- Email: [email protected]
The progression of vascular calcification and serum osteoprotegerin levels in patients on long-term hemodialysis
The Progression of Vascular Calcification and Serum Osteoprotegerin Levels in Patients on Long-Term Hemodialysis Kosaku Nitta, MD, PhD, Takashi Akiba, MD, PhD, Keiko Uchida, MD, PhD, Akira Kawashima, MD, PhD, Wako Yumura, MD, PhD, Takashi Kabaya, MD, and Hiroshi Nihei, MD, PhD ● Background: The aortic calcification index (ACI), estimated on abdominal computed tomographic scans, has been associated with the extent of arteriosclerosis in hemodialysis patients. However, the contribution of biochemical markers to the progression of vascular calcification in patients undergoing hemodialysis is not fully understood. Methods: We examined the relationship between coronary risk factors; metabolic factors, including serum osteoprotegerin (OPG) concentration; and progression of vascular calcification in 26 dialysis patients. Results: Mean patient age was 52.6 ⴞ 8.7 (SD) years, and mean duration of dialysis therapy was 7.7 ⴞ 5.8 years. ACI was measured twice in each patient, and the mean interscan period was 4.9 ⴞ 0.3 years. Mean ACI changed from 22.2 ⴞ 24.2 to 33.9 ⴞ 28.8 overall, and mean change in ACI (⌬ACI) was 12.0 ⴞ 9.9. Patients were divided into 2 groups: slow progressors, with ⌬ACI of 4.1 ⴞ 3.2 (n ⴝ 13), and rapid progressors, with ⌬ACI of 19.8 ⴞ 7.9 (n ⴝ 13). Serum fasting glucose and CRP levels of rapid progressors were high, and their serum albumin and intact parathyroid hormone levels were low. Multiple regression analyses showed that serum OPG levels were independently associated with vascular calcification in the hemodialysis patients studied. Conclusion: Rapid progression of vascular calcification was associated with dose of calcium carbonate prescribed and serum OPG concentration. The clinical significance of these observations remains to be determined. Am J Kidney Dis 42:303-309. © 2003 by the National Kidney Foundation, Inc. INDEX WORDS: Renal dialysis; vascular calcification; arteriosclerosis; osteoprotegerin (OPG); calcium (Ca).
C
ARDIOVASCULAR mortality is greater in hemodialysis patients than nonuremic persons. An epidemiological study has shown that damage to large arteries is a major contributory factor to the high cardiovascular morbidity and mortality of patients with end-stage renal disease (ESRD),1 and recent clinical studies2,3 have shown that sclerosis of the abdominal aorta reflects early arteriosclerosis and correlates significantly with coronary arteriosclerosis. Atherosclerosis is manifested by 2 major features: thickening and stiffening of arterial walls.4 The increased stiffness causes an increase in systolic blood pressure and pulse pressure and a decrease in diastolic blood pressure, thereby causing increased left ventricular afterload and altered coronary perfusion. Osteoprotegerin (OPG) is a decoy receptor that blocks the interaction of the receptor activator of nuclear factor-B with its ligand, thereby inhibiting osteoclast differentiation and activity.5 OPG and the OPG ligand (OPGL) constitute a complex mediator system involved in regulation of the resorption process in bone, which probably is responsible for the homeostatic mechanism of bone turnover. Recent experimental evidence has suggested that changes in this system may form the basis of certain metabolic bone diseases, such as osteoporosis and osteopetro-
sis.5,6 Recent studies have shown that parathyroid hormone (PTH) enhances production of the osteoclastogenic factor OPGL and inhibits the synthesis of the soluble receptor OPG, which blocks the biological effect of OPGL.7-9 However, the relationship between vascular calcification and serum OPG level is not fully understood. The purpose of the present study is to evaluate the contribution of cardiac risk factors to the progression of vascular calcification in hemodialysis patients and examine the relationship between the aortic calcification index (ACI) and the following clinical parameters: age, underlying diseases, blood pressure, and serum levels of albumin, cholesterol, triglyceride, C-reactive pro-
From the Department of Medicine, Kidney Center, Tokyo Women’s Medical University; and the Dialysis Unit, Minami Senju Hospital, Tokyo, Japan. Received December 30, 2002; accepted in revised form April 1, 2003. Supported in part by a grant from the Renal Osteodystrophy Foundation in Japan. Address reprint requests to Kosaku Nitta, MD, Department of Medicine, Kidney Center, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected] © 2003 by the National Kidney Foundation, Inc. 0272-6386/03/4202-0011$30.00/0 doi:10.1016/S0272-6386(03)00655-3
American Journal of Kidney Diseases, Vol 42, No 2 (August), 2003: pp 303-309
303
304
NITTA ET AL
tein (CRP), calcium (Ca), phosphorus (P), intact PTH (iPTH), and OPG. Results show evidence of a significant relationship between vascular calcification and serum OPG levels in patients undergoing hemodialysis. PATIENTS AND METHODS
Subjects Of 180 patients undergoing regular hemodialysis between 1995 and 2002 at the Dialysis Unit of Minami Senju Hospital (Tokyo, Japan), 49 patients agreed to have their ACI estimated. We excluded patients who had severe illnesses, patients with obvious acute inflammation, and those who did not comply with their drug therapy. Twelve patients had liver cirrhosis, and 28 patients had congestive heart failure. Thirty-eight patients had a common cold at the time of blood sampling. Twenty-three patients did not comply adequately with their antihypertensive drug regimen, and 30 patients frequently forgot to take Ca carbonate. Twenty-three patients were excluded from the study for the following reasons: refusal of follow-up computed tomographic (CT) scan, 3 patients; death, 2 patients; transfer to another dialysis unit, 8 patients; and incomplete blood sampling, 10 patients. Repeated ACI estimation was possible in 26 patients (14 men, 12 women). Mean patient age was 52.6 ⫾ 8.7 (SD) years, and mean duration of dialysis therapy was 7.7 ⫾ 5.8 years. Hemodialysis using hollow-fiber dialyzers, such as cellulose triacetate and polysulfone, was performed 3 times weekly (4 h/d). Patients had been on hemodialysis therapy for at least 3 months before the study, and the same membrane and same dialysis procedure had been used for at least 2 months before the study. Dialysate potassium concentration was 2.0 mEq/L, and Ca concentration was 3.0 mEq/L. Blood flow rate was 200 mL/min, and dialysate flow rate was 500 mL/min. Residual urine volume of hemodialysis patients was less than 100 mL/d. CT scans and blood sampling were performed in the morning before the first weekly hemodialysis session. Each subject gave informed consent to participate in the study. This study was approved by the institutional ethics committee of the hospital.
