variable at the same time. In this analysis, the variables with the greatest influence can be known, the form of relationships between variables, related directly or indirectly to other variables.

Research Subjects Characteristic Distribution
This study was followed by 113 patients as research subjects who had met the inclusion and exclusion criteria. The majority of the study subjects were men (59.3%) with a median age of 57 years. Based on the results of laboratory tests, the median serum calcium level was 9.8 mg / dl, the mean serum phosphate level was 5.39 mg / dl. From the results of lateral abdominal X-ray examination in this study the majority of patients having severe calcification of 43.4%, no calcifications of 38.1%), and only 18,6% having mild calcification. (Table 1)

Vessel Calcification
Man having more severe calcification (47.8%), compared with women who showed the same results in severe calcification and no calcification (each of 37.0%). Based on the statistical test, it was concluded that there was no significant difference between gender and the degree of calcification (p = 0.211). (Table 2) Subjects with no calcification were 49.0 ± 13.020 years of age, lower than the average age of mild and severe degrees (54.81 ± 9.19 and 59.47 ± 8.615). Based on the statistical test, it was concluded that there were significant differences between mean of age to the degree of calcification (p = 0.001) ( Table 2). In Table 3 there was a significant difference in age at normal levels compared to severe degrees (p = 0.001).
The subjects who have no calcifications had underwent hemodialysis for 52.09 ± 21.725, the average HD duration of mild calcification degree was 51.81 ± 29.115, while the average length of HD degrees of severe calcification was 69.06 ± 36.030. It was concluded that there was a difference in the average duration of HD to the degree of calcification (p = 0.004). In table 3 there is a significant difference between the no calcification and severe calcification (p = 0.007) and between mild calcification and severe calcification (p = 0.007).
10 subjects with no calcification had a history of DM more than of mild and severe group (8 and 6 people) so it was concluded that there was a correlation between the history of DM and the degree of calcification (p = 0.049).
Subjects who did not have calcifications had an average serum calcium level of 9,467 ± 0,843 mg / dl, the average level of mild calcium was 9,638 ± 0.708 mg / dl, while the average level of calcium in severe calcification group was 5.412 ± 0.645 mg / dl. It can be concluded that there are significant differences between the average calcium level and the degree of calcification (p = 0.005). In table 3 there is a significant difference between the degree of absence of calcification and severe calcification (p = 0.002) and between mild calcification and severe calcification (p = 0.063).
The serum phosphate levels of no calcification group of patients were 5.419 ± 0.626mg / dl, level of 5.290 ± 0.656 mg / dl in mild calficiation group, and level of 10.047 ± 0.774mg / dl in severe calcification group. Based on the statistical test it was concluded that there was no difference in the average phosphate level to the degree of calcification (p = 0.723).

Correlation Between Calcium Serum Level and Phosphate Level
From this analysis it was found that serum calcium and serum phosphate had a significant relationship (p = 0.001) with a fairly strong relationship strength (r = 0.324) ( Based on the statistical test, it was concluded that there was a relationship between the history of DM and the degree of calcification (p = 0.049). The oxidation process and oxidative stress of the tissue formed as a product of increased glucose levels are a risk factor for increasing the degree of calcification. This situation also occurs in the study of Joachim, et al. 10,11 The results of this study are in line with the research conducted by Coll et al where there was a significant relationship between DM patients with the occurrence of vascular calcification (p = 0.007). 9 In this study, it was found that serum calcium had a significant relationship with the occurrence of vascular calcification, in severe and without calcification. Calcium plays a major role in regulating contraction and relaxation of vascular smooth muscle by maintaining extracellular calcium and phosphate concentration. 13 In patients with chronic kidney disease, there is a relationship between impaired mineral metabolism (serum calcium and phosphorus levels), bone abnormalities (renal osteodystrophy) and vascular calcification. 1  Advice on Tables

Conclusions
The conclusion of this study is that the duration of hemodialysis, diabetes mellitus, and calcium levels significantly influence the occurrence of calcification in the abdominal blood vessels. The prevalence of vascular calcification in hemodialysis patients who experienced severe calcification from this study was 49 people (43.4%), there were no