Associations of Bone Turnover Markers with Cognitive Function in Patients Undergoing Hemodialysis

Background Patients undergoing hemodialysis experience a greater risk of cognitive impairment than the general population, but limited data elucidates the biomarkers on this. We evaluated the association of bone turnover markers on cognitive function among 251 prevalent hemodialysis enrollees in a cross-sectional study. Methods 251 hemodialysis patients (median age = 57.8, 55% men) and 37 control subjects (mean age = 61.2, 56% men) without a prior stroke or dementia diagnosis were enrolled. Serum concentrations of 8 bone markers were analyzed as the association of cognitive function (Montreal Cognitive Assessment (MoCA) and Cognitive Abilities Screening Instrument (CASI)) using linear regression analysis. Results A lower cognitive function was noted in hemodialysis patients compared to control subjects. The receptor activator of nuclear factor kappa-B ligand (RANKL) was the only bone marker found to be associated with cognitive function (MoCA and CASI tests) in hemodialysis patients without a prior stroke or dementia diagnosis. In stepwise multiple linear regression analysis, the association remained significant in MoCA (β = 1.14, 95% CI 0.17 to 2.11) and CASI (β = 3.06, 95% CI 0.24 to 5.88). Short-term memory (β = 0.52, 95% CI 0.01 to 1.02), mental manipulation (β = 0.51, 95% CI 0.05 to 0.96), and abstract thinking (β = 0.57, 95% CI 0.06 to 1.09) were the significant subdomains in the CASI score related to RANKL. Conclusions Serum RANKL levels were potentially associated with better cognitive function in hemodialysis patients. Further large-scale and prospective studies are needed to confirm our findings.

explain the cognitive impairment and dementia on them; hence, the possibility of identifying novel mechanisms and disease biomarkers is suggested.
Osteoporosis and low bone mineral density (osteopenia) have been associated with cognitive impairment and dementia [6][7][8][9]. This raises the possibility that factors related to bone regulation and function may influence cognitive performance. Because bone-related peptides are secreted into the circulation, there has been growing interest in their influences on the central nervous system [10]. Several bone turnover markers were found to be associated with cognitive function in general population, such as Dickkopf-related protein 1 (DKK1) [11,12], Osteocalcin (OC) [13,14], Osteopontin (OPN) [15], Osteoprotegerin (OPG) [16], and Leptin [17]. These findings suggested that there might be a convergence in mechanisms between bone and neurodegeneration and that bone may possess endocrine properties which aid in maintaining cognitive well-being.
The discovery of novel risk markers for cognitive impairment on ESKD patients can help us to predict the trait and has the potential to provide us with a better understanding of the pathogenesis. Recent technological advances have enabled the simultaneous measurement of multiple proteins to provide new opportunities for unbiased discovery of novel pathophysiologic pathways of disease, as well as for the identification of novel clinically relevant biomarkers. We aimed to explore the potential biomarkers of cognitive function by using a Luminex bead-based multiplex assay on ESKD patients. In the present study, we used these data from a study of 251 prevalent hemodialysis patients to compare the association between cognitive function and bone turnover biomarkers.

Materials and Methods
2.1. Subjects. From August 2016 to January 2017, we recruited participants from 2 hemodialysis units (Kaohsiung Municipal Hsiao-Kang Hospital and Kaohsiung Medical University Hospital) in Taiwan. Eligible participants were those who had an age > 30 years and who were receiving hemodialysis for at least 90 days. Participants with cerebrovascular disease (N = 39) or dementia (N = 6) were excluded. A total of 293 individuals were screened, and neuropsychological tests were performed on them. Those subjects who failed to complete the neuropsychological tests were excluded from the final analysis (N = 45) (Supplementary Figure 1). All participants received regular hemodialysis three times per week through the use of automated volumetric machines. Each hemodialysis session lasted 3.5-4 hours and involved using high-flux dialyzers. The blood flow rate was controlled between 250 and 300 ml/min, and the dialysate flow was maintained at 500 ml/min, and single pool Kt/V was more than 1.2 per week. Informed consent was obtained from all subjects. The study protocol was approved by the Institutional Review Board of Kaohsiung Medical University (KMUHIRB-E(I)-20160095).  [18][19][20] and Montreal Cognitive Assessment (MoCA) [21] (Supplementary Table 1). The CASI is a 40-item global cognitive test that assesses a broad range of cognitive domains. Nine cognitive evaluation domains were evaluated including attention, orientation, language, list-generating fluency, mental manipulation, abstraction/judgment, drawing, long-term memory, and short-term memory. The MoCA is a 30point screening tool and encompasses multiple cognitive domains such as language, attention, visuospatial abilities and executive function, concentration and working memory, short-term memory, and orientation to time and place. The MoCA has been translated and adapted into several languages and is available freely on the Internet (http://www.mocatest.org). The tasks include cube copying, clock drawing, naming, digit span backward and forward, serial subtraction, selective attention, sentence repetition, phonemic word fluency, verbal abstraction, a 5-word learning and delayed recall task, and spatial and temporal orientation. Besides, we also performed the Center for Epidemiologic Studies Depression Scale (CES-D) to determine depression symptoms' score, as part of a broader neurocognitive assessment.

