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Increased circulating levels of osteoclastogenesis inhibitory factor (osteoprotegerin) in patients with chronic renal failure
Increased Circulating Levels of Osteoclastogenesis Inhibitory Factor (Osteoprotegerin) in Patients With Chronic Renal Failure Junichiro J. Kazama, MD, PhD, Takashi Shigematsu, MD, PhD, Kazuki Yano, PhD, Eisuke Tsuda, PhD, Masakazu Miura, PhD, Yoshiko Iwasaki, MS, Yoshindo Kawaguchi, MD, Fumitake Gejyo, MD, Kiyoshi Kurokawa, MD, and Masafumi Fukagawa, MD, PhD ● Skeletal resistance to parathyroid hormone (PTH) is one of the major abnormalities underlying bone diseases in uremia, the mechanism of which has not yet been fully elucidated. Osteoclastogenesis inhibitory factor (OCIF), or osteoprotegerin, is a natural decoy receptor for osteoclast differentiation factor (ODF), produced by osteoblasts in response to PTH. To elucidate the kinetics and roles of OCIF in chronic renal failure, serum OCIF levels were measured in 46 predialysis patients and 21 dialysis patients by means of enzyme-linked immunosorbent assay (ELISA). Serum OCIF levels in predialysis patients increased as renal function declined (OCIF ⴝ 1.178 ⴙ 0.233 ⴛ creatinine; r 2 ⴝ 0.413; P < 0.0001). Twenty-four– hour creatinine clearance and 1/OCIF in predialysis patients showed a clear positive correlation and a straight line regression (1/OCIF ⴝ 0.443 ⴙ 0.004 ⴛ creatinine clearance; r 2 ⴝ 0.425; P < 0.0001). In dialysis patients, serum OCIF levels were significantly elevated (5.18 ⴞ 1.48 ng/mL) to a level that would inhibit 50% osteoclast formation in vitro. These findings suggest that OCIF accumulates in serum of patients with renal dysfunction. Because serum levels of OCIF with the ability to bind ODF in vitro (active OCIF) correlated well with those of OCIF detected by standard ELISA (active OCIF ⴝ 0.251 ⴙ 0.877 ⴛ OCIF; r 2 ⴝ 0.829; P < 0.0001), OCIF accumulated in serum may be a candidate uremic toxin responsible for the skeletal resistance to PTH seen in chronic renal failure. Further studies with serum parameters and bone histological evaluation are needed to assess this possibility. © 2002 by the National Kidney Foundation, Inc. INDEX WORDS: Osteoclastogenesis inhibitory factor (OCIF); osteoprotegerin (OPG); bone; parathyroid hormone (PTH); renal failure.
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KELETAL RESISTANCE to parathyroid hormone (PTH) is one of the major abnormalities underlying bone diseases in uremia.1,2 Recent findings suggest that two- to threefold higher PTH concentrations than normal usually are needed to maintain the normal bone-turnover rate in chronic dialysis patients.3 Control of PTH levels has become much easier because of progress in therapeutic modalities; however, this in turn has resulted in the development of lowturnover or adynamic bone diseases4 in a large number of patients because of skeletal resistance to PTH, or relative hypoparathyroidism.5 Although resistance to vitamin D action,6,7 possible downregulation of PTH receptors,8 and overestimation of the number of active PTH molecules,9 among other mechanisms,10,11 have been implicated in the skeletal resistance to PTH in uremia, the precise pathogenesis of this abnormality has not been fully elucidated. Osteoclastogenesis inhibitory factor (OCIF),12 also named osteoprotegerin (OPG),13 is a newly identified circulating factor that inhibits osteoclast maturation. This molecule is a natural decoy receptor for osteoclast differentiation factor (ODF),14,15 produced by osteoblasts in response to PTH and other factors. Expression of OCIF has been found in many
organs, including bone, kidney, and small intestine13,14; however, little information exists on the degradation pathways of OCIF or its kinetics and role in chronic renal failure. To elucidate the role of OCIF in uremic bone
From the Niigata University Medical School, Niigata; National Sakura Hospital, Chiba; Snow Brand Milk Product Co Ltd, Tochigi; Mitsubishi Chemical BCL; Tokyo Teishin Hospital; Jikei Medical School, Tokyo; Tokai University School of Medicine, Kanagawa; and Kobe University School of Medicine, Kobe, Japan. For the ROD-21 Clinical Research Group, Japan. Received September 5, 2000; accepted in revised form October 5, 2001. Supported in part by a program project grant from the Ministry of Health, Japan (M.F.) and a Special Grant for Medical Research from the Ministry of Post and Telecommunications, Japan (M.F.). Presented in part at the 32nd Annual Meeting of the American Society of Nephrology, Miami Beach, FL, November 1-8, 1999. Published in abstract form (J Am Soc Nephrol 10:599A, 1999). Address reprint requests to Masafumi Fukagawa, MD, PhD, Associate Professor and Chief, Division of Nephrology and Dialysis Center, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected] © 2002 by the National Kidney Foundation, Inc. 0272-6386/02/3903-0009$35.00/0 doi:10.1053/ajkd.2002.31402
American Journal of Kidney Diseases, Vol 39, No 3 (March), 2002: pp 525-532
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diseases, we measured serum concentrations of OCIF in predialysis and chronic dialysis patients with a recently developed enzyme-linked immunosorbent assay (ELISA) system.16 Our findings show that OCIF accumulates in serum of uremic patients and is capable of binding ODF. Thus, elevated circulating OCIF levels could potentially modulate bone metabolism, thus contributing to the PTH resistance observed in uremia. PATIENTS AND METHODS
Serum Samples For the pilot study, serum samples were obtained from chronic dialysis patients at facilities affiliated with Niigata University (Niigata, Japan) and Kobe University (Kobe, Japan; n ⫽ 162; 108 men, 54 women; age, 55.71 ⫾ 28.45 years) and age- and sex-matched healthy volunteers (n ⫽ 212; 130 men, 82 women; age, 53.45 ⫾ 26.87 years) with creatinine levels less than 1.0 mg/dL and no abnormal urinalysis findings. For the main study, serum samples were obtained from 46 predialysis patients (26 men, 20 women) aged older than 50 years (63.71 ⫾ 8.95 years; range, 50 to 82 years) with various degrees of renal failure at Niigata University Hospital and National Sakura Hospital (Sakura, Japan). Causes of renal failure were chronic glomerulonephritis in 29 patients, hypertensive nephrosclerosis in 10 patients, diabetic nephropathy in 3 patients, and other causes in 4 patients. Renal function in these patients was estimated by a standard 24-hour creatinine clearance test (58.8 ⫾ 42.6 mL/min/1.73 m2). Patients had no history of renal replacement therapy or renal transplantation and were never administered oral active vitamin D sterols. Serum samples also were obtained for the main study from 21 chronic hemodialysis patients (14 men, 7 women) aged older than 50 years (60.14 ⫾ 6.81 years; range, 50 to 70 years) with a minimum 5-year history of hemodialysis therapy at Sumiyoshi Clinic Hospital (Mito, Japan). Causes of end-stage renal failure were chronic glomerulonephritis in 15 patients, hypertensive nephrosclerosis in 3 patients, and other causes in 3 patients. No patients had been treated by continuous ambulatory peritoneal dialysis or renal transplantation. This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the institutional ethical committee, and written informed consent was obtained from all participants.
