Downregulation of parathyroid hormone receptor gene expression and osteoblastic dysfunction associated with skeletal resistance to parathyroid hormone in a rat model of renal failure with low turnover bone

Yoshiko Iwasaki-Ishizuka1, Hideyuki Yamato2, Tomoko Nii-Kono3, Kiyoshi Kurokawa4 and Masafumi Fukagawa3

1 Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, 870-1201, 2 Kureha Special Laboratory Co., Ltd, Fukushima, 974-8232, 3 Division of Nephrology and Dialysis Center, Kobe University School of Medicine, Kobe, 650-0017 and 4 Institute of Medical Sciences, Tokai University School of Medicine, Kanagawa, 259-1193, Japan

Correspondence and offprint requests to: Masafumi Fukagawa, MD, PhD, Division of Nephrology and Dialysis Center, Kobe University School of Medicine, Kobe, 650-0017, Japan. Email: fukagawa{at}med.kobe-u.ac.jp



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Adynamic bone disease (ABD), which is characterized by reduced bone formation and resorption, has become an increasingly common manifestation of bone abnormalities in patients with end-stage renal failure. It has been recognized that skeletal resistance to parathyroid hormone (PTH) underlies the pathogenesis of ABD; however, the mechanisms of such resistance remain unclear.

Methods. We established a rat model simulating ABD under chronic renal failure conditions by thyroparathyroidectomy and partial nephrectomy (TPTx-Nx). TPTx-Nx rats were infused subcutaneously with a physiological dose of PTH. We analysed bone histomorphometric parameters and demonstrated gene expression using semi-quantitative reverse transcription–polymerase chain reaction.

Results. Reduced bone formation was observed in this model, simulating ABD. The reduction was dependent on the degree of renal dysfunction. Bone formation rate was 6.4±2.7 µm3/m2/year in TPTx-5/6Nx rats and 22.7±7.2 µm3/m2/year in TPTx rats (P<0.05). Osteoblast surface was also significantly depressed (P<0.05) in TPTx-5/6Nx (3.8±2.7%) compared with TPTx rats (15.9±8.6). The expression of PTH/parathyroid hormone-related peptide (PTHrP) receptor and alkaline phosphatase genes was reduced significantly in TPTx-Nx compared with TPTx rats (P<0.05). Reduced bone formation in TPTx-Nx rats was ameliorated by intermittent injection of pharmacological doses of PTH.

Conclusions. Renal dysfunction without secondary hyperparathyroidism induces osteoblast dysfunction and reduces bone formation. Skeletal resistance to PTH develops in renal failure even at low or normal PTH levels, possibly through downregulation of PTH/PTHrP receptor and dysfunction of osteoblasts.

Keywords: adynamic bone disease; animal model; bone histomorphometry; renal failure; skeletal resistance to parathyroid hormone



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients with end-stage renal disease develop various kinds of abnormalities in bone and mineral metabolism, widely known as renal osteodystrophy. Renal osteodystrophy is classified into several types depending on the status of bone turnover [1,2]. Until recently, high turnover bone disease due to enhanced parathyroid hormone (PTH) secretion from hyperplastic parathyroid glands was the main cause of renal osteodystrophy. For the treatment of this abnormality, many new therapeutic modalities to suppress PTH secretion have been developed. However, to the surprise of most nephrologists, normalization of PTH levels in these patients leads to adynamic bone disease (ABD) with reduced bone formation and resorption [3]. It has been suggested that PTH levels 2–3 times greater than normal are needed to maintain normal bone turnover under uraemic conditions. Recently, there has been an increase in the prevalence of ABD in dialysis patients [4], and low PTH levels are frequently seen in chronic renal failure patients with ABD [4]. In addition, it has been reported that ABD is associated with increased fracture rate [5], brittle calcium control [6] and increased vascular calcification [7]. Since vascular calcification is currently recognized as a risk factor for mortality in patients with end-stage renal disease [8], elucidating the mechanisms underlying the development of ABD is essential to improve quality of life and prognosis.

Low bone turnover despite a normal PTH level suggests skeletal resistance to PTH in a uraemic state, which was originally proposed as a cause for the blunted calcaemic action of PTH. Although several mechanisms have been suggested for this abnormality [9], nevertheless the pathogenesis of ABD has not been fully elucidated, mainly due to a lack of appropriate rat models and in vitro systems simulating this abnormality.

