Serum osteoprotegerin is an independent marker of central arterial stiffness as assessed using carotid–femoral pulse wave velocity in hemodialysis patients: a cross sectional study

Background Cardiovascular morbidity and mortality are highly prevalent in patients with end-stage renal disease, and osteoprotegerin (OPG) may be an important link between bone loss and vascular calcification. This study was conducted to evaluate the relationship between central arterial stiffness and serum OPG levels in hemodialysis (HD) patients. Methods Blood samples were collected from 120 HD patients, and the carotid–femoral pulse wave velocity (cfPWV) value was measured using a validated tonometry system. The cfPWV value of > 10 m/s was used to define the high artery stiffness group. Serum OPG levels were analyzed categorically into tertiles. Results Of the 120 HD patients, 53 (44.2%) were defined as the high arterial stiffness group, who had higher values of systolic blood pressure (p = 0.038), serum calcium (p = 0.007), and OPG (p <  0.001) levels and a higher prevalence of diabetes mellitus (DM, p = 0.001). Increasing tertiles of serum OPG levels were significantly associated with greater height (p = 0.011), male gender (p = 0.008), higher cfPWV values (p = 0.020), and lower intact parathyroid hormone (iPTH, p = 0.049) levels. Multivariable linear regression analysis showed that cfPWV value was independently associated with DM (β = 1.83, p = 0.008) and increasing tertiles of serum OPG levels (β = 0.89 and 1.63 for tertile 2 and tertile 3, respectively, p for trend = 0.035) in HD patients. Multivariable logistic regression analysis revealed that, in addition to age, DM, low iPTH levels, and high serum calcium levels, increasing tertiles of serum OPG levels (OR = 5.34 for tertile 2; OR = 7.06 for tertile 3; p for trend = 0.002) were an independent predictor of high arterial stiffness in HD patients. Serum calcium levels positively correlated with cfPWV value only in the highest OPG tertile group (r = 0.408, p = 0.009). Conclusion A positive association was detected between serum OPG levels and central arterial stiffness in HD patients, and patients with high serum OPG levels may have greater influence of calcium load on central arterial stiffening.

Background Cardiovascular (CV) disease is highly prevalent in patients with end-stage renal disease (ESRD), accounting for approximately 20-60% of all-cause death [1][2][3]. In addition to traditional risk factors for atherosclerosis, patients with chronic kidney disease (CKD) and ESRD are susceptible to vascular calcification, which is characterized by medial and intimal layer calcification [4,5]. Both the abovementioned conditions subsequently contribute to increased arterial stiffness and risk of premature CV disease death. Thus, exploring the potential mechanisms of vascular calcification in patients with ESRD is of paramount importance. Aging, inflammation, oxidative stress, diabetes mellitus (DM), and calcium and phosphorus loading are well-established factors associated with vascular calcification [5,6]. Moreover, cumulative evidence has demonstrated that abnormal bone mineralization is also involved in the pathogenesis of vascular calcification.
Osteoprotegerin (OPG), a soluble glycoprotein that binds to receptor activator of nuclear factor kappa-B ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), is not only well-known to play a critical role in bone homeostasis, but it has also been implicated in the complex process of vascular remodeling. The stimulation of OPG/RANK/RANKL signaling, through inflammation and reactive oxygen species, has been reported to be involved in the pathogenesis of vascular calcification [7]. This connection is of particular interest in patients with ESRD due to the accelerated vascular calcification and the markedly elevated plasma OPG levels in this special population [8]. However, this complex pathogenesis has not yet been completely understood.
Carotid-femoral pulse wave velocity (cfPWV) is a noninvasive gold standard method for measuring large artery stiffness [9,10], which is known to be associated with CV mortality in patients with ESRD [11]. In this study, we determined whether serum OPG levels are associated with central arterial stiffness in chronic hemodialysis (HD) patients, independent of the abovementioned well-established vascular risk factors.