Multiplex Analysis of Serum Biomarkers.
Serum was obtained using a serum separation tube (SST) after blood centrifugation at 1500 rpm for 10 min at 4°C. Samples were aliquoted and stored at -80°C until use. The MILLIPLEX MAP technology (Luminex) combines the principle of a sandwich immunoassay with the fluorescent bead-based technology allowing individual and multiplex analyses of different analytes in a single microtiter well. Defrosted serum samples were analyzed using a multiplex immunobead assay (Millipore, St. Charles, MO, US) to simultaneously quantify multiple potential biomarkers. Human RANKL Single Plex (receptor activator of nuclear factor kappa-Β ligand (RANKL)) and MILLIPLEX MAP Human Bone Panel (Dickkopf-related protein 1 (DKK1), Fibroblast growth factor 23 (FGF23), Leptin, Osteocalcin (OC), Osteopontin (OPN), Osteoprotegerin (OPG), and Sclerostin (SOST)) were used for determining the serum levels of these molecules (Supplementary Table 2). The multiplex immunobead assays were done in 96-well microplate format according to the manufacturer's instructions. A filter-bottom, 96-well microplate (Millipore, Billerica, MA) was blocked for 10 minutes with PBS/bovine serum albumin. Experiments generating standard curves involved a 7-point standard curve using 5-fold serial dilutions from premixed standards provided in the kits. 50 microliters of standards of patient sera was added per well in duplicate and mixed with 50 microliters of the bead mixture. The microplate was incubated for 1 hour at room temperature on a microtiter shaker. Wells were then washed thrice with a washing buffer using a vacuum manifold. Phycoerythrin-(PE-) conjugated secondary antibody was added to the appropriate wells, and the wells were incubated for 45 minutes in the dark with constant shaking. Wells were washed twice, assay buffer was added to each well, and samples were acquired on Luminex xMAP technology

Control Subjects of the General Population.
A voluntary nondialysis comparison group of 37 independently people was recruited by advertisement from the community. Eligibility criteria for the comparison group were the same as for the hemodialysis subjects, with additional exclusions of diagnosed CKD. Sociodemographic data and medical history were obtained for all participants by self-report records. Biochemical data were performed in the same laboratory. Bone turnover biomarkers were measured using Luminex beadbased multiplex assay, and neuropsychological tests (CASI, MoCA, and CES-D) for control subjects were also assessed.
2.6. Statistical Analysis. Data were presented as mean ± standard deviation (SD) or percentages. The Kolmogorov-Smirnov test was applied to test for normal distribution. Student's t-tests or Mann-Whitney U tests were applied to compare means of continuous variables. Categorical data were tested using the chi-squared test. Data were logtransformed to approximate normal distributions. Variance Inflation Factor (VIF) calculation was performed to address the issue of collinearity by using principal component analysis. Screened multiple biomarkers associated with cognitive function were evaluated using linear regression models adjusted for age and sex (equation: cognitive function = β0 + β1 * log − transformed RANKL + β2 * age + β3 * sex).
We plotted the distribution of log-transformed biomarker values with a neuropsychiatric test score. Correlations between serum bone markers and neuropsychiatric test scores were calculated by Spearman's rank correlation coefficients. The tertile levels of serum bone markers and cognitive function tests (CASI and MoCA) were also analyzed. To identify independent determinants of serum bone markers, multivariate linear regression analysis was performed to assess the relationships among log-transformed biomarkers levels with scores on the MoCA and CASI controlling age, sex, education level, depression scale (CES-D), comorbidities (diabetes mellitus, hypertension, dyslipidemia, and coronary artery disease), clinical laboratory data (hemoglobin, albumin, blood urea nitrogen, ion calcium, phosphorus, parathyroid hormone, alkaline phosphatase, C-reactive protein, and Kt/V), and hemodialysis duration. Relevant demographic parameters were also analyzed by a backward stepwise selection with P values for independent variables to enter and to stay in the models set at 0.2 and, subsequently, a final elimination step at P < 0:05. Furthermore, hierarchical selection procedures were also performed to construct a powerful covariate set for adjustment in a sensitivity analysis.
Results were reported as a beta coefficient (β) with 95% CIs, and a two-tailed P < 0:05 was considered statistically significant. All statistical analyses were performed using SAS version 9.4 and STATA version 14.

