1 Hospital Curry Cabral, Lisbon, Portugal, 2 Service de Néphrologie et de Médecine Interne, CHU, Amiens, France, 3 Hospital Militar Principal, Lisbon, Portugal, 4 Laboratoire de Physiologie, Faculté de Médecine Necker, Paris, France, 5 Division of Nephrology, University of Ulm, Ulm, Germany, 6 Musculoskeletal Diseases Center (151), Jerry L. Pettis VA Medical Center, Loma Linda, CA, USA and 7 INSERM U 507, Division of Nephrology, Hôpital Necker, Paris, France
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Abstract |
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Methods. We conducted a prospective, open study in 24 chronic haemodialysis patients who were randomized to dialysis treatment with either cellulosic (CELL group, n=11) or polyacrylonitrile (AN-69 group, n=13) membrane for 9 months. Repeated determinations of plasma parameters reflecting bone turnover were done in all patients, and a bone biopsy in a subgroup at the start and end of study.
Results. At the start, mean plasma intact parathyroid hormone levels were comparable between the two groups and they did not vary significantly at 9 months of treatment. Similarly, plasma bone-specific alkaline phosphatase and osteocalcin (markers of bone formation), and cross-laps (marker of bone resorption) remained unchanged. However, plasma insulin-like growth factor-I (IGF-I) progressively decreased from 169 to 119 ng/ml in AN-69 group (P<0.01), whereas it remained unchanged in CELL group. In addition, the levels of IGF binding protein (IGFBP)-1 and IGFBP-2 were increased while the levels of IGFBP-5 were decreased in AN-69 group. In the five patients of each group who had repeat bone biopsies, histomorphometric analysis showed a decrease in osteoblast surface, osteoclast surface and osteoclast number in AN-69 group at 9 months, compared with baseline values measured at the start of the study. In contrast, all three parameters significantly increased in the CELL group at 9 months (P<0.001 for the difference between each of the three parameters).Bone formation rate decreased by 31% in the AN-69 group, but increased by 50% in CELL group. However, this latter difference was not statistically significant. Plasma interleukin (IL)-6 and soluble IL-6 receptor levels did not change in the two groups of patients who had undergone bone biopsy.
Conclusion. Dialysis with CELL membrane was associated with increased bone turnover whereas the use of AN-69 membrane was associated with decreased bone turnover, suggesting a beneficial effect of the latter on high-turnover uraemic bone disease. However, as the number of patients with repeat bone biopsies was small, these findings need to be confirmed in a larger study. Further studies are also needed to evaluate whether or not the changes in IGF system components play a role in decreased bone cell activity in patients on dialysis using the AN-69 polyacrylonitrile membrane.
Keywords: renal osteodystrophy; haemodialysis; membrane; bone markers; bone biopsy
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Introduction |
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Last but not least, some treatments given to uraemic patients exert marked effects on the skeleton such as corticosteroids and immunosuppressive drugs. Renal replacement therapy is part of such treatment-related factors. Thus, a low calcium concentration of the dialysis fluid may lead to a stimulation of parathyroid hormone (PTH) release during and after the dialysis whereas a high calcium concentration inhibits PTH secretion. The bacteriologic quality of the dialysate has been suggested to play a role in the occurrence of carpal tunnel syndrome, probably via endotoxin release and excessive cytokine generation, and the type of haemodialysis membrane has also been shown to influence the prevalence of carpal tunnel syndrome and destructive arthropathy in chronic dialysis patients [1].
In contrast, not much is known about a possible contribution to uraemic osteodystrophy of the dialysis procedure and, in particular, the type of haemodialysis membrane used for renal replacement therapy. Therefore, we decided some years ago to test this hypothesis in a retrospective study. We found evidence of higher bone turnover in patients on long-term haemodialysis treatment with cellulosic membranes (Cuprophan) than in patients dialysed with highly permeable, synthetic membranes (polyacrylonitrile AN-69 or high-flux polysulfone) for prolonged time periods [2]. Additional indirect evidence in favour of a possible contribution of the dialysis technique to bone complications is the observation of an association between plasma cytokine levels, known to be modified by the dialysis procedure, and bone cell activity in uraemic patients [3].
