Different type and dosage of heparin were not associated with the progression of coronary artery calcification in haemodialysis patients

ABSTRACT Aim Several studies have verified that unfractionated heparin (UFH) and low molecular heparin (LWMH) can induce bone loss, and bone mineral density has been inversely associated with vascular calcification in some clinical researches. But few have focused on the relationship between types and dosages of heparin and the progression of vascular calcification. We observed the progression of coronary artery calcification (CAC) in maintenance haemodialysis (MHD) patients who were treated with UFH and LMWH. Methods This was a prospective prevalent cohort study of MHD patients. Computed tomography was performed at enrolment and 2 years after enrolment, and CAC score was obtained. Demographic and clinical data, baseline and time‐average laboratory indices were collected. Multiple linear regression and logistic regression were used to estimate the influencing factors of progression of CAC. Results In this study, (i) we initially enrolled 69 HD patients, and then 56 patients finished the follow‐up. (ii) Among the total 56 patients, 27 patients (48.2%) were treated with UFH, 14 (25.0%) with LMWH and 15 (26.8%) with both. The median baseline CAC scores of three groups (UFH, LMWH and both users) were 91.0 (1.0, 1052.0), 134.0 (0, 1292.0) and 250.5 (27.0, 1139.0), respectively, with no significant difference (P = 0.663); the median CAC progression scores were 42.0 (0, 364.0), 172.0 (7.0, 653.0) and 118.5 (0, 434.0), respectively, with no significant difference (P = 0.660). (iii) Pearson and spearman correlation analysis shown that the progression of CAC was not associated with cumulative dosage of heparin used. (iv) After adjusted for diabetes mellitus, time‐averaged intact parathyroid hormone, phosphate and alkaline phosphatase, logistic regression analysis showed using different types of heparin was not an independent risk factor for CAC progression; and multiple linear regression analysis showed that the type of heparin used was not associated with CAC progression. Conclusion There were no significant differences in the effects of the types and dosages of heparin on CAC progression in patients on haemodialysis.

who had been on UFH or low molecular heparin (LMWH), Monreal et al. 7 found that the incidence of fractures was significantly higher in patients treated with UFH than in patients treated with LMWH. Although LMWH had relatively fewer side effects than UFH, 9 long-term use of LMWH was not risk-free. 10 In vivo, Meng et al. treated rats with different doses of UFH and LMWH, they observed that UFH-induced bone loss in chronic kidney disease (CKD) rats with secondary hyperparathyroidism was mainly caused by inhibition of osteoblast activity and promotion of osteoclast activity. 11 In vitro, the mechanism of UFH caused osteoporosis was that UFH promoted osteoclastogenesis by inhibiting the activity of osteoprotegerin. 12,13 The relationship among abnormalities of serum biochemical markers, renal bone disease and vascular calcification in ESRD patients had been established. Chen et al. 14 indicated that low bone density is an independent risk factor of vascular calcification in non-dialysis CKD patients. Tangvoraphonkchai and Davenport reported that lower BMD at the femoral neck was associated with greater aortic pulse wave velocity, reinforcing the hypothesis of a link between bone disease and vascular disease in dialysis patients. 15 Aleksova et al. 16 showed that trabecular bone score, an instrument for measurement of bone microarchitecture integrity, was inversely related to vascular calcification in dialysis patients, which means that patients with low trabecular bone scores tended to have more severe vascular calcification.
Unfractionated heparin and LMWH have remained the most widely used anticoagulant in HD. 17,18 Previous studies have observed the phenomenon that long-term, high-dose using of heparin can cause bone density decrease and osteoporosis. 7,9,19,20 And in pre-dialysis CKD and dialysis patients, some studies indicated that bone density is inversely related to vascular calcification. 15,16 Taking all these together, we hypothesized that high dosage or long-time usage of UFH and LWMH may accelerate the progression of vascular calcification in HD patients. This was confirmed by Meng et al. 11 in rats in 2013, in this study, they had four groups: normal rats, CKD control rats, low-dose UFH CKD rats and highdose UFH CKD rats. They found that calcium and phosphorus contents in the thoracic aorta in the high-dose UFH CKD rats were higher than those in the low-dose UFH CKD group and CKD control group.
To date, the effects of long-term use of UFH or LMWH on the occurrence and progression of vascular calcification in patients on MHD were not reported. In this study, we analyzed types and cumulative dosages of heparin on progression of CAC in HD patients in a prospective cohort.

Study design and subjects
This was a single-centre prospective cohort study with MHD patients. Enrolled patients were received multi-slice spiral computed tomography to evaluate CAC at the time of enrolment and the end of 2 years follow-up period. The influences of type and dosage of heparins on progression of CAC were analyzed using logistic model and linear regression model. This study was approved by the Ethics Committee of Peking University People's Hospital (IRB00001052-11055).
We enrolled MHD patients with ESRD in our dialysis center from February 2012 to January 2015. Inclusion criteria for participants: (i) age >18 years old, dialysis vintage ≥6 months and stable clinical condition; (ii) use UFH and/or LMWH as anticoagulant during dialysis. Exclusion criteria: (i) conditions making computed tomography (CT) technically impossible or unreliable (such as severe cardiac arrhythmias); (ii) patients with acute complications, such as heart failure, severe infection, malignant tumour and life expectancy less than 3 months.

