Association of restless legs syndrome with oxidative stress and inflammation in patients undergoing hemodialysis

Accepted Manuscript Title: Association of restless legs syndrome with oxidative stress and inflammation in patients undergoing hemodialysis Author: Terumi Higuchi, Masanori Abe, Mari Mizuno, Toshio Yamazaki, Hiroko Suzuki, Masari Moriuchi, Osamu Oikawa, Erina Okawa, Hideyuki Ando, Kazuyoshi Okada PII: DOI: Reference:

S1389-9457(15)00746-7 http://dx.doi.org/doi:10.1016/j.sleep.2015.03.025 SLEEP 2761

To appear in:

Sleep Medicine

Received date: Revised date: Accepted date:

14-1-2015 6-3-2015 15-3-2015

Please cite this article as: Terumi Higuchi, Masanori Abe, Mari Mizuno, Toshio Yamazaki, Hiroko Suzuki, Masari Moriuchi, Osamu Oikawa, Erina Okawa, Hideyuki Ando, Kazuyoshi Okada, Association of restless legs syndrome with oxidative stress and inflammation in patients undergoing hemodialysis, Sleep Medicine (2015), http://dx.doi.org/doi:10.1016/j.sleep.2015.03.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Article Association of restless legs syndrome with oxidative stress and inflammation in patients undergoing hemodialysis

Terumi Higuchi a, Masanori Abe

b, *

, Mari Mizuno c, Toshio Yamazaki a, Hiroko Suzuki b, Masari

Moriuchi b, Osamu Oikawa b, Erina Okawa a, Hideyuki Ando d, Kazuyoshi Okada b

a

b

Department of Nephrology, Keiai Hospital, Tokyo, 173-0036, Japan Division of Nephrology, Hypertension and Endocrinology, Department of Internal Medicine,

Nihon University School of Medicine, 173-8610, Tokyo, Japan c

Department of Nephrology, Keiai Clinic, Tokyo, 173-0036, Japan

d

Department of Cardiology, Keiai Hospital, Tokyo, 173-0036, Japan

*Corresponding author. Division of Nephrology, Hypertension and Endocrinology, Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchi Kami-chou, Itabashi-ku, 173-8610 Tokyo, Japan. Tel: +81 3 3972 8111; Fax: +81 3 3972 8311. E-mail address: [email protected] (M. Abe).

Highlights   

Restless legs syndrome is a common comorbidity in patients undergoing hemodialysis. Restless legs syndrome was associated with oxidative stress and inflammation. Serum 8-OHdG level was a significant independent predictor of RLS severity.

ABSTRACT

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Background: Restless legs syndrome (RLS) is a common comorbidity in patients undergoing hemodialysis, but clinical factors predictive of RLS risk and severity have not been identified. The aims of this multicenter cross-sectional study were to document RLS prevalence and severity in patients undergoing hemodialysis and to identify associated risk factors. Methods: One-hundred and fifty-nine stable patients on maintenance hemodialysis were enrolled (113 men, 46 women; mean age: 68 ± 11 years; mean duration of dialysis: 54 ± 60 months). Diagnosis of RLS was based on criteria proposed by the International Restless Legs Syndrome Study Group (IRLSSG), and RLS severity was assessed using the IRLSSG Severity Scale. Potential factors associated with RLS and IRLSSG Severity Scale score were assessed by univariate and multivariate regression analyses. Results: RLS affected 22% of the study population. The RLS subgroup had a significantly longer duration of hemodialysis and higher cardiothoracic ratio compared to the non-RLS subgroup. The RLS subgroup also had significantly higher serum levels of high-sensitivity C-reactive protein, interleukin-6, ferritin, N-terminal pro-B-type natriuretic peptide, and 8-hydroxy-2′-deoxyguanosine (8-OHdG), and a significantly lower transferrin saturation level compared to the non-RLS subgroup, suggesting a chronic inflammatory state and associated oxidative stress in comorbid patients. Serum 8-OHdG level was an independent risk factor for high IRLSSG Severity Scale score. Conclusion: This study confirms the high prevalence of RLS among hemodialysis patients and identifies the oxidative stress marker serum 8-OHdG as a significant independent predictor of RLS severity. Further studies are needed to identify detailed risk factors and the pathophysiological role of oxidative stress in RLS.

Keywords: Hemodialysis

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N-terminal pro-B-type natriuretic peptide Oxidative stress Restless legs syndrome 8-Hydroxy-2′-deoxyguanosine 1．Introduction Restless legs syndrome (RLS) is a movement disorder characterized by a compulsive and uncomfortable urge to move the legs that worsens at night and during periods of inactivity [1]. The reported prevalence of RLS is 5–10% of the general population in Western countries [2,3], with lower prevalence in Asian populations [4]. Diagnostic criteria established by the International Restless Legs Syndrome Study Group (IRLSSG) [5] are now widely accepted. RLS frequently occurs in individuals with chronic kidney disease, and especially in patients with end-stage kidney disease (ESKD) [6–8]. Several studies using IRLSSG criteria reported that the prevalence of RLS was 7–22% in dialysis patients [9–12], substantially higher than in the general population. The pathophysiology of RLS is unclear. It has been suggested that dopaminergic dysfunction may be responsible for RLS as small doses of levodopa [13] relieve symptoms and dopamine antagonists exacerbate symptoms [14]. Iron deficiency, anemia, hyperphosphatemia, and smoking have been implicated as risk factors for RLS in the dialysis population, but clinical characteristics and particular comorbidities associated with RLS have not been confirmed [15]. The prevalence and risk factors for RLS are clinically significant because RLS may indicate incorrect dialysis treatment and contribute to reduced quality of life [10,16]. Moreover, it was reported that RLS enhanced cardiovascular disease risk and mortality in patients on long-term hemodialysis [17], increases not completely attributable to well-known cardiovascular risk factors such as hypertension, diabetes, smoking, obesity, and physical inactivity. Additional possible RLS risk factors specific to ESKD conditions are uremia, mineral bone metabolism disorders,

