Osteoporos Int DOI 10.1007/s00198-014-2934-8

ORIGINAL ARTICLE

Associations between the levels of sclerostin, phosphate, and fibroblast growth factor-23 and treatment with vitamin D in hemodialysis patients with low intact PTH level Y. Asamiya & A. Yajima & S. Shimizu & S. Otsubo & K. Tsuchiya & K. Nitta

Received: 7 March 2014 / Accepted: 15 September 2014 # International Osteoporosis Foundation and National Osteoporosis Foundation 2014

Abstract Summary Serum sclerostin levels could be closely associated with serum phosphate and fibroblast growth factor-23 levels in hemodialysis patients with low intact parathyroid hormone (PTH) levels. Further study is required to indicate whether these close associations are present in patients with spontaneously low PTH levels without any vitamin D treatment. Introduction Intact parathyroid hormone (iPTH) is involved in the interaction between sclerostin and phosphate/fibroblast growth factor-23 (FGF23) in animal models. However, their relationship in patients on hemodialysis (HD) is unclear. Methods Data of 102 HD patients were collected regarding clinical and laboratory parameters and mineral bone disorder Y. Asamiya (*) : A. Yajima : K. Tsuchiya : K. Nitta Department of Medicine, Kidney Center, Tokyo Women’s Medical University, 8-1 Kawada-chou, Shinjuku-ku, Tokyo 162-8666, Japan e-mail: [email protected] A. Yajima Department of Medicine, Division of Nephrology, Otsuki Municipal Central Hospital, 1225 Otsukimachihanasaki, Otsuki, Yamanashi 401-0015, Japan A. Yajima Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Kita 13 Nishi 7 kita-ku, Sapporo 060-8586, Japan S. Shimizu Medical Research Institute, Tokyo Women’s Medical University, 8-1 Kawada-chou, Shinjuku-ku, Tokyo 162-8666, Japan S. Otsubo Department of Blood Purification, Sangenjaya Hospital, 1-25-5 Sangenjaya, Setagaya-ku, Tokyo 154-0024, Japan K. Tsuchiya Department of Blood Purification, Kidney Center, Tokyo Women’s Medical University, 8-1 Kawada-chou, Shinjuku-ku, Tokyo 162-8666, Japan

medications. The patients were divided into subgroups according to the iPTH level (A, <70 pg/mL; B, 70–150 pg/ mL; C, 150–300 pg/mL; and D, ≥300 pg/mL). Results The sclerostin level was significantly and positively correlated with phosphate and log of FGF23 levels in subgroups A, B, and combined A and B. Multiple linear regression analysis in the combined A and B subgroup revealed that male sex (t=3.24, P=0.01; 95 % confidence interval [CI] 11.78 to 50.43) and phosphate level (t=2.13, P=0.04; 95 % CI, 1.08 to 36.91) were independent factors for serum sclerostin level. The log of serum FGF23 level (t=1.90, P=0.06, 95 % CI −1.85 to 63.50) appeared to be an important factor for serum sclerostin level. The frequency of patients using vitamin D treatment was not significantly different among subgroups A (93.1 %), B (88.0 %), C (85.2 %), and D (90.5 %). Conclusion Serum sclerostin levels were associated with serum phosphate and FGF23 levels in patients with low iPTH levels. Further study is required to indicate whether these close associations are present in patients with spontaneously low iPTH levels without vitamin D treatment. Keywords Fibroblast growth factor-23 . Parathyroid hormone . Phosphate . Sclerostin

Introduction Sclerostin, a product of the SOST gene, is mainly expressed by osteocytes and plays an important role in regulating bone formation by inhibiting Wnt–β-catenin signaling [1–5]. Sclerostin also stimulates osteocyte support of osteoclast activity in a receptor activator of the NF-κB ligand (RANKL)dependent pathway [6]. Moreover, sclerostin at least partly regulates bone matrix mineralization through a signaling pathway involving phosphate regulators—the phosphateregulating neutral endopeptidase on chromosome X (PHEX)

Osteoporos Int

and the matrix extracellular phosphoglycoprotein (MEPE) axis—by targeting late-osteoblasts and preosteocytes [7, 8]. Sclerostin has also been reported to interact closely with fibroblast growth factor 23 (FGF23) [9], vitamin D [9, 10], and parathyroid hormone (PTH) [11–13]. Given these roles, sclerostin appears to be a key factor in chronic kidney disease (CKD), as this condition is frequently associated with abnormalities in bone remodeling, bone mineralization, and systematic mineral metabolism. In patients with CKD, sclerostin has been associated with progression of renal osteodystrophy [14], bone turnover [15], and serum PTH levels [15]. However, there is little understanding of the role of sclerostin in CKD, and even a consensus regarding whether a low level of serum sclerostin is desirable in CKD patients has not been reached [16]. However, FGF23 has been proven to play an extremely important role in CKD mineral and bone disorder (CKD-MBD) [17–19]. Nevertheless, the association between sclerostin and FGF23 is still not fully understood. Compared with that in other countries [20, 21], the recommended target range in the national guidelines in Japan for the intact PTH (iPTH) level in hemodialysis (HD) patients is very low (60–180 pg/mL) [22], although it was revised to 60– 240 pg/mL in 2012 [23]. Current investigation of Japanese patients may facilitate the understanding of the role of sclerostin in patients with low PTH levels and in patients who underwent treatment for hyperparathyroidism using therapies such as vitamin D administration. Recently, a study of an experimental animal model of adynamic bone disease associated with CKD and hypoparathyroidism showed that an increase in sclerostin expression in bone is associated with an increase in dietary phosphate intake through a PTH-independent mechanism [24]. With regard to this important association, which is largely unclear in humans, we hypothesize the presence of a relationship between sclerostin and phosphate and phosphate regulators, especially in individuals with a low PTH level. To test this hypothesis, we examined the association between serum sclerostin levels and serum phosphate levels, FGF23 levels, bone formation/resorption parameters, and medications for CKD-MBD, including vitamin D, through the analysis of differences in the serum iPTH levels of HD patients. We then compared the serum sclerostin levels between HD patients and controls with normal renal function to further understand the characteristics of serum sclerostin levels in HD patients. Methods Study participants One hundred and two outpatients receiving maintenance HD treatment at one hospital (Towa Hospital, Tokyo, Japan) and

