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NDT Advance Access originally published online on April 11, 2008
Nephrology Dialysis Transplantation 2008 23(9):2761-2767; doi:10.1093/ndt/gfn143
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Cinacalcet does not affect longitudinal growth but increases body weight gain in experimental uraemia

Kumiko Nakagawa1, Eduardo C. Pérez2, Jun Oh1, Fernando Santos2, Aman Geldyyev3, Marie -L. Gross3, Franz Schaefer1 and Claus P. Schmitt1

1 University Hospital for Pediatric & Adolescent Medicine, University of Heidelberg, Germany 2 Department of Pediatrics, Hospital Universitario Central de Asturias, University of Oviedo, Spain 3 Institute of Pathology, University of Heidelberg, Germany

Correspondence and offprint requests to: Claus Peter Schmitt, Division of Pediatric Nephrology, University Hospital for Pediatric and Adolescent Medicine, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany. Tel: +49-6221-56-39313; Fax: +49-6221-56-4203; E-mail: claus.peter.schmitt{at}med.uni-heidelberg.de



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Cinacalcet (CIN) efficiently suppresses parathyroid hormone (PTH) secretion by the activation of the calcium-sensing receptor (CaR). Epiphyseal chondrocytes also express the CaR and its activation promotes cell proliferation and differentiation in vitro. Hence, the impact of CIN on the growth plate function requires assessment before routine administration in children.

Methods. We treated subtotally nephrectomized (SNX) and sham-operated, ad lib and pair-fed Sprague-Dawley rats with CIN (15 mg/kg day) or solvent (S) for 14 days p.o. and assessed whole body and tibia length gain, growth plate morphology, osseous front advance (OFA) (calcein staining) and chondrocyte proliferation rate [5-bromo-2'-deoxyuridine (BrdU) staining].

Results. Total body length gain did not differ after 7 and 14 days (SNX + CIN 2.9 ± 0.6, SNX + S 3.0 ± 0.7; sham + CIN 4.2 ± 0.4, sham + S 4.5 ± 0.4; sham pair-fed + CIN 3.3 ± 0.5, sham pair-fed + S 3.5 ± 0.6 cm/14 days; P = n.s.). Tibia length, the height of the total growth plate and the hypertrophic zone, OFA and chondrocyte proliferation rate were similar with CIN and S. Serum Ca2+ declined with CIN treatment; PTH was 61% lower in CIN- compared to S-treated SNX (P < 0.05). Food intake was similar, whereas body weight gain (21.6 ± 8.7 versus 12.7 ± 11.2 g) and body weight gain per food intake (141 ± 50 versus 77 ± 70 g/kg) improved in CIN- versus S-treated SNX animals (P < 0.05).

Conclusion. CIN treatment does not impact on growth plate chondrocyte function in uraemic rats, but improves food efficiency and body weight gain.

Keywords: calcimimetic agents; calcium receptor; growth plate chondrocyte; longitudinal growth; rats



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Calcimimetic agents efficiently reduce plasma parathyroid hormone (PTH) and calcium phosphate product in patients with uraemic hyperparathyroidism [1] and are likely to improve the clinical outcome [2]. In growing children, insufficient control of hyperparathyroidism is particularly harmful and may result in bone deformities and epiphyseal slipping [3]. One-third of adult patients with a paediatric onset of end-stage renal disease (ESRD) have clinical symptoms, and one out of six patients is disabled due to bone disease [4]. Moreover, the persistent increase in calcium phosphate product and PTH observed in many of the paediatric patients with prolonged ESRD essentially contributes to cardiovascular calcifications in early adulthood and a dramatically increased mortality [5,6]. Calcimimetic agents may therefore be of great benefit in paediatric CKD (chronic kidney disease) patients; specific paediatric side effects however need to be excluded prior to routine clinical administration.