Assay of Serum OCIF Levels Serum OCIF was assayed by ELISA, as previously described.16 In brief, monoclonal antibodies against human OCIF (OPG), designated clone OI-19, were adhered to a 96-well plate. Samples were incubated in the wells, and OCIF bound to OI-19 was visualized with peroxidaselabeled monoclonal antibody against human OCIF, designated clone OI4, and tetramethylbenzidine (TMB) substrate. Quantitative evaluation was performed by measuring absorption at 450 nm with a microplate reader (Nippon InterMed,
Tokyo, Japan). As reported previously, interassay and intraassay variations were less than 5%.16
Active OCIF ELISA Antithioredoxin antibody solution was diluted with 0.1 mmol/L of sodium bicarbonate (pH 9.6) to 1/5,000, and 100 L of the solution was added to each well of the 96-well plates. Plates were maintained at 4°C overnight, after which the solution in each well was replaced with 300 L of blocking solution, and plates were incubated for 2 hours at room temperature. After washing three times with phosphatebuffered saline (PBS) containing 0.1% Tween 20 (PBS-P), 100 L of 100 ng/mL of thioredoxin-fused murine ODF dissolved in the first buffer was added to each well. Plates were incubated for 2 hours at room temperature, washed six times with PBS-P, and100 L of recombinant OCIF standard or test sample, prepared by serial dilution with the first buffer, was added to each well. Plates were incubated for 2 hours at room temperature, then washed six times with PBS-P, after which 100 L of peroxidase (POD)-labeled anti-OCIF polyclonal antibody, diluted 2,000-fold with the second buffer, was added to each well. After incubation for an additional 2 hours at room temperature, plates were washed six times with PBS-P, 100 L of TMB substrate reagent was added to each well, and plates were incubated for 30 minutes at room temperature. TMB stop buffer (100 L) was added to each well, and absorbance at 450 nm was measured with a microplate reader.
In Vitro Bioassay by Osteoclast-Like Cell Formation Osteoclast-like cell formation assay was performed as described previously.12 Briefly, spleen cells (1 ⫻ 105 cells) prepared from normal male ddy mice (6 to 15 weeks old) and ST2 cells (4 ⫻ 103 cells) were cocultured on a 96-well plate in ␣-minimal essential medium (␣-MEM) supplemented with 10% fetal calf serum (FCS) for 1 week in the presence of 10 nmol/L of 1,25-(OH)2D3 and 100 nmol/L of dexamethasone, and osteoclast-like cell formation was evaluated in the presence or absence of partially inactivated OCIF by measuring tartrate-resistant acid phosphatase (TRAP) activity as follows. One hundred microliters of 50 mmol/L of citrate buffer, pH 4.5, containing 5.5 mmol/L of pnitrophenol phosphate and 10 mmol/L of sodium tartrate was added to each well at the plate. After incubation for 15 minutes at room temperature, 20 L of 0.1 N NaOH was added to each well, and absorbance at 405 nm was measured by a microplate reader. OCIF used in this assay was heat treated as follows. Tubes containing OCIF solution were incubated for 0, 10, 20, 30, 40, and 50 minutes at 80°C. Tubes were quickly chilled on ice after heat treatment and stored at ⫺30°C until use. Suppression of osteoclast-like cell formation (TRAP activity) by OCIF without heat treatment was defined as 100% residual activity.
Statistical Analyses All data are expressed as mean ⫾ SD. Correlations among data were examined by single regression analysis using Stat
SERUM OCIF (OPG) LEVELS IN RENAL FAILURE
Fig 1. Serum OCIF levels increased with age in healthy volunteers (■; n ⴝ 212), as previously reported. Levels in dialysis patients (䊐; n ⴝ 162) also increased in an age-dependent manner, but were higher than those in healthy volunteers regardless of age. *P < 0.01.
View 5.0 (SAS Institute Inc, Cary, NC) by Power Macintosh G3. P less than 0.05 is considered significant.