30 age-, sex-, and menopausal status-matched controls with normal renal function were enrolled in this study between April and June 2011. The controls were recruited from among the medical staff working at the hospital who did not have any disease, as well as from among the outpatients presenting with mild hypertension or mild benign prostatic hypertrophy who had not undergone any pharmacological treatment known to interfere with bone remodeling and/or serum phosphate, calcium, or vitamin D levels. The inclusion criteria were ability to provide informed consent and age >18 years. The exclusion criteria were (1) primary or metastatic bone disease; (2) bone fracture; (3) diseases known to affect bone metabolism, such as rheumatoid arthritis, hypercortisolism, and hyperthyroidism; (4) acute bacterial or viral infection; (5) liver dysfunction; (6) any malignant disease; (7) inability to walk without assistance (a condition that increases serum sclerostin level); (8) HD therapy for ≤3 months before study initiation; (9) parathyroidectomy within 5 years of study initiation; (10) history of renal transplantation; or (11) pregnancy. The HD patient group consisted of 76 men and 26 women (24 postmenopausal and 2 premenopausal women), and the control group consisted of 18 men and 12 women (11 postmenopausal women and 1 premenopausal woman). All study participants were of East Asian origin. They had no past history of fracture or radiographic evidence of vertebral or rib fracture. The HD patients underwent HD therapy thrice a week (4.0–4.5 h/session) using hollow fiber dialyzers and a standard dialysate (2.5 mEq/L of calcium and 27.5 mEq/L of bicarbonate). All patients were treated according to the guidelines for the management of CKD-MBD developed by the Japanese Society for Dialysis Therapy (JSDT) [22], which recommended target ranges of 8.4–10.0 mg/dL for adjusted calcium, 3.5–6.0 mg/dL for phosphate, and 60–180 pg/mL for iPTH. Recommendations for hyperparathyroidism treatment include the use of calcium carbonate, sevelamer hydrochloride, lanthanum carbonate, vitamin D (alfacalcidol, calcitriol, or maxacalcitol), and cinacalcet hydrochloride. However, parathyroid intervention therapy should be recommended in patients with severe hyperparathyroidism (persistently high serum levels of iPTH of >500 pg/mL), associated with hyperphosphatemia (serum phosphate >6.0 mg/dL) and/or hypercalcemia (serum-adjusted calcium >10.0 mg/dL) that is refractory to medical therapy. None of the patients received any other medication that might affect calcium or bone metabolism, such as bisphosphonates, glucocorticoids, raloxifene, estrogens, androgen, vitamin K, aluminum-based phosphate binders, paricalcitol, or doxercalciferol. Written informed consent for participation was obtained from all participants, and approval for the study was obtained by the institutional review board of the study site, in accordance with the Declaration of Helsinki.

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Measurement of clinical and laboratory parameters

Study design

Height and body weight after HD therapy were measured for calculation of body mass index (BMI) as weight/height2 (kg/ m2). Data regarding current CKD-MBD treatment were collected from review of patient records. Blood samples were collected from HD patients in the nonfasting state, before the first HD treatment of the week and from control subjects in the nonfasting state, and then stored at −80 °C within 30 min of centrifugation (3,000×g for 15 min) until needed for the measurement of serum parameters. Serum albumin, calcium, phosphorus, and C-reactive protein levels were measured using an automated analyzer (TBA120FR; Toshiba Medical Systems, Tochigi, Japan), and the following equation was used to calculate the corrected calcium level: serum calcium (mg/dL) + (4–albumin [g/dL]) [25]. Hemoglobin A1C was measured by latex immunoassay (Diabetes Mellitus Test Analyzer; Kyowa Medex, Tokyo, Japan). Serum iPTH level was measured by electrochemiluminescence immunoassay (ECLIA) using the Modular Analytics E170 system (Roche Diagnostics, Mannheim, Germany; intra-assay coefficient of variation [CV] <15 %). We selected the measurement of serum bone-specific alkaline phosphatase (BAP) by chemiluminescence enzyme immunoassay (CLEIA; Beckman Coulter, Brea, California, USA; intra-assay CV <10 %) as a bone formation marker because it depends on the rate of release from bone osteoblasts, and its concentration is not modified by variation in glomerular filtration rate [21]. We selected the measurement of serum tartrate-resistant acid phosphatase isoform type5b (TRAP5b) by enzyme immunoassay (EIA; Nittobo Medical, Tokyo, Japan; intra-assay CV <15 %) as a bone resorption marker because its concentration is not affected by renal dysfunction and is shown as a useful marker of osteoclastic activity and bone resorption in CKD-MBD [26]. Serum sclerostin level was measured by sandwichtype enzyme-linked immunosorbent assay (ELISA) using commercial reagents (Biomedica Medizinprodukte, Vienna, Austria), as described previously [27]. The intra-assay CV of the measurement ranged from 4 to 6 %, the inter-assay CV ranged from 5 to 7 %, and the detection limit of the assay was 0.2 ng/mL. Conversion factors for unit are the following: 1 pg/mL=0.044 pmol/ L (sclerostin in pg/mL to pmol/L, ×0.044, and in ng/mL to pmol/L, ×44). Serum FGF23 level was measured by sandwich-type ELISA for human FGF-23 (Kainos Laboratories, Inc., Tokyo, Japan), which measures biologically active, full-length FGF23 using two monoclonal antibodies for FGF23, as described previously [28]. The intra-assay CV of the measurement was <10 %, and the range of measurement was 3–800 pg/mL.