Demographic and Cardiac Risk Factors Age was calculated at the time of the first ACI estimation. Smoking was noted if the patient was a current cigarette smoker. Blood pressure was measured before dialysis sessions during the follow-up period, and the mean of at least 5 measurements was used in the first and second ACI estimates. Diabetes was noted if the patient was being treated for it medically or had a fasting blood glucose level of 126 mg/dL (7.0 mmol/L) or greater.
Determination of ACI by CT Scan We performed a prospective longitudinal study using CT scans to detect aortic calcification. As previously described,10,11 the abdominal aorta was examined on consecu-
tive, sequential, 8-mm section, noncontrast CT scans, and ACI was calculated as the proportion of aortic circumference covered by calcification. This method was used to morphometrically quantify arteriosclerosis in the crosssection showing the most extensive aortosclerosis. Arithmetic mean values of 3 measurements were calculated and used for analysis. Change in ACI (⌬ACI) was obtained by subtracting the baseline ACI value from the follow-up ACI value. The second assessment was performed 4.5 to 5.8 years after the first determination. ACI was independently checked by 2 observers, and reproducibility was absolute for the patients examined. The sequence of the CT scans and their orientation have been standardized, and the quality of scans primarily depends on the hardware used. To optimize reproducibility, all scans in the cross-sectional study were made by the same investigator using the same CT equipment, as previously described.11 Because 12 was the median ⌬ACI value in our sample population, it was chosen to divide patients into rapid progressors, with ⌬ACI of 12 or greater (n ⫽ 13), and slow progressors, with ⌬ACI less than 12 (n ⫽ 13).
Biochemical Analysis Blood chemistry values were determined once a month. Whole blood was used for hematocrit; EDTA plasma, for fasting blood glucose and lipids; and serum, for the other biochemical assays, including albumin, Ca, P, and CRP. Albumin, Ca, P, total cholesterol, triglyceride, and CRP levels were measured by means of automated methods. Serum OPG was measured by means of a sandwich enzyme immunoassay purchased from Immunodiagnostik (Bensheim, Germany) that uses 2 highly specific antibodies against human OPG. The detection antibody was a biotinlabeled polyclonal antiserum OPG antibody obtained from a goat immunized with recombinant human OPG. Intra-assay and interassay coefficients of variation were 8% and 12%, respectively. Normal values from a large number of healthy subjects of different ages obtained by the same assay method recently have been reported.12 Plasma osteopontin (OPN) concentrations were measured by means of a conventional enzyme immunoassay, as previously described.13 Serum iPTH was measured by means of radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Values reported for albumin, Ca, P, total cholesterol, triglyceride, fasting blood glucose, and CRP during the follow-up period are the means of at least 5 determinations. Serum iPTH and OPG values are the means of 2 determinations during the follow-up period.
Statistical Analysis All values are expressed as mean ⫾ SD. Differences between mean values of the 2 groups were tested by analysis of variance. Pearson’s correlation coefficient was used to test associations between 2 kinds of parameters. Group comparisons of categorical variables were made by chisquare analysis. A multiple regression analysis was applied to identify independent determinants of ACI. A probability less than 0.05 is considered significant. Statistical analyses
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
305
Fig 1. Progression of aortic calcification in a long-term dialysis patient. (A) Baseline scan showing mild calcification in the abdominal aorta (ACI ⴝ 36). (B) Follow-up scan 5.1 years later showing aortic calcification had progressed (ACI ⴝ 76).
were performed using the StatView statistical software package (StatView, version 5; SAS Institute, Cary, NC).
RESULTS
Background Characteristics None of the patients had undergone parathyroidectomy. Mean patient age was 52.6 ⫾ 8.7 years, and mean duration of dialysis therapy was 7.7 ⫾ 5.8 years. Fourteen patients (54%) were men, 6 patients (23%) had diabetes, and 7 patients (27%) were current smokers. ACI Assessment ACI was estimated twice in each patient, and the mean interscan period was 4.9 ⫾ 0.3 years.
Fig 2. ⌬ACI in each patient. Patients were divided into 2 groups at the median ⌬ACI value of 12: rapid progressors had ⌬ACI of 12 or greater (n ⴝ 13), and slow progressors had ⌬ACI less than 12 (n ⴝ 13). Different columns indicate the magnitude of ⌬ACI.
Mean ACI changed from 22.2 ⫾ 24.2 to 33.9 ⫾ 28.8 overall, and mean ⌬ACI was 12.0 ⫾ 9.9. A representative case is shown in Fig 1 (patient no. 26 in Fig 2). Dialysis Patients Grouped According to ⌬ACI Patients were divided into 2 groups based on the rate of ⌬ACI (Fig 2). Because 12 was the median ⌬ACI value in our sample population, it was chosen to divide patients into rapid progressors, with ⌬ACI of 12 or greater (n ⫽ 13), and slow progressors, with ⌬ACI less than 12 (n ⫽ 13). No significant differences between these groups were found in background characteristics, except for the percentage of patients with
306
NITTA ET AL Table 1.