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inflammation, oxidative stress, and malnutrition. Systemic inflammation has been linked to poor sleep quality in hemodialysis patients [18], but evidence connecting RLS directly to inflammation and oxidative stress are still lacking. One of the most abundant oxidative DNA products, 8-hydroxy-2′-deoxyguanosine (8-OHdG), is formed by hydroxylation at the C8 position of deoxyguanosine by reactive oxygen species (ROS) [19]. Serum levels of 8-OHdG are increased in several conditions involving excess ROS generation, such as diabetes, cancer, radiation injury, smoking, normal aging [19], and kidney disease [20]. Moreover, elevated blood 8-OHdG was also observed in amyotrophic lateral sclerosis [21], a neurodegenerative disease in which oxidative stress has been proposed as a key pathogenic mechanism. In this study, we measured the prevalence of RLS in a group of stable hemodialysis patients and compared clinical data and laboratory findings in patients with and without RLS to identify possible causes and risk factors. Furthermore, we evaluated the potential association of RLS with inflammatory and oxidative stress markers.

2．Methods 2.1. Study design All patients treated at three outpatient dialysis clinics or hospitals between March and August 2014 were considered for this observational cross-sectional study. Enrollment criteria were (1) age ≥ 20 years or ≤ 85 years, and (2) on hemodialysis for at least 6 months. Exclusion criteria were as follows: (1) age of < 20 years or > 85 years; (2) recent infectious disease, cancer, or drug/alcohol abuse; (3) treatment with steroids or immunosuppressants; (4) currently hospitalized; (5) severe neurological, hepatic, lung, or cardiac diseases; (6) current treatment with intravenous iron therapy; or (7) recent hospitalization or unwillingness to participate in the study. Demographic and clinical data were recorded using a closed-question data collection

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instrument. Patients were analyzed according to the presence or absence of RLS. Diagnosis of RLS was made based on an interview conducted by three trained physicians. Cardiovascular disease comorbidities were defined as previous ischemic heart disease or aortic disease, conditions requiring percutaneous coronary intervention or surgical revascularization, previous cerebrovascular disease, or peripheral artery disease treated previously or currently. The study protocol was approved by the Keiai Hospital Ethics Committee and written informed consent was obtained from all patients. This study was conducted in compliance with the Declaration of Helsinki (revised in October 2008). 2.2. RLS Questionnaire The RLS diagnosis was established based on the minimum criteria defined by the International Restless Legs Syndrome Study Group (IRLSSG): (1) an urge to move the legs, usually accompanied or caused by uncomfortable and unpleasant sensations in the leg; (2) the urge to move or unpleasant sensations begin or worsen during periods of rest or inactivity, such as lying or sitting; (3) the urge to move or unpleasant sensations are partially or totally relieved by movement, such as walking or stretching, for at least as long as the activity continues; and (4) the urge to move or unpleasant sensations are worse in the evening or at night [5]. Patients reporting all four IRLSSG criteria necessary for RLS diagnosis were requested to answer the 10-question IRLSSG Severity Scale [22]. 2.3. Study procedure Blood cell counts, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, serum creatinine, serum urea nitrogen, electrolytes, uric acid, serum iron, total iron binding capacity (TIBC), serum ferritin, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglyceride, total protein, and albumin were measured by routine clinical chemistry procedures using commercial kits. Transferrin saturation (TSAT) was

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calculated from serum iron and TIBC levels. High-sensitivity C-reactive protein (hs-CRP) and serum β2-microglobulin were measured by latex agglutination, intact parathyroid hormone by radioimmunoassay, interleukin-6 (IL-6) by a chemiluminescent enzyme immunoassay (SRL, Inc., Tokyo,