Longitudinal bone growth is based on proliferating chondrocytes, transforming into hypertrophic cells, which secrete matrix vesicles with a high calcium content and cartilaginous matrix and concomitant invasion of bone cell precursors and vessels into the metaphyseal end of the growth plate. This process is regulated by multiple local and systemic factors, including PTH-related protein (PTHrP), fibroblast growth factors (FGFs), Indian hedgehog (Ihh), bone morphogenetic proteins (BMPs), growth hormone (GH), insulin-like growth factor I (IGF-I), oestrogens, androgens, glucocorticoids, thyroid hormone, vitamin D3 metabolites [7] and leptin [8]. Chondrocyte differentiation is under the control of extracellular calcium concentrations [9], possibly via L-type calcium channels. Verapamil inhibits growth plate chondrocyte proliferation and differentiation in vitro [10]. In addition, calcium-sensing receptor (CaR) expression has now been demonstrated on epiphyseal chondrocytes [11]. Exposure of rat metatarsal bones to the calcimimetic R-568 promotes chondrocyte proliferation and differentiation [12]. Calcimimetics may moreover interfere with longitudinal growth via a reduction in plasma testosterone levels (CIN package insert). Androgens promote growth through local conversion to oestrogens [13] and by a direct action on the growth plate androgen receptor [14]. In the present study we therefore investigated the impact of calcimimetics on growth plate morphology, chondrocyte proliferation and longitudinal bone growth rate (LBR).



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Male Sprague-Dawley rats (Charles River Laboratories Inc., Sulzfeld, Germany) were kept individually in a light- (12-h on/off cycles), temperature- (24°C) and humidity- (70%) controlled room. Except for pair-fed controls, the animals had free access to a standard diet (ssniff R/M-H; Ssniff Co., Soest, Germany) that contained 12.8 MJ/kg, 19% protein, 1.0% calcium, 0.7% phosphorous, 0.2% magnesium, 0.9% potassium, and 1000 IU/kg vitamin D3, and tap water.

Initial body weight was 80–90 g. They were allowed to adapt to the environment for 7 days. The day prior to the first operation, the animals were assigned to the different experimental groups according to their body weight. A two-stage subtotal nephrectomy (SNX) was performed. The right kidney was removed in the first session under general anaesthesia with isoflurane. Seven days after uninephrectomy, we selectively removed cortical tissue from the hypertrophied left kidney corresponding to 66% of the weight of the previously excised right kidney. Particular care was taken to preserve adrenals without damage. Control animals were sham operated (renal decapsulation) in two consecutive sessions. The animal experimentation and handling were in accordance with the German law for protection of animals.

Experimental design
Uraemic and sham-operated rats were randomized for treatment with cinacalcet (CIN) (15 mg/kg day; AMGEN, München, Germany) or standard food. CIN was homogenously mixed to the food pellets and offered from 8 p.m. on. After consumption of the daily dose of CIN, standard diet was given. Sham animals were ad lib and pair fed, respectively; the number of animals per group was 14 in SNX and 8 in sham animals. The duration of uraemia and treatment was 14 days.

LBR was determined by measuring osseous front advance (OFA) that was estimated by calcein labelling. To this end, 30 mg/kg body weight of calcein (Sigma, St. Louis, MO, USA) was intraperitoneally injected 48 h before sacrifice. Chondrocyte proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) labelling. BrdU (Sigma) was injected at doses of 100 mg/kg body weight, 17, 9 and 1 h prior to sacrifice.

Snout-to-tip tail length was measured under isoflurane anaesthesia during second-stage operation, after 7 and again after 14 days. Food intake and body weight were measured daily. Retrobulbar blood sampling (150 µl) was performed pre-treatment and 7 days after second-stage nephrectomy. At sacrifice, blood was sampled by aortic puncture. On Day 7, CIN was administered 12 h before blood sampling and on Day 14 CIN was given 24 h before aortic puncture.

Tissue processing
Both tibiae were isolated immediately after sacrifice, soft tissues removed and tibia lengths measured with a sliding mechanical calliper. Then, the proximal ends of the tibiae including the growth plate were dissected out. The right tibia was fixed in 40% ethanol (4°C) for analysis of calcein labelling and BrdU immunohistochemistry. The left tibia was fixed in 4% neutral formalin at 4°C for morphometric analysis and CaR immunohistochemistry. Both tibiae were dehydrated in graded solutions of ethanol and embedded in methylmethacrylate as formerly described [15,16].

Measurement of LBR
LBR was measured in 10-µm-thick frontal sections of proximal end of tibiae obtained using a rotary microtome (HM355S®, Microm, Barcelona, Spain) fitted with tungsten carbide blades. Sections were examined under an Olympus incident light fluorescence microscope (Olympus BX41®) coupled to a digital camera (Olympus DP11®, Olympus Optical España, Barcelona, Spain) to detect calcein label. Images were captured and the distance between the chondro-osseous junction and the calcein label was measured using an image analysis system (Scion Image®, Scion Corporation, Frederick, MD, USA). The average value of these measurements was considered the OFA during the 48 h prior to sacrifice, representing the LBR in each animal.