Age (y) Sex (men/women) Duration of dialysis (y) Diabetes mellitus (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Smoking (%) Ca carbonate (g/day) 1,25(OH)D3 (g/day) Baseline ACI (%) Follow-up ACI (%) ⌬ACI (%)
Characteristics of Dialysis Patients by Subgroup Rapid Progressors ⌬ ACI ⱖ 12 (n ⫽ 13)
Slow Progressors ⌬ ACI ⬍ 12 (n ⫽ 13)
P
53.2 ⫾ 8.1 8/5 7.4 ⫾ 6.2 4 (30.7) 161.0 ⫾ 8.2 85.5 ⫾ 8.5 4 (30.7) 2.5 ⫾ 0.9 0.12 ⫾ 0.19 30.5 ⫾ 24.9 49.7 ⫾ 26.1 19.8 ⫾ 7.9
52.1 ⫾ 9.6 6/7 8.0 ⫾ 5.7 2 (15.4) 150.2 ⫾ 17.6 89.7 ⫾ 8.9 3 (23.0) 1.4 ⫾ 1.0 0.17 ⫾ 0.19 14.0 ⫾ 21.4 18.1 ⫾ 22.7 4.1 ⫾ 3.2
NS NS NS 0.001 NS NS NS 0.013 NS NS 0.005 ⬍0.0001
NOTE. Values expressed as mean ⫾ SD or number (percent). Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; ⌬ACI, follow-up ACI minus baseline ACI; NS, not significant.
diabetes and prescribed doses of Ca carbonate (Table 1). Laboratory Factors in the 2 Subgroups Rapid progressors had significantly greater serum Ca, fasting glucose, and CRP levels than slow progressors and lower serum albumin and iPTH levels (Table 2). Median serum OPG level in dialysis patients (237.9 ⫾ 11.2 pg/mL) was significantly greater than that in age-matched Table 2.
Total cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglyceride (mg/dL) Fasting blood glucose (mg/dL) Albumin (g/dL) Hematocrit (%) Ca (mg/dL) P (mg/dL) Ca ⫻ P product (mg/dL)2 PTH (pg/mL) OPG (pg/mL) OPN (ng/mL) CRP (mg/dL)
control subjects (66.6 ⫾ 38.4 pg/mL). Serum OPG concentrations of rapid progressors were significantly greater than those of slow progressors (Fig 3; P ⫽ 0.014), and their plasma OPN levels (872.4 ⫾ 122.8 ng/mL) also were significantly greater than those in slow progressors (621.4 ⫾ 78.9 ng/mL; P ⫽ 0.018). As shown in Fig 4, progression of vascular calcification was significantly associated with serum OPG concentration (r ⫽ 0.54; P ⫽ 0.004). There were no
Comparison of Biochemical Markers in Dialysis Patients Rapid Progressors ⌬ACI ⱖ 12 (n ⫽ 13)
Slow Progressors ⌬ACI ⬍ 12 (n ⫽ 13)
P
164.1 ⫾ 33.7 102.4 ⫾ 28.6 117.9 ⫾ 51.7 122.1 ⫾ 37.9 3.8 ⫾ 0.3 29.9 ⫾ 1.9 9.8 ⫾ 0.8 6.2 ⫾ 0.5 60.9 ⫾ 12.9 128.2 ⫾ 107.0 298 ⫾ 116.0 872.4 ⫾ 122.8 0.3 ⫾ 0.06
154.8 ⫾ 21.3 92.1 ⫾ 16.7 93.9 ⫾ 49.0 100.6 ⫾ 11.6 4.0 ⫾ 0.1 30.7 ⫾ 1.8 8.6 ⫾ 0.7 6.0 ⫾ 0.5 51.7 ⫾ 13.6 249.6 ⫾ 155.9 177.3 ⫾ 62.2 621.0 ⫾ 78.9 0.19 ⫾ 0.07
NS NS NS 0.046 0.02 NS 0.036 NS NS 0.048 0.014 0.018 ⬍0.0001
NOTE. Values expressed as mean ⫾ SD. To convert total and LDL cholesterol in mg/dL to mmol/L, multiply by 0.02586; triglycerides in mg/dL to mmol/L, multiply by 0.01129; glucose in mg/dL to mmol/L, multiply by 0.05551; albumin in g/dL to g/L, multiply by 10; Ca in mg/dL to mmol/L, multiply by 0.2495; P in mg/dL to mmol/L, multiply by 0.3229; iPTH in pg/mL to ng/L, multiply by 1. Abbreviations: NS, not significant; LDL, low-density lipoprotein.
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
307
Fig 3. Comparison of serum OPG concentrations in the rapid progressor and slow progressor groups described in Fig 2.
differences in other lipid levels, Ca ⫻ P product, or hematocrit. Multiple regression analyses were performed to adjust the roles of different pathogenic factors in the vascular calcification in all patients, and serum OPG level was found to be an independent variable associated with vascular calcification (P ⫽ 0.019). DISCUSSION
Vascular calcification is strongly associated with an increased risk for cardiovascular events and mortality in uremic patients.14,15 As in nonuremic persons, calcification in the arterial intima in patients with ESRD occurs in association with atherosclerotic plaques.16 Goodman et al17
recently showed a high prevalence of coronary artery calcification among young adults on dialysis therapy, especially those who had been on dialysis therapy for more than 10 years. Thus, the extent of arterial stiffness may be associated with progression of vascular calcification in patients undergoing long-term dialysis. We examined the relationship between extent of vascular calcification and metabolic factors in long-term dialysis patients, and results showed greater serum fasting glucose and CRP levels and lower serum albumin concentrations in rapid progressors. Multiple risk factors have been implicated in the development of vascular calcifications, and in previous studies, cardiovascular
Fig 4. Correlation between serum OPG concentrations and ACI of 26 hemodialysis patients.