RESULTS
In healthy controls, serum OCIF levels were similar (0.945 ⫾ 0.263 ng/mL) to those reported previously16 and increased in an age-dependent manner (OCIF ⫽ 0.226 ⫹ 0.020 ⫻ age; r2 ⫽ 0.529; P ⬍ 0.0001). Age-dependent increases in serum OCIF levels also were observed in ageand sex-matched dialysis patients (OCIF ⫽ 0.121 ⫹ 0.051 ⫻ age; r2 ⫽ 0.126; P ⬍ 0.0001), as well as healthy controls. However, OCIF levels in dialysis patients were higher than those of healthy controls for all ages (Fig 1). Further
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studies were performed in predialysis and dialysis patients aged older than 50 years. In predialysis patients, serum OCIF concentrations increased progressively with serum creatinine concentrations. These two parameters showed a significant positive correlation (OCIF ⫽ 1.178 ⫹ 0.233 ⫻ creatinine; r2 ⫽ 0.413; P ⬍ 0.0001; Fig 2A). Serum OCIF levels in dialysis patients further increased to more than threefold (5.18 ⫾ 1.48 ng/mL) those of healthy controls (Fig 2B). These elevated levels corresponded to the level that would inhibit 50% of osteoclast formation in vitro of OCIF for the suppression of osteoclastogenesis.12 To assess renal clearance of OCIF, 1/OCIF was plotted against 24-hour creatinine clearance. As shown in Fig 3, they showed a clear positive correlation and a straight regression line (1/OCIF ⫽ 0.443 ⫹ 0.004 ⫻ creatinine clearance; r2 ⫽ 0.425; P ⬍ 0.0001). These findings suggest that the kidney is the main site of OCIF clearance. Although results of OCIF ELISA correlated well with those of the bioassay (r2 ⫽ 0.81; P ⬍ 0.001), it was not clear whether OCIF detected by the latter still had the ability to bind ODF. To clarify this issue, active OCIF ELISA was constructed with trx-ODF as the capture substance with the POD-labeled polyclonal antibody; the detection range for OCIF was 0.625 to 10 ng/mL
Fig 2. Serum levels of OCIF and creatinine (Cr) in (A) predialysis patients and (B) dialysis patients. (A) Serum OCIF levels in predialysis patients (E) showed a clear positive correlation with serum creatinine levels (OCIF ⴝ 1.178 ⴙ 0.233 ⴛ Cr; P < 0.0001; r 2 ⴝ 0.413). (B) Serum OCIF concentrations in dialysis patients (F) were elevated to more than threefold those of age-matched healthy controls to a concentration of OCIF that inhibits 50% of osteoclast formation in vitro. Mean ⴞ SD in healthy volunteers is shown as a reference.
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Fig 3. Renal function and 1/OCIF. Creatinine clearance (Ccr) per 24 hours in predialysis patients (E) and the reciprocal number of serum OCIF showed a clear positive correlation and a straight line regression (1/ OCIF ⴝ 0.443 ⴙ 0.004 ⴛ Ccr; P < 0.0001; r 2 ⴝ 0.425), suggesting that the kidney is the major site for OCIF clearance. For comparison, data from chronic dialysis patients without renal function are plotted (F) as Ccr ⴝ 0.
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(OD450 ⫽ 0.113 ⫹ 0.103 ⫻ active OCIF; r2 ⫽ 0.99; P ⬍ 0.0001; Fig 3A). The minimum detection limit (0.625 ng/mL) was determined as the lowest concentration of recombinant OCIF showing an absorbance twice that of the blank. Coefficients of variation ranged from 2.2% to 6.1% (intra-assay) and 2.6% to 10.1% (interassay) among different ELISA formats (data not shown). OCIF bioactivity was decreased in a timedependent manner by heat treatment (residual activity ⫽ 102.829 ⫺ 1.663 ⫻ time; r2 ⫽ 0.993; P ⫽ 0.017; Fig 4B). Furthermore, residual bioactivity correlated better with the value determined from active OCIF ELISA (residual activity ⫽ ⫺11.568 ⫹ 1.572 ⫻ active OCIF; r2 ⫽ 0.853; P ⫽ 0.0086; Fig 4C). Active OCIF levels in normal and uremic sera correlated well with those detected by standard ELISA (active OCIF ⫽ 0.251 ⫹ 0.877 ⫻ OCIF; r2 ⫽0.829; P ⬍ 0.0001; Fig 4D). Thus, it is possible that elevated serum OCIF levels in
Fig 4. Active OCIF ELISA. (A) Dose-response curve of active OCIF ELISA (OD450 ⴝ 0.113 ⴙ 0.103 ⴛ active OCIF; r 2 ⴝ 0.99; P < 0.0001). (B) Time-dependent decrease in bioactivity by heat treatment. Heat treatment decreased the activity of OCIF evaluated by osteoclast-like cell formation assay. Suppression of osteoclast-like cell formation by OCIF without heat treatment was defined as 100% residual activity. (Residual activity ⴝ 102.829 ⴚ 1.663 ⴛ time; r 2 ⴝ 0.993; P ⴝ 0.017). (C) Correlation between active OCIF ELISA and in vitro bioactivity was significant. (Residual activity ⴝ ⴚ11.568 ⴙ 1.572 ⴛ active OCIF; r 2 ⴝ 0.853; P ⴝ 0.0086). (D) Comparison of active OCIF ELISA and standard OCIF ELISA. Results of these two assays correlated highly, suggesting that OCIF molecules accumulated in uremic serum maintained the ability to bind ODF. (Active OCIF ⴝ 0.251 ⴙ 0.877 ⴛ OCIF; r 2 ⴝ 0.829; P < 0.0001).