Chondrocyte-proliferating index
The index of proliferating chondrocytes was estimated in 5-µm-thick frontal sections of the proximal end of tibiae. After a 30-min incubation in prewarmed 100% acetone to remove MMA from the tissue sections, hydrated sections were incubated in HCl (2N, 60', 37°C), thoroughly washed in water, rinsed in a 0.1 M Tris–hydrochloric buffer, and treated with trypsin (1 mg/ml, 1% Cl2Ca; 60 min, 37°C). After several washes in a Tris–hydrochloric buffer, endogenous peroxidase activity was inactivated by a 30-min treatment in 10% H2O2. Samples were incubated with horse serum (25%, 75 min; Sigma), followed by a 48-h incubation with monoclonal antibody to BrdU (1:20; Dakopatts, Glostrup, Denmark) in moist chamber at 4°C. Then, sections were incubated (30 min, room temperature) with antimouse secondary conjugated antibody (EnVision + system HRP®, DakoCytomation, Carpinteria, CA, USA). The final reaction product was revealed with 3-3'-diaminobenzidine (DAB; Sigma), 20 mg of DAB in 50 ml of 0.05 M Tris–hydrochloric buffer plus 50 µl of 30% hydrogen peroxide. Preparations were lightly counterstained with alzian blue and mounted with Eukitt. The number of BrdU-positive cells per column of chondrocytes was counted as well as the number of columns per field. Six fields per section and two sections per animal were measured. For each animal, proliferative activity was expressed as the mean number of BrdU-labelled cells per column.

Growth plate height and hypertrophic zone height
For morphometric analysis of growth plate, heights of growth cartilage and its hypertrophic zone were measured in 5-µm-thick sections stained with alzian blue and safranine at twenty randomly selected locations on each section, using the above mentioned image analysis system.

CaR immunohistochemstry
Immunohistochemical staining for CaR was performed in formalin-fixed sections. After deplastination in acetone and rehydration, hyaluronidase (Sigma, St. Louis, MO, USA) (2 mg/ml in PBS, pH = 5, 30 min, 37°C) was used for antigen retrieval. Then, sections were treated with 10% hydrogen peroxide and 25% goat serum and incubated overnight at 4°C with 1/30 solution of CaR antibody (Affinity BioReagents, Inc, Golden, CO, USA) in PBS. After a 30-min incubation with antirabbit secondary conjugated antibody (En Vision + TM®; Dako Corporation), the final reaction product was revealed with DAB (Sigma), 20 mg of DAB in 50 ml of 0.05 M Tris–hydrochloric buffer plus 50 µl of 30% hydrogen peroxide. Sections were counterstained with methyl green.

Biochemical measurements
Blood ionized calcium was measured using an ion selective electrode system (Ionometer 2 EH-F; Fresenius Medical Care, Bad Homburg, Germany). The results were corrected for pH 7.4; the mean intra- and interassay coefficients of variation were 2.5%. Serum electrolytes were analysed using a multichannel analyzer (ADVIA 2400; Siemens Medical Diagnostics). Creatinine was determined by an enzymatic method, coefficient of variation <3%. PTH was measured 24 h after solvent (S) and CIN administration in all SNX animals in duplicates using a Rat BioActive Intact PTH ELISA (Immutopics Inc., San Clemente, CA, USA). The assay is specific for full-length biologically active intact 1-84 form of rat PTH. It has no cross-reactivity with N-terminal 1-34, mid-region and C-terminal 39-84 and other non-(1-84) fragments. The mean intra- and interassay coefficients of variation were 9 and 12%, respectively.

Statistics
Data are given as mean ± standard deviation (SD). The overall effects of the different treatment options were analysed by ANOVA, followed by Bonferroni's t-test for multiple comparison of the different treatment groups. P < 0.05 was accepted as statistically significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
A similar degree of uraemia was achieved in both groups of SNX rats, with serum creatinine values three to four times above sham-operated controls (Table 1). CIN treatment significantly reduced ionized and total calcium levels. On Day 7, 12 h after CIN administration, ionized calcium was reduced by 0.35 ± 0.11 in SNX, 0.26 ± 0.06 in sham ad lib and 0.19 ± 0.11 mmol/l in sham pair-fed animals as compared to the respective pretreatment levels on Day 1 (each P < 0.05), whereas in controls, ionized calcium levels remained unchanged. In SNX rats, PTH measured 24 h after CIN administration was 208 ± 194 pg/ml as compared to 531 ± 216 pg/ml in SNX controls (P < 0.05).