308
calcification in patients with ESRD has been associated variably with age; dialysis vintage; race; diabetes; serum Ca, P, high-density lipoprotein cholesterol, and triglyceride concentrations; and markers of inflammation.16-20 However, no consistent and/or constant associations have been established between vascular remodeling and common risk factors. We also show an association between dose of Ca-based P binders prescribed and extent of vascular calcification, and this finding was consistent with findings of Guerin et al16 and Goodman et al,17 who reported an association between oral Ca dose and risk for vascular calcification. Rapid progressors in the present study had lower serum iPTH levels than slow progressors. Much attention recently has been focused on adynamic bone disease (ABD). Although the precise pathological mechanism of ABD is not fully understood, hypoparathyroidism is considered one of the causative factors.21,22 Diabetes mellitus is thought to be responsible for apparent absolute or relative hypoparathyroidism,23 and in the present study, patients with diabetes accounted for 30.7% of rapid progressors, a significantly greater percentage than for slow progressors. Thus, it is likely that the greater doses of Ca carbonate prescribed for dialysis patients with ABD induce the progression of vascular calcification. OPG recently has been identified as a novel cytokine that increases the mineral density and volume of bone tissue in vitro by decreasing the number of active osteoclasts in vitro. Overexpression of OPG in transgenic mice results in osteopetrosis,5 whereas OPG-deficient mice develop severe osteoporosis.6 These experimental findings suggest a counterregulatory function of OPG that prevents further bone loss. Kazama et al24 recently showed that serum OPG levels of uremic patients were elevated, independent of their serum iPTH levels, and suggested that circulating OPG is an independent factor affecting bone metabolism in uremic patients. Hass et al25 showed that a combination of OPG and iPTH can be used as a marker for the noninvasive diagnosis of renal osteodystrophy in dialysis patients. Coen et al26 showed lower serum OPG levels in patients with ABD than in patients with predominant hyperparathyroidism and found a significant negative correlation between serum OPG
NITTA ET AL
and iPTH levels. However, the relationship between serum OPG level and vascular calcification has not been fully understood. The present study has shown significantly greater serum OPG concentrations in rapid progressors than slow progressors and a significant association between rapid progression of vascular calcification and serum OPG concentrations. Multiple regression analysis showed serum OPG level as an independent variable associated with vascular calcification. Dhore et al27 reported that OPN was upregulated when calcification or bone formation was detected in human atherosclerotic plaques, and OPG was present in nondiseased vessel walls and early atherosclerotic lesions. Jono et al28 recently reported significantly greater serum OPG levels in patients with significant stenosis of the coronary arteries than in those without stenosis. Increases in severity of coronary artery disease were associated with significant increases in serum OPG levels. These findings suggest that determination of serum OPG levels could be useful in the diagnosis of vascular calcification, as well as low-turnover bone disease, in patients undergoing hemodialysis. Because our sample size was small and we have no proper control data for patients not on dialysis therapy, a larger patient group must be studied to confirm our findings. However, results of this study show that the extent of vascular calcification may increase in association with chronic inflammatory responses and administration of relatively high Ca carbonate doses to dialysis patients with low bone turnover. They also suggest that greater serum OPG concentrations may be associated with progression of vascular calcification in dialysis patients. The clinical significance of these observations remains to be determined. ACKNOWLEDGMENT The authors thank Takashi Oba for OPG measurement; Hiroyuki Kimura for ACI assessment; and John P McCormick, PhD, for manuscript review.
REFERENCES 1. Lindner A, Charra B, Sherrad DJ, Scribner BH: Accelerated atherosclerosis in prolonged maintenance hemodialysis. N Engl J Med 290:697-701, 1974 2. Ibels LS, Alfrey AC, Huffer WE, Craswell PW, Anderson JT, Weil R III: Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med 66:790-796, 1979
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
3. Ansari A, Kaupke CJ, Vaziri ND, Miller R, Barbari A: Cardiac pathology in patients with end-stage renal disease maintained on hemodialysis. Int J Artif Organs 16:31-36, 1993 4. O’Rourke M: Mechanical principles in arterial disease. Hypertension 26:2-9, 1995 5. Simonet WS, Lacey DL, Dunstan CR, et al: Osteoprotegerin: A novel secreted protein involved in the bone density. Cell 89:309-319, 1997 6. Misuno A, Amizuka N, Irie K, et al: Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/ osteoprotegerin. Biochem Biophys Res Commun 247:610615, 1998 7. Horwood NJ, Elliot J, Martin JM, Gillespie MT: Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoblastic stroma cells. Endocrinology 139:4743-4746, 1998 8. Lee SK, Lorenzo JA: Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: Correlation with osteoclast-like cell formation. Endocrinology 140:3552-3561, 1999 9. Onyia JE, Miles RR, Yang X, Halladay DL, Onyia JE: 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 10. Kabaya T, Nitta K, Kimura H, et al: An increased serum level of parathyroid hormone is a risk factor for aortosclerosis in hemodialysis patients. Nephron 86:213214, 2000 11. Nitta K, Akiba T, Kawashima A, et al: Serum levels of macrophage colony-stimulating factor and aortic calcification in hemodialysis patients. Am J Nephrol 21:465-470, 2001 12. Blacher J, Guerin AP, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness on survival in end-stage renal disease. Circulation 99:2434-2439, 1999 13. Nitta K, Ishizuka T, Horita S, et al: Soluble osteopontin and vascular calcification in hemodialysis patients. Nephron 89:455-458, 2001 14. Guerin AP, Blacher J, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness on survival of patients in end-stage renal failure. Circulation 103:987-992, 2001 15. Schwartz U, Buzello M, Ritz E, Stein G: Morphology of coronary atherosclerosis lesions in patients with end-stage renal failure. Nephrol Dial Transplant 15:218-223, 2000
309