To clarify the pathogenesis of skeletal resistance to PTH in ABD, we have established a rat model simulating ABD under uraemic conditions and attempted to analyse bone turnover in these rats.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All animal experiments were approved by the Animal Care and Use Committee of the Biomedical Research Laboratories, Kureha Chemical Industry Co., Ltd.

Rat model of chronic renal failure without secondary hyperparathyroidism
Male Sprague–Dawley rats (Nippon Bio Supply Center Co., Ltd, Tokyo, Japan) were purchased at 9 weeks of age and housed in individual cages at constant room temperature with a 12 h light and dark cycle. The protocol is outlined in Figure 1. Following an acclimation period of 7 days, rats underwent thyroparathyroidectomy (TPTx) by blunt dissection under pentobarbital anaesthesia. Serum calcium concentrations were measured before and 2 days after surgery. A decrease in serum calcium concentration of ≥2 mg/dl from the pre-operative level was taken as evidence of successful extirpation of the parathyroid glands. One week after TPTx, the rats underwent two-stage 1/2, 3/4 or 5/6 subtotal nephrectomy (Nx) at 1 week intervals to produce chronic renal failure. All the TPTx rats with or without Nx received continuous infusion of rat 1–34 PTH (0.1 µg/kg/h, Peninsula Laboratories, Talyo Way, San Carios, CA) using a subcutaneously (s.c.) implanted Alzet osmotic mini pump (Model 2002; Alza Corp., Palo Alto, CA; pumps exchanged every 2 weeks), and subcutaneous L-thyroxine (Sigma Chemical Company, St Louis, MO) 4 µg/kg body weight three times per week, beginning on the second day after TPTx. Administration was continued until completion of the study. Six rats with normal thyroid–parathyroid function and renal function underwent a sham operation. From the beginning of the study up to second stage nephrectomy, the animals were fed on a normal diet containing 0.6% calcium. From completion of the second nephrectomy to the end of study, the animal feed was switched to a diet containing 2% calcium and 1% phosphorus (TD-92095, Harlan Teklad, Madison, WI). The amount of food consumption was equalized among groups by pair-feeding; the amount of food given to TPTx and sham rats was adjusted every day to the amount consumed by TPTx-Nx rats on the previous day. Water was provided ad libitum. Animals were weighed each week throughout the experiment. A 24 h urine sample was collected from each rat using a metabolic cage before sacrifice. Six weeks after the second Nx, the rats were sacrificed (without fasting on the previous day) and the right tibiae were removed for histomorphometric study.



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Fig. 1. Experimental protocol for producing a rat model of chronic failure without hyperparathyroidism. Sham = sham-operated rats; TPTx = thyroparathyroidectomy; Nx = nephrectomy; TPTx-1/2Nx = thyroparathyroidectomized and 1/2 nephrectomized rats; TPTx-3/4Nx = thyroparathyroidectomized and 3/4 nephrectomized rats; TPTx-5/6Nx = thyroparathyroidectomized and 5/6 nephrectomized rats.

 
Effect of intermittent PTH injection on bone formation in TPTx-Nx rats
Forty-five male Sprague–Dawley rats underwent TPTx with or without 5/6 Nx as described. Six weeks after the second nephrectomy, TPTx-5/6 Nx rats were divided into four groups of six animals each. Three groups were treated intermittently with rat 1–34 PTH at doses of 10, 30 and 90 µg/kg given s.c. three times per week. One group was injected with saline as vehicle control. All rats received continuous s.c. infusion of rat 1–34 PTH and s.c. L-thyroxine as described above. After 4 weeks, all the rats were sacrificed and the right tibiae were removed for histomorphometric study (Figure 2).



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Fig. 2. Experimental protocol of intermittent PTH treatment in TPTx-Nx rats. TPTx-Nx rats were divided into four groups 6 weeks after the second nephrectomy, and were given PTH or vehicle injection three times a week for 4 weeks. Vehicle = saline injection; PTH 10 µg/kg = rat 1–34 PTH 10 µg/kg injection; PTH 30 µg/kg = rat 1–34 PTH 30 µg/kg injection; PTH 90 µg/kg = rat 1–34 PTH 90 µg/kg injection.