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  		Table 1 Serum biochemistry

 
Effect of CIN on body weight gain
At start of treatment, body weight was 158 ± 10 and 159 ± 11 g in SNX, 168 ± 9 and 170 ± 9 in sham ad lib and 171 ± 10 and 173 ± 10 in sham pair-fed rats (all P = n.s.) treated with S and CIN, respectively. Food intake was significantly reduced in SNX animals but not influenced by the treatment (Table 2). Total body weight gain, body weight gain per 100 g body weight and body weight gain per food intake, however, were significantly improved in SNX rats treated with CIN compared with S-treated SNX controls (each P < 0.05). Of note, body weight gain and body weight gain per food intake were even higher in CIN-treated SNX animals as compared to respective sham pair-fed control animals.


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  		Table 2 Food consumption, water intake and body weight gain

 
Effect of CIN on growth
Initial body length did not differ between treatment groups. Sham ad lib animals grew significantly better than uraemic animals and pair-fed controls. CIN had no effect on growth rate in the rats. Body length gain did not differ between the S and CIN treatment groups after 1 and 2 weeks (Figure 1). After 2 weeks, body length gain was 3.0 ± 0.7 versus 2.9 ± 0.6, 4.5 ± 0.4 versus 4.2 ± 0.4 and 3.5 ± 0.6 versus 3.3 ± 0.5 cm in SNX, sham ad lib and sham pair-fed rats treated with S and CIN, respectively (all P = n.s.). Accordingly, tibia length determined at sacrifice was less in SNX animals but not influenced by the treatment (Table 3).


Figure 1
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  		Fig. 1 Body length gain on Days 7 and 14 compared to Day 0. Growth velocity did not differ in cinacalcet-treated rats (dotted lines) as compared to respective controls (full lines). Independent of treatment, sham ad lib-fed animals grew significantly better then SNX animals and pair-fed sham controls (*P < 0.05).

 

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  		Table 3 Tibia length and proximal tibia growth plate histomorphometry

 
Effect of CIN on growth plate morphology and function
Total growth plate and hypertrophic zone heights were increased in SNX rats compared to sham pair-fed animals (P = 0.09 and 0.04) and slightly higher in SNX compared to sham ad lib-fed rats (P = n.s.) (Table 3). CIN treatment had no impact on growth plate and hypertrophic zone morphology and sizes (Figure 2a).


Figure 2
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  		Fig. 2 Pairs of representative images of sections from subtotally nephrectomized rats treated with cinacalcet (left) or solvent (right) showing growth cartilage morphology after alzian blue and safranine staining (a), calcein labelling of newly formed bone at the proximal tibial metaphysis (b), immunostaining of BrdU-labelled nuclei as an index of chondrocyte proliferative activity (c) and calcium-sensing receptor immunostaining (d). White lines in (b) identify the chondro-osseous junction and the proximal end of calcein-labelled bone. Magnification bar: 100 µ.

 
LBR (Figure 2b) was assessed by calcein staining during 48 h before sacrifice. Mean LBR tended to be lower in SNX compared with sham rats fed ad lib (Table 3, Figure 2b). CIN treatment had no effect on SNX and sham ad lib-fed animals, but reduced bone growth in pair-fed controls by 24.3% (P < 0.05).

Chondrocyte-proliferating indices assessed by the mean number of BrdU-positive cells per column (Figure 2c) were not different among S-treated groups. CIN again had no effect on chondrocyte proliferation in SNX and sham ad lib-fed rats, and reduced the proliferation rate in pair-fed controls by 18.6% (P = n.s.).

Effect of CIN on chondrocyte CaR expression
The immunocytochemical signal of CaR was detected in most hypertrophic chondrocytes. Immunostaining was strong and homogeneous in chondrocytes at the upper hypertrophic zone and rather diffuse in chondrocytes located close to the ossification zone. No difference in CaR immunoreactivity pattern and abundance was seen in CIN-treated animals. Figure 2d gives representative examples in SNX rats.



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Longitudinal bone growth is a highly coordinated process involving a variety of hormones and cytokines. In an ex vivo murine model, interference with chondrogenesis by the activation of the CaR with the calcimimetic agent R-568 improved longitudinal growth [12]. The present study however could not extend the growth-promoting effect of calcimimetics to the in vivo situation. CIN did not impact on longitudinal growth rate, growth plate morphology and function in young SD rats. In uraemic rats, however, CIN exerted anabolic effects; CIN-treated animals had significantly improved body weight gain.