16. Guerin AP, London GM, Marchais SJ, Metivier F: Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transplant 15:1014-1021, 2000 17. Goodman WG, Goldin J, Kuizon BD, et al: Coronaryartery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 342:14781483, 2000 18. Braun J, Oldendorf M, Moshage W, Heidler R, Zeitler E, Luft FC: Electron beam computed tomography in the evaluation of cardiac calcification in chronic dialysis patients. Am J Kidney Dis 27:394-401, 1996 19. Kimura K, Saika Y, Otani H, Fujii R, Mune M, Yukawa S: Factors associated with calcification of the abdominal aorta in hemodialysis patients. Kidney Int Suppl 71:S238-S241, 1999 20. Shoji T, Nishzawa Y, Kawagishi T, et al: Intermediatedensity lipoprotein as an independent risk factor for aortic atherosclerosis in hemodialysis patients. J Am Soc Nephrol 9:1277-1284, 1998 21. Quarles LD, Lobaugh B, Murphy G: Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab 75:145-150, 1992 22. Hercz G, Pei Y, Greenwood C, et al: Aplastic osteodystrophy without aluminum: The role of “suppressed” parathyroid function. Kidney Int 44:860-866, 1993 23. Mucsi I, Hercz G: Adynamic bone disease: Pathogenesis, diagnosis and clinical relevance. Curr Opin Nephrol Hypertens 7:356-361, 1997 24. Kazama JJ, Shigematsu T, Yano K, et al: Increased circulating levels of osteoclastogenesis inhibitory factor (osteoprotegerin) in patients with chronic renal failure. Am J Kidney Dis 39:525-532, 2002 25. Haas M, Leko-Mohr Z, Roschger P, et al: Osteoprotegerin and parathyroid hormone as markers of high-turnover osteodystrophy and decreased bone mineralization in hemodialysis patients. Am J Kidney Dis 39:580-586, 2002 26. Coen G, Ballanti P, Balducci A, et al: Serum osteoprotegerin and renal osteodystrophy. Nephrol Dial Transplant 17:233-238, 2002 27. Dhore CR, Cleutjens JP, Lutgens E, et al: Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol 21: 1998-2003, 2001 28. Jono S, Ikari Y, Shioi A, et al: Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. Circulation 106:1192-1194, 2002
C
ARDIOVASCULAR mortality is greater in hemodialysis patients than nonuremic persons. An epidemiological study has shown that damage to large arteries is a major contributory factor to the high cardiovascular morbidity and mortality of patients with end-stage renal disease (ESRD),1 and recent clinical studies2,3 have shown that sclerosis of the abdominal aorta reflects early arteriosclerosis and correlates significantly with coronary arteriosclerosis. Atherosclerosis is manifested by 2 major features: thickening and stiffening of arterial walls.4 The increased stiffness causes an increase in systolic blood pressure and pulse pressure and a decrease in diastolic blood pressure, thereby causing increased left ventricular afterload and altered coronary perfusion. Osteoprotegerin (OPG) is a decoy receptor that blocks the interaction of the receptor activator of nuclear factor-B with its ligand, thereby inhibiting osteoclast differentiation and activity.5 OPG and the OPG ligand (OPGL) constitute a complex mediator system involved in regulation of the resorption process in bone, which probably is responsible for the homeostatic mechanism of bone turnover. Recent experimental evidence has suggested that changes in this system may form the basis of certain metabolic bone diseases, such as osteoporosis and osteopetro-
sis.5,6 Recent studies have shown that parathyroid hormone (PTH) enhances production of the osteoclastogenic factor OPGL and inhibits the synthesis of the soluble receptor OPG, which blocks the biological effect of OPGL.7-9 However, the relationship between vascular calcification and serum OPG level is not fully understood. The purpose of the present study is to evaluate the contribution of cardiac risk factors to the progression of vascular calcification in hemodialysis patients and examine the relationship between the aortic calcification index (ACI) and the following clinical parameters: age, underlying diseases, blood pressure, and serum levels of albumin, cholesterol, triglyceride, C-reactive pro-
From the Department of Medicine, Kidney Center, Tokyo Women’s Medical University; and the Dialysis Unit, Minami Senju Hospital, Tokyo, Japan. Received December 30, 2002; accepted in revised form April 1, 2003. Supported in part by a grant from the Renal Osteodystrophy Foundation in Japan. Address reprint requests to Kosaku Nitta, MD, Department of Medicine, Kidney Center, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected] © 2003 by the National Kidney Foundation, Inc. 0272-6386/03/4202-0011$30.00/0 doi:10.1016/S0272-6386(03)00655-3
American Journal of Kidney Diseases, Vol 42, No 2 (August), 2003: pp 303-309
303
304
NITTA ET AL
tein (CRP), calcium (Ca), phosphorus (P), intact PTH (iPTH), and OPG. Results show evidence of a significant relationship between vascular calcification and serum OPG levels in patients undergoing hemodialysis. PATIENTS AND METHODS
Subjects Of 180 patients undergoing regular hemodialysis between 1995 and 2002 at the Dialysis Unit of Minami Senju Hospital (Tokyo, Japan), 49 patients agreed to have their ACI estimated. We excluded patients who had severe illnesses, patients with obvious acute inflammation, and those who did not comply with their drug therapy. Twelve patients had liver cirrhosis, and 28 patients had congestive heart failure. Thirty-eight patients had a common cold at the time of blood sampling. Twenty-three patients did not comply adequately with their antihypertensive drug regimen, and 30 patients frequently forgot to take Ca carbonate. Twenty-three patients were excluded from the study for the following reasons: refusal of follow-up computed tomographic (CT) scan, 3 patients; death, 2 patients; transfer to another dialysis unit, 8 patients; and incomplete blood sampling, 10 patients. Repeated ACI estimation was possible in 26 patients (14 men, 12 women). Mean patient age was 52.6 ⫾ 8.7 (SD) years, and mean duration of dialysis therapy was 7.7 ⫾ 5.8 years. Hemodialysis using hollow-fiber dialyzers, such as cellulose triacetate and polysulfone, was performed 3 times weekly (4 h/d). Patients had been on hemodialysis therapy for at least 3 months before the study, and the same membrane and same dialysis procedure had been used for at least 2 months before the study. Dialysate potassium concentration was 2.0 mEq/L, and Ca concentration was 3.0 mEq/L. Blood flow rate was 200 mL/min, and dialysate flow rate was 500 mL/min. Residual urine volume of hemodialysis patients was less than 100 mL/d. CT scans and blood sampling were performed in the morning before the first weekly hemodialysis session. Each subject gave informed consent to participate in the study. This study was approved by the institutional ethics committee of the hospital.
Demographic and Cardiac Risk Factors Age was calculated at the time of the first ACI estimation. Smoking was noted if the patient was a current cigarette smoker. Blood pressure was measured before dialysis sessions during the follow-up period, and the mean of at least 5 measurements was used in the first and second ACI estimates. Diabetes was noted if the patient was being treated for it medically or had a fasting blood glucose level of 126 mg/dL (7.0 mmol/L) or greater.