The goal of the present prospective, randomized study in chronic haemodialysis patients was to confirm our hypothesis, based on retrospective and indirect evidence, that the use of a relatively biocompatible, highly permeable synthetic dialysis membrane should be associated with a lower bone turnover than the use of a less biocompatible, low permeability cellulosic membrane. For this purpose, patients with comparable degrees of mild to moderate hyperparathyroidism were allocated to a 9-month treatment period with either cellulosic or polyacrylonitrile AN-69 membrane. Possible changes of their bone metabolism during this time period were evaluated by circulating bone markers, and a subgroup of patients also underwent repeat bone histomorphometric examination and detailed analysis of circulating IGF system components.
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Patients and methods |
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Twenty patients of each centre were randomly assigned to one of two treatment arms, the goal being to have comparable initial plasma iPTH values in each group. A step-by-step procedure was used, allocating randomly the first pair of patients with the highest baseline plasma iPTH to either arm, then the second pair with the next lower iPTH values and so on until the pair with the lowest iPTH values was reached. Unfortunately, the number of patients in the two centres who fulfilled the criterion of baseline iPTH values between 100 and 500 pg/ml and who agreed to participate at study was too small, so that the range of iPTH values had to be enlarged secondarily to values comprised between 16 and 1265 pg/ml.
The two treatment arms consisted of haemodialysis treatment for a period of 9 months either with cellulosic membrane (cellulose acetate or cellulose diacetate, CELL group) or with polyacrylonitrile AN-69 membrane (AN-69 group), under otherwise stably maintained dialysis conditions.
All the patients underwent bimonthly local monitoring of plasma total calcium, phosphorus, total protein, iPTH and total alkaline phosphatases. In addition, plasma samples were taken and stored at the start and at time points 6 and 9 months of study (and also at 12 months in Lisbon centre) for subsequent determinations of iPTH, bone-specific alkaline phosphatase, osteocalcin, total and free insulin-like growth factor-I (IGF-I), IGF-II, IGF binding protein (IGFBP)-1 to -6, and cross-laps concentrations. Plasma interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) levels were determined at the start and at time points 6, 9, and 12 months.
In addition, the patients of the Lisbon centre underwent a bone biopsy immediately before the start of study and again at the end of study, which is within a maximum of 2 months after the 9-month treatment period.
Participating patients
In the Amiens centre, 14 Caucasian chronic haemodialysis patients of the initially recruited 20 subjects completed the study, six in the CELL group and eight in the AN-69 group. Among the five patients who left the study prematurely: one died, one moved to another centre and three received kidney grafts. The baseline dialysis treatment schedule during the 6 months prior to study was 3x45 h per week. Oral calcium carbonate supplements were prescribed to the majority of patients in the Amiens centre and the dose was maintained stable unless serum phosphorus increased. Alfacalcidol therapy of 0.25 µg/day or less was given to two patients and maintained stable throughout the 9 month-study period. The dialysate calcium concentration was 1.5 mmol/l in all patients.
In the Lisbon centre, only a total of 10 patients completed the study, five in the CELL group and five in the AN-69 group. It was actually impossible to obtain a total number of 20 patients with repeat bone biopsies at a 9 month interval. Sixteen patients were initially recruited. Six left prematurely: three received kidney transplants, two were transferred to another centre, and one died of myocardial infarction. The baseline dialysis treatment schedule during the 6 months prior to study was 3x4 h per week. The dialysate calcium concentration was 1.5 mmol/l in all patients. Calcitriol therapy of 0.25 µg/day or less was given to one patient and this dose was maintained throughout the 9 month study period. Oral calcium carbonate supplements were prescribed to the majority of the patients and the dose was maintained stable in all of them throughout the study.
Assays
All blood samples were non-fasting morning samples. Plasma total calcium, phosphorous, total alkaline phosphatase, albumin, C-reactive protein (CRP), and transferrin were determined by standard biochemical methods.
Plasma iPTH levels were measured using an immunoradiometric assay (Nichols Institute Diagnostics, Paris, France), with normal values comprised between 10 and 65 pg/ml and intra- and inter-assay coefficients of variation of less than 7%.