First, the serum sclerostin levels between all HD patients and controls were compared. Second, low iPTH level and poorly controlled hyperparathyroidism were involved in low bone turnover and high bone turnover, respectively, and were both closely associated with the serum sclerostin level [11–13]. Thus, as the difference in the iPTH level appears to be an influential factor for understanding the effects on the sclerostin level, the HD patients were classified according to the iPTH level: subgroup A, <70 pg/mL (n = 29); subgroup B, 70– 150 pg/mL (n=25); subgroup C, 150–300 pg/mL (n= 27); and subgroup D, ≥300 pg/mL (n=21). Moreover, subgroups A and B were analyzed together as the low iPTH subgroup (<150 pg/mL, n =54), as these levels were less than the target range of iPTH levels recommended by the Kidney Disease Outcomes Quality (K/DOQI) guidelines [20]. As the four subgroups classified according to the iPTH levels are relatively small, which limited the interpretability of the multivariate analysis, the combined subgroups A and B were also assessed together with the low iPTH subgroup. Statistical analysis Descriptive statistics were calculated as frequencies, means±standard deviations (SD), or medians and interquartile ranges (IQRs), according to the distribution of the variables examined. Differences between categorical variables were tested using the 2×2 chi-squared test. Normal distribution was assessed using the Shapiro– Wilk test. After identification of skewed distribution among serum FGF23 levels, the data were log transformed to allow further statistical analysis. Two groups were compared using the unpaired t test or the Mann– Whitney U test. As sclerostin production is suppressed by iPTH secretion [11–13], the patients were divided into the following subgroups based on the iPTH level: subgroup A, <70 pg/mL; subgroup B, 70–150 pg/mL; subgroup C, 150–300 pg/mL; and subgroup D, ≥300 pg/ mL. These four subgroups were compared using nonrepeated measure analysis of variance (ANOVA) or the Kruskal–Wallis H test. Correlation analysis of serum sclerostin levels was performed to identify associations between serum sclerostin levels and significant clinical and laboratory variables. Variables with a P value of <0.2 in univariate analysis were entered into multiple linear regression analysis to identify the independent factor(s) associated with the sclerostin level, using the stepwise forward-selection method. Using categorical data in multiple regression models, the variables were represented by dummy variables coded as 0 for female

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individuals and 1 for male individuals for the variable of sex and as 0 for the absence of the variable (e.g., absence of disease) and 1 for the presence of the variable for all other variables. A P value of <0.05 was considered statistically significant. All statistical analyses were performed using version 10.0 JMP software (SAS for Windows, SAS Institute, Cary, NC, USA).

Results Comparison of serum sclerostin levels between all HD patients and controls The clinical profiles of the HD patients and controls are shown in Table 1, and the distribution of their serum sclerostin levels in Fig. 1. The serum sclerostin levels of the HD patients were extremely high and varied widely compared to those of the controls (median 15.2, IQR 11.3–22.3 ng/mL vs median 1.15, IQR 0.93–1.46 ng/mL, respectively; P<0.001). Three patients had undergone parathyroidectomy >5 years ago. The differences in the levels of hemoglobin and phosphate between the two groups were inevitable because of the difference in the renal function. Characteristics of HD patients in the subgroups classified according to the iPTH level The data on the clinical and laboratory parameters and current treatment for CKD-MBD of HD patients classified according to the iPTH level are shown in Table 2. The levels of serum BAP, TRAP5b, and log of FGF23 were significantly higher in patients with high iPTH. With regard to the frequency of patients using vitamin D treatment, those in subgroup A (93.1 %), subgroup B (88.0 %), subgroup C (85.2 %), and subgroup D (90.5 %) did not significantly differ. Univariate analysis of serum sclerostin levels in the subgroups of HD patients according to iPTH level Correlation analysis and 2×2 chi-squared test for serum sclerostin levels are shown in Table 3a, b, respectively. The serum sclerostin level was significantly and positively correlated with the serum phosphate level in subgroup A (r=0.65, P<0.001, 95 % confidence interval [CI] 0.37 to 0.82), subgroup B (r=0.49, P=0.01, 95 % CI 0.12 to 0.74), and the subgroup with iPTH <150 pg/mL (r=0.56, P<0.001, 95 % CI 0.24 to 0.79; Fig. 2a), as well as with the log of serum FGF23 level in subgroup A (r=0.57, P=0.01, 95 % CI 0.25 to 0.77), subgroup B (r=0.63, P<0.001, 95 % CI 0.31 to 0.82), and the subgroup with iPTH <150 pg/mL (r = 0.59,