Associations of Serum RANKL Levels and Cognitive
Impairment in Hemodialysis Participants. The scatter plot with cubic spline demonstrated the association of serum RANKL and the cognitive function score on MoCA and CASI scores. There was a positive association between serum log-transformed RANKL levels and cognitive function tests (MoCA and CASI) ( Supplementary Figures 2A and 2B). The tertile levels of log-transformed RANKL also demonstrated a positive correlation between higher RANKL levels and better cognitive function tests in CASI and MoCA (Supplementary Figure 3). The positive association was found in different CASI domains, including long-term memory, short-term memory, attention, mental manipulation, abstract thinking, and name fluency (Supplementary Figure 4). However, after adjusting for age, sex, and education levels, the serum RANKL levels were positively correlated with cognitive function test scores on MoCA, CASI, and CASI domains of short-term memory, mental manipulation, abstract thinking, and name fluency (Figure 3). In multiple linear regression models adjusted for age, sex, education levels, depression scales (CES-D), and comorbidities (diabetes mellitus, hypertension, dyslipidemia, and coronary artery disease), higher levels of log-transformed RANKL were associated with better MoCA scores (β coefficient 1. 12 Table 3).

Discussion
A multiplex bead-based Luminex assay was used to investigate the association between multiple circulating bone turnover markers and the 2 different cognitive function tests in hemodialysis individuals. The main finding of this study was that serum levels of RANKL were able to classify independently the presence of clinically overt cognitive impairment in hemodialysis patients. Using a linear regression model with age and sex adjustment for multiple biomarker screening, serum RANKL levels were associated with the higher cognitive function, which is defined by MoCA and CASI. In multiple linear regression analysis, serum RANKL levels were positively correlated to the cognitive function test  4 Disease Markers (MoCA and CASI), especially in short-term memory, mental manipulation, and abstract thinking. Other bone turnover makers, including DKK1, FGF23, Leptin, OC, OPN, OPG, and SOST, were statistically insignificantly associated with cognitive function after age and sex adjustment during the screening process. Although one cross-sectional study previously reported that the higher serum FGF23 levels were associated with poor cognitive performance [22], it is unable to determine the associations in our study. Patients undergoing hemodialysis have more prothrombotic factors, such as endothelial dysfunction, abnormal vascular reactivity, and atherosclerosis, leading to the cause of cognitive impairment. The prevalence of dementia is between 5 and 9% of the population, but there is limited prevalence data in patients with ESKD [23]. The reported prevalence of cognitive impairment among patients with ESKD, as assessed using neuropsychological tests, varies from 16 to 38% [5]. Patients undergoing hemodialysis were found to have more cognitive impairment than the general population, particularly in the domains of orientation and attention and executive function [1]. In the present study, the neuropsychological test scores, including MoCA and CASI, were all lower in hemodialysis participants than in reference control subjects.
Serum RANKL levels were found to be associated with cognitive function test scores in this study. Regarding this finding, RANKL may be considered to have an important role in the pathogenesis of cognitive function in hemodialysis patients. The OPG/RANK/RANKL system is an important pathway in bone biology and skeletal health. This system also plays an important role in other tissues [24,25], such as the brain, heart, and lungs. The OPG/RANK/RANKL axis has been implicated in the pathophysiology of various cardiovascular disorders that involve a vascular component, such as atherosclerosis and diabetes [26]. However, conflicting results of the OPG/RANK/RANKL axis on cardiovascular disease were reported [27][28][29][30]. RANKL was detected in atherosclerotic lesions, potentially promoting inflammation, endothelial cell activation, and matrix degradation [31]. RANKL was reported to exhibit several properties with relevance to atherogenesis, induction of chemotactic properties in monocytes, and induction of matrix metalloproteinase (MMP) activity in vascular smooth muscle cells (SMC) [32,33]. Raised serum levels of RANKL have been reported in patients with heart failure [31,34,35] and predicted the risk of cardiovascular disease in healthy individuals [36]. However, other studies did not find any relation between serum levels of RANKL and cardiovascular disease [27][28][29]. Besides,