To assess bone metabolism more specifically, bone-specific alkaline phosphatase (bAP; Hybritech, Köln, Germany) and osteocalcin (CIS, Dreieich, Germany) were determined by RIA. For quantification of degradation products of C-terminal telopeptides of type I collagen, plasma cross-laps were determined by Cross-laps One Step Elisa kit (Osteometer BioTech, Herlev, Denmark). In a personal study, plasma cross-laps levels were determined in 140 healthy men and 140 healthy women with normal renal function, aged 6080 years. The range of values found in these individuals was 1.255.40 nmol/l for men and 1.357.40 nmol/l for women (unpublished results). In addition, they were also determined in healthy pre-menopausal women aged 4050 years. In these women, the levels were lower, in the range of 0.652.85 nmol/l (unpublished results). Circulating IL-6 and sIL-6R were determined using commercially available kits (Biosource International, CliniSciences, Montrouge, Paris).
Plasma total IGF-I was measured with a new IRMA kit (IGF-I RIACT, Cis Bio, Gif sur Yvette, France), using two monoclonal antibodies directed against two different epitopes of the IGF-I molecule. No extraction step was performed, but the bound IGF-I was displaced from IGFBPs by acidification. An excess IGF-II was then added to the acid-treated serum to prevent re-association of IGF-I with its carrier proteins when buffer was added. In our laboratory, the within-run CV (SD/mean of 10 measurements of three serum pools) was 1.8% at 52 ng/ml, 3.7% at 289 ng/ml, and 4.1% at 676 ng/ml, while the between-run CV (the same three serum pools measured in duplicate over six non-consecutive days) was 5.8% at 56 ng/ml, 4.5% at 299 ng/ml, and 4.1% at 700 ng/ml. The detection limit, defined as the concentration at the mean+3SD of 20 measurements of the zero calibrator, was 1 ng/ml. Serial dilutions of eight sera with high (three acromegalic patients) or medium (five patients with renal failure on haemodialysis) IGF I concentrations gave recovery values of 89.2104.5%.
All the other components of the circulating IGF system were measured as previously described [4]. Free IGF-I levels were determined by a specific RIA (DSL, Frankfurt, Germany) with intra- and inter-assay coefficients of variation of less than 11%. There was no cross-reaction with IGF-II, insulin, proinsulin, and growth hormone. IGF-II was measured by a specific RIA (DSL, Frankfurt, Germany) with intra- and inter-assay coefficients of variation of less than 7.5 and 10.5%. There was no cross-reaction with IGF-I, insulin, proinsulin, and IGFBP-2, -3, -4, and -5. IGFBP-1 was measured by enzyme immunoassay (ELISA, Mediagnost, Tuebingen, Germany) with intra- and inter-assay coefficients of variation of less than 4 and 8% and no cross-reaction with other IGFBPs. IGFBP-2 was measured by a specific RIA with intra- and inter-assay coefficients of variation of less than 9 and 8% and no cross-reaction with IGFBP-1, -3, -4, -5, and -6 (DSL, Frankfurt, Germany). IGFBP-3 was determined by a specific RIA with intra- and inter-assay coefficients of variation of less than 3.5 and 7.5% and no cross-reaction with other IGFBPs (Mediagnost, Tuebingen, Germany). IGFBP-4 and -5 were measured by specific RIA as previously described (see Ref. [3]) with intra- and inter-assay coefficients of variation of less than 10% and no cross-reaction with other IGFBPs. IGFBP-6 was determined by a specific RIA with intra- and inter-assay coefficients of variation of less than 11 and 10% and no cross-reaction with IGFBP-1, -2, -3, -4 and -5 (DSL, Frankfurt, Germany).