Determination of ACI by CT Scan We performed a prospective longitudinal study using CT scans to detect aortic calcification. As previously described,10,11 the abdominal aorta was examined on consecu-
tive, sequential, 8-mm section, noncontrast CT scans, and ACI was calculated as the proportion of aortic circumference covered by calcification. This method was used to morphometrically quantify arteriosclerosis in the crosssection showing the most extensive aortosclerosis. Arithmetic mean values of 3 measurements were calculated and used for analysis. Change in ACI (⌬ACI) was obtained by subtracting the baseline ACI value from the follow-up ACI value. The second assessment was performed 4.5 to 5.8 years after the first determination. ACI was independently checked by 2 observers, and reproducibility was absolute for the patients examined. The sequence of the CT scans and their orientation have been standardized, and the quality of scans primarily depends on the hardware used. To optimize reproducibility, all scans in the cross-sectional study were made by the same investigator using the same CT equipment, as previously described.11 Because 12 was the median ⌬ACI value in our sample population, it was chosen to divide patients into rapid progressors, with ⌬ACI of 12 or greater (n ⫽ 13), and slow progressors, with ⌬ACI less than 12 (n ⫽ 13).
Biochemical Analysis Blood chemistry values were determined once a month. Whole blood was used for hematocrit; EDTA plasma, for fasting blood glucose and lipids; and serum, for the other biochemical assays, including albumin, Ca, P, and CRP. Albumin, Ca, P, total cholesterol, triglyceride, and CRP levels were measured by means of automated methods. Serum OPG was measured by means of a sandwich enzyme immunoassay purchased from Immunodiagnostik (Bensheim, Germany) that uses 2 highly specific antibodies against human OPG. The detection antibody was a biotinlabeled polyclonal antiserum OPG antibody obtained from a goat immunized with recombinant human OPG. Intra-assay and interassay coefficients of variation were 8% and 12%, respectively. Normal values from a large number of healthy subjects of different ages obtained by the same assay method recently have been reported.12 Plasma osteopontin (OPN) concentrations were measured by means of a conventional enzyme immunoassay, as previously described.13 Serum iPTH was measured by means of radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Values reported for albumin, Ca, P, total cholesterol, triglyceride, fasting blood glucose, and CRP during the follow-up period are the means of at least 5 determinations. Serum iPTH and OPG values are the means of 2 determinations during the follow-up period.
Statistical Analysis All values are expressed as mean ⫾ SD. Differences between mean values of the 2 groups were tested by analysis of variance. Pearson’s correlation coefficient was used to test associations between 2 kinds of parameters. Group comparisons of categorical variables were made by chisquare analysis. A multiple regression analysis was applied to identify independent determinants of ACI. A probability less than 0.05 is considered significant. Statistical analyses
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
305
Fig 1. Progression of aortic calcification in a long-term dialysis patient. (A) Baseline scan showing mild calcification in the abdominal aorta (ACI ⴝ 36). (B) Follow-up scan 5.1 years later showing aortic calcification had progressed (ACI ⴝ 76).
were performed using the StatView statistical software package (StatView, version 5; SAS Institute, Cary, NC).
RESULTS
Background Characteristics None of the patients had undergone parathyroidectomy. Mean patient age was 52.6 ⫾ 8.7 years, and mean duration of dialysis therapy was 7.7 ⫾ 5.8 years. Fourteen patients (54%) were men, 6 patients (23%) had diabetes, and 7 patients (27%) were current smokers. ACI Assessment ACI was estimated twice in each patient, and the mean interscan period was 4.9 ⫾ 0.3 years.
Fig 2. ⌬ACI in each patient. Patients were divided into 2 groups at the median ⌬ACI value of 12: rapid progressors had ⌬ACI of 12 or greater (n ⴝ 13), and slow progressors had ⌬ACI less than 12 (n ⴝ 13). Different columns indicate the magnitude of ⌬ACI.
Mean ACI changed from 22.2 ⫾ 24.2 to 33.9 ⫾ 28.8 overall, and mean ⌬ACI was 12.0 ⫾ 9.9. A representative case is shown in Fig 1 (patient no. 26 in Fig 2). Dialysis Patients Grouped According to ⌬ACI Patients were divided into 2 groups based on the rate of ⌬ACI (Fig 2). Because 12 was the median ⌬ACI value in our sample population, it was chosen to divide patients into rapid progressors, with ⌬ACI of 12 or greater (n ⫽ 13), and slow progressors, with ⌬ACI less than 12 (n ⫽ 13). No significant differences between these groups were found in background characteristics, except for the percentage of patients with
306
NITTA ET AL Table 1.
Age (y) Sex (men/women) Duration of dialysis (y) Diabetes mellitus (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Smoking (%) Ca carbonate (g/day) 1,25(OH)D3 (g/day) Baseline ACI (%) Follow-up ACI (%) ⌬ACI (%)
Characteristics of Dialysis Patients by Subgroup Rapid Progressors ⌬ ACI ⱖ 12 (n ⫽ 13)
Slow Progressors ⌬ ACI ⬍ 12 (n ⫽ 13)
P
53.2 ⫾ 8.1 8/5 7.4 ⫾ 6.2 4 (30.7) 161.0 ⫾ 8.2 85.5 ⫾ 8.5 4 (30.7) 2.5 ⫾ 0.9 0.12 ⫾ 0.19 30.5 ⫾ 24.9 49.7 ⫾ 26.1 19.8 ⫾ 7.9
52.1 ⫾ 9.6 6/7 8.0 ⫾ 5.7 2 (15.4) 150.2 ⫾ 17.6 89.7 ⫾ 8.9 3 (23.0) 1.4 ⫾ 1.0 0.17 ⫾ 0.19 14.0 ⫾ 21.4 18.1 ⫾ 22.7 4.1 ⫾ 3.2
NS NS NS 0.001 NS NS NS 0.013 NS NS 0.005 ⬍0.0001
NOTE. Values expressed as mean ⫾ SD or number (percent). Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; ⌬ACI, follow-up ACI minus baseline ACI; NS, not significant.
diabetes and prescribed doses of Ca carbonate (Table 1). Laboratory Factors in the 2 Subgroups Rapid progressors had significantly greater serum Ca, fasting glucose, and CRP levels than slow progressors and lower serum albumin and iPTH levels (Table 2). Median serum OPG level in dialysis patients (237.9 ⫾ 11.2 pg/mL) was significantly greater than that in age-matched Table 2.