Extracellular calcium is crucial for physiological chondrogenesis and bone growth. Increased extracellular calcium concentrations promote collagen X expression [9] and removal of extracellular calcium by EGTA blocks the maturational process [17]. Cytosolic calcium concentrations are regulated by release from intracellular stores and entry through the plasma membrane. They increase with chondrocyte maturation [18,19]. Calcium influx is under the control of voltage-gated calcium channels. Blockade by verapamil dose dependently inhibits longitudinal bone growth, chondrocyte proliferation and differentiation in fetal rat metatarsalia cultures [10]. Activation of the CaR also increases cytosolic calcium concentrations in chondrocytes by IP3-dependent calcium release from intracellular stores and by increased calcium influx into chondrocytes, and promotes chondrogenesis and bone growth [12]. Whether calcium channels are involved in this process as demonstrated for parafollicular thyroid cells [20] is not known at present.

In contrast to these in vitro findings, our analysis of growth plate morphology and function did not reveal any growth-promoting activity of calcimimetic agents in vivo. Total growth plate and hypertrophic zone heights, LBR and chondrocyte proliferation indices were not increased in CIN-treated animals. Potential explanations include the marked decline in serum calcium levels, which could still be demonstrated 24 h after CIN administration. Different from the culture model with constant medium calcium concentrations, reduced extracellular calcium may antagonize the chondrogenesis-promoting potential of CIN. In addition, the well-described decline in plasma PTH levels in response to calcimimetics in rats [21,22] may also interfere with growth plate homeostasis. The pace of chondrocyte differentiation depends on a balance of interactions between chondrocyte PTHrP/PTH1R and extracellular calcium signalling [23]. PTHrP- [24] and PTH1R-null mice [25] exhibit short limbs. CaR-null mice also develop growth retardation, but do not allow delineating the precise role of the CaR on growth plate function, since the animals suffer from severe rickets due to excessive PTH secretion and die early [26]. CaR/PTH double knock-out mice and CaR/Gcm2-deficient mice are rescued from severe hyperparathyroidism and exhibit a normal skeletal phenotype, suggesting that the CaR has no essential, non-redundant role in osteo- and chondrogenesis [27,28]. In addition, growth retardation has not been usually reported in children with autosomal dominant hypocalcaemia secondary to activating mutations of CaR [29–31].

SNX in young rats is a well-established model of uraemia that has repeatedly been used to demonstrate growth-promoting effects not only of growth hormone [32,33] but also of PTH [34]. In the present study, a similar degree of uraemia with a fourfold increase in creatinine was achieved in all groups. Total growth plate and hypertrophic zone heights were higher in SNX compared to sham pair-fed controls, but not influenced by CIN treatment. Enlargement of the growth plate and its hypertrophic stratum in uraemic rats has been reported previously and may depend not only on the severity and duration of renal insufficiency [35] but also on the degree of hyperparathyroidism [36]. The increase in serum phosphate levels in CIN-treated animals is in line with a previous study with SNX rats [37] and in contrast to the effect of R-568 on serum phosphate concentrations in experimental uraemia. It may be explained by the reduction in plasma PTH concentrations, an essential regulator of renal phosphate reabsorption [38]. The CIN-induced decline in serum calcium and the concomitant 17% increase in serum phosphate levels did not result in any changes in body length gain, growth plate morphology and function. Extension of the treatment period should not result in different findings. Significant differences in cumulative body length gain can only be expected when epiphyseal growth velocity is modified or when a substance accumulates in bone, both of which are not the case in our study. Still, our results do not yet provide the rationale for administration of the calcimimetic compound in children with CKD; clinical trials are mandatory.

Cachexia frequently develops in uraemia and is characterized by anorexia, energy expenditure, loss of protein stores and muscle wasting. As expected, SNX animals in this study had significantly reduced food intake, and reduced body weight gain and food conversion ratio. Food intake did not change in CIN-treated SNX animals, suggesting that CIN-associated nausea was not relevant. Body weight gain per food intake, however, was significantly improved. Potential explanations include activation of the CaR expressed in peripheral organs such as the muscle, which may inhibit protein degradation processes, namely the caspase-3/ubiquitin proteasome system [39] or the IGF-1/myostatin-dependent regulation of muscle mass [40]. Alternatively, central CaR activation may interact with downstream intracellular signalling pathways triggered by leptin and neuropeptide Y via the melanocortin type 4 receptor