 
Serum and urine biochemistry
Serum samples were stored at –70°C until biochemical or hormonal assays. Urine samples were stored at –20°C for later analyses. Serum calcium, phosphorus, alkaline phosphatase activity and urinary calcium were measured using an autoanalyser (Hitachi 736, Hitachi Co. Ltd, Hitachi-City, Japan). Serum and urine creatinine concentrations were determined using Wako kit 277-1050 and urea nitrogen levels were measured using Wako kit 279-36201 (Wako Pure Chemicals, Tokyo, Japan). Serum PTH levels were measured with an immunoradiometric assay kit for rat PTH (Immutopics, San Clemente, CA). Serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were measured by a radioreceptor assay using vitamin D receptors derived from calf thymocytes.

Bone histomorphometry
Fourteen and 7 days prior to sacrifice, all animals were injected s.c. with calcein (8 mg/kg body weight, Wako Pure Chemical Industries, Osaka, Japan) for labelling. Rats were sacrificed by exsanguination via cardiac puncture. Blood samples were collected for subsequent biochemical determinations. The right proximal tibia was removed from each rat, fixed in 70% ethanol and embedded in glycol-methacrylate (Wako Pure Chemical Industries) without decalcification. Then, serial sections (5 µm in thickness) were cut longitudinally using a microtome (Model 2050; Reichert Jung, Buffalo, NY), and the sections were stained further with toluidine blue O to discriminate between mineralized and unmineralized bone and to identify cellular components. Unstained sections (5 µm) were used to visualize calcein labelling under a fluorescent light microscope. Histomorphometric analysis of secondary spongiosa of the proximal metaphysis between 1.2 and 3.6 mm distal to the growth plate–epiphyseal junction was performed using a semi-automated system (Osteoplan II; Carl Zeiss, Thornwood, NY), and measurements were made at 200x magnification. Dynamic parameters were determined as follows. Single-labelled and double-labelled surfaces as well as total bone surface (BS) in the secondary spongiosa were traced at 200x magnification. Then, single-labelled surface (sLS/BS, %) and double-labelled surface (dLS/BS, %) were calculated as a percentage of the total bone surface. Labelling width was determined as the average distance between the double labels. Mineral apposition rate (MAR, µm/day) was calculated by dividing the labelling width by the number of days between the two calcein administrations. Bone formation rate per bone surface (BFR/BS, µm3/µm2/year) was the product of (sLS/2 + dLS) xMAR/BS. Parameters for bone resorption were determined as follows. Trabecular osteoclast surface (Oc.S/BS, %) and eroded surface (ES/BS, %) were determined. The nomenclature, symbols and units used in this study are those recommended by the American Society for Bone Mineral Research (ASBMR) Nomenclature Committee [10].

RNA extraction and reverse transcription–polymerase chain reaction (RT–PCR)
After sacrifice, the left tibia was removed. The proximal metaphysis was separated from the diaphysis. The bone was flushed with phosphate-buffered saline to remove all the bone marrow and then frozen in liquid nitrogen. Left proximal metaphysis and diaphysis of the tibia without the marrow were ground to a fine powder using a mortar and pestle under liquid nitrogen in RNase-free conditions. Total RNA was extracted using an ISOGEN kit (Wako Pure Chemicals Industries) following the manufacturer's instructions. Total RNA (2 µg) from bone powder was used as the template for cDNA synthesis in a 20 µl volume using an RT–PCR kit (Takara Shuzo, Shiga, Japan) according to the manufacturer's instructions. The reaction mixture contained 1 U/µl Moloney murine leukaemia virus reverse transcriptase; 1x RNA PCR buffer; 5 mM MgCl2; 250 µM each of dATP, dCTP, dGTP and dTTP; and 1 µM random 9mer oligonucleotide. The mixture was incubated at 30°C for 10 min, 42°C for 30 min and 100°C for 5 min, and finally stored at 4°C. The PCR mixture totalled 50 µl and contained 1 µl of cDNA, 0.05 U/µl native Pfu DNA polymerase (Clontech, Palo Alto, CA), 20 mM Tris–HCl (pH 8.0), 2 mM MgCl2, 6 mM (NH4)2SO4, 10 mM KCl, 0.1% Triton X-100, 10 µg bovine serum albumin, 200 µM dNTP and 0.5 µM each of forward and reverse oligonucleotide primer. PCRs were carried out using various primers (Table 1). ß-Actin was used as an internal control for efficacy of the extraction, reverse transcription and PCR steps. The thermal cycling protocol was 95°C for 50 s, 60°C for 50 s and 72°C for 1 min, for 32 cycles (Thermal Cycler, Takara Shuzo). The amplification reaction products (10 µl) were analysed using agarose gel electrophoresis and visualized by ethidium bromide staining. Semi-quantitative analysis was performed using NIH-Image version 1.62.


