Parathyroid hormone-independent osteoclastic resorptive bone disease: a new variant of adynamic bone disease in haemodialysis patients

Anca Gal-Moscovici and Mordecai M. Popovtzer

Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Osteitis fibrosa cystica (OFC) caused by secondary hyperparathyroidism is the pre-eminent form of uraemic osteodystrophy. In recent years, however, new bone abnormalities have been described. Among them adynamic bone disease (ABD) has become a focus of growing interest. Marked suppression of dynamic bone measurements with normal or near-normal static bone-forming parameters are the hallmarks of this disorder. Depressed parathyroid hormone (PTH) levels, frequently evident in this entity, have been linked causally with low bone turnover.

Methods. We reviewed bone biopsy specimens from 96 patients with end-stage renal disease undergoing chronic haemodialysis.

Results. We found OFC in 50% of our patients, 20% had mixed bone disease, 24% showed bone morphology of ABD and a minority (6%) had osteomalacia, mostly due to aluminium accumulation. In the patients that were affected by ABD there was a distinct subgroup with bone morphology featuring a striking increase in osteoclast number and osteoclast surface, whereas the osteoid volume, osteoid thickness, osteoblast surface, tetracycline uptake and bone formation rates were diminished as in ordinary ABD. Similarly the PTH levels in this subgroup were low or undetectable.

Conclusion. We describe patients undergoing chronic haemodialysis with static and dynamic bone forming parameters, indistinguishable from that of ABD, but differing from the classic ABD by the presence of increased osteoclastic bone resorption. The suppressed PTH levels in this subgroup suggests that factors other than PTH activate osteoclasts in some patients on chronic haemodialysis. Uraemic cytokines and/or toxic metabolites, including ß-microglobulin, may be involved in this disorder. The precise nature of this bone abnormality remains to be defined by further studies.

Keywords: adynamic bone disease; ß2-microglobulin; bone histomorphometry; bone disease in dialysis; cytokines; renal osteodystrophy



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Renal osteodystrophy, the skeletal complication of end-stage renal disease, is a multifactorial disorder of bone remodelling.

The classic histological form of renal osteodystrophy is osteitis fibrosa cystica (OFC), which is attributed to secondary hyperparathyroidism [1,2]. 1{alpha},25-dihydroxycholecalciferol (1{alpha},25(OH)2D3) deficiency as well as phosphate retention, have been implicated as a major factor in the pathogenesis of secondary hyperparathyroidism. Additional factors, however, including various cytokines, may also play a role in this bone disorder [2,3]. Langub et al. [4] have demonstrated that interleukin-6 is involved in the bone pathology in this form of uraemic osteodystrophy.

Therapeutic regimens aimed to reduce parathyroid hormone (PTH) secretion by the administration of 1{alpha},25(OH)2D3, on one hand decreased the frequency and the severity of osteitis fibrosa, but on the other hand contributed to the pathogenesis of other forms of renal osteodystrophy and particularly to the pathogenesis of adynamic bone disease (ABD) [5,6].

ABD is most commonly described in patients with end-stage renal disease undergoing chronic haemodialysis, with relatively suppressed parathyroid function [7]. Excessive suppression of parathyroid gland activity may be secondary to overzealous employment of calcium and vitamin D derivatives or surgical ablation of parathyroid tissue. Histologically the bone picture in these patients is characterized by absence of osteoblastic and osteoclastic activity [8].

Various cytokines such as interleukin-1, tumour necrosis factor {alpha}, interleukin-6 and interleukin-11 as well as their soluble receptors have been recently considered as factors participating in the pathogenesis of renal osteodystrophy. Imbalance between stimulators and inhibitors of bone remodelling, have been proposed to play a role in the pathogenesis of ABD. In this regard, overproduction of ß2 microglobulin (ß2M), nitric oxide or deficiency of bone morphogenic protein may also adversely influence bone turnover [911].

In the present study we reviewed the histomorphometric data of bone biopsies performed in patients undergoing chronic dialysis. Emerging from this analysis is a variant of ABD in a subgroup of our patients with low PTH level. This variant of adynamic bone is characterized by suppressed bone formation but increased osteoclastic bone resorption.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Bone specimens were obtained from 96 patients (53 males, 43 females; age range 10–65 years) undergoing chronic haemodialysis for a mean (±SD) 6±2.8 (range 3–18 years).