Patients
The cross-sectional study was conducted at a medical center in Hualien between March and July in 2015 and was approved by the Institutional Review Board of Tzu-Chi Hospital. Patients aged more than 20 years and who had undergone HD for at least 3 months with standard 4-h dialysis three times per week were included. The same type of high-flux polysulfone disposable artificial kidney (FX class dialyzer, Fresenius Medical Care, Bad Homburg, Germany) was used by all patients. Patients who had any infection, acute myocardial infarction, stroke, peripheral arterial occlusive diseases, pulmonary edema, arrhythmias and atrial fibrillation at the time of enrollment or who refused to participate were excluded from the study. DM was defined based on a history of antidiabetic drug use, and hypertension was defined based on a history of or receiving antihypertensive agents. Coronary artery disease (CAD) was diagnosed through coronary angiography. The drug use history was collected by a review of medical records, which included the use of angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), β-blockers, and statins.

Anthropometric analysis and blood pressure measurement
Body weights were measured in light clothing after HD, and height was measured to the nearest half centimeter without shoes. Body mass index (BMI) was calculated as body weight (kilograms) divided by height squared (meters). Blood pressure was measured before the HD session using standard mercury sphygmomanometers.

Measurement of arterial stiffness
PWV was evaluated from the time taken for the arterial pulse waveform to propagate from the carotid to the femoral artery, using applanation tonometry (Sphygmo-Cor system, AtCor Medical, Australia). A transcutaneous recording of the pressure pulse waveform in the underlying artery was performed in the supine position after a minimum 10-min rest before the HD session. The cfPWV was calculated using the distance and mean time difference between the two superficial artery sites. A detailed explanation of this device has been provided elsewhere [12]. Patients with cfPWV > 10 m/s were defined as the high arterial stiffness group, according to the European Society of Hypertension and the European Society of Cardiology (ESH-ESC) 2013 Guidelines [10].

Biochemical determinations
Blood samples were collected from the patients for analysis before the HD session. Fasting blood samples (approximately 5 mL) were immediately centrifuged at 3000 g for 10 min. The obtained serum samples were stored at 4°C and used for biochemical analyses within 1 h of collection. Serum levels of blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, calcium, phosphorus, and C-reactive protein were measured using an autoanalyzer (Siemens Advia 1800, Siemens Healthcare GmbH, Henkestr, Germany). Corrected serum calcium levels were calculated as follows: corrected calcium (mg/dL) = serum calcium (mg/dL) + 0.8 [4serum albumin (mg/dL)], and the upper normal limit was chosen as the reference value according to the K/DOQI recommendations [13]. The levels of intact parathyroid hormone (i-PTH) were measured by an autoanalyzer (Diagnostic Systems Laboratories, Webster, Texas, USA), and levels less than two times the upper limit and more than nine times the upper normal limit were defined as low and high parathyroid hormone levels, which are 130 and 595 pg/mL, respectively [14]. Serum OPG levels were measured using a commercially available enzyme-linked immunosorbent assays (ELISA) (eBioscience Inc., San Diego, CA, USA). The fractional clearance index for urea (Kt/V) was measured before and immediately after dialysis using a formal, single-compartment dialysis urea kinetic model.

Statistical analysis
The normality of the distributions of continuous variables was assessed using the Kolmogorov-Smirnov test. Variables with normal distribution were expressed as mean ± standard deviation and analyzed by the Student's independent t-test, whereas those not normally distributed were expressed as medians and interquartile ranges and analyzed by the Mann-Whitney U test. Categorical variables were expressed as absolute (n) and relative frequency (%) and analyzed by the χ 2 test or Fisher's exact test. Serum OPG levels were analyzed categorically into tertiles, and characteristics of the study population in different OPG groups were evaluated for linear trend using one-way analysis of variance (ANOVA) or the Cochran-Armitage test for trend. The values of cfPWV, HD duration, glucose levels, iPTH levels, and serum OPG levels showed skewed distribution and were logarithmically transformed to achieve normality. Univariable correlations between cfPWV and potential explanatory variables were assessed by Pearson's correlation coefficient. Multivariable linear and logistic regression analyses were conducted using cfPWV values and the presence of high arterial stiffness (defined as cfPWV > 10 m/s) as dependent variables and potential predictors (age, sex, DM, systolic blood pressure, corrected serum calcium, phosphorus, iPTH, and OPG levels) as independent variables. Data were analyzed using SPSS for Windows (version 19.0; SPSS Inc., Chicago, IL, USA). A p value < 0.05 was considered as statistically significant.