SERUM OCIF (OPG) LEVELS IN RENAL FAILURE
patients with renal failure could still bind ODF in vitro, which is the main function of OCIF. DISCUSSION
The calcemic action of PTH is blocked in uremic patients, referred to as increased skeletal resistance to PTH.1 Bone histomorphometric studies have shown that parameters associated with osteoclastic bone resorption generally were suppressed compared with values estimated from serum PTH levels in healthy controls.3,17-21 Because increased skeletal resistance to PTH was first recognized in the early 1970s, it has been regarded as one of the major mechanisms of pathogenesis of secondary hyperparathyroidism in chronic renal failure.22,23 Recent progress in new therapeutic modalities, such as calcitriol pulse therapy,17 has made it possible to suppress serum PTH to near normal levels in a large proportion of uremic patients; however, this has resulted in a growing number of adynamic bone diseases.4 Thus, increased skeletal resistance to PTH remains a common abnormality underlying both high- and low-turnover bone diseases associated with uremia. Recently, mechanisms of osteoclastogenesis and osteoclast activation have been clarified at the molecular level.24 Humoral factors, such as PTH, vitamin D, and cytokines, bind to osteoblasts and induce a membrane-bound glycoprotein called ODF25 that is identical to TNF-related activation-induced cytokine (TRANCE),26 osteoprotegerin ligand (OPGL),15 or receptor activator of NF-B ligand (RANKL).13 ODF binds to its receptor expressed on the surface of osteoclast precursor cells27 and promotes differentiation into mature osteoclasts. ODF also acts on mature osteoclasts to induce activation.28,29 These actions of ODF on osteoclastic lineage result in increased bone resorption. Thus, any of these steps from ligand binding to osteoclastogenesis can be disturbed in uremia, leading to skeletal resistance to PTH. OCIF is a circulating glycoprotein with a molecular weight of 60 kd that belongs to the tumor necrosis factor-␣ receptor superfamily; however, it lacks the transmembrane domain.12,13 Circulating or local OCIF binds to ODF as a decoy receptor to disturb the binding of ODF and its functional receptors expressed on the surface of osteoclast lineage cells. As a result, OCIF
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inhibits osteoclastogenesis and osteoclastic bone resorption in vitro.30,31 In addition, OCIF also was found to promote osteoclastic apoptosis, which shortens osteoclastic survival in vitro.32 Although these findings suggest an important role of OCIF as a local and paracrine factor, several reports have supported the same role of circulating OCIF in bone metabolism at high concentrations. Intravenous injection of OCIF in normal rats caused transient hypocalcemia because of suppression of osteoclastic bone resorption in vivo.33 Another group showed the inhibitory effects of chimeric OCIF on bone resorption induced by PTH and other factors in mice.34 These findings suggest that a basal amount of circulating OCIF produced by many organs, including bone, kidney, intestines, and vascular endothelial cells, also could modulate general bone and mineral metabolism in addition to local OCIF produced by osteoblasts. Supporting this possibility, a slight elevation in serum OCIF levels was reported in postmenopausal osteoporotic women.16 As shown here, serum OCIF levels were increased in patients with renal failure. Our data clearly suggest that OCIF accumulation may be caused in part by decreased renal clearance. A recent study reported that serum OCIF levels returned to normal within 2 weeks after renal transplantation, further supporting a major role of the kidney in OCIF clearance.35 Considering the molecular weight of OCIF, it is likely to be metabolized or cleaved in serum or the kidney before renal clearance. At present, no information is available on this issue, and we could not measure detectable levels of OCIF in urine with available assays (data not shown). Nevertheless, such mechanisms should be clarified in the near future. OCIF levels increased in an age-dependent manner in both healthy volunteers and dialysis patients. In healthy volunteers, a decline in renal function by aging may be a major contributing factor; however, other mechanisms should be considered in dialysis patients without residual renal function to explain this phenomenon. Another possibility is that OCIF production may be stimulated in response to high PTH levels; however, recent studies showed that PTH suppressed OCIF production in vitro and in vivo.36,37 We could not find a significant correla-
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tion between OCLF levels and such serum parameters as PTH, calcium, and phosphate in the small number of patients included on this study. However, this possibility should be examined in a large homogenous group of patients with the same serum calcium and phosphorus levels, without variations in therapeutic modalities. Stimulation of OCIF production by uremia or hemodialysis alone or by several agents used for treatment may be induced in dialysis patients. Specifically, an increase in OCIF production by bone needs to be ruled out. Although it has been reported that estrogen38 stimulated and glucocorticoid inhibited OCIF production in vitro39 and in humans,40 the effect of calcitriol, one of most frequently used agents in uremic patients, on OCIF production is still controversial.41-43 Circulating OCIF may not be in its intact form, but may be fragmented into shorter pieces, especially in uremia. Nevertheless, our findings suggest that OCIF molecules accumulated in uremic serum maintained the ability to bind ODF. Thus, it is possible that accumulated OCIF in serum of uremic patients prevents osteoclastogenesis in vivo. Our preliminary analysis of serum parameters and bone histological characteristics supports this possibility44; however, larger studies are needed. Finally, blocking excess OCIF action, possibly by removal of OCIF or by OCIF antagonists, may be a promising strategy to normalize skeletal resistance to PTH in uremia. If this can be achieved, ideal maintenance of bone turnover, as well as parathyroid function, will become a reality for chronic dialysis patients. ACKNOWLEDGMENT The authors thank Dr Takeshi Kurosawa, Sumiyoshi Clinic Hospital, Mito, Japan, for cooperation in serum sampling from chronic dialysis patients. We also thank members of the ROD-21 Clinical Research Group for thoughtful discussions.