P<0.001, 95 % CI 0.12 to 0.47; Fig. 2b). Analysis of the correlation among the parameters also indicated a positive correlation between the serum phosphate level and log of serum FGF23 level in subgroup A (r=0.73, P<0.001, 95 % CI 0.49 to 0.86), subgroup B (r=0.73, P<0.001, 95 % CI 0.48 to 0.88), and the subgroup with iPTH <150 pg/mL (r=0.71, P<0.001, 95 % CI 0.54 to 0.82; Fig. 2c). In these three subgroups, the serum sclerostin level was significantly higher in male patients than that in female patients. In subgroup C, the serum sclerostin level was lower in patients taking medication containing cinacalcet hydrochloride than that in patients not taking medication containing cinacalcet hydrochloride (P=0.01). In subgroup D, the serum sclerostin level was significantly and negatively correlated with the serum BAP level (r=−0.60, P=0.01, 95 % CI −0.82 to −0.22). Multivariate linear regression analysis of serum sclerostin levels The results of multivariate linear regression analysis of serum sclerostin levels in the low iPTH subgroup (<150 pg/mL, 54 patients) are shown in Table 4. The variables with a P value of <0.2 from the univariate analysis for serum sclerostin level (correlation analysis and the 2 × 2 chi-squared test), including duration of HD, phosphate level, log of FGF23 level, and gender were inserted as explanatory variables in the model of the multivariate linear regression analysis. Thereafter, these were selected using the forward stepwise method. Finally, we observed that gender (t=3.24, P=0.01, 95 % CI 11.78 to 50.43) and phosphate level (t=2.13, P= 0.04, 95 % CI 1.08 to 36.91) were significant independent factors for the serum sclerostin level. The log of serum FGF23 level (t=1.90, P=0.06, 95 % CI −1.85 to 63.50) appeared to be an important factor for the serum sclerostin level. Correlation analysis between the levels of sclerostin and mineral/bone markers in all HD patients Correlation analysis between the levels of serum sclerostin and mineral/bone markers was performed in all HD patients. Serum sclerostin level was negatively correlated with the serum BAP level (r=−0.25, P=0.01, 95 % CI −0.43 to −0.06) and TRAP5b level (r=−0.23, P=0.02, 95 % CI −0.41 to −0.04). Serum sclerostin levels did not show a significant association with iPTH levels; however, the serum sclerostin level was positively correlated with the serum phosphate level (r=0.42, P<0.001, 95 % CI 0.25 to 0.57) and log of FGF23 level (r=0.31, P=0.002, 95 % CI 0.12 to 0.47). The positive correlation of sclerostin level with phosphate level (r = 0.56,

Osteoporos Int Table 1 Clinical profiles of hemodialysis patients and controls Parameters

HD patients (n=102)

Controls (n=30)

P value

Age (years) Male sex, n (%) HD duration (years) Body mass index (kg/m2) Primary kidney disease Diabetes mellitus, n (%) CGN, n (%) Nephrosclerosis, n (%) PKD, n (%) Unknown, n (%) Serum parameters Hemoglobin (g/dL) Albumin (g/dL) Calcium (mg/dL)a

66.4±8.9 76 (74.5) 8.1±6.3 21.4±3.7

65.2±10.1 18 (60.0) – 20.9±2.7

0.54 0.11 – 0.59

44 (43.1) 25 (24.5) 13 (12.7) 4 (3.9) 16 (15.7)

– – – – –

– – – – –

10.6±1.0 3.8±0.3

13.0±1.3 4.1±0.4

<0.001 <0.001

9.1±0.7 5.2±1.4 241±112 0.3±0.3

9.3±0.3 3.4±0.8 – 0.2±0.3

0.13 <0.001 – 0.19

5.7±1.0 190±185 14.9±6.0 434±301 3.7±0.8 16.7±8.2

– – – – 1.6±0.2 1.26±0.47

– – – – <0.001 <0.001

88 (86.3) 2.9±1.5 35 (34.3) 2.55±1.56 17 (16.7) 0.69±0.29 91 (89.2) 58 (56.9) 0.45±0.25 1 (1.0) 0.5 32 (31.4)

– – – – – – – – – – – –

– – – – – – – – – – – –

9.8±4.7 24 (23.5) 27.7±9.2

– – –

– – –

Phosphate (mg/dL) ALP (U/L) CRP (mg/dL) Hemoglobin A1C (%) Intact PTH (pg/mL) BAP (μg/L) TRAP5b (mU/dL) Log of FGF23 (pg/mL) Sclerostin (ng/mL) Current treatments for CKD-MBD Calcium carbonate, n (%) Dose (g/day) Sevelamer-HCl, n (%) Dose (g/day) Lanthanum, n (%) Dose (g/day) Vitamin D, n (%) Alfacalcidol, n (%) Dose (μg/day) Calcitriol, n (%) Dose (μg/day) Maxacalcitol, n (%) Dose (μg/week) Cinacalcet HCl, n (%) Dose (mg/day)

Note: Data are expressed as mean±standard deviation or number (%). Conversion factors for units are the following: albumin and hemoglobin in g/dL to g/L, ×10; calcium in mg/dL to mmol/L, ×0.2495; phosphate in mg/dL to mmol/L, ×0.3229. No conversion necessary for intact PTH in pg/mL and ng/L HD hemodialysis, CGN chronic glomerulonephritis, PKD polycystic kidney disease, ALP alkaline phosphatase, CRP C-reactive protein, PTH parathyroid hormone, BAP bone-specific alkaline phosphatase, TRAP5b 4 tartrate-resistant acid phosphatase isoform type 5b, FGF23 fibroblast growth factor-23, sevelamer-HCl sevelamer hydrochloride, cinacalcet HCl cinacalcet hydrochloride a

Corrected calcium=serum calcium (mg/dL)+(4–albumin [g/dL]) [25].

P<0.001, 95 % CI 0.24 to 0.79) and log of FGF23 level (r=0.59, P<0.001, 95 % CI 0.12 to 0.47) was observed

only in the iPTH <150 pg/mL subgroup (n=54) and not in the iPTH ≥150 pg/mL subgroup (n=48).