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Table 1. PCR primer sequences and their product sizes

 
Statistical analysis
All data are expressed as the mean±SD. Means of groups were compared by analysis of variance (ANOVA), and significance of difference was determined by post hoc testing using Fisher's PLSD test.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Renal function and bone turnover in TPTx-Nx model rats
Although there was no difference in the amount of food consumed among all groups (average intake: 23.8 g/day), TPTx-Nx rats had significant lower body weights compared with TPTx rats. The degree of decrease in body weight was dependent on renal function (570.3±12.5 g in sham rats, 568.5±20.3 g in TPTx rats, 494.0±22.5 g in TPTx-1/2Nx rats, 480.6±38.0 g in TPTx-3/4Nx rats, 420.6±51.4 g in TPTx-5/6Nx rats). The groups did not differ in serum albumin concentration (data not shown). Significant increases of serum creatinine, urea nitrogen and inorganic phosphate were observed in TPTx-Nx rats (Table 2). Creatinine clearance was significantly decreased in TPTx-Nx rats compared with TPTx rats. The changes of these parameters were dependent on the extent of nephrectomy. Urinary calcium excretion (mg/24 h) was 0.36±0.06 in intact, 0.34±0.10 in TPTx, 0.47±0.17 in TPTx-1/2Nx, 0.54±0.14 in TPTx-3/4Nx and 0.63±0.16 in TPTx-5/6Nx rats. In contrast, there were no significant differences in serum calcium, 1,25(OH)2D3 and PTH concentrations among these groups, whereas 1,25(OH)2D3 appeared slightly decreased in the TPTx-5/6Nx group.


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Table 2. Creatinine clearance and serum biochemical parameters

 
The parameters of bone turnover are summarized in Table 3. The TPTx-5/6Nx group showed suppressed osteoblast surface, osteoid surface and mineralized surface compared with TPTx groups. The bone formation rate was reduced to 72% of that of the TPTx group. The decline of the bone formation rate depended on the degree of renal dysfunction. In contrast, parameters of bone resorption such as Oc.S and ES/BS were significantly decreased only in the TPTx-5/6Nx group. The changes in parameters of bone resorption were slight compared with the parameters of bone formation. There were no differences in serum parameters and bone histomorphometric parameters between sham-operated groups and the TPTx group. The growth plate did not differ among all the groups.


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Table 3. Histomorphometric analysis

 
Effect of intermittent PTH treatment
Intermittent injections of pharmacological doses of PTH ameliorated the decreased bone formation in TPTx-Nx rats (Figure 3). Bone formation rate increased in a dose-dependent manner. On the other hand, bone resorption parameters did not change following intermittent PTH injection (data not shown).



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Fig. 3. Effect of intermittent PTH treatment on bone formation. (A) Osteoblast surface per bone surface (Ob.S/BS); (B) osteoid surface per bone surface (OS/BS); (C) mineralized surface per bone surface (MS/BS); (D) bone formation rate (BFR/BS). TPTx rats, vehicle = TPTx-Nx rats given saline injection; PTH 10 = TPTx-Nx rats given rat 1–34 PTH 10 µg/kg injection; PTH 30 = TPTx-Nx rats given rat 1–34 PTH 30 µg/kg injection; PTH 90 = TPTx-Nx rats given rat 1–34 PTH 90 µg/kg injection. *P < 0.05 vs TPTx rats; #P < 0.05 vs vehicle rats.