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characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: Increased serum concentration in postmenopausal women with osteoporosis. J Bone Miner Res 14:518-527, 1999 17. Goodman WG, Ramirez JA, Belin TR, Chon Y, Gales B, Segre GV, Salusky IB: Development of adynamic bone in patients with secondary hyperparathyroidism after intermittent calcitriol therapy. Kidney Int 46:1160-1166, 1994 18. Hutchinson AJ, Whitehouse RW, Boulton HF, Adams JE, Mawer EB, Freemont TJ, Gokal R: Correlation of bone histology with parathyroid hormone, vitamin D and radiology in end-stage renal disease. Kidney Int 44:1071-1077, 1993 19. Salusky IB, Ramirez JA, Oppenheim W, Gales B, Segre GV, Goodman WG: Biochemical markers of renal osteodystrophy in pediatric patients undergoing CAPD/ CCPD. Kidney Int 45:253-258, 1994 20. Torres A, Lorenzo V, Hernandez D, Rodriguez D, Conception J, Rodriguez A, Hernandez A, De Bonis E, Darias E, Gonzalez Pasada J: Bone disease in predialysis, hemodialysis and CAPD patients: Evidence for a better response to PTH. Kidney Int 47:1434-1442, 1995 21. Qi Q, Monier-Faugere MC, Geng Z, Malluche HH: Predictive value of serum parathyroid hormone levels for bone turnover in patients on chronic maintenance dialysis. Am J Kidney Dis 26:622-631, 1995 22. Llach F, Massry SG, Singer FR, Kurokawa K, Kaye JH, Coburn JW: Skeletal resistance to endogenous parathyroid hormone in patients with early renal failure. A possible cause for secondary hyperparathyroidism. J Clin Endocrinol Metab 41:339-345, 1975 23. Bover J, Jara A, Trinidad P, Rodriguez M, Felsenfeld AJ: Dynamics of skeletal resistance to parathyroid hormone in the rat: Effect of renal failure and dietary phosphorus. Bone 25:279-285, 1999 24. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ: Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345-357, 1999 25. Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Morinaga T, Toyama Y, Yabe Y, Higashio K, Suda T: Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures. Biochem Biophys Res Commun 246:199-204, 1998 26. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E, Bartlett FS III, Frankel WN, Lee SY, Choi Y: TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 272:25190-25194, 1997 27. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L: A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175-179, 1997 28. Fuller K, Wong B, Fox S, Choi Y, Chambers TJ: TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997-1001, 1998 29. Burgess TL, Qian Y, Kaufman S, Ring BD, Van G,
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Capparelli C, Kelley M, Hsu H, Boyle WJ, Dunstan CR, Hu S, Lacey DL: The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527-538, 1999 30. Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, Higashio K: Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun 234:137-142, 1997 31. Hakeda Y, Kobayashi Y, Yamaguchi K, Yasuda H, Tsuda E, Higashio K, Miyata T, Kumegawa M: Osteoclastogenesis inhibitory factor (OCIF) directly inhibits boneresorbing activity of isolated mature osteoclasts. Biochem Biophys Res Commun 251:796-801, 1998 32. Akatsu T, Murakami T, Nishikawa M, Ono K, Shinomiya N, Tsuda E, Mochizuki S, Yamaguchi K, Kinosaki M, Higashio K, Yamamoto M, Motoyoshi K, Nagata N: Osteoclastogenesis inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclast. Biochem Biophys Res Commun 250:229-234, 1998 33. Yamamoto M, Murakami T, Nishikawa M, Tsuda E, Mochizuki S, Higashio K, Akatsu T, Motoyoshi K, Nagata N: Hypocalcemic effect of osteoclastogenesis inhibitory factor/osteoprotegerin in the thyroparathyroidectomized rat. Endocrinology 139:4012-4015, 1998 34. Morony S, Capparelli C, Lee R, Shimamoto G, Boone T, Lacey DL, Dunstan CR: A chimeric form of osteoprotegerin inhibits hypercalcemia and bone resorption induced by IL-1beta, TNF-alpha, PTH, PTHrP, and 1,25(OH)2D3. J Bone Miner Res 14:1478-1485, 1999 35. Sato T, Tominaga Y, Iwasaki Y, Kazama JJ, Shigematsu T, Inagaki H, Watanabe I, Katayama A, Haba T, Uchida K, Fukagawa M: Osteoprotegerin levels before and after renal transplantation. Am J Kidney Dis 38:S175-S177, 2001 (suppl 1) 36. Lee S-K, Lorenzo JA: Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: Correlation with osteoblast-like cell formation. Endocrinology 140:3552-3561, 1999 37. Onyia JE, Miles RR, Yang X, Halladay DL, Hale J, Glasebrook A, Seno G, Churgay L, Chandrasekhar S, Martin TJ: In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene. J Bone Miner Res 15:863-871, 2000 38. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg TC, Riggs BL: Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140:4367-4370, 1999 39. Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S: Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: Potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:4382-4389, 1999 40. Sakai N, Kusano E, Ando Y, Yano K, Tsuda E, Asano Y: Glucocorticoid decreases circulating osteoprotegerin (OPG): Possible mechanism for glucocorticoid induced osteoporosis. Nephrol Dial Transplant 16:479-482, 2001 41. Horwood NJ, Elliot J, Martin TJ, Gillespie MT:
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Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 139:4743-4746, 1998 42. Hofbauer LC, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S: Osteoprotegerin production by human osteoblast lineage cells is stimulated by vitamin D, bone morphogenetic protein-2, and cytokines. Biochem Biophys Res Commun 250:776-781, 1998
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43. Nagai M, Sato N: Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation. Biochem Biophys Res Commun 21:719-723, 1999 44. Kazama JJ, Fukagawa M, Shigematsu T, Maruyama H, Kawaguchi Y, Kurokawa K, Gejyo F: Increased circulating osteoprotegerin modifies bone metabolism in uremic patients. J Am Soc Nephrol 11:565A, 2000 (abstr)
S
KELETAL RESISTANCE to parathyroid hormone (PTH) is one of the major abnormalities underlying bone diseases in uremia.1,2 Recent findings suggest that two- to threefold higher PTH concentrations than normal usually are needed to maintain the normal bone-turnover rate in chronic dialysis patients.3 Control of PTH levels has become much easier because of progress in therapeutic modalities; however, this in turn has resulted in the development of lowturnover or adynamic bone diseases4 in a large number of patients because of skeletal resistance to PTH, or relative hypoparathyroidism.5 Although resistance to vitamin D action,6,7 possible downregulation of PTH receptors,8 and overestimation of the number of active PTH molecules,9 among other mechanisms,10,11 have been implicated in the skeletal resistance to PTH in uremia, the precise pathogenesis of this abnormality has not been fully elucidated. Osteoclastogenesis inhibitory factor (OCIF),12 also named osteoprotegerin (OPG),13 is a newly identified circulating factor that inhibits osteoclast maturation. This molecule is a natural decoy receptor for osteoclast differentiation factor (ODF),14,15 produced by osteoblasts in response to PTH and other factors. Expression of OCIF has been found in many
organs, including bone, kidney, and small intestine13,14; however, little information exists on the degradation pathways of OCIF or its kinetics and role in chronic renal failure. To elucidate the role of OCIF in uremic bone