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% of patients

a

60

median 1.15 (interquartile range 0.93–1.46) minimum 0.52, maximum 2.49

40 20 0 0.5 5

b % of patients

Fig. 1 Distribution of serum sclerostin levels in a 30 controls and b 102 hemodialysis patients. a Median level 1.15 ng/mL (IQR 0.93–1.46 ng/mL), lowest level 0.52 ng/mL, highest level 2.49 ng/mL; b median level 15.2 ng/mL (IQR 11.3–22.3 ng/ mL), lowest level 2.75 ng/mL, highest level 39.7 ng/mL. Serum sclerostin levels in hemodialysis patients were markedly high and varied widely compared with those of controls (P<0.001). Conversion of sclerostin in ng/mL to pmol/L, ×44. IQR interquartile range

1.0 10

1.5 15

2.0 20

60

2.5 25

3.0 ng/ mL 30

median 15.2 (interquartile range 11.3–22.3) minimum 2.75, maximum 39.7

40 20 0

5

Discussion This examination of the relationship between serum sclerostin level and clinical/laboratory data and current CKD-MBD treatment yielded several important findings. The most important finding was that the serum phosphate and serum FGF23 levels appear to be closely associated with the serum sclerostin level in CKD patients with low iPTH levels. PTH has been observed to directly inhibit sclerostin transcription and sclerostin expression in osteocytes in animal models [11, 12], and the serum PTH level has been found to be negatively correlated with serum sclerostin level in nonCKD postmenopausal women (r=−0.474, P=0.035) [13]. These observations suggest that the serum sclerostin level is likely to be directly influenced by PTH function. Therefore, in the present study, we focused on identifying the factor that regulates serum sclerostin level by analyzing the differences in serum iPTH levels of HD patients. The finding of a significant and positive association between serum sclerostin levels and serum phosphate levels in HD patients with low iPTH levels (subgroups according to the iPTH level of <70, 70–150, and <150 pg/mL [combined]) was consistent with a study of 90 CKD patients who had not received dialysis therapy (r=0.26, P=0.016), had relatively low iPTH levels (median, 54.5 pg/mL; IQR, 36.7–97.3 pg/ mL), had serum 25 OH vitamin D levels of 20.6±8.7 ng/mL, and did not have any data on whether they received vitamin D treatment [29]. In addition, in a CKD model rat, with very low PTH levels after parathyroidectomy without vitamin D treatment, Ferreira et al. interestingly observed that a highphosphate diet increases the expression of sclerostin in bone as well as the circulating sclerostin level [24].

10

15

20

25

30

35

40 ng/mL

With regard to the frequency of patients who received vitamin D treatment (alfacalcidol, calcitriol, or maxacalcitol), there was no significant difference between the subgroups according to the iPTH level of <70 pg/mL (93.1 %), 70– 150 pg/mL (88.0 %), 150–300 pg/mL (85.2 %), and ≥300 pg/mL (90.5 %); however, the frequencies of patients who received alfacalcidol or maxacalcitol were different, probably because the treatment agents for hyperparathyroidism were selected based on the drug’s power, and maxacalcitol has a higher beneficial effect than alfacalcidol. Because the serum iPTH level target range of the Japanese guideline for CKD-MBD is lower [22] than that of other countries [20, 21], many patients received any vitamin D treatment for hyperparathyroidism. The lower target range of the iPTH level in Japan may be because this value is based on the results of a retrospective analysis of a nationwide survey and on expert opinion, due to the paucity of high-level evidence [30]. Therefore, further study is required to investigate whether a similar positive association with serum sclerostin level and serum phosphate level or serum FGF 23 level is also observed in patients with spontaneously low PTH levels without any vitamin D treatment. Thus far, the mechanism underlying the positive association between serum sclerostin levels and serum phosphate levels under the condition of low serum iPTH levels remains unclear. To our knowledge, the presence of a signaling pathway with a direct connection between sclerostin and phosphate, such as a pathway composed of receptors of a phosphate or any phosphate-combining molecule on the surface of osteocytes that promotes the production of sclerostin, has not been identified. However, the interactions that link sclerostin and several phosphate regulators including FGF23, dentin

Osteoporos Int Table 2 The clinical and laboratory parameters and current treatment for CKD-MBD in the intact PTH subgroups of hemodialysis patients iPTH subgroups Variables

<70 A (n=29)

70–150 B (n=25)

150–300 C (n=27)

≥300 D (n=21)

Pa

<150 (combined A and B) (n=54)

Age (years)

68.3±7.9

64.1±9.3

66.6±9.0

66.2±9.6

0.39

66.4±8.7

Male sex, n (%) HD duration (years) Serum parameters Albumin (g/dL) Calcium (mg/dL)b Phosphate (mg/dL) ALP (U/L) CRP (mg/dL) Intact PTH (pg/mL) BAP (μg/L) TRAP5b (mU/dL) Log of FGF23 (pg/mL) Sclerostin (ng/mL) Current treatments for CKD-MBD Calcium carbonate, n (%) Dose (g/day) Sevelamer-HCl, n (%) Dose (g/day)