 
Gene expression of PTH/PTHrP receptor and bone metabolic markers
To determine the effect of renal dysfunction without secondary hyperparathyroidism on gene expression, we performed RT–PCR using RNA samples extracted from the proximal metaphysis and diaphysis of tibia separately. The expression of parathyroid/parathyroid hormone-related peptide (PTH/PTHrP) receptor in the proximal metaphysis decreased significantly dependent on renal dysfunction in TPTx-Nx rats compared with TPTx rats, and the expression of the alkaline phosphatase (ALP) gene was also reduced in TPTx-Nx rats (Figure 4a–c). On the other hand, the expression of the PTH/PTHrP receptor and ALP gene in tibial diaphysis did not change in all groups (data not shown).



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Fig. 4. (a) Expression of osteoblastic regulatory genes. RNA was extracted from proximal metaphysis of rat tibia and subjected to RT–PCR followed by gel electrophoresis. Upper panel: PTH/PTHrP receptor. Middle panel: alkaline phosphatase. Lower panel: ß-actin. Lane 1, sham-operated rats; lane 2, TPTx rats; lane 3, TPTx-1/2Nx rats; lane 4, TPTx-3/4Nx rats; lane 5, TPTx-5/6Nx rats. (b and c) The relative expression of the PTH/PTHrP receptor and ALP gene in tibial proximal metaphysis. Each value is the ratio to the expression of the ß-actin gene. *P < 0.05 vs TPTx rats; #P < 0.05 vs TPTx-1/2Nx rats; +P < 0.05 vs TPTx-3/4Nx rats.

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, our first aim was to establish a rat model simulating ABD under uraemic conditions. In animals, Nx is known to induce an increase in PTH secretion. High turnover bone disease has also been observed in renal failure rats with hyperparathyroidism. Therefore, to establish a rat model of renal failure without the effect of secondary hyperparathyroidism, we combined Nx with TPTx and supplemented physiological doses of PTH and thyroxine. Studies conducted to date using Nx combined with parathyroidectomy and PTH infusion [11,12] did not provide direct data of bone turnover. Moreover, the model of Szabo et al. [13] manifested osteomalacia, a pathological condition different from ABD. Referring to these past studies, we tried various PTH infusion rates and diet compositions, and succeeded in developing a novel experimental model for studying ABD, using a PTH infusion rate of 0.1 µg/kg/h (the same as that used by Szabo et al. [12]) with a diet containing 2% calcium and 1% phosphorus.

In our ABD model (TPTx-5/6Nx rats), osteoblast and labelled surface were observed, although reduced, and the bone formation rate was significantly reduced. On the other hand, the pathological picture of ABD obtained from bone biopsies is characterized by barely discernible osteoblast and labelled surface, with a dramatic reduction in bone formation. The difference in ABD pathology in our animal model and clinical disease may be explained by the fact that we studied the period up to 6 weeks after Nx during which renal dysfunction is mild. In addition, when comparing the results in Table 3 and Figure 3, bone turnover in our model continues to decrease from 6 weeks after Nx (TPTx-5/6Nx group in Table 3) to 10 weeks after Nx (vehicle group in Figure 3). Even at 10 weeks after Nx, the presence of osteoblast and labelled surface can be observed. Therefore, the pathology shown in our model may represent that of lowered bone turnover at the early stage of renal failure, and the progression leading to the full pathological picture of clinical ABD. In addition, urinary calcium excretion increased depending on the degree of renal failure. Considering these findings together with the histomorphometric results, the increased urinary calcium excretion indicates lowered calcium buffering capacity of the bone as a result of lowered bone turnover. The serum calcium levels did not change in TPTx and TPTx-Nx rats, probably because the residual renal function of the rats and high calcium content in the diet contributed to maintaining calcium homeostasis despite lowered calcium buffering capacity in bone.