Bone biopsy and bone histomorphometry
Two transiliac bone biopsies, using the trephine of Bordier-Meunier with an internal diameter of 8 mm, were obtained in all Lisbon patients, one at the start of study and the other after 912 months, and analysed as reported previously [2]. For bone tetracyclin labelling, patients first received an oral dose of 250 mg doxytetracyclin twice daily for 3 days, then stopped taking tetracyclin for 10 days, and then resumed tetracyclin for another 3 days. Bone biopsy was performed 5 days after the last dose. Normal bone histomorphometric values are from de Vernejoul et al. [5]. The following bone histomorphometric parameters, expressed according to standardized nomenclature, were measured in trabecular bone: trabecular bone volume (BV/TV) expressed as the per cent of trabecular space (normal values 19.3±1.3%); osteoblast surface (Ob.S/BS) expressed as the osteoid surface covered with plump osteoblasts and expressed as the per cent of trabecular bone surface (normal values 5.4±1.9%); osteoclast surface (Oc.S/BS) defined as the fraction of trabecular bone surface covered with osteoclasts (normal values 1.39±0.77%); osteoclast number (N.Oc/mm2) per mm2 of tissue section (normal values 1.11±0.69); aluminium surface (Al.S/BS) defined as the fraction of trabecular bone surface covered by aluminium (normal values, 0%); mineral apposition rate (MAR) (normal values 0.63±0.17 µm/day); mineralizing surface (MS) (normal values 14.2±1.3%); and bone formation rate (BFR) (normal values 0.3±0.05 µm2/µm/day).
Statistical evaluation
Statistics were computed using SPSS. Results have been expressed as mean±SEM. Data were analysed by Student's paired or unpaired t test and one-way analysis of variance (ANOVA), followed by NewmanKeuls test. The analysis was separated for the individual subgroups. Correlations between variables (Pearson's correlation coefficient) were assessed using univariate linear regression analysis. P<0.05 was accepted as statistically significant.
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Results |
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Biochemical markers of bone metabolism
As shown in Table 2, plasma total calcium and phosphorus levels were stable and comparable in all groups throughout the three time periods of study. Moreover, no significant changes in circulating bone-specific markers were observed between both groups. At baseline, iPTH levels were comparable between AN-69 group and CELL group patients and did not significantly change during the follow-up period of 9 months. Similarly, plasma concentrations of bone-specific alkaline phosphatase, osteocalcin, and cross-laps remained unchanged during this observation period.
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IGF system components
In the AN-69 group, plasma IGF-I levels progressively decreased whereas no change was found in the CELL group (Table 2 and Figure 1
). The differing behaviour of plasma IGF-I levels with time between AN-69 and CELL groups observed in the whole patient group was also seen when considering the Lisbon group patients alone, namely a decrease in total IGF-I levels in AN-69-treated patients, but no change in CELL-treated patients (Figure 2
). Table 3
summarizes data on circulating IGF system components in the five AN-69-group patients and the five CELL-group patients of the Lisbon centre, compared with reference values of healthy control subjects. Similar to total IGF-I concentrations, AN-69 patients revealed a moderate decrease in free IGF-I levels reaching significance at 6 months. In contrast with the total and free IGF-I levels, IGF-II levels remained unchanged in both groups. To address the question whether changes in the different IGFBPs may account for the fluctuations in free and total IGF-I levels in the AN-69 but not in the CELL groups, we further determined IGFBP-1 to -6 levels by specific immunoassays. Interestingly, in AN-69 patients plasma levels of IGFBP-1 increased continuously, reaching 2-fold higher levels after 12 months compared with baseline values. Furthermore, IGFBP-2 levels were moderately higher at 12 months whereas IGFBP-5 levels decreased with a nadir at 9 months (1.7-fold lower levels, P<0.05). In contrast, in CELL-treated patients, levels of IGFBP-1 remained unchanged but those of IGFBP-5 significantly increased, with a maximum of 1.6-fold value at 9 months (P<0.05).
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Cytokine and inflammatory parameters
No differences between groups were observed in the change with time of plasma cytokine parameters IL-6 and sIL-6R in the patients of the Lisbon centre except a slightly, significantly (P<0.05) lower IL-6 level in AN-69-group patients than in CELL group patients at the 12 months time point. Plasma IL-6 levels for the 0, 6, 9- and 12-month study time points were 6.6±1.0, 5.9±1.4, 8.3±3.0, and 7.2±0.5 pg/ml in AN-69 group, and 15.1±7.4, 15.7±5.8, 11.7±5.0, and 18.6±5.05 pg/ml in CELL group, respectively. Plasma sIL-6R levels were 81.2±7.9, 69.0±5.6, 68.2±8.9, and 63.8±8.1 ng/ml in AN-69 group, and 85.6±7.0, 77.2±15.3, 77.2±15.3, and 84.0±9.9 ng/ml in CELL group, respectively. Of interest, when comparing all available plasma levels from Lisbon centre patients at all time points (n=40), a significant correlation was found between the plasma levels of sIL-6R and those of osteocalcin (r=0.54, P<0.001), of bone-specific alkaline phosphatase (r=0.60, P<0.002), and of cross-laps (r=0.48, P<0.002).