21 (72.4) 9.4±7.7

16 (64.0) 5.3±4.5

21 (77.8) 8.1±4.9

18 (85.7) 9.6±6.9

0.38 0.05

37 (68.5) 7.5±6.7

3.7±0.3 9.3±0.8 4.8±1.5 203±69 0.3±0.3 38±17 12.4±3.9 299±154 3.5±0.8 18.5±9.3

3.9±0.3 9.1±0.5 5.2±1.2 259±145 0.2±0.1 103±24 14.5±4.5 416±205 3.4±0.8 15.8±7.9

3.8±0.3 8.9±0.7 5.2±1.1 246±126 0.4±0.3 217±48 16.4±7.3 450±266 3.7±0.8 16.3±7.3

3.9±0.3 9.1±0.9 5.8±1.6 264±90 0.3±0.3 470±205 17.0±7.1 619±466 4.1±0.7 15.5±7.9

0.21 0.28 0.09 0.18 0.16 – 0.02 0.01 0.04 0.53

3.8±0.3 9.2±0.7 5.0±1.4 229±113 0.3±0.2 68±39 13.3±4.3 353±187 3.5±0.8 17.3±8.7

23 (79.3) 2.9±1.8 5 (17.2) 3.30±1.88

23 (92.0) 2.6±1.1 10 (40.0) 1.83±1.14

23 (85.2) 3.0±1.2 9 (33.3) 2.97±1.37

19 (90.5) 3.1±1.7 11 (52.4) 2.52±1.78

0.53 0.78 0.07 0.27

46 (85.2) 2.8±1.5 15 (27.8) 2.32±1.54

6 (20.7) 0.63±0.21 27 (93.1) 23 (79.3) 0.53±0.33 1 (3.4) 0.5 3 (10.3) 10.0±4.3 4 (13.8) 21.4±7.1

3 (12.0) 0.83±0.63 22 (88.0) 16 (64.0) 0.39±0.16 0 (0) – 6 (24.0) 7.9±1.0 6 (24.0) 26.8±12.7

5 (18.5) 0.60±0.14 23 (85.2) 10 (37.0) 0.42±0.17 0 (0) – 13 (48.1) 9.4±5.4 6 (22.2) 26.8±4.4

3 (14.3) 0.83±0.14 19 (90.5) 9 (42.9) 0.37±0.17 0 (0) – 10 (47.6) 11.3±5.3 8 (38.1) 32.1±9.0

0.83 0.55 0.80 0.01 0.24 – – 0.01 0.60 0.26 0.29

9 (16.7) 0.69±0.37 49 (90.7) 39 (72.2) 0.47±0.28 1 (1.9) 0.5 9 (16.7) 8.6±2.5 10 (18.5) 24.6±10.7

Lanthanum, n (%) Dose (g/day) Vitamin D, n (%) Alfacalcidol, n (%) Dose (μg/day) Calcitriol, n (%) Dose (μg/day) Maxacalcitol, n (%) Dose (μg/week) Cinacalcet HCl, n (%) Dose (mg/day)

Note: Data are expressed as mean±standard deviation. Conversion factors for units are the following: calcium in mg/dL to mmol/L, ×0.2495; phosphate in mg/dL to mmol/L, ×0.3229. No conversion necessary for intact PTH in pg/mL and ng/L a

Four subgroups (A–D) were compared using nonrepeated measure analysis of variance (ANOVA) or the Kruskal–Wallis H test

b

Corrected calcium=serum calcium (mg/dL)+(4–albumin [g/dL]) [25]

CKD-MBD chronic kidney disease–mineral bone disorder, HD hemodialysis, PTH parathyroid hormone, ALP alkaline phosphatase, CRP C-reactive protein, BAP bone-specific alkaline phosphatase, TRAP5b tartrate-resistant acid phosphatase isoform type 5b, FGF23 fibroblast growth factor-23, sevelamer-HCl sevelamer hydrochloride, cinacalcet HCl cinacalcet hydrochloride

matrix protein 1 (DMP1), PHEX, MEPE, and processed forms of peptides (the acidic serine- and aspartic acid-rich motif [ASARM] peptide) have been found [7–9, 31–33]. These phosphate regulators play an important role in regulating bone turnover, bone mineralization, and renal mineral homeostasis [7, 8, 31–33] and interact closely with each other. For example, ASARM peptide has been observed to regulate FGF23 expression by the competitive displacement of a cell surface

PHEX–DMP1–integrin interaction [34]. In an animal study, Ryan et al. found that the serum intact FGF23 level decreased in the absence of sclerostin [9], suggesting that sclerostin alters FGF23 processing by inhibiting PHEX [9, 31]. In addition, Rowe indicated that in osteoblasts, sclerostin directly interacts with the binding substance of PHEX and DMP1 as well as the MEPE/ASARM peptide, and thus regulates FGF23 expression [32].

0.9 0.01 0.07 0.9 0.25 0.67 0.9 <0.001

−0.26 to 0.46 0.37 to 0.82 −0.41 to 0.32 −0.42 to 0.32 −0.11 to 0.58 −0.62 to 0.05 −0.56 to 0.13 0.25 to 0.77

0.11 0.65 −0.05 −0.06 0.27 −0.33 −0.24 0.57

Nonuse, n Use, n Alfacalcidol Nonuse, n

Male, n Female, n Non-DM patient, n DM patient, n Calcium carbonate Nonuse, n Use, n Sevelamer-HCl Nonuse, n Use, n Lanthanum

18.5±9.9 18.7±7.4

19.2±13.2

6

18.2±10.0 20.1±5.5 0.68

24 5

23 6

17.9±9.5 18.7±9.5

6 23

0.9

0.86

0.9

0.02

21.1±8.9 11.9±7.3 18.5±9.6 18.6±9.3

21 8 18 11

P

9

22 3

15 10

2 23

16 9 14 11

−0.40 to 0.39 0.12 to 0.74 −0.04 to 0.66 −0.39 to 0.40 −0.17 to 0.58 −0.32 to 0.47 −0.40 to 0.39 0.31 to 0.82

15.6±8.0 17.8±8.7 0.66

14.6±8.0 17.6±7.9 0.37

7.7±7.1 16.5±7.8 0.14

13.1±5.7

r

P

150–300 C

0.70 0.22 0.77 0.73 0.70 0.61 0.25 0.48

17

22 5

18 9

4 23

P 21 6 13 14

−0.45 to 0.31 −0.15 to 0.57 −0.43 to 0.33 −0.44 to 0.32 −0.31 to 0.44 −0.47 to 0.29 −0.56 to 0.17 −0.50 to 0.25