Demographic and clinical data
Baseline demographics were collected, including age, gender, dialysis vintage, the presence or absence of diabetes and body mass index (BMI). Laboratory indices were tested at baseline and every 3 months thereafter, and then averaged as the time-average value, including serum corrected calcium (cCa), phosphate (P), intact parathyroid hormone (iPTH), alkaline phosphatase (ALP), albumin (ALB), uric acid (UA), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (T-Chol), serum creatinine (Scr), carbon dioxide combining power (CO2CP), haemoglobin (Hgb), total urea clearance (Kt/V). We also recorded the use of the drug during the follow-up period, such as calcium-based phosphate binders, non-calcium-based phosphate binders, vitamin D analogue and Cinacalcet.
The type and dose of anticoagulants used for each dialysis during follow-up were recorded and the cumulative dosage used during the period was calculated. Patients were grouped as UFH users (UFH was used in more than 90% dialysis sessions), LMWH users (LMWH was used in more than 90% dialysis sessions), and UFH/LMWH users.

Evaluation of CAC
Computed tomography scans were performed at enrolment and 2 years after enrolment in the department of radiology of our hospital, CAC scores assessed blindly by two radiologists according to the method previously described by Agaston et al. 21 CAC progression score was defined as the difference in follow-up CAC scores minus baseline CAC scores, reflecting the progression of CAC during the 2 years follow-up period. To describe the various rate of progression of calcification, subjects were also classified as the progression of CAC score <100, 100-500, and >500.

Statistical methods
Continuous variables were expressed as mean AE standard deviation or median (25th, 75th), and categorical data were expressed as number and percentages. As the number in each group is small, differences in mean and median values among UFH, LWMH or both users were tested using Kruskal-Wallis test, which is a method of nonparametric test. Categorical variables among groups were compared using χ² test. Pearson and Spearman correlation analysis were used to examine the relationship between the cumulative dosage of heparin substances and the progression of CAC, the relationship between the types of heparin substances and the progression of CAC. In the one-way analysis of variance, there were significant differences of the proportion of DM, timeaveraged iPTH, and time-averaged P among groups of CAC progression. We put these factors and the type of heparin into the models of multivariate linear regression analysis and logistic regression analysis, which were used to analyze the independent influencing factors for CAC progression. P value <0.05 was considered to be statistically significant. Statistical analysis was performed using SPSS software, version 22.0 (IBM Corp., Armonk, NY, USA).

RESULT Demographic data and clinical characteristics
We initially enrolled 69 HD patients, but during the followup period 13 patients were excluded, the baseline characteristics of these patients were shown in Table 1. Reasons of elimination: three patients only had baseline CT scans, three patients received kidney transplantation, three patients died during follow-up, two patients had CT that could not assess coronary calcification score due to severe motion artefacts or stents, one patient received bypass surgery and one patient transferred to other hospital. Finally, 56 patients were included in the present study, including 37 males (66.1%), with an average age of 52.3 AE 13.7 years and a median dialysis duration of 39.0 (13.5, 80.8) months. Primary causes of ESRD were predominantly chronic glomerulonephritis (n = 28, 50.0%), followed by diabetic nephropathy (n = 12, 21.4%), chronic tubulointerstitial nephropathy (n = 5, 8.9%), hypertensive nephropathy (n = 4, 7.1%), and others (n = 7, 12.5%). There were 13 patients (23.2%) complicated with diabetes ( Table 2).

Usage of heparin
Of the 56 patients, 27 (48.2%) were UFH users, 14 (25.0%) were LMWH user and 15 (26.8%) were both users. The cumulative dosages of UFH and LMWH in the three groups were shown in Table 3. There were no significant differences in baseline demographics of age, dialysis vintage, proportion of diabetes and laboratory indices of serum cCa, P and iPTH among the three groups. Serum ALP level was lower in both users than in UFH users or LMWH users (P = 0.018), Kt/V level was higher in LMWH users than UFH users or both users (P = 0.015) ( Table 2).

Coronary artery calcification
Baseline CT scans showed a total of 43 patients (76.8%) with CAC. Among the 13 patients (23.2%) without CAC at baseline, 3 patients developed CAC after 2 years of followup. The baseline CAC scores of the UFH, LMWH and both UFH/LMWH users were 91.0 (1.0, 1052.0) (mean value 511.4), 134 (0,1292.00) (mean value 574.9) and 250.5 (27.0, 1139.0) (mean value 651.9), respectively, and no significant difference in baseline CAC scores were found among the three groups (P = 0.663, Table 3). After 2 years, the progression CAC scores in the three groups were 42.0 (0, 364.0) (mean value 351.2), 172.0 (7.0, 653.0) (mean value 349.5) and 118.5 (0, 434.0) (mean value 216.1), respectively. No significant difference in CAC progression was found among the three groups (P = 0.660, Table 3). The detailed changes between baseline and 2 years later CAC score by group were shown in Fig 1. And the delta CAC scores of each patient in three groups were shown in Fig. 2.