Population characteristics
A total of 120 chronic HD patients were enrolled in our study. The mean age of the patients was 63.3 ± 13.7 years, and 53 (44.2%) of them had high arterial stiffness. The clinical and laboratory characteristics of the 120 chronic HD patients with low or high arterial stiffness are presented in Table 1. Compared with the low arterial stiffness group, patients in the high arterial stiffness group had higher systolic blood pressure (p = 0.038), cfPWV (p < 0.001), corrected serum calcium (p = 0.007), and OPG levels (p < 0.001). In addition, the proportion of DM was higher in the high arterial stiffness group (p = 0.001).
The clinical characteristics of the study population were stratified by tertiles of serum OPG levels ( Table 2). Increasing tertiles of serum OPG levels showed a significant association with greater height (p = 0.011), male gender (p = 0.008), higher cfPWV (p = 0.020), and lower iPTH levels (p = 0.049).

Factors correlated with cfPWV
The continuous variables were found to correlate with cfPWV values among the 120 HD patients, as shown in Table 3. The cfPWV values showed a positive correlation with systolic blood pressure (r = 0.196, p = 0.032), serum glucose (r = 0.182, p = 0.047), and OPG levels (r = 0.281, p = 0.002).
The factors associated with cfPWV values were analyzed among the 120 HD patients. After multivariable adjustment, increasing tertiles of serum OPG levels (β = 0.89 and 1.63 for tertile 2 and tertile 3, respectively, p for trend = 0.035) and the presence of DM (β = 1.83, p = 0.008) were the two independent factors that were associated with cfPWV values. Notably, low iPTH levels were also marginally associated with cfPWV values (β = 1.29, p = 0.055), compared to iPTH levels in the normal range (Table 4).
Furthermore, we analyzed the correlation between corrected calcium levels and cfPWV values based on the tertiles of serum OPG levels, as shown in Fig. 1. The corrected calcium levels showed a positive correlation with cfPWV values in the tertile 3 group (r = 0.408, p = 0.009), but not in the tertile 1 or 2 group.