16.5±7.3 15.5±8.4 0.80

17.4±7.8 14.1±6.2 0.27

16.8±4.5 16.2±7.8 0.87

18.0±6.9

r

≥300 P

12

18 3

10 11

2 19

18 3 13 8

≥300 D

−0.15 0.36 −0.34 0.32 −0.22 −0.60 −0.32 0.27

−0.55 to 0.30 −0.08 to 0.69 −0.67 to 0.11 −0.12 to 0.66 −0.59 to 0.24 −0.82 to −0.22 −0.66 to 0.13 −0.18 to 0.63

−0.33 to 0.52 −0.46 to 0.40

95 % CI

15.5±7.0

16.3±7.9 10.4±6.3 0.24

14.6±9.6 16.3±6.2 0.65

18.6±8.1 15.2±8.0 0.57

P 16.7±7.8 8.1±3.5 0.08 17.0±9.3 13.0±4.4 0.27

0.52 0.11 0.13 0.15 0.35 0.01 0.16 0.23

−0.39 to 0.37 0.12 0.61 −0.43 to 0.33 −0.03 0.89

95 % CI

P 17.0±8.0 13.7±3.3 0.34 15.0±8.0 17.4±6.8 0.41

150–300 C

−0.08 0.25 −0.06 −0.07 0.08 −0.10 −0.23 −0.14

−0.34 to 0.45 −0.01 0.9 −0.13 to 0.61 −0.05 0.79

95 % CI

P 18.3±8.7 11.5±3.9 0.04 13.6±6.5 18.6±9.0 0.12

(b) The 2×2 chi-squared test for the serum sclerostin level (ng/mL) Intact PTH subgroups <70 A 70–150 B

0.55 <0.001 0.80 0.76 0.16 0.09 0.20 0.01

−0.01 0.49 0.36 0.01 0.24 0.09 −0.01 0.63

0.75 0.18

−0.68 to −0.07 0.07 − 0.19 to 0.52 0.28

−0.42 0.02 0.19 0.34

Age HD duration Serum parameters Calciuma Phosphate ALP CRP Intact PTH BAP TRAP5b Log of FGF23

P

r

95 % CI

r

Variable

P

70–150 B

(a) Correlation analysis for serum sclerostin level Intact PTH subgroups <70 A

Table 3 Univariate analysis of serum sclerostin in the intact PTH subgroups of hemodialysis patients

P

0.50 <0.001 0.33 0.9 0.9 0.23 0.24 <0.001

−0.27 to 0.51 0.24 to 0.79 −0.14 to 0.39 −0.16 to 0.23 −0.30 to 0.08 −0.43 to −0.06 −0.41 to −0.04 0.12 to 0.47

−0.75 to −0.13 −0.10 to 0.63

95 % CI

15

45 9

39 15

8 46

37 17 32 22

15.6±9.5

17.1±9.1 18.4±73 0.68

16.8±9.4 18.5±7.1 0.55

15.4±9.7 17.6±8.6 0.51

P 19.9±8.8 11.7±5.6 <0.001 16.4±8.6 18.6±8.9 0.35

<150 (combined A and B)

0.10 0.56 0.14 0.01 −0.01 −0.17 −0.16 0.59

−0.15 0.29 0.25 0.07

r

D <150 (combined A and B)

Osteoporos Int

Corrected calcium=serum calcium (mg/dL)+[4–albumin (g/dL)] [25] a

PTH parathyroid hormone, HD hemodialysis, ALP alkaline phosphatase, CRP C-reactive protein, BAP bone-specific alkaline phosphatase, TRAP5b tartrate-resistant acid phosphatase isoform type 5b, FGF23 fibroblast growth factor-23, CI confidence interval, DM diabetes mellitus, sevelamer-HCl sevelamer hydrochloride, cinacalcet HCl cinacalcet hydrochloride, r correlation coefficient

17.8±9.3 15.1±5.5 0.38 44 10 17.0±8.8 13.1±5.8 0.28 13 8 18.1±6.7 9.8±6.3 0.01 21 6 15.8±8.8 15.9±4.7 0.9 0.28 19.3±9.5 13.8±7.0 25 4

19 6

17.8±9.1 14.6±6.8 0.32 45 9 15.2±6.7 15.8±9.3 0.87 11 10 17.7±7.8 14.8±6.7 0.31 14 13 16.0±8.7 15.3±5.5 0.84 19 6 19.1±9.2 13.4±10.3 0.32 26 3

Use, n Maxacalcitol Nonuse, n Use, n Cinacalcet HCl Nonuse, n Use, n

Table 3 (continued)