From the Niigata University Medical School, Niigata; National Sakura Hospital, Chiba; Snow Brand Milk Product Co Ltd, Tochigi; Mitsubishi Chemical BCL; Tokyo Teishin Hospital; Jikei Medical School, Tokyo; Tokai University School of Medicine, Kanagawa; and Kobe University School of Medicine, Kobe, Japan. For the ROD-21 Clinical Research Group, Japan. Received September 5, 2000; accepted in revised form October 5, 2001. Supported in part by a program project grant from the Ministry of Health, Japan (M.F.) and a Special Grant for Medical Research from the Ministry of Post and Telecommunications, Japan (M.F.). Presented in part at the 32nd Annual Meeting of the American Society of Nephrology, Miami Beach, FL, November 1-8, 1999. Published in abstract form (J Am Soc Nephrol 10:599A, 1999). Address reprint requests to Masafumi Fukagawa, MD, PhD, Associate Professor and Chief, Division of Nephrology and Dialysis Center, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected] © 2002 by the National Kidney Foundation, Inc. 0272-6386/02/3903-0009$35.00/0 doi:10.1053/ajkd.2002.31402
American Journal of Kidney Diseases, Vol 39, No 3 (March), 2002: pp 525-532
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diseases, we measured serum concentrations of OCIF in predialysis and chronic dialysis patients with a recently developed enzyme-linked immunosorbent assay (ELISA) system.16 Our findings show that OCIF accumulates in serum of uremic patients and is capable of binding ODF. Thus, elevated circulating OCIF levels could potentially modulate bone metabolism, thus contributing to the PTH resistance observed in uremia. PATIENTS AND METHODS
Serum Samples For the pilot study, serum samples were obtained from chronic dialysis patients at facilities affiliated with Niigata University (Niigata, Japan) and Kobe University (Kobe, Japan; n ⫽ 162; 108 men, 54 women; age, 55.71 ⫾ 28.45 years) and age- and sex-matched healthy volunteers (n ⫽ 212; 130 men, 82 women; age, 53.45 ⫾ 26.87 years) with creatinine levels less than 1.0 mg/dL and no abnormal urinalysis findings. For the main study, serum samples were obtained from 46 predialysis patients (26 men, 20 women) aged older than 50 years (63.71 ⫾ 8.95 years; range, 50 to 82 years) with various degrees of renal failure at Niigata University Hospital and National Sakura Hospital (Sakura, Japan). Causes of renal failure were chronic glomerulonephritis in 29 patients, hypertensive nephrosclerosis in 10 patients, diabetic nephropathy in 3 patients, and other causes in 4 patients. Renal function in these patients was estimated by a standard 24-hour creatinine clearance test (58.8 ⫾ 42.6 mL/min/1.73 m2). Patients had no history of renal replacement therapy or renal transplantation and were never administered oral active vitamin D sterols. Serum samples also were obtained for the main study from 21 chronic hemodialysis patients (14 men, 7 women) aged older than 50 years (60.14 ⫾ 6.81 years; range, 50 to 70 years) with a minimum 5-year history of hemodialysis therapy at Sumiyoshi Clinic Hospital (Mito, Japan). Causes of end-stage renal failure were chronic glomerulonephritis in 15 patients, hypertensive nephrosclerosis in 3 patients, and other causes in 3 patients. No patients had been treated by continuous ambulatory peritoneal dialysis or renal transplantation. This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the institutional ethical committee, and written informed consent was obtained from all participants.
Assay of Serum OCIF Levels Serum OCIF was assayed by ELISA, as previously described.16 In brief, monoclonal antibodies against human OCIF (OPG), designated clone OI-19, were adhered to a 96-well plate. Samples were incubated in the wells, and OCIF bound to OI-19 was visualized with peroxidaselabeled monoclonal antibody against human OCIF, designated clone OI4, and tetramethylbenzidine (TMB) substrate. Quantitative evaluation was performed by measuring absorption at 450 nm with a microplate reader (Nippon InterMed,
Tokyo, Japan). As reported previously, interassay and intraassay variations were less than 5%.16
Active OCIF ELISA Antithioredoxin antibody solution was diluted with 0.1 mmol/L of sodium bicarbonate (pH 9.6) to 1/5,000, and 100 L of the solution was added to each well of the 96-well plates. Plates were maintained at 4°C overnight, after which the solution in each well was replaced with 300 L of blocking solution, and plates were incubated for 2 hours at room temperature. After washing three times with phosphatebuffered saline (PBS) containing 0.1% Tween 20 (PBS-P), 100 L of 100 ng/mL of thioredoxin-fused murine ODF dissolved in the first buffer was added to each well. Plates were incubated for 2 hours at room temperature, washed six times with PBS-P, and100 L of recombinant OCIF standard or test sample, prepared by serial dilution with the first buffer, was added to each well. Plates were incubated for 2 hours at room temperature, then washed six times with PBS-P, after which 100 L of peroxidase (POD)-labeled anti-OCIF polyclonal antibody, diluted 2,000-fold with the second buffer, was added to each well. After incubation for an additional 2 hours at room temperature, plates were washed six times with PBS-P, 100 L of TMB substrate reagent was added to each well, and plates were incubated for 30 minutes at room temperature. TMB stop buffer (100 L) was added to each well, and absorbance at 450 nm was measured with a microplate reader.
In Vitro Bioassay by Osteoclast-Like Cell Formation Osteoclast-like cell formation assay was performed as described previously.12 Briefly, spleen cells (1 ⫻ 105 cells) prepared from normal male ddy mice (6 to 15 weeks old) and ST2 cells (4 ⫻ 103 cells) were cocultured on a 96-well plate in ␣-minimal essential medium (␣-MEM) supplemented with 10% fetal calf serum (FCS) for 1 week in the presence of 10 nmol/L of 1,25-(OH)2D3 and 100 nmol/L of dexamethasone, and osteoclast-like cell formation was evaluated in the presence or absence of partially inactivated OCIF by measuring tartrate-resistant acid phosphatase (TRAP) activity as follows. One hundred microliters of 50 mmol/L of citrate buffer, pH 4.5, containing 5.5 mmol/L of pnitrophenol phosphate and 10 mmol/L of sodium tartrate was added to each well at the plate. After incubation for 15 minutes at room temperature, 20 L of 0.1 N NaOH was added to each well, and absorbance at 405 nm was measured by a microplate reader. OCIF used in this assay was heat treated as follows. Tubes containing OCIF solution were incubated for 0, 10, 20, 30, 40, and 50 minutes at 80°C. Tubes were quickly chilled on ice after heat treatment and stored at ⫺30°C until use. Suppression of osteoclast-like cell formation (TRAP activity) by OCIF without heat treatment was defined as 100% residual activity.