Japan),

8-hydroxy-2′-deoxyguanosine

(8-OHdG)

by

a

specific

enzyme-linked

immunosorbent assay (SRL, Inc.), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) by an electro-chemiluminescence immunoassay. All of these clinical variables were measured at the initiation of HD. Single pool Kt/Vurea (Kt/V) for assessing hemodialysis dose and protein catabolic rate (PCR), and creatinine generation rate (%CGR) for assessing nutritional status, blood urea nitrogen, and serum creatinine levels were obtained pre-dialysis and immediately post-dialysis [23,24]. Geriatric nutritional risk index (GNRI) was also measured [25]. Erythropoietin responsiveness index (ERI), defined as the average weekly erythropoietin-stimulating agent (ESA) dose divided by clinical dry weight and average blood hemoglobin (ERI = weekly ESA dose (U)/dry weight (kg)/hemoglobin (g/dL)) [26] was calculated to normalize the amount of ESA to the severity of anemia. Cardiothoracic ratio (CTR) was measured by chest X-ray after HD treatment. All parameters were re-evaluated once per month for three consecutive months in each patient following the questionnaire investigation of RLS. The data of each patient are reported as the mean of three measurement times and those data were analyzed. Intravenous iron therapy was not performed during this 3-month period because it may affect serum 8-OHdG [27]. 2.4. HD procedure In all patients, HD was performed three times per week for 4 h at a blood flow rate of 200–250 mL/min and a dialysate flow rate of 500 mL/min using high-flux dialyzer membranes such as polysulfone (PSu; APS-SA; Asahi Medical Co., Ltd., Tokyo, Japan) or polyester-polymer alloy (FDX-GW; Nikkiso, Tokyo, Japan). The surface area of the membrane was selected according to

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the body weight of the patient. Hemodiafiltration was performed by the post-dilution method using 8–10 L sterile bicarbonate solution per session. The glucose concentration of the dialysate was 100 mg/dL. Heparin was administered at 2600–6000 U per 4-h HD session for anticoagulation. The volume of ultrafiltration was kept constant based on clinical dry weight during each session. 2.5. Statistical analysis Data were expressed as mean ± SD or median (interquartile range) as was appropriate. Continuous variables were compared using Student’s t-test or Mann-Whitney U test and categorical variables were compared by the chi-square or Fisher’s exact test as was appropriate to the data distribution. For statistical analysis and data description, patients were stratified according to the presence or absence of RLS. Patients with RLS (RLS subgroup) were compared to patients without RLS (non-RLS subgroup). Furthermore, we investigated possible factors associated with RLS prevalence. Univariate logistic regression analysis was performed using presence of RLS as the dependent variable and all clinical and nutritional variables as independent variables. A multiple logistic regression analysis was then performed to identify risk factors for RLS, using all parameters that had p < 0.1 in univariate analysis as independent variables. Correlations between variables were assessed by Spearman’s correlation test. Univariate linear regression analysis was performed using RLS Severity Score as the dependent variable and all clinical and nutritional variables as independent variables. Continuous variables that were not normally distributed were log-transformed. A multiple linear regression model was built with RLS Severity Score as the dependent variable and all parameters with p < 0.1 by univariate analyses entered as independent variables. Statistical significance was set at p < 0.05. All analyses were performed using the Statistical Package for the Social Sciences 11.5 for Windows (SPSS Inc., Chicago, IL).

3. Results

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3.1. Clinical characteristics of all study patients and subgroups defined by the presence or absence of restless legs syndrome In total, 197 patients were screened, and 159 patients were enrolled. Of the 159 patients, 2 had already received a diagnosis of RLS and received treatment with dopaminergic agonists (Fig. 1). No evidence of RLS was found in 124 patients, while positive response to all 4 IRLSSG questions proposed for the diagnosis of RLS were obtained from 33 patients. This yielded an RLS prevalence rate of 22.0% in our dialysis population. Ultimately the 2 currently treated patients were excluded, giving a total of 157 patients (111 men, 46 women; mean age, 68 ± 11 years (range, 38–85 years)) included in the analysis. Baseline characteristics and medications used by the entire patient group and subgroups with and without RLS are shown in Table 1. There was no significant difference in alcohol or coffee intake between the RLS and non-RLS subgroups, nor any difference in the rates of antidepressants and benzodiazepines use. There was no significant difference in ESKD etiology or in diabetes rate between the two subgroups. However, mean HD duration was longer in the RLS group. Although there was no significant difference in BMI or clinical dry weight, CTR was significantly greater in the RLS group than the non-RLS group. The mean IRLSSG Severity Scale score in the RLS group was 19.1 ± 8.9. 3.2. Comparison of hemodialysis-related factors between RLS and non-RLS subgroups Table 2 shows hemodialysis dose and hemodialysis-related nutritional assessments in all patients and for the RLS and non-RLS subgroups. There were no significant differences in dialysis mode, ERI, Kt/V, and nutritional assessment parameters (PCR, GNRI, or %CGR) between subgroups. 3.3. Markers of inflammation and oxidative stress in RLS patients Table 3 shows clinical and laboratory values of all patients and those with and without RLS. Hs-CRP and IL-6 levels were significantly higher in the RLS subgroup than the non-RLS subgroup.