0.85 18.4±8.4 23

16

17.3±8.8 0.21

10

13.4±7.5 0.12

9

15.5±9.3 0.9

39

18.0±8.5 0.37

Osteoporos Int

With regard to the association between serum sclerostin levels and the log of serum FGF23 levels in HD patients, there was a significant and positive correlation in the low iPTH subgroups (<70, 70–150, and <150 pg/mL), consistent with the result of a study of 40 adult patients with CKD stage 3 or 4 (r=0.362, P=0.022) [35], although there was no significant correlation in the subgroups with iPTH levels of 150–300 and ≥300 pg/mL. Therefore, further study is required to identify whether the interaction between sclerostin and the phosphate regulators including FGF23 and others could potentially provide insight for understanding the association between serum sclerostin and serum phosphate levels in HD patients with low iPTH levels. Recently, sclerostin has been reported to interact closely with vitamin D [9, 10]. The serum concentration of 1α,25dihydroxyvitamin D increased in the absence of sclerostin in an animal study [9], and the serum concentration of sclerostin increased after supplementation with vitamin D (700 IU/day) plus calcium (500 mg/day) in 66 healthy men aged ≥65 years [10]. However, the effects of vitamin D treatment on the serum sclerostin level in CKD-MBD have not been understood. In the present study, the frequency of any vitamin D treatment did not have a significant implication on serum sclerostin level. However, a prospective study design is needed to determine the effects of vitamin D treatment on serum sclerostin level. The reason for the markedly high serum sclerostin level in HD patients in the present study, compared with that of subjects without renal dysfunction, may be attributable to the presence of renal failure, as indicated by a report that serum sclerostin level was associated with the progression of decreasing renal function [29]. In addition, abnormal mineral and bone metabolism and use of vitamin D treatment may be associated with serum sclerostin level, as shown by reports [35, 10]. For instance, severe hyperparathyroidism usually presents as a complication with high bone turnover, increased bone resorption, and increased born formation. Increased bone formation is negatively associated with the serum sclerostin level [36]. Furthermore, severe hyperparathyroidism often presents a s a co m pl i c a t i on w i t h h i gh F G F 23 l e ve l s a n d hyperphosphatemia, as observed in the present study (Table 2), which are positively associated with the serum sclerostin level. Thus, mixed opposing actions and complex interactions may be present, which affect the serum sclerostin level. With regard to the correlation between serum sclerostin and BAP levels, the reason for the difference in the presence of the correlation (such as in the subgroup with iPTH ≥300 pg/mL in the present study and a report on patients without CKD [36]) or the absence of a correlation (such as in the subgroups with iPTH <300 pg/mL) remains to be elucidated. Thus, we believe that the effect of the sclerostin level in CKD should be interpreted with caution, while considering the effects of

Osteoporos Int

b 50 40

30 20 10 0 0 2 4 6 8 10 Serum phosphate (mg/dL)

Serum sclerostin (ng/mL)

a Serum sclerostin (ng/mL)

50 40

30 20 10

0 0.0 2.0 4.0 6.0 Log of serum FGF23 (pg/mL)

c Log of serum FGF23 (pg/mL)

Fig. 2 Correlation analysis between a the serum sclerostin and serum phosphate levels, b the serum sclerostin and log of serum FGF23 levels, and c the log of serum FGF23 and serum phosphate levels in 54 hemodialysis patients with low iPTH levels. a r=0.56, P<0.001, 95 % CI 0.24 to 0.79; b r=0.59, P<0.001, 95 % CI 0.12 to 0.47; c r=0.71, P<0.001, 95 % CI 0.54 to 0.82. Conversion factors for units are the following: phosphate in mg/dL to mmol/L, ×0.3229. FGF23 fibroblast growth factor23, iPTH intact parathyroid hormone, CI confidence interval

6.0 4.0 2.0 0.0

0 2 4 6 8 10 Serum phosphate (mg/dL)

influential factors for sclerostin level and the interactions among them, and that it is essential to analyze the iPTH level following stratification, due to its strong effect on the status of bone remodeling. The optimum serum sclerostin level in CKD patients remains unclear. In populations without CKD, the inhibition of sclerostin activity appears to be an effective treatment for osteoporosis [37, 38]. Therefore, it is important to examine whether the suppression of serum sclerostin levels in CKD patients is effective for treating osteoporosis, as in the case of populations without CKD. Moreover, further studies on Table 4 Multiple linear regression analysis of serum sclerostin in the low intact PTH subgroups (<150 pg/mL, 54 HD patients) Variable

t

P value

95 % CI

Male sex Phosphate Log of FGF23 HD duration R2

3.24 2.13 1.90 1.46 0.52

0.01 0.04 0.06 0.15

11.78 to 50.43 1.08 to 36.91 −1.85 to 63.50 −0.73 to 4.58

PTH parathyroid hormone, FGF23 fibroblast growth factor-23, DM diabetes mellitus, cinacalcet HCl cinacalcet hydrochloride, BAP bonespecific alkaline phosphatase, CRP C-reactive protein, HD hemodialysis, CI confidence interval

whether the control of serum phosphate level within the normal range by diet therapy and/or by using phosphate binders in HD patients with low iPTH levels has an effect on decreasing the serum sclerostin level and improving decreased bone formation would be interesting. This study had several limitations that should be considered when reviewing the results. First, only a small number of patients were examined. Second, the use of a cross-sectional design did not allow for the determination of causality in the relationships of serum sclerostin levels with serum phosphate levels or serum FGF23 levels; for the determination of the effects of treatment with vitamin D on serum sclerostin levels; or for the identification of the mechanism responsible for the significant differences in serum sclerostin levels observed between male and female subjects in this study, as well as in several previous studies of both non-CKD subjects [39] and CKD patients [40]. Third, the results obtained for Japanese patients undergoing treatment in accordance with the different target ranges for iPTH recommended by the guidelines cannot be generalized to a wider population. Fourth, we did not examine the use of daily physical activity as an approximate indicator of mechanical loading [36], which also influences the sclerostin levels. In conclusion, this study yielded several significant findings that have important research indications and clinical

Osteoporos Int

implications, particularly the presence of an association between serum sclerostin levels and serum phosphate and serum FGF23 levels in CKD patients with low iPTH levels. Identifying the precise mechanism underlying the signaling pathway associated with sclerostin, phosphate, and FGF23 regulation under the condition of low PTH levels and the effect of vitamin D treatment on serum sclerostin levels requires further investigation. Acknowledgments We thank Mr. Tsutomu Ishizuka for his technical support in the measurement of serum sclerostin and serum FGF23 levels. Conflicts of interest Yukari Asamiya, Aiji Yajima, Satoru Shimizu, Shigeru Otsubo, Ken Tsuchiya, and Kosaku Nitta declare that they have no conflict of interest.

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