Statistical Analyses All data are expressed as mean ⫾ SD. Correlations among data were examined by single regression analysis using Stat
SERUM OCIF (OPG) LEVELS IN RENAL FAILURE
Fig 1. Serum OCIF levels increased with age in healthy volunteers (■; n ⴝ 212), as previously reported. Levels in dialysis patients (䊐; n ⴝ 162) also increased in an age-dependent manner, but were higher than those in healthy volunteers regardless of age. *P < 0.01.
View 5.0 (SAS Institute Inc, Cary, NC) by Power Macintosh G3. P less than 0.05 is considered significant.
RESULTS
In healthy controls, serum OCIF levels were similar (0.945 ⫾ 0.263 ng/mL) to those reported previously16 and increased in an age-dependent manner (OCIF ⫽ 0.226 ⫹ 0.020 ⫻ age; r2 ⫽ 0.529; P ⬍ 0.0001). Age-dependent increases in serum OCIF levels also were observed in ageand sex-matched dialysis patients (OCIF ⫽ 0.121 ⫹ 0.051 ⫻ age; r2 ⫽ 0.126; P ⬍ 0.0001), as well as healthy controls. However, OCIF levels in dialysis patients were higher than those of healthy controls for all ages (Fig 1). Further
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studies were performed in predialysis and dialysis patients aged older than 50 years. In predialysis patients, serum OCIF concentrations increased progressively with serum creatinine concentrations. These two parameters showed a significant positive correlation (OCIF ⫽ 1.178 ⫹ 0.233 ⫻ creatinine; r2 ⫽ 0.413; P ⬍ 0.0001; Fig 2A). Serum OCIF levels in dialysis patients further increased to more than threefold (5.18 ⫾ 1.48 ng/mL) those of healthy controls (Fig 2B). These elevated levels corresponded to the level that would inhibit 50% of osteoclast formation in vitro of OCIF for the suppression of osteoclastogenesis.12 To assess renal clearance of OCIF, 1/OCIF was plotted against 24-hour creatinine clearance. As shown in Fig 3, they showed a clear positive correlation and a straight regression line (1/OCIF ⫽ 0.443 ⫹ 0.004 ⫻ creatinine clearance; r2 ⫽ 0.425; P ⬍ 0.0001). These findings suggest that the kidney is the main site of OCIF clearance. Although results of OCIF ELISA correlated well with those of the bioassay (r2 ⫽ 0.81; P ⬍ 0.001), it was not clear whether OCIF detected by the latter still had the ability to bind ODF. To clarify this issue, active OCIF ELISA was constructed with trx-ODF as the capture substance with the POD-labeled polyclonal antibody; the detection range for OCIF was 0.625 to 10 ng/mL
Fig 2. Serum levels of OCIF and creatinine (Cr) in (A) predialysis patients and (B) dialysis patients. (A) Serum OCIF levels in predialysis patients (E) showed a clear positive correlation with serum creatinine levels (OCIF ⴝ 1.178 ⴙ 0.233 ⴛ Cr; P < 0.0001; r 2 ⴝ 0.413). (B) Serum OCIF concentrations in dialysis patients (F) were elevated to more than threefold those of age-matched healthy controls to a concentration of OCIF that inhibits 50% of osteoclast formation in vitro. Mean ⴞ SD in healthy volunteers is shown as a reference.
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Fig 3. Renal function and 1/OCIF. Creatinine clearance (Ccr) per 24 hours in predialysis patients (E) and the reciprocal number of serum OCIF showed a clear positive correlation and a straight line regression (1/ OCIF ⴝ 0.443 ⴙ 0.004 ⴛ Ccr; P < 0.0001; r 2 ⴝ 0.425), suggesting that the kidney is the major site for OCIF clearance. For comparison, data from chronic dialysis patients without renal function are plotted (F) as Ccr ⴝ 0.
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(OD450 ⫽ 0.113 ⫹ 0.103 ⫻ active OCIF; r2 ⫽ 0.99; P ⬍ 0.0001; Fig 3A). The minimum detection limit (0.625 ng/mL) was determined as the lowest concentration of recombinant OCIF showing an absorbance twice that of the blank. Coefficients of variation ranged from 2.2% to 6.1% (intra-assay) and 2.6% to 10.1% (interassay) among different ELISA formats (data not shown). OCIF bioactivity was decreased in a timedependent manner by heat treatment (residual activity ⫽ 102.829 ⫺ 1.663 ⫻ time; r2 ⫽ 0.993; P ⫽ 0.017; Fig 4B). Furthermore, residual bioactivity correlated better with the value determined from active OCIF ELISA (residual activity ⫽ ⫺11.568 ⫹ 1.572 ⫻ active OCIF; r2 ⫽ 0.853; P ⫽ 0.0086; Fig 4C). Active OCIF levels in normal and uremic sera correlated well with those detected by standard ELISA (active OCIF ⫽ 0.251 ⫹ 0.877 ⫻ OCIF; r2 ⫽0.829; P ⬍ 0.0001; Fig 4D). Thus, it is possible that elevated serum OCIF levels in
Fig 4. Active OCIF ELISA. (A) Dose-response curve of active OCIF ELISA (OD450 ⴝ 0.113 ⴙ 0.103 ⴛ active OCIF; r 2 ⴝ 0.99; P < 0.0001). (B) Time-dependent decrease in bioactivity by heat treatment. Heat treatment decreased the activity of OCIF evaluated by osteoclast-like cell formation assay. Suppression of osteoclast-like cell formation by OCIF without heat treatment was defined as 100% residual activity. (Residual activity ⴝ 102.829 ⴚ 1.663 ⴛ time; r 2 ⴝ 0.993; P ⴝ 0.017). (C) Correlation between active OCIF ELISA and in vitro bioactivity was significant. (Residual activity ⴝ ⴚ11.568 ⴙ 1.572 ⴛ active OCIF; r 2 ⴝ 0.853; P ⴝ 0.0086). (D) Comparison of active OCIF ELISA and standard OCIF ELISA. Results of these two assays correlated highly, suggesting that OCIF molecules accumulated in uremic serum maintained the ability to bind ODF. (Active OCIF ⴝ 0.251 ⴙ 0.877 ⴛ OCIF; r 2 ⴝ 0.829; P < 0.0001).