Discussion
This study demonstrated that cfPWV was independently associated with DM and serum OPG levels in our sample of HD patients. In addition, older age, high serum OPG levels, DM, low iPTH levels, and high serum calcium levels were identified as independent predictors for high arterial stiffness. Moreover, in patients in the highest OPG tertile groups, serum calcium levels positively correlated with cfPWV values.
OPG is not only a well-known factor that is involved in the bone remodeling process, but it also plays an important role in vascular calcification. OPG is highly expressed in vascular smooth muscle cells and endothelial cells of arteries and has been regarded as a vascular protective factor by inhibiting vascular calcification; OPG-deficient mice were shown to exhibit an accelerated arterial calcification [15]. However, several observational studies have demonstrated that serum OPG levels appear to be elevated in patients with vascular damage and were considered as a strong vascular risk factor in a variety of different populations, including healthy people and patients with DM, hypertension, coronary artery disease, and CKD [12,[16][17][18][19][20][21]. In addition, elevated serum OPG levels were found to be associated with CV events and mortality in patients with ESRD [22,23]. This paradoxical phenomenon may be partially explained by the compensatory response of serum OPG levels in mitigating further vascular calcification [5]. Consistent with previous studies, our patients in the high arterial stiffness group showed significantly higher serum OPG levels that, after being categorized into tertiles, demonstrated a significant trend with central arterial stiffness after complete adjustment of potential confounding factors, analyzed as either continuous or dichotomous outcome variable. Values for continuous variables are shown as mean ± standard deviation; variables not normally distributed are shown as median and interquartile range; values for categorical variables are presented as number (%). characteristics of the study population in different OPG groups were evaluated for linear trend using one-way analysis of variance (ANOVA) or the Cochran-Armitage test for trend OPG osteoprotegerin, HD hemodialysis, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, cfPWV carotid-femoral pulse wave velocity, BUN blood urea nitrogen, PTH parathyroid hormone, Kt/V fractional clearance index for urea, CAD coronary artery disease, ACEIs angiotensin-converting enzyme inhibitors, ARBs angiotensin receptor blockers; CCBs, calcium channel blockers *p < 0.05 is considered as statistically significant Hypercalcemia has been regarded as a promoter in the development of vascular calcification in patients with ESRD [4,24]. In our study, we found that serum calcium levels were an independent predictor for arterial stiffness among our chronic HD patients. In addition, a positive correlation was found between serum calcium levels and cfPWV only in patients in the highest OPG tertile groups. These unique findings indicate that patients with high serum OPG levels may experience a greater influence of calcium load on central arterial stiffening.
Some studies have demonstrated that vascular calcification is accelerated in both high and low bone turnover disease [24][25][26]. In our study, patients with low serum iPTH levels, which indicated low bone remodeling, exhibited an increased risk of developing high arterial stiffness compared to those with iPTH levels in the normal range. However, the elevated serum iPTH levels were not significantly associated with high arterial stiffness in our study, which could be attributed to the limited sample size in this subgroup. DM is associated with an increased activity of renin-angiotensin-aldosterone system, oxidative stress, formation of advanced glycation end-products (AGEs), enhanced expression of angiotensin type I receptor in vascular tissue, and impaired PI3-kinase-dependent signaling, all of which promote the development of wall hypertrophy and fibrosis [27,28]. Therefore, patients The data of cfPWV, HD duration, glucose, iPTH and OPG showed skewed distribution and were log-transformed before analysis Data were analyzed using Pearson's correlation analysis cfPWV carotid-femoral pulse wave velocity, HD hemodialysis, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, BUN blood urea nitrogen, iPTH intact parathyroid hormone, CRP C-reactive protein, OPG osteoprotegerin, Kt/V fractional clearance index for urea *p < 0.05 is considered as statistically significant  with diabetic ESRD exhibit more advanced atherosclerotic changes of arteries that result in arterial stiffness [29]. In our study, up to two thirds of patients in the arterial stiffness group had DM. Moreover, DM was found to be the major predictor of arterial stiffness in our chronic HD patients. Such an association between DM and arterial stiffness in chronic HD patients has also been demonstrated in previous studies [29][30][31][32]. Several limitations must be acknowledged in our study. First, the sample size was relatively small. Second, measurements of blood pressure and cfPWV values before the HD session may be influenced by the hydration status. Third, we could not obtain the causal relationship between serum OPG levels and arterial stiffness in this cross-sectional study. Finally, we did not include certain medications in our analysis that may have an influence on arterial stiffness, such as phosphate binders and vitamin D analogs.

Conclusions
Our study has demonstrated that, in addition to age, DM, hypercalcemia, and low serum iPTH levels, which are well-known factors contributing to arterial stiffness, a strong and positive correlation exists between serum OPG levels and arterial stiffness in patients with chronic HD. Moreover, positive correlations were found between serum calcium levels and cfPWV in patients in the highest OPG tertile groups, indicating that patients with high serum OPG levels may experience a significantly greater influence of calcium load on central arterial stiffening. Further studies are warranted to determine whether serum OPG is a surrogate marker or plays a causal role directly in mediating against vascular injury in patients with ESRD.