The patient samples were drawn from a wide haemodialysis population. CAPD patients and patients taking steroids were not included. The patients included in the study presented a variety of clinical symptoms or laboratory abnormalities, including non-specific musculo-skeletal problems, spontaneous fractures, hypercalcaemia, excessive PTH or suspected aluminium overload. All patients were evaluated between 1994 and 1999.

Bone biopsies and bone histomorphometry
Prior to bone biopsy the patients received a double tetracycline labelling of bone. The labelling schedule consisted of a 3-day oral administration of tetracycline hydrochloride (250 mg t.i.d.) followed by a free interval of 17 days and subsequently a 3-day oral administration of Declocycline (300 mg t.i.d.). Bone biopsy was performed 3 days thereafter. Bone samples were taken from the anterior iliac crest using an 8 mm Bordier trephine biopsy needle. Iliac bone samples were fixed in Carson-Milloning's solution, dehydrated in ethanol and embedded in methymethacrylate. Serial sections of 5 µm and 10 µm thickness were cut with a microtome, model FinesseTM Microtomes Shandon. Sections (5 µm thick) were stained with modified Masson. Unstained sections (10 µm thick) were prepared for fluorescence light microscopy. Separate sections were also stained with aurin tricarboxylic acid for the identification of aluminium and Prussian blue for the detection of iron.

Measurements of bone mass were performed at a x20/0.4 magnification. An automatic image analyser (Leica 2500 IW-F) equipped with a JVC camera and coupled with a Leica orthoplan microscope was used. Measurements of bone parameters and nomenclature correspond to those established by the committee of the American Society of Bone and Mineral Research. Seventy-five fields were measured for each biopsy.

Results were compared with histomorphometric parameters of healthy control subjects. The reference values are those of Malluche and Faugere [12].

The following bone parameters were studied: osteoblast surface Ob.S/BS, the percentage of trabecular surfaces covered by osteoblasts; osteoid surface OS/BS, the percentage of trabecular surface covered by osteoid; osteoid volume OV/BV, the percentage of trabecular bone volume occupied by osteoid; osteoclast surface Oc.S/BS, the percentage of trabecular surface corresponding to the eroded trabecular surface covered by osteoclasts; osteoclast number Oc.N/TA, the number of osteoclasts per bone area is the ratio of the number of osteoclast per area unit of bone tissue; osteoid thickness (O.Th) was measured at equidistant points on trabecular bone and expressed in microns; eroded surface (ES/BS), the percentage of trabecular surface covered by lacunae (including ‘active’ lacunae with osteoclasts and lacunae in reversal phase, i.e. the period in which the resorption lacunae are filled with inactive-looking mononuclear cells); double-labelled tetracycline surface (DL.S/BS), the percentage of trabecular surface covered by double-labelled tetracycline; mineralized surface (MS/BS) is given by the total extent of double labels plus half the extent of single labels; bone formation rate (BFR/BS) corresponds to the amount of new bone mineralized at the tissue level per µm2 of trabecular bone surface area per day, and was calculated using the formula MS/BSxMAR (µm3/µm2/day=BFR/BS) appositional rate; mineral appositional rate (MAR µm/day) obtained by measuring the mean distance between tetracycline labels and dividing by the number of days between doses of tetracycline.

The following classifications of renal osteodystrophy were made: osteitis fibrosa, if bone formation was increased (BFR >0.93 µm3/µm2/day) and if there was significant marrow fibrosis (>0.5% of trabecular surface covered by fibrosis); osteomalacia, if bone formation was low and there was an excess of unmineralized osteoid (osteoid thickness >9 µm); mixed bone disease was defined as an association of low to normal BFR excess osteoid and marrow fibrosis; adynamic bone if osteoid was reduced, no marrow fibrosis and low bone formation was present (lower than the lowest value seen in normal individuals).

Hormonal determinations
PTH levels were measured using a N-tactTM PTH SP IRMA-kit for quantitative determination of biologically active intact h PTH 1–84 in serum by immunoradiometric assay (Dia-Sorin-Stillwater, Minnesota, USA). ß2M was measured by ELISA, Abbot Automatic, normal range <2000 µg/ml.

Statistical analysis
All results are presented as means±standard error. Statistical analysis of the data was done using one-way analysis of variance. P value <0.05 was considered significant. Additionally the different groups defined on the basis of histological results were compared using the Mann-Whitney U-test.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The distribution of the different forms of renal osteodystrophy in our patients is shown in Figure 1Go. The majority (50%) showed high turnover bone disease compatible with OFC. One quarter (24%) of the patients showed a bone picture of ABD characterized by acellular trabeculae and low turnover rate. Mixed bone disease (MBD) was found in 20% of the patients whereas a minority of patients (6%) showed features of osteomalacia (OM), mostly due to aluminium accumulation.