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Although serum iron and TIBC did not differ significantly between subgroups, serum ferritin level was significantly higher in the RLS group. In contrast, TSAT was significantly lower in the RLS subgroup compared to the non-RLS subgroup. Finally, both NT-proBNP and 8-OHdG levels were significantly higher in the RLS subgroup. 3.4. Regression analysis to determine the risk factors for RLS To identify risk factors for RLS, univariate analysis was performed in all patients (Table 4). Presence of RLS was significantly associated with HD duration, CTR, NT-proBNP, Hs-CRP, IL-6, and 8-OHdG. Furthermore, multivariate logistic regression analysis showed that Hs-CRP and 8-OHdG were significant and independent risk factors for RLS. 3.5. Relationship between IRLSSG Severity Scale and clinical parameters To identify factors associated with RLS severity, univariate linear regression analysis was performed using IRLSSG Severity Scale score as the dependent variable and all clinical and nutritional variables as independent variables in the RLS group (Table 5). As shown in Fig. 2, there was a significant positive correlation between IRLSSG Severity Scale and HD duration (p = 0.014), hs-CRP (p = 0.021), serum ferritin (p = 0.0005), and 8-OHdG levels (p < 0.0001), while there was no correlation between IRLSSG Severity Scale and serum albumin level (p = 0.64). 3.6. Multivariate regression analysis Multivariate linear regression analysis was performed to identify independent predictors of IRLSSG Severity Scale score in RLS patients (Table 6). Multiple stepwise regression analysis was performed using IRLSSG Severity Scale score as the dependent variable and HD duration, log hs-CRP, ferritin, and log 8-OHdG as independent variables. Although ferritin and 8-OHdG levels were independent predictors of RLS severity in the unadjusted model (R 2 = 0.726), only 8-OHdG level was an independent predictor of IRLSSG Severity Scale score in the adjusted model (R 2 = 0.743).

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4. Discussion RLS is a frequent source of distress in patients on long-term dialysis [10]. The prevalence of RLS reported in our study is similar to that of previous studies using IRLSSG criteria [7,11,28]. RLS has been reported as a common comorbidity in other disease as well, including diabetes mellitus in a population with normal kidney function [29,30]. The etiology of RLS in ESKD, the indication for HD, is still not well defined. However, a role of kidney insufficiency in RLS pathogenesis is suggest by the resolution or improvement of symptoms after kidney transplantation and recurrence after graft failure [31,32]. The association between RLS and declining kidney function in transplant patients and patients with chronic kidney disease further suggests a link between RLS and chronic kidney insufficiency [8,32]. Pizza et al. reported that residual diuresis was a predictor of risk for RLS in ESKD patients on HD [33]. This might be the reason why HD duration was a risk factor for RLS in our study, since our RLS patients had relatively long dialysis durations and anuria. RLS negatively impacts outcome in ESKD patients undergoing long-term hemodialysis, with prominent adverse effects on sleep quality [16,34] and quality of life [10,16]. Considering that sleep-deprived patients are at greater risk for immunological and cardiovascular diseases [35], disordered sleep can be an important factor in reducing the life expectancy of ESKD patients [36]. Cardiovascular disease is the main cause of morbidity and mortality in patients with ESKD undergoing hemodialysis, with CVD-related mortality 10- to 20-times higher than in the general population [31,32]. This increased incidence of cardiovascular events is not explained by common cardiovascular risk factors such as hypertension, diabetes, smoking, obesity, and physical inactivity. Risk factors specific to ESKD, such as uremia, mineral metabolism disorders, inflammation, oxidative stress and malnutrition [37], may also contribute to RLS. An association between RLS

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and CVD or cardiovascular risk factors was reported in the general population [38–41]. Furthermore, it was reported that RLS severity was associated with cardiovascular events and higher short-term mortality at 1.5 year follow-up in hemodialysis patients [17], with RLS prevalence and cardiovascular events associated with lower albumin and higher inflammatory markers, CRP, and fibrinogen levels [17]. To our knowledge, however, ours is the first study to find an association between 8-OHdG and RLS in hemodialysis patients and to establish serum 8-OHdG as an independent predictor of RLS severity. Chronic inflammation and malnutrition have also been reported in patients on maintenance hemodialysis [42–45]. Increased levels of C-reactive protein and IL-6, often observed in hemodialysis patients, reflect the increased release of proinflammatory cytokines, which promote cardiovascular disease by contributing to endothelial dysfunction, oxidative stress, insulin resistance, and adhesion molecule stimulation [46–48]. Moreover, the links among inflammation, malnutrition, and atherosclerosis have enabled the identification of malnutrition-inflammation-cachexia syndrome (MICS), which is associated with poor outcome in dialysis patients [49]. Although MICS was initially associated with improper dialysis dose and poor efficacy, we found no significant difference in Kt/V between RLS and non-RLS subgroups. This may reflect the good clearance achieved by hemodialysis techniques using high-flux membranes and adequate dialysis dose as assessed by Kt/V. In the present study, hs-CRP better reflected RLS severity than IL-6 did, but the interindividual variability of IL-6 was greater than that of hs-CRP. Further long-term investigation would be needed to clarify which is more predictive of RLS severity. It was reported that serum 8-OHdG, an abundant oxidative product of cellular DNA, was correlated with the severity of renal anemia and serum ferritin levels [27,50]. Therefore, inflammation and oxidative stress were suggested as stronger triggers for RLS than Kt/V itself. On the other hand, our study showed that serum 8-OHdG levels did not correlate with nutritional status,