Bone histomorphometry
At the start of the study, the mean values of the three static parameters of bone formation and bone resorption measured in the 10 patients of the Lisbon centre were elevated compared with values in healthy subjects: osteoblast surface (Ob.S/BS, n=10), 12.4±3.2%; osteoclast surface (Oc.S/BS; n=10), 7.0±2.3%; osteoclast number (N.Oc; n=10), 3.34±0.75/mm2. At the 9 months time point, the values of these bone formation and resorption parameters had significantly decreased in AN-69 group patients compared with baseline; in contrast, same values had significantly increased in CELL group patients. Figure 2 shows the increase and decrease of the three parameters in the former and the latter group, respectively.
Dynamic parameters of bone turnover in these patients are shown in Table 4. Mean MAR and BFR values decreased in AN-69 group by 17 and 31%, respectively, whereas mean MS did not change. In contrast, in the CELL group mean BFR and MS values increased by 50 and 48%; however, MAR remained unchanged. Although several of these changes were marked, none of them reached the level of statistical significance, presumably because of the small sample size.
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Discussion |
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The second important finding of this study was the progressive decrease of total and free plasma IGF-I concentrations in the AN-69 patient group whereas no significant change occurred in the CELL group. This latter observation is in keeping with the well-known role of IGF-I in the modulation of bone turnover, in that high IGF-I levels stimulate bone formation whereas low levels are associated with a reduction of bone turnover [4]. It remains to be established whether the changes in serum levels of IGFBPs are the cause or consequence of the decrease in IGF-I levels in AN-69 group patients. Both IGFBP-1 and -2 are known to inhibit IGF-I action [6] whereas IGFBP-5 plays a key role in regulating bone formation [7]. Clinical studies in patients with metabolic bone diseases revealed that IGFBP-1 and -2 levels are negatively, but IGFBP-5 levels positively correlated with bone mineral density and histological indices of bone formation [4,8]. Our finding of a positive correlation between bone formation rate and plasma IGFBP-5, and a negative relation between bone formation rate and plasma IGFBP-4 in all Lisbon patients together is in agreement with these reports. Of note, no correlation was found between plasma iPTH and any of the IGF system components.
Our observations raise a number of intriguing questions. The first is why there were no concomittant changes of circulating markers considered to reflect bone activity (except the above mentioned changes of IGF system components). One would have expected the same directional changes of these markers as the ones indicated by histomorphometrically assessed bone parameters. One possibility is that circulating markers are less reliable than bone biopsy findings to assess changes of bone turnover in individual patients. This is particularly true in the case of normal or low bone turnover [9]. Another possibility is the small number and the low degree of homogeneity of the present patient population at the start of the study. This, together with the relatively weak sensitivity of available bone turnover markers in the circulation, may have prevented us from observing significant differences when in fact changes of moderate degree did occur. A third possibility is that whereas plasma markers are thought to reflect the average turnover of the entire skeleton bone biopsy can only inform, strictly speaking, about the focal condition at the site of bone tissue sampling. As bone biopsy usually provides better information about spongious bone than about cortical bone, one could speculate that the observed differences in osteoblast and osteoclast numbers and surface between the two groups of patients mainly reflect differences in spongious bone.
We also failed to observe a change of the plasma cytokine IL-6 or its circulating receptor sIL-6R. IL-6, which is produced by osteoblasts, is an established factor of osteoclast activation, independent of the OPG/OPG-L (osteoprotegerin and osteoprotegerin-ligand) system [10]. In our patients, IL-6 was only correlated with CRP (data not shown), indicating its implication in the inflammatory state of uraemia. However, sIL-6R was correlated with markers of bone formation (bAP and osteocalcin) and bone resorption (cross-laps), which is in favour of a role in uraemic osteodystrophy. Circulating levels of cytokines probably do not reflect well the situation at the bone tissue level. The stimulation of other cytokines by cellulosic dialysis membranes, but not by synthetic membranes, and the subsequent interference with cellular and subcellular processes involving the skeletal action of PTH, vitamin D and other hormones and cytokines is also possible.