Influencing factors of the progression of CAC
The proportion of patients combined with diabetes in groups of CAC progression scores between 100-500 and >500 was higher than that in the group of progression scores <100 (P = 0.025). There were no significant differences in baseline laboratory indices among the three groups. The timeaverage serum P, iPTH, and ALP levels in the progression scores >500 group were higher than those with progression scores between 100-500 and <100 (P = 0.001, 0.043, 0.025, respectively, Table 4). Pearson and Spearman correlation analysis showed that progression of CAC was not associated with the type (correlation index: 0.086, P: 0.530) and cumulative dosage (correlation index: 0.052, P: 0.747) of heparin used (Fig. 3).

DISCUSSION
In this perspective cohort, we did not find the significant differences in the effects of types and cumulative dosages of heparin on the CAC progression of HD patients in our centre.
A total of 56 HD patients using UFH, LMWH or both of UFH and LMWH were finished the follow-up in the study, CT were performed twice, 2 years apart. Results showed that there was no significant difference in baseline and progression of CAC score among the three heparin type groups.   Also, we did not find a significant association between heparin dosages and progression of CAC score. These results were consistent in our logistic model and linear model, after adjusted recognized risk factors of CAC, including diabetes and some laboratory indices. To obtain stable regression model, the time-average value of laboratory indices, which can reflect patients' actual condition, were used. According to literatures, 6,7,[14][15][16] it was reasonable if we confirm that UFH can promote more progression of CAC than LWMH or a high cumulative dosage of heparin substances was relative to fast progression of CAC, but our results were negative. There were several reasons that we got these negative results. (i) The shorter period of observation. Vascular calcification in CKD is a complication of slow onset and slow progression, 22 There was a meta-analysis indicated that LMWH for 3-6 months may not increase the risk of fractures in participants with venous thromboembolism and underlying CVD, but longer exposure for up to *P < 0.05. † For the pairwise comparison, the percentage of DM were different between the progression <100 and progression >500 groups (P < 0.05), and the progression <100 and 100-500 groups, and no different between the progression 100-500 and > 500 groups (P > 0.05). ‡ The baseline T-Chol were different between the progression <100 and progression >500 groups (P = 0.024), and no different between other groups (P = 0.092, P = 0.397). § The timeaverage P level were different between the progression <100 and progression >500 groups (P = 0.001), and the progression 100-500 and progression > 500 groups (P = 0.002), and there was no difference between progression <100 and 100-500 group (P = 0.634). ¶ The time-average iPTH levels were different between the progression <100 and progression > 500 groups (P = 0.037), and no difference between other groups (P = 0.495, 0.064); † † The time-average ALP levels were different between the progression <100 and progression >500 groups (P = 0.021), and no difference between other groups (P = 0.632, 0.332). Alb, albumin; ALP, alkaline phosphatase; BMI, body mass index; CAC: coronary artery calcification; cCa, corrected calcium; DM, diabetes mellitus; Hb, haemoglobin; HDL-C, high-density lipoprotein cholesterol; iPTH, intact parathyroid hormone; Kt/V, total urea clearance; LDL-C, low-density lipoprotein cholesterol; LMWH, low molecular heparin; P, phosphate; Scr, serum creatinine; TG, triglyceride; T-Chol, total cholesterol; UA, uric acid; UFH, unfractionated heparin, 25-OH-VD, 25-hydroxy Vitamin D3.
2 years may adversely affect BMD. 20 In last year, Meng et al. 23 published another study, they divided rats into normal group, CKD control group, UFH group, and LMWH group, and they did not found the different effects of UFH and LMWH on vascular calcification in rats in 3-week observation. For the effects of UFH and LMWH on bone metabolism, some previous studies have suggested that LMWH causes less bone loss and osteoporosis than UFH. 24 Our results also found that some factors were related to the progression of CAC, including diabetes, higher time-averaged iPTH, higher time-averaged P. These findings were consistent with previous studies. 22,32,34 Interestingly, there were 10 patients had no CAC during baseline and follow-up period. Dive into the data of these patients, most of them were younger, had a shorter dialysis vintage and without diabetes. As aging and progression of diseases, some of them might develop vascular calcification in their future days. Among them, there were a 61 years old male who had been on HD for 3 years and a 48 years old female who had been on HD for 5 years, did not have CAC. Further study was needed to elucidate why not all HD patients develop CAC in their disease course.
The limitation of our study was the relatively small sample size, which may have impact on the stability of statistical results. And the processes of osteoporosis and vascular calcification are slow, two-year follow-up period might not enough to be observed.
In conclusion, after 2 years observation, we did not find significant differences in baseline and progression of CAC scores among the three groups of UFH, LMWH and both users after adjusted by DM, time-averaged iPTH, P and ALP. In the future, studies with large sample sizes and long follow-up period are still needed to explore the effects of heparin substances on vascular calcification in HD patients.

DISCLOSURES
We have no conflict of interest to report.