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consistent with a previous study [27]. However, long-term studies are needed to clarify the association between worsening nutritional status, as assessed by GNRI, %CGR and PCR, and worsening RLS severity scale, since continuous inflammation leads to malnutrition. Serum NT-proBNP is the strongest prognostic biomarker for cardiovascular and all-cause mortality in patients with hemodialysis [51–53]. In the present study, CTR and NT-proBNP levels were higher in the RLS group despite no significant difference in dry weight and BMI. Higher CRP, 8-OHdG, and NT-proBNP levels in the RLS subgroup may explain the poor outcomes observed in these patients and demonstrates the importance of diagnosis and therapeutic management. While several recent studies have examined risk factors for RLS in dialysis patients, results are conflicting. Anemia, serum ferritin, serum iron, calcium, phosphate, PTH, creatinine, and urea levels have all been causally linked to RLS in dialysis patients. However, recent studies have failed to confirm such correlations. Anemia has been linked to RLS in uremia [9], and combined intravenous iron with ESA has been shown to improve symptoms [54], but previous studies as well as the present study failed to show this association with iron deficiency anemia in uremic patients [55,56]. This may be due to routine use of ESA and intravenous iron therapy in modern hemodialysis management, resulting in a very low prevalence of iron deficiency or anemia in our dialysis patients. Iron deficiency in dialysis patients is defined by Japanese Society for Dialysis Therapy (JSDT) guidelines as serum ferritin level < 100 ng/mL and TSAT < 20% [57]. In patients that fulfilled these criteria, saccharated ferric oxide (40 mg iron) was administered intravenously 13 times via the dialysis circuit at the end of each dialysis session to maintain ferritin levels above 100 ng/mL and TSAT levels above 20%, according to the recommendations of our dialysis units. Given these criteria, two patients were treated with intravenous iron therapy during the present study period and excluded from the present analysis since intravenous iron treatment reportedly affects serum 8-OHdG levels [27]. Although serum iron and ERI did not differ between RLS and non-RLS

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subgroups, serum ferritin was higher and TSAT lower in RLS patients, suggesting defective iron utilization due to inflammation and oxidative stress in RLS patients. Several limitations of this report should be noted. First, the cross-sectional design does not allow for conclusions about the direction of these relationships. A second limitation is that we show the associations of RLS with only the average clinical values over 3 months. Although we evaluated the variability of CTR and other parameters during the three measurements, there were no associations between the presence of RLS or RLS severity and those variability values. Third, although it was anticipated that ERI would increase when defective iron utilization continued for a longer duration, ERI was not associated with RLS severity in our study, possibly because of the relatively short observational period. To clarify the association between RLS severity scale and survival, prospective long-term studies are needed. In conclusion, 21% of patients on hemodialysis in this study were diagnosed with RLS. Moreover, the RLS subgroup had higher hs-CRP, ferritin, NT-proBNP, and serum 8-OHdG levels than hemodialysis patients without RLS, strongly suggesting contributions of chronic inflammation and oxidative stress to RLS pathogenesis. Furthermore, serum 8-OHdG level was identified as an independent predictor of RLS severity. These findings support the important role of uremia-related factors in addition to known risk factors such as iron deficiency, mineral bone metabolism disorders, inadequate dialysis, and duration of dialysis in the pathogenesis of dialysis-associated RLS. Further studies are needed to clarify the pathophysiological role of inflammation and oxidative stress in RLS.

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Conflict of interest The authors declare that there are no conflicts of interest. References 1. Merlino G, Valente M, Serafini A et al. Restless legs syndrome: diagnosis, epidemiology, classification and consequences. Neurol Sci 2007; 28: 37-46.

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48. Sande FM, Kooman JP, Leunissen KML. The predictive value of C-reactive protein in end-stage renal disease: Is it clinically significant? Blood Purif 2006; 24: 335-341. 49. Kalantar-Zadeh K, Ikizler A, Block G, et al. Malnutrition-inflammation complex syndrome in dialysis patients: causes and consequences. Am J Kidney Dis 2003; 42: 864-881. 50. Maruyama Y, Nakayama M, Yoshimura K, et al. Effect of repeated intravenous iron administration in haemodialysis patients on serum 8-hydroxy-20-deoxyguanosine levels. Nephrol Dial Transplant 2007; 22: 1407-1412. 51. Jung Oh H, Lee MJ, Lee HS, Park JT, Han SH, Yoo TH, et al. NT-proBNP: Is It a More Significant Risk Factor for Mortality Than Troponin T in Incident Hemodialysis Patients? Medicine (Baltimore) 2014; 93: e241. 52. Shafi T, Zager PG, Sozio SM, Grams ME, Jaar BG, Christenson RH, et al. Troponin I and NT-proBNP and the association of systolic blood pressure with outcomes in incident hemodialysis patients: the Choices for Healthy Outcomes in Caring for ESRD (CHOICE) Study. Am J Kidney Dis 2014; 64: 443-451. 53. Locatelli F, Hannedouche T, Martin-Malo A, Jacobson SH, Vanholder R, Ronco C, et al. The relationship of NT-proBNP and dialysis parameters with outcome of incident haemodialysis patients: results from the membrane permeability outcome study. Blood Purif. 2013;35:216-223. 54. Benz RL, Pressman MR, Hovick ET, Peterson DD. A preliminary study of the effects of correction of anemia with recombinant human erythropoietin therapy on sleep, sleep disorders, and daytime sleepiness in hemodialysis patients (The SLEEPO study). Am J Kidney Dis 1999; 34: 1089-1095. 55. Siddiqui S, Kavanagh D, traynor J, Mak M, Deighan C, Geddes C. Risk factors for restless legs syndrome in dialysis patients. Nephron Clin Pract 2005; 101: c155-c160. 56. Miranda M, Araya F, Castillo JL, Duran C, Gonzalez F, Aris L. Restless legs syndrome: a

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clinical study in adult general population and in uremic patients. Rev Med Chil 2001; 129: 179-186. 57. Tsubakihara Y, Nishi S, Akiba T, et al. 2008 Japanese Society for Dialysis Therapy: Guidelines for Renal Anemia in Chronic Kidney Disease. Ther Aphel Dial 2010; 14: 240-275.