The second question is why plasma total IGF-I concentrations decreased with highly biocompatible AN-69 membrane, but not with less biocompatible CELL membrane. As the AN-69 membrane is known to adsorb numerous plasma proteins onto its surface, in contrast to cellulosic membrane [11], it is possible that circulating IGF-I is regularly removed during dialysis sessions with the former, but not the latter type of membrane. The results of a recent study appear to plead against such a possibility as no acute change of plasma IGF-1 concentration were observed during a dialysis session with AN-69 membrane [11]. Moreover, plasma IGF-1 is not necessarily modified by other types of biocompatible membranes. Thus, in a previous study aimed at examining the long-term effect of membrane biocompatibility on nutritional parameters, no difference in pre-dialysis serum total IGF-I values was observed between two groups of chronic haemodialysis patients treated for 18 months with either cellulosic membrane or polymethylmethacrylate (PMMA) membrane [12]. However, the physical properties and biocompatibility characteristics of the latter are different from those of AN-69 membrane. Another possibility would be an aggravation in the nutritional status of our patients treated with AN-69 membrane, compared with CELL membrane. However, there was no clinical or biochemical evidence in support of this theory. In contrast, highly biocompatible membranes have been recently shown to offer an advantage over cellulosic membranes in terms of morbidity and mortality [1315].
The third question is what may be the relative importance of the IGF system, compared with other endocrine systems such as the PTHvitamin D axis, for the metabolic activity of the skeleton in uraemic patients. In case of severe secondary hyperparathyroidism, high bone turnover is mainly a result of this cause and associated conditions such as insufficient calcium intake and vitamin D deficiency. However, at present parathyroid overfunction is generally better controlled than 1020 years ago, and other factors may come into play which were previously less important or simply neglected. Thus, it has become clear that bone turnover is regulated by numerous factors other than calcium, phosphate, PTH, and vitamin D. The role of numerous growth factors and cytokines in the regulation of bone metabolism has been progressively elucidated during recent years. Disturbances of the activity of several of these factors may play a role in uraemic bone disease as well [16]. To what extent such disturbances may be reflected by changes in circulating concentrations of such factors or only by local changes in concentration or activity, remains to be seen. The IGF system is one of them, and changes of circulating IGF and IGFBPs such as plasma IGFBP-4 and IGFBP-5 have been shown to be associated with abnormal bone turnover in uremia [4,17]. Thus, total and free IGF-I was lower in haemodialysis patients with osteopenia than in those without osteopenia [4], in accord with other studies in osteoporosis patients without renal failure [8,18,19]. Recently, Garnero et al. reported that in 435 healthy post-menopausal women decreased serum concentrations of IGF-I were strongly associated with an increased risk of osteoporotic fractures independently of bone mineral density [20]. In this study, however, serum IGF-I concentrations explained less than 6% of the inter-individual variance of bone mineral density at any skeletal sites. Studies in smaller groups of renal failure patients found no relationship between IGF-I levels and bone metabolism. Coen et al. failed to identify a correlation between plasma IGF-I levels and bone formation parameters in pre-dialysis patients [21]. In dialysis patients, Weinreich et al. were unable to find a difference in plasma IGF-I, IGF-II, IGFBP-2, or IGFBP-3 levels between two groups having either severe secondary hyperparathyroidism or adynamic bone disease [22]. The conflicting results of the links between serum IGF-I and bone metabolism could be because of different underlying metabolic diseases such as diabetes or obesity and differences in nutritional status [23, 24]. It remains to be determined whether IGFBP-5 is a more sensitive bone marker than IGF-I concentrations.
In conclusion, dialysis with CELL membrane was associated with stimulation of bone turnover whereas the use of AN-69 membrane slowed down bone cell activity, suggesting a beneficial effect of the latter on uraemic bone disease. However, the issue whether the observed association between the decrease in plasma IGF-I and the reduction of histologic parameters of bone formation and resorption is causal or coincidental requires further study. Moreover, the results of the present work need to be confirmed in a larger patient population.
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Acknowledgments |
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Notes |
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References |
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