Total cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglyceride (mg/dL) Fasting blood glucose (mg/dL) Albumin (g/dL) Hematocrit (%) Ca (mg/dL) P (mg/dL) Ca ⫻ P product (mg/dL)2 PTH (pg/mL) OPG (pg/mL) OPN (ng/mL) CRP (mg/dL)
control subjects (66.6 ⫾ 38.4 pg/mL). Serum OPG concentrations of rapid progressors were significantly greater than those of slow progressors (Fig 3; P ⫽ 0.014), and their plasma OPN levels (872.4 ⫾ 122.8 ng/mL) also were significantly greater than those in slow progressors (621.4 ⫾ 78.9 ng/mL; P ⫽ 0.018). As shown in Fig 4, progression of vascular calcification was significantly associated with serum OPG concentration (r ⫽ 0.54; P ⫽ 0.004). There were no
Comparison of Biochemical Markers in Dialysis Patients Rapid Progressors ⌬ACI ⱖ 12 (n ⫽ 13)
Slow Progressors ⌬ACI ⬍ 12 (n ⫽ 13)
P
164.1 ⫾ 33.7 102.4 ⫾ 28.6 117.9 ⫾ 51.7 122.1 ⫾ 37.9 3.8 ⫾ 0.3 29.9 ⫾ 1.9 9.8 ⫾ 0.8 6.2 ⫾ 0.5 60.9 ⫾ 12.9 128.2 ⫾ 107.0 298 ⫾ 116.0 872.4 ⫾ 122.8 0.3 ⫾ 0.06
154.8 ⫾ 21.3 92.1 ⫾ 16.7 93.9 ⫾ 49.0 100.6 ⫾ 11.6 4.0 ⫾ 0.1 30.7 ⫾ 1.8 8.6 ⫾ 0.7 6.0 ⫾ 0.5 51.7 ⫾ 13.6 249.6 ⫾ 155.9 177.3 ⫾ 62.2 621.0 ⫾ 78.9 0.19 ⫾ 0.07
NS NS NS 0.046 0.02 NS 0.036 NS NS 0.048 0.014 0.018 ⬍0.0001
NOTE. Values expressed as mean ⫾ SD. To convert total and LDL cholesterol in mg/dL to mmol/L, multiply by 0.02586; triglycerides in mg/dL to mmol/L, multiply by 0.01129; glucose in mg/dL to mmol/L, multiply by 0.05551; albumin in g/dL to g/L, multiply by 10; Ca in mg/dL to mmol/L, multiply by 0.2495; P in mg/dL to mmol/L, multiply by 0.3229; iPTH in pg/mL to ng/L, multiply by 1. Abbreviations: NS, not significant; LDL, low-density lipoprotein.
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
307
Fig 3. Comparison of serum OPG concentrations in the rapid progressor and slow progressor groups described in Fig 2.
differences in other lipid levels, Ca ⫻ P product, or hematocrit. Multiple regression analyses were performed to adjust the roles of different pathogenic factors in the vascular calcification in all patients, and serum OPG level was found to be an independent variable associated with vascular calcification (P ⫽ 0.019). DISCUSSION
Vascular calcification is strongly associated with an increased risk for cardiovascular events and mortality in uremic patients.14,15 As in nonuremic persons, calcification in the arterial intima in patients with ESRD occurs in association with atherosclerotic plaques.16 Goodman et al17
recently showed a high prevalence of coronary artery calcification among young adults on dialysis therapy, especially those who had been on dialysis therapy for more than 10 years. Thus, the extent of arterial stiffness may be associated with progression of vascular calcification in patients undergoing long-term dialysis. We examined the relationship between extent of vascular calcification and metabolic factors in long-term dialysis patients, and results showed greater serum fasting glucose and CRP levels and lower serum albumin concentrations in rapid progressors. Multiple risk factors have been implicated in the development of vascular calcifications, and in previous studies, cardiovascular
Fig 4. Correlation between serum OPG concentrations and ACI of 26 hemodialysis patients.
308
calcification in patients with ESRD has been associated variably with age; dialysis vintage; race; diabetes; serum Ca, P, high-density lipoprotein cholesterol, and triglyceride concentrations; and markers of inflammation.16-20 However, no consistent and/or constant associations have been established between vascular remodeling and common risk factors. We also show an association between dose of Ca-based P binders prescribed and extent of vascular calcification, and this finding was consistent with findings of Guerin et al16 and Goodman et al,17 who reported an association between oral Ca dose and risk for vascular calcification. Rapid progressors in the present study had lower serum iPTH levels than slow progressors. Much attention recently has been focused on adynamic bone disease (ABD). Although the precise pathological mechanism of ABD is not fully understood, hypoparathyroidism is considered one of the causative factors.21,22 Diabetes mellitus is thought to be responsible for apparent absolute or relative hypoparathyroidism,23 and in the present study, patients with diabetes accounted for 30.7% of rapid progressors, a significantly greater percentage than for slow progressors. Thus, it is likely that the greater doses of Ca carbonate prescribed for dialysis patients with ABD induce the progression of vascular calcification. OPG recently has been identified as a novel cytokine that increases the mineral density and volume of bone tissue in vitro by decreasing the number of active osteoclasts in vitro. Overexpression of OPG in transgenic mice results in osteopetrosis,5 whereas OPG-deficient mice develop severe osteoporosis.6 These experimental findings suggest a counterregulatory function of OPG that prevents further bone loss. Kazama et al24 recently showed that serum OPG levels of uremic patients were elevated, independent of their serum iPTH levels, and suggested that circulating OPG is an independent factor affecting bone metabolism in uremic patients. Hass et al25 showed that a combination of OPG and iPTH can be used as a marker for the noninvasive diagnosis of renal osteodystrophy in dialysis patients. Coen et al26 showed lower serum OPG levels in patients with ABD than in patients with predominant hyperparathyroidism and found a significant negative correlation between serum OPG
NITTA ET AL
and iPTH levels. However, the relationship between serum OPG level and vascular calcification has not been fully understood. The present study has shown significantly greater serum OPG concentrations in rapid progressors than slow progressors and a significant association between rapid progression of vascular calcification and serum OPG concentrations. Multiple regression analysis showed serum OPG level as an independent variable associated with vascular calcification. Dhore et al27 reported that OPN was upregulated when calcification or bone formation was detected in human atherosclerotic plaques, and OPG was present in nondiseased vessel walls and early atherosclerotic lesions. Jono et al28 recently reported significantly greater serum OPG levels in patients with significant stenosis of the coronary arteries than in those without stenosis. Increases in severity of coronary artery disease were associated with significant increases in serum OPG levels. These findings suggest that determination of serum OPG levels could be useful in the diagnosis of vascular calcification, as well as low-turnover bone disease, in patients undergoing hemodialysis. Because our sample size was small and we have no proper control data for patients not on dialysis therapy, a larger patient group must be studied to confirm our findings. However, results of this study show that the extent of vascular calcification may increase in association with chronic inflammatory responses and administration of relatively high Ca carbonate doses to dialysis patients with low bone turnover. They also suggest that greater serum OPG concentrations may be associated with progression of vascular calcification in dialysis patients. The clinical significance of these observations remains to be determined. ACKNOWLEDGMENT The authors thank Takashi Oba for OPG measurement; Hiroyuki Kimura for ACI assessment; and John P McCormick, PhD, for manuscript review.