Fig. 1. Flowchart of the distribution of patients from assessment of eligibility to completion of study period.

Fig. 2. Correlation between IRLSSG Severity Scale and (A) hemodialysis duration, (B) high-sensitivity C-reactive protein, (C) ferritin, and (D) 8-OHdG. IRLSSG, International Restless Legs Syndrome Study Group; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

Table 1. Demographic and baseline characteristics of the all patients, with and without restless legs syndrome.

Variables

All patients

RLS group

Non RLS group

p value

N (male/female) Age (y) Duration on dialysis (m) Clinical dry weight (kg) Body mass index (kg/m2) Smoking (%) Drinking (daily and regular) (%) Coffee intake (daily and regular) (%) Diabetes (%) Cardiovascular disease comorbidity Myocardial infarction Angina pectoris Cerebral vascular disease Peripheral artery disease Pre-dialysis Systolic BP (mmHg) Pre-dialysis diatolic BP(mmHg)

157 (111/46) 68 ± 11 54 ± 60 58.3 ± 11.7 22.0 ± 4.0 12.7 17.8 16.6 58.6 26 (16.6) 6 (3.8) 6 (3.8) 9 (5.7) 5 (3.2) 145 ± 17 79 ± 12

33 (24/9) 71 ± 8 76 ± 88 59.0 ± 12.1 22.4 ± 3.7 12.1 15.2 15.2 51.5 6 (18.2) 1 (3.0) 2 (6.1) 2 (6.1) 1 (3.0) 146 ± 18 80 ± 9

124 (87/37) 68 ± 11 48 ± 49 58.1 ± 11.7 22.2 ± 4.0 12.9 19.3 16.9 60.4 20 (16.1) 4 (3.2) 5 (4.0) 7 (5.6) 4 (3.2) 145 ± 17 79 ± 13

NS NS 0.009 NS NS NS NS NS NS NS NS NS NS NS NS NS

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Pre-dialysis heart rate (bpm) Cardiothoracic ratio (%) Medications Antihypertensive agents Antidepressant Benzodiazepines Antipsychotics Antiepileptics Antidiabetics Active vitamin D Phosphate binders Cause of ESKD Diabetic nephropathy Chronic glomerular nephritis Hypertensive nephrosclerosis Vasculitis Cystic disese Unknown IRLSSG Severity Scale score

75 ± 10 51.1 ± 5.3

75 ± 10 54.1 ± 5.4

75 ± 10 50.3 ± 5.1

NS 0.013

105 (67) 4 (2.5) 46 (29.2) 0 (0) 0 (0) 53 (33.7) 52 (33) 157 (100)

23 (70) 1 (3.0) 12 (36.4) 0 (0) 0 (0) 11 (33.3) 13 (39) 33 (100)

82 (66) 3 (2.4) 34 (27.5) 0 (0) 0 (0) 42 (33.9) 38 (31) 124 (100)

NS NS NS NS NS NS NS NS

83 (52.8) 30 (19.1) 34 (21.7) 3 (1.9) 3 (1.9) 4 (2.6) —

16 (48.6) 8 (24.2) 8 (24.2) 0 (0) 0 (0) 1 (3.0) 19.1 ± 8.9

67 (54.0) 22 (17.7) 26(37.1) 3 (2.4) 3 (2.4) 3 (2.4) —

NS NS NS — — NS —

Data are expressed as mean ± SD or n (%). BP, blood pressure; ESKD, end-stage kidney disease; IRLSSG, International Restless Legs Syndrome Study Group; RLS, restless legs syndrome; NS, not significant.

Table 2. Dose of hemodialysis and hemodialysis-related nutritional assessments.

Dialysis mode Hemodialysis Hemodiafiltration ERI Kt/V PCR GNRI %CGR

All patients (N = 157)

RLS group (N = 33)

Non RLS group (N = 124)

p value

116 (73.9) 41 (26.1) 7.1 [3.2–13.2] 1.32 ± 0.19 0.83 ± 0.15 93.2 ± 7.0

24 (72.2) 9 (27.8) 7.8 [3.9–16.2] 1.36 ± 0.20 0.86 ± 0.14 94.0 ± 5.5

92 (74.2) 32 (25.8) 6.9 [3.2–12.5] 1.31 ± 0.19 0.83 ± 0.15 92.9 ± 7.3

NS NS NS NS NS NS

95.8 ± 26.1

98.2 ± 30.2

95.2 ± 25.1

NS

Data are expressed as mean ±SD, median [interquartile range] or N (%). ERI, erythropoietin responsiveness index;

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GNRI, geriatric nutritional risk index; %CGR, % creatinine generation rate; RLS, restless legs syndrome; NS, not significant.