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Fig. 1.  Histological classification and distribution of renal osteodystrophy in 96 patients undergoing chronic haemodialysis. OFC, osteitis fibrosa cystica; MBD, mixed bone disease; OM, osteomalacia; ABD, adynamic bone disease.

 
When we examined the group of patients manifesting ABD (Figure 2Go) we observed that approximately one quarter of these patients showed suppressed bone forming parameters similar to all other patients in this group, but differed from others by the presence of enhanced active bone resorption. This group we named adynamic bone disease-variant form (ABD-V).



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Fig. 2.  Patients affected by ABD. A new ABD variant was observed in 26% of the patients showing typical ABD. ABD, adynamic bone disease.

 
Table 1Go presents the static bone-forming parameters of the patients with a bone picture of ABD-V, typical ABD and OFC and compared with individuals with normal renal function (normal).


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Table 1.  Static bone forming parameters of the different forms of renal osteodystrophy found in our patients and compared with normal values. ABD-V, adynamic bone disease variant; ABD, adynamic bone disease; OFC, osteitis fibrosa cystica

 
Osteoid volume, osteoid surface and osteoblastic surface, were significantly suppressed in the ABD-V group when compared to the OFC and normal groups, but were similar to those found in patients affected by typical ABD. The bone volume in the ABD-V group was higher than that in the ABD group, but the difference was not statistically significant. It is of interest that all patients in the ABD-V group had a history of secondary hyperparathyroidism and had undergone subtotal or total parathyroidectomy some years before the performance of bone biopsy, whereas the majority of patients with typical ABD were never affected by secondary hyperparathyroidism. It is noteworthy that only four patients in this group (typical ABD) had previously shown secondary hyperparathyroidism that was reversed with conservative treatment.

The dynamic bone-forming parameters are shown in Table 2Go. Bone formation was suppressed in the two ABD groups when compared to values in the normal and in the OFC group (P<0.005).


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Table 2.  Dynamic bone forming parameters of the different forms of renal osteodystrophy found in our patients and compared with normal values. ABD-V, adynamic bone disease variant; ABD, adynamic bone disease; OFC, osteitis fibrosa cystica

 
In the ABD group we could distinguish two subgroups according to bone resorbing parameters. In one subgroup the osteoclastic surface and number (Table 3Go) were close to zero, indicating absence of osteoclastic bone resorption characteristic of classical ABD. The other subgroup that we subsequently named ABD-V showed a significant increase in osteoclastic activity. In this subgroup there was a twofold increase in osteoclastic number and osteoclastic surface when compared with values of normal bone. The PTH level in the patients affected by ABD-V was as low as that observed in patients with typical ABD (PTH in ABD-V 3.03±0.49 pmol/l, in typical ABD 1.4±0.2 pmol/l, normal range 2–6 pmol/l), whereas patients with OFC showed significantly higher PTH levels of 63±4.5 pmol/l (P<0.0025 OFC vs ABD, ABD-V). The duration of haemodialysis treatment was significantly longer (P<0.05 ABD-V vs ABD and OFC) in the ABD-V group 11.34±1.3 (range 8–16 years) when compared with patients having typical ABD 5.12±1.8 (range 3–8 years) and OFC 6.8±1.54 (range 4–9 years). Similarly, the ß2M level (36000 µg/ml) was significantly higher in the ABD-V group (P<0.025 ABD-V vs ABD, OFC) than that in the OFC and ABD groups (19000 µg/ml, normal range <2000 µg/ml).


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Table 3.  Resorbing bone parameters in the different forms of renal osteodystrophy found in our patients and compared with normal values. ABD-V, adynamic bone disease variant; ABD, adynamic bone disease; OFC, osteitis fibrosa cystica

 



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The distribution of the different forms of renal osteodystrophy found in our patients undergoing chronic haemodialysis shows that ABD is the second in magnitude after OFC.

Absence of secondary hyperparathyroidism was a common feature in our patients who presented with ABD. Reduced parathyroid activity in haemodialysis patients has been associated with overtreatment with calcium salts, vitamin D, the presence of diabetes or following parathyroidectomy. In our patients presenting with ABD and suppressed PTH levels, the histological picture with regard to bone resorbing parameters was not uniform. A subgroup of these patients showed a bone picture different from that which we observed in the classical ABD by the presence of increased osteoclastic bone resorption. This subgroup of patients also differed from others with ABD by a longer duration of dialysis treatment and higher levels of serum ß2M.