Disease Markers
an inverse correlation between circulating RANKL and OPG levels was also found [28]. RANKL concentrations also displayed inverse associations with multiple cardiovascular disease risk factors, including smoking, diabetes, and antihypertensive treatment [28]. Furthermore, a significant negative correlation between serum RANKL values and coronary artery calcification score was found in hemodialysis patients [37]. Some basic studies also reported that RANKL was important for dendritic cell survival and, therefore, antigen surveillance, T cell memory formation, and immunomodulatory effects [38]. RANKL also regulates endothelial cell survival and proliferation; disruption along the OPG/RANK/RANKL axis could result in endothelial dysfunction and impaired angiogenesis [38].
RANKL and RANK are also expressed in the central nervous system [39]. In an adult mouse model of ischemic stroke, enhancing RANKL/RANK signaling in wild-type animals by recombinant RANKL is able to significantly reduce the infarct volume [40]. RANKL/RANK is a novel signal involved in the regulation of microglial inflammation through Toll-like receptor (TLR) 4 [40], which is the main damage-associated molecular pattern (DAMP) receptor in the ischemic brain [41]. Both RANKL and RANK are expressed in activated microglia and macrophages (M/M) of ischemic brain tissue, and enhancement of the RANKL/-RANK signal using recombinant RANKL (rRANKL) has been shown to reduce ischemic injury in mice [40]. Besides, microglial cells decrease in RANK signaling during inflammation. Pretreatment of the BV2 microglial cell line with rRANKL decreased the ability of Toll-like receptor 3 (TLR3)/Toll-like receptor 4 (TLR4) signaling to activate the expression of TLR3, myeloid differentiation primary response gene 88 (MyD88), and TIR-domain-containing adaptor-inducing interferon-(IFN-) β (TRIF). This suppressed BV2 microglial activation, which was confirmed by the reduction of the inflammatory markers iNOS and COX2. Therefore, keeping the expression of adaptor proteins at physiological levels by RANK activation is important to prevent the overactivation of TLR signaling in microglial cells [42]. As we have known, ESKD patients experience more inflammation processes, which may be associated with the decline in neurocognitive performance. RANK signaling plays an important role in the regulation of microglial responses to inflammatory stimuli. This indicated that RANKL could potentially be beneficial in brain damage.
The strength of the present study is that we conducted several detailed neuropsychological assessments. In spite of different sensitivities and specificities of cognitive function tests on MoCA and CASI, similar results using different cognitive function tests may eliminate the bias from the diagnostic tools. We measured multiple bone turnover biomarkers simultaneously using the Luminex bead-based multiplex assay to evaluate the potential biomarkers on cognitive function. However, some limitations still should be addressed. First, our sample came from a single geographic site and patients in this study were receiving hemodialysis; thus, these findings may not be generalizable to patients receiving peritoneal dialysis. Second, brain imaging was not available to ascertain the etiology of cognitive impairment. Thus, we excluded hemodialysis patients with dementia and cerebrovascular disease to decrease potential bias. Third, the concentration of RANKL in cerebrospinal fluid is likely to be more important than serum concentrations with respect to cognitive

Conclusion
Serum levels of RANKL were found potentially associated with cognitive function, especially short-term memory,

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
The funding sources did not play any role in the design or conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Acknowledgments
The study was funded by grants from the Ministry of Science

Supplementary Materials
The MILLIPLEX MAP technology (Luminex) method. Supplementary Table 1: description of the neuropsychiatric test on patients with hemodialysis in the study. Supplementary Table 2: list of bone turnover biomarkers measured by bead system multiplex Luminex immunoassays. Supplementary Table 3: multiple linear regression analysis of cognitive function test (Montreal Cognitive Assessment and Cognitive Abilities Screening Instrument). Supplementary Table 4: association between log-transformed serum RANKL concentrations and cognitive function test in hemodialysis participants using linear regression analysis adjusted for hierarchically selected covariates. Supplementary Figure 1