Table 3. Laboratory features of hemodialysis patients in the whole study population and patients with or without RLS.

Variables White blood cell (/μL) Hemoglobin (g/dL) Platelet (× 104/μL) Serum urea nitrogen (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Sodium (mEq/L) Pottassium (mEq/L) Corrected Ca (mg/dL) Phosphate (mg/dL) Alkaline phosphatase (U/L) Intact-PTH (pg/mL) Total protein (g/dL) Albumin (g/dL) Total cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglyceride (mg/dL) Iron (μg/mL) TIBC (μg/mL) TSAT (%) Ferritin (ng/mL) β2-microglobulin (mg/L) NT-proBNP (pg/mL) Hs-CRP (mg/dL)

All patients (N = 157) 5955 ± 1716 11.0 ± 1.1

RLS group (N = 33) 6472 ± 1738 11.2 ± 0.9

Non RLS group (N = 124) 5817 ± 1691 11.0 ± 1.1

18.1 ± 6.1 58.2 ± 14.1 9.47 ± 2.66 6.7 ± 1.3 142 ± 3 4.8 ± 0.7 9.2 ± 0.8 5.4 ± 1.3 236 [188–293] 134 [85–193] 6.6 ± 0.5 3.7 ± 0.4 153 ± 28 43 ± 17 125 ± 73 65 ± 23 256 ± 51 26.0 ± 10.4 83 [45-130] 27.0 ± 7.4 7850 [3192–13840] 0.13 [0.05–0.34]

18.2 ± 5.7 62.1 ± 13.1 9.43 ± 2.78 6.9 ± 1.4 141 ± 3 4.8 ± 0.7 9.2 ± 0.7 5.1 ± 1.2 255 [204–317] 148 [82–212] 6.7 ± 0.4 3.7 ± 0.3 154 ± 23 44 ± 16 142 ± 89 66 ± 18 262 ± 36 22.6 ± 7.9 118 [75-179] 27.8 ± 7.7 10600 [3822–21870] 0.26 [0.11–0.41]

18.1 ± 6.2 57.3 ± 14.3 9.48 ± 2.65 6.7 ± 1.2 142 ± 3 4.8 ± 0.7 9.2 ± 0.7 5.5 ± 1.4 219 [184–284] 133 [87–191] 6.6 ± 0.5 3.7 ± 0.4 152 ± 30 40 ± 18 121 ± 67 65 ± 24 254 ± 55 26.9 ± 10.8 79 [40-123] 26.8 ± 7.3 6650 [3083–12550] 0.11 [0.05–0.29]

p value NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.037 0.045 NS 0.012 0.021

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Interleukin-6 (pg/mL) 8-OHdG (ng/mL)

5.10 [3.34–8.55] 0.39 [0.32–0.49]

7.65 [4.02–10.8] 0.45 [0.38–0.62]

4.77 [3.23–7.59] 0.37 [0.29–0.48]

0.034 0.002

Data are expressed as mean ± SD or median [interquartile range]. 8-OHdG, 8-hydroxy-2′-deoxyguanosineCa, calcium; HDL, high density lipoprotein; Hs-CRP, high sensitivity C-reactive protein; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PTH, parathyroid hormone; RLS, restless legs syndrome; TIBC, total iron binding capacity; TSAT, transferrin saturation; NS, not significant.

Table 4. Univariate and multivariate logistic regression analysis for risk factors of RLS.

Variables Age Hemodialysis duration Female sex Diabetes Cardiothoracic ratio ERI Kt/V PCR GNRI %CGR White blood cell Hemoglobin Platelet Serum urea nitrogen Creatinine Uric acid Sodium Potassium

OR 1.028 1.008 1.134 1.490 1.150 1.006 3.873 5.253 1.012 1.006 1.0002 0.969 0.978 1.009 1.010 1.104 0.972 1.164

Univariate 95% CI Lower Upper 0.990 1.071 1.002 1.015 0.493 2.789 0.684 3.238 1.063 1.254 0.962 1.046 0.461 35.041 0.385 73.937 0.955 1.076 0.991 1.022 0.9999 1.0004 0.654 1.439 0.907 1.046 0.976 1.042 0.869 1.172 0.808 1.518 0.861 1.097 0.660 2.067

p value 0.162 0.0014 0.775 0.312 0.0004 0.765 0.218 0.219 0.686 0.381 0.051 0.678 0.965 0.573 0.893 0.538 0.653 0.601

OR

Multivariate * 95% CI Lower Upper

p value

1.002

0.994

1.009

0.683

1.074

0.970

1.188

0.167

1.000

1.000

1.000

0.447

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Corrected calcium Phosphate Alkaline phosphatase Intact PTH Total protein Albumin Total cholesterol HDL cholesterol Triglyceride β2-microglobulin Iron TIBC TSAT Ferritin NT-proBNP Hs-CRP Interleukin-6 8-OHdG

1.684 0.860 1.002 1.003 1.771 1.059 1.001 0.985 1.003 1.016 1.001 1.003 0.962 1.003 1.0001 7.465 1.088 165.78