An increased osteoclastic bone resorption in the absence of parathyroid hyperactivity is not a common finding in patients with renal bone disease. A similar finding was documented by Lafage-Proust et al. [13] in a report of non-dialysed uraemic patients maintained on a diet with severe phosphorus restriction. This finding implies that factors other than PTH may play a role in bone resorption in patients undergoing chronic haemodialysis. The nature of these factors remains to be determined. Theoretically among factors involved in the activation of osteoclastic bone resorption, in the ABD-V subgroup ß2M could play an essential role [14]. Long-term haemodialysis treatment has been shown to be associated with ß2M deposition in the skeletal system. This abnormality is not apparent until after 5 years of haemodialysis.

The accumulation of ß2M is due to a combination of increased synthesis, lack of removal and conditions that favour its polymerization [1517]. During the dialysis session ß2M, which is expressed on the surface of all nucleated cells, is released from intragranular stores of degranulating granulocytes. In addition, complement activation products C5a and C56-9 lead to increased transcription, synthesis and release of ß2M by granulocytes [18]. It is noteworthy that in addition to the increased synthesis of ß2M induced by the bioincompatible dialysis membrane, its accumulation is also due to the loss of its excretion by the native kidneys in patients with end-stage renal disease [16,18].

Sprague and Popovtzer [10] showed already in 1992 that ß2-M induces a dose- and time-dependent cell-mediated calcium efflux from neonatal mouse calvariae that involves osteoclast stimulation. Later, a study conducted by Petersen and Kang [14] showed in vivo that ß2M causes bone resorption in doses that may be achieved in end-stage renal disease. This occurred without any regulation of osteoblast activity. However, as there is no statistical correlation between the serum concentration of ß2M and the occurrence of skeletal amyloidosis, its pathogenesis cannot be explained entirely by an increase in the serum ß2M level [17]. In this regard, recent studies concerning the composition of ß2M isolated from patients undergoing long-term dialysis show that the dominant constituent of amyloid is generated by the modification of ß2M with AGEs by the Maillard reaction (AGE-ß2M) [19]. Miyata et al. [20] showed that AGE-ß2M but not normal ß2M stimulated macrophages to secrete inflammatory cytokines such as TNF-{alpha} and IL-1ß.

Both TNF-{alpha} and IL-1ß stimulate bone resorption by activating the osteoclasts. Both TNF-{alpha} and IL-1ß potently stimulate monocytes/macrophages and osteoblast to secrete IL-6, which in turn induce the differentiation of osteoclasts from precursors. Furthermore, both TNF-{alpha} and IL-1ß inhibit bone formation. These findings suggest that AGE-ß2M functions as a stimulator of bone resorption and as an inhibitor of bone formation [19,20].

Based on the above consideration we propose that in our subgroup of patients undergoing long-term dialysis, with suppressed PTH levels, the bone abnormality of the variant form of ABD showing suppressed bone forming parameters but increased osteoclastic resorption, can be explained by the involvement of AGE-ß2M in their bone remodelling. As alluded to above, AGE-ß2M acts as osteoclastic stimulator and osteoblast inhibitor.

During the dialysis session the contact of blood with the dialysis membrane activates the polymerization of ß2M and its glycation, thus increasing the production of AGE-ß2M. AGE-ß2M activates the macrophage-monocytic cells to secrete potent osteoclastogenic cytokines TNF-{alpha} and IL-6 which are responsible for the increased osteoclastic bone resorption. In addition, there is a concomitant secretion by the same cells of IL-1ß, which together with TNF inhibit osteoblastic function, leading to an arrest of bone formation. This entire process is maintained and perpetuated by the products of matrix degradation. This theoretical proposal, however, needs further investigation.

Undoubtedly, long-term dialysis plays a potential role in the pathogenesis of AGE-ß2M-related complications. We suggest that in the context of ABD, the bone picture of suppressed formation and increased osteoclastic resorption labelled by us as a variant form of ABD, may represent a new entity of renal osteodystrophy possibly related to AGE-ß2M action.



   Notes
 
Correspondence and offprint requests to: Gal-Moscovici, MD, Nephrology and Hypertension Services, Hadassah University Hospital, PO Box 12000, Jerusalem 91120, Israel. Email: galanca{at}hadassah.org.il Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 29.11.00
Accepted in revised form: 27.11.01





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