REFERENCES 1. Lindner A, Charra B, Sherrad DJ, Scribner BH: Accelerated atherosclerosis in prolonged maintenance hemodialysis. N Engl J Med 290:697-701, 1974 2. Ibels LS, Alfrey AC, Huffer WE, Craswell PW, Anderson JT, Weil R III: Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med 66:790-796, 1979
VASCULAR CALCIFICATION AND OSTEOPROTEGERIN
3. Ansari A, Kaupke CJ, Vaziri ND, Miller R, Barbari A: Cardiac pathology in patients with end-stage renal disease maintained on hemodialysis. Int J Artif Organs 16:31-36, 1993 4. O’Rourke M: Mechanical principles in arterial disease. Hypertension 26:2-9, 1995 5. Simonet WS, Lacey DL, Dunstan CR, et al: Osteoprotegerin: A novel secreted protein involved in the bone density. Cell 89:309-319, 1997 6. Misuno A, Amizuka N, Irie K, et al: Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/ osteoprotegerin. Biochem Biophys Res Commun 247:610615, 1998 7. Horwood NJ, Elliot J, Martin JM, Gillespie MT: Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoblastic stroma cells. Endocrinology 139:4743-4746, 1998 8. Lee SK, Lorenzo JA: Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: Correlation with osteoclast-like cell formation. Endocrinology 140:3552-3561, 1999 9. Onyia JE, Miles RR, Yang X, Halladay DL, Onyia JE: 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 10. Kabaya T, Nitta K, Kimura H, et al: An increased serum level of parathyroid hormone is a risk factor for aortosclerosis in hemodialysis patients. Nephron 86:213214, 2000 11. Nitta K, Akiba T, Kawashima A, et al: Serum levels of macrophage colony-stimulating factor and aortic calcification in hemodialysis patients. Am J Nephrol 21:465-470, 2001 12. Blacher J, Guerin AP, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness on survival in end-stage renal disease. Circulation 99:2434-2439, 1999 13. Nitta K, Ishizuka T, Horita S, et al: Soluble osteopontin and vascular calcification in hemodialysis patients. Nephron 89:455-458, 2001 14. Guerin AP, Blacher J, Pannier B, Marchais SJ, Safar ME, London GM: Impact of aortic stiffness on survival of patients in end-stage renal failure. Circulation 103:987-992, 2001 15. Schwartz U, Buzello M, Ritz E, Stein G: Morphology of coronary atherosclerosis lesions in patients with end-stage renal failure. Nephrol Dial Transplant 15:218-223, 2000
309
16. Guerin AP, London GM, Marchais SJ, Metivier F: Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transplant 15:1014-1021, 2000 17. Goodman WG, Goldin J, Kuizon BD, et al: Coronaryartery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 342:14781483, 2000 18. Braun J, Oldendorf M, Moshage W, Heidler R, Zeitler E, Luft FC: Electron beam computed tomography in the evaluation of cardiac calcification in chronic dialysis patients. Am J Kidney Dis 27:394-401, 1996 19. Kimura K, Saika Y, Otani H, Fujii R, Mune M, Yukawa S: Factors associated with calcification of the abdominal aorta in hemodialysis patients. Kidney Int Suppl 71:S238-S241, 1999 20. Shoji T, Nishzawa Y, Kawagishi T, et al: Intermediatedensity lipoprotein as an independent risk factor for aortic atherosclerosis in hemodialysis patients. J Am Soc Nephrol 9:1277-1284, 1998 21. Quarles LD, Lobaugh B, Murphy G: Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab 75:145-150, 1992 22. Hercz G, Pei Y, Greenwood C, et al: Aplastic osteodystrophy without aluminum: The role of “suppressed” parathyroid function. Kidney Int 44:860-866, 1993 23. Mucsi I, Hercz G: Adynamic bone disease: Pathogenesis, diagnosis and clinical relevance. Curr Opin Nephrol Hypertens 7:356-361, 1997 24. Kazama JJ, Shigematsu T, Yano K, et al: Increased circulating levels of osteoclastogenesis inhibitory factor (osteoprotegerin) in patients with chronic renal failure. Am J Kidney Dis 39:525-532, 2002 25. Haas M, Leko-Mohr Z, Roschger P, et al: Osteoprotegerin and parathyroid hormone as markers of high-turnover osteodystrophy and decreased bone mineralization in hemodialysis patients. Am J Kidney Dis 39:580-586, 2002 26. Coen G, Ballanti P, Balducci A, et al: Serum osteoprotegerin and renal osteodystrophy. Nephrol Dial Transplant 17:233-238, 2002 27. Dhore CR, Cleutjens JP, Lutgens E, et al: Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol 21: 1998-2003, 2001 28. Jono S, Ikari Y, Shioi A, et al: Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. Circulation 106:1192-1194, 2002