0.936 0.627 0.999 0.998 0.740 0.368 0.987 0.961 0.998 0.962 0.983 0.995 0.917 0.999 1.0000 2.712 1.026 13.292

3.078 1.160 1.006 1.006 4.391 3.208 1.015 1.009 1.008 1.072 1.016 1.010 1.003 1.007 1.0001 24.718 1.166 2764.3

0.082 0.339 0.056 0.191 0.205 0.917 0.829 0.242 0.145 0.554 0.918 0.421 0.098 0.073 0.0003 <0.0001 0.0044 <0.0001

2.175

0.972

4.869

0.058

1.000

0.995

1.005

0.979

0.942 1.001 1.000 5.667 1.013 33.99

0.881 0.996 1.000 1.118 0.919 1.069

1.008 1.007 1.000 28.738 1.117 >999.999

0.082 0.695 0.057 0.036 0.789 0.045

8-OHdG, 8-hydroxy-2′-deoxyguanosine; %CGR, % creatinine generation rate; ERI, erythropoietin responsiveness index; GNRI, geriatric nutritional risk index; HDL, high-density lipoprotein; Hs-CRP, high-sensitivity C-reactive protein; Kt/V, single pool Kt/Vurea; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PTH, parathyroid hormone; RLS, restless legs syndrome; TIBC, total iron binding capacity; TSAT, transferrin saturation. * c statistics = 0.810.

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Table 5. Univariate linear regression analysis for IRLSSG Severity Scale. Univariate Variables

β

Age Hemodialysis duration Female sex Diabetes CVD comorbidity Cardiothoracic ratio Log ERI Kt/V PCR GNRI %CGR White blood cell Hemoglobin Platelet Serum urea nitrogen Creatinine Uric acid Sodium Potassium Corrected calcium Phosphate Alkaline phosphatase Intact PTH Total protein Albumin Total cholesterol HDL cholesterol Triglyceride β2-microglobulin

0.091 0.420 0.290 -4.315 4.770 0.462 0.152 0.443 15.41 -0.019 0.023 0.180 0.298 0.154 -0.082 -0.284 -0.367 0.197 -0.006 -0.120 -0.049 -0.050 -0.005 3.185 -2.934 0.045 -0.020 0.007 0.189

Lower -0.298 0.090 -3.309 -10.53 -3.350 -0.139 -0.319 -16.86 -4.370 -0.634 -0.080 -0.001 -3.256 -0.451 -0.368 -1.474 -2.833 -0.824 -5.133 -5.520 -2.840 -0.030 -0.030 -4.420 -15.80 -0.103 -0.193 -0.028 -0.238

95% CI Upper 0.479 0.670 3.892 1.911 12.91 1.064 0.623 17.85 35.19 0.595 0.126 0.003 3.854 0.759 0.203 0.904 2.098 1.219 5.121 2.360 2.741 0.030 0.019 10.79 9.932 0.193 0.153 0.043 0.615

p value 0.639 0.014 0.869 0.167 0.240 0.127 0.453 0.954 0.122 0.949 0.652 0.210 0.865 0.607 0.560 0.628 0.763 0.693 0.998 0.410 0.971 0.850 0.662 0.399 0.645 0.539 0.813 0.685 0.374

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Iron TIBC TSAT Ferritin Log NT-proBNP Log hs-CRP Log interleukin-6 Log 8-OHdG

-0.005 -0.046 0.310 0.064 0.585 7.343 2.105 45.16

-0.184 -0.132 -0.03 0.031 -4.821 2.792 -8.373 32.14

0.174 0.04 0.59 0.098 5.993 11.894 12.585 58.16

0.954 0.285 0.118 0.0005 0.826 0.0025 0.684 <0.0001

8-OHdG, 8-hydroxy-2′-deoxyguanosine; %CGR, % creatinine generation rate; CVD, cardiovascular disease; ERI, erythropoietin responsiveness index; GNRI, geriatric nutritional risk index; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; Kt/V, single pool Kt/Vurea; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PTH, parathyroid hormone; RLS, restless legs syndrome; TIBC, total iron binding capacity; TSAT, transferrin saturation.

Table 6. Multiple linear regression analysis for IRLSSG Severity Scale Unadjusted (R2 = 0.726) Variables

β

SE

HD duration Ferritin

-0.003 0.035

0.012 0.014

95% CI Lower Upper -0.029 0.022 0.004 0.065

Adjusted* (R2 = 0.743) p value

β

SE

0.780 0.026

-0.002 0.028

0.014 0.016

Lower -0.031 -0.004

95% CI Upper 0.027 0.061

p value 0.885 0.083

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Log hs-CRP Log 8-OHdG

2.358 35.19

1.600 6.601

-2.64 21.66

-0.919 48.71

5.635 <0.0001

1.319 37.71

1.869 7.213

-2.531 22.87

5.171 52.58

0.486 <0.0001

8-OHdG, 8-hydroxy-2′-deoxyguanosine; HD, hemodialysis; hs-CRP, high-sensitivity C-reactive protein. * Adjusted for age, gender, presence of diabetes, and history of cardiovascular disease.

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