The reduction of bone formation in TPTx-5/6Nx rats demonstrated by bone histomorphometry was supported by the reduction in gene expression. A significant decrease in ALP gene expression was revealed by RT–PCR analysis. ALP is well known as a differentiation marker of osteoblasts. These data thus suggest that renal dysfunction induces not only lowered osteoblast function but also reduced osteoblast differentiation. The reduction of bone formation was ameliorated by injection of pharmacological doses of intermittent PTH in the present study. Some studies so far have examined the effects of continuous and intermittent administration of PTH. Generally, continuous infusion of PTH increases bone formation and resorption with a net decrease in bone volume, and intermittent injection of PTH increases the bone apposition rate accompanied by an increase in formation surface without any increase in resorption surface. Although our rat model was infused with a physiological dose of PTH after TPTx, additional intermittent injection of pharmacological doses of PTH in TPTx-Nx rats increased the bone formation rate without changing bone resorption parameters. A recent study has reported increased bone resorption as well as bone formation by short-term PTH intermittent treatment [14]. However, increased bone resorption was not observed by intermittent PTH injection in our rat model. Buxton et al. [15] examined the effect of intermittent PTH injection in patients with low turnover osteoporosis. They observed increased serum level of soluble receptor activator of nuclear factor-{kappa}B ligand (sRANKL) and decreased osteoprotegrin (OPG), and suggested that the augmented bone resorption by intermittent PTH treatment is due to enhanced osteoblast-induced osteoclast formation. Previously, we have reported the accumulation of OPG, an inhibitor of osteoclast formation, in patients with renal failure [16]. Therefore, the OPG accumulated in blood as a result of renal failure may inhibit the sRANKL-induced increase in bone resorption by intermittent PTH administration. As a result, we could not observe any change in bone resorption in our animal model.

We also found that the reduction of PTH/PTHrP receptor gene expression was dependent on renal dysfunction in TPTx-Nx rats compared with TPTx rats. These observations suggest that skeletal resistance to PTH underlies ABD in this rat model. Although a reduction in PTH/PTHrP receptor gene expression has been reported previously in humans and in rats with hyperparathyroidism [17,18], whether this downregulation occurs in bones of animals with low or normal levels of PTH and renal failure was unclear. Downregulation of PTH receptor, a reduced production of bone morphogenic protein-7 (BMP-7, also called osteogenetic protein) and the presence of C-terminal fragments of PTH [19] may conceivably account for PTH resistance in low-turnover bone, as in the case of hyperparathyroidism. However, we infused 1–34 PTH in the present study, therefore the C-terminal fragment is probably not responsible. Since administration of BMP-7 has been reported to improve bone formation [20], the involvement of BMP-7 is possible. We confirmed the downregulation of PTH receptor in this study. At least in our model, the decreased PTH response observed in low bone turnover may be mediated by the downregulation of PTH receptors that occurs in the early stage of renal failure when changes in serum calcium and 1,25(OH)2D3 levels are not yet detectable. Although this hypothesis may seem different from the classic definition of skeletal resistance to PTH (i.e. decreased calcaemic response to PTH) indicated by changes in serum calcium levels, the initial decrease in PTH response through reduced PTH receptors may progress further, leading to a decreased calcaemic response. Therefore, downregulation of PTH receptors is regarded as a part of the phenomenon of decreased PTH response resulting from renal dysfunction.

In our rat model with renal failure, bone formation decreased depending on the degree of renal function. Since serum PTH concentrations were similar among all the groups, bone formation may be affected by renal function in this model. Elevated serum levels of uraemic toxins are well documented as the common phenomenon accompanying progression of renal disease. Circulating uraemic toxins have also been proposed to facilitate progression of chronic renal failure. More recently, it has been reported that uraemic sera decreased the PTH receptor mRNA level in osteoblastic cultured cells [21]. In our model too, it is highly possible that uraemic toxins may suppress osteoblastic function and reduce bone formation through downregulation of the PTH/PTHrP receptor. Furthermore, it is possible that the lesser weight gain (nutritional status) as a result of lowered renal function could have contributed to the differences that were observed. The effect of under-nutrition has to be examined further.

In summary, we established a rat model simulating low-turnover bone disease secondary to renal failure, using TPTx and PNx. In this model, decreased osteoblasts and lowered osteoblast function were dependent on renal dysfunction. The progression of osteoblast dysfunction seems to parallel deterioration of renal function, which might suggest accumulation of uraemic toxins. Furthermore, demonstration of the role of uraemic toxins in osteoblast dysfunction and identification of the specific uraemic toxins may reveal the pathogenesis and mechanism of progression of ABD.



   Acknowledgments
 
This work was supported in part by grants from the Renal Osteodystrophy Foundation. Part of this paper was presented at the American Society of Nephrology, San Diego, 2003, and appeared as abstract #SA-PO0779.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 3.04
Accepted in revised form: 8. 4.05





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