SERUM OCIF (OPG) LEVELS IN RENAL FAILURE
patients with renal failure could still bind ODF in vitro, which is the main function of OCIF. DISCUSSION
The calcemic action of PTH is blocked in uremic patients, referred to as increased skeletal resistance to PTH.1 Bone histomorphometric studies have shown that parameters associated with osteoclastic bone resorption generally were suppressed compared with values estimated from serum PTH levels in healthy controls.3,17-21 Because increased skeletal resistance to PTH was first recognized in the early 1970s, it has been regarded as one of the major mechanisms of pathogenesis of secondary hyperparathyroidism in chronic renal failure.22,23 Recent progress in new therapeutic modalities, such as calcitriol pulse therapy,17 has made it possible to suppress serum PTH to near normal levels in a large proportion of uremic patients; however, this has resulted in a growing number of adynamic bone diseases.4 Thus, increased skeletal resistance to PTH remains a common abnormality underlying both high- and low-turnover bone diseases associated with uremia. Recently, mechanisms of osteoclastogenesis and osteoclast activation have been clarified at the molecular level.24 Humoral factors, such as PTH, vitamin D, and cytokines, bind to osteoblasts and induce a membrane-bound glycoprotein called ODF25 that is identical to TNF-related activation-induced cytokine (TRANCE),26 osteoprotegerin ligand (OPGL),15 or receptor activator of NF-B ligand (RANKL).13 ODF binds to its receptor expressed on the surface of osteoclast precursor cells27 and promotes differentiation into mature osteoclasts. ODF also acts on mature osteoclasts to induce activation.28,29 These actions of ODF on osteoclastic lineage result in increased bone resorption. Thus, any of these steps from ligand binding to osteoclastogenesis can be disturbed in uremia, leading to skeletal resistance to PTH. OCIF is a circulating glycoprotein with a molecular weight of 60 kd that belongs to the tumor necrosis factor-␣ receptor superfamily; however, it lacks the transmembrane domain.12,13 Circulating or local OCIF binds to ODF as a decoy receptor to disturb the binding of ODF and its functional receptors expressed on the surface of osteoclast lineage cells. As a result, OCIF
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inhibits osteoclastogenesis and osteoclastic bone resorption in vitro.30,31 In addition, OCIF also was found to promote osteoclastic apoptosis, which shortens osteoclastic survival in vitro.32 Although these findings suggest an important role of OCIF as a local and paracrine factor, several reports have supported the same role of circulating OCIF in bone metabolism at high concentrations. Intravenous injection of OCIF in normal rats caused transient hypocalcemia because of suppression of osteoclastic bone resorption in vivo.33 Another group showed the inhibitory effects of chimeric OCIF on bone resorption induced by PTH and other factors in mice.34 These findings suggest that a basal amount of circulating OCIF produced by many organs, including bone, kidney, intestines, and vascular endothelial cells, also could modulate general bone and mineral metabolism in addition to local OCIF produced by osteoblasts. Supporting this possibility, a slight elevation in serum OCIF levels was reported in postmenopausal osteoporotic women.16 As shown here, serum OCIF levels were increased in patients with renal failure. Our data clearly suggest that OCIF accumulation may be caused in part by decreased renal clearance. A recent study reported that serum OCIF levels returned to normal within 2 weeks after renal transplantation, further supporting a major role of the kidney in OCIF clearance.35 Considering the molecular weight of OCIF, it is likely to be metabolized or cleaved in serum or the kidney before renal clearance. At present, no information is available on this issue, and we could not measure detectable levels of OCIF in urine with available assays (data not shown). Nevertheless, such mechanisms should be clarified in the near future. OCIF levels increased in an age-dependent manner in both healthy volunteers and dialysis patients. In healthy volunteers, a decline in renal function by aging may be a major contributing factor; however, other mechanisms should be considered in dialysis patients without residual renal function to explain this phenomenon. Another possibility is that OCIF production may be stimulated in response to high PTH levels; however, recent studies showed that PTH suppressed OCIF production in vitro and in vivo.36,37 We could not find a significant correla-
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tion between OCLF levels and such serum parameters as PTH, calcium, and phosphate in the small number of patients included on this study. However, this possibility should be examined in a large homogenous group of patients with the same serum calcium and phosphorus levels, without variations in therapeutic modalities. Stimulation of OCIF production by uremia or hemodialysis alone or by several agents used for treatment may be induced in dialysis patients. Specifically, an increase in OCIF production by bone needs to be ruled out. Although it has been reported that estrogen38 stimulated and glucocorticoid inhibited OCIF production in vitro39 and in humans,40 the effect of calcitriol, one of most frequently used agents in uremic patients, on OCIF production is still controversial.41-43 Circulating OCIF may not be in its intact form, but may be fragmented into shorter pieces, especially in uremia. Nevertheless, our findings suggest that OCIF molecules accumulated in uremic serum maintained the ability to bind ODF. Thus, it is possible that accumulated OCIF in serum of uremic patients prevents osteoclastogenesis in vivo. Our preliminary analysis of serum parameters and bone histological characteristics supports this possibility44; however, larger studies are needed. Finally, blocking excess OCIF action, possibly by removal of OCIF or by OCIF antagonists, may be a promising strategy to normalize skeletal resistance to PTH in uremia. If this can be achieved, ideal maintenance of bone turnover, as well as parathyroid function, will become a reality for chronic dialysis patients. ACKNOWLEDGMENT The authors thank Dr Takeshi Kurosawa, Sumiyoshi Clinic Hospital, Mito, Japan, for cooperation in serum sampling from chronic dialysis patients. We also thank members of the ROD-21 Clinical Research Group for thoughtful discussions.
REFERENCES 1. Massry SG, Coburn JW, Lee DB, Jowsey J, Kleeman CR: Skeletal resistance to parathyroid hormone in renal failure. Studies in 105 human subjects. Ann Intern Med 78:357-364, 1973 2. Hruska KA, Teitelbaum SL: Renal osteodystrophy. N Engl J Med 333:166-174, 1995 3. Quarles LD, Lobaugh B, Murphy G: Intact parathyroid hormone overestimates the presence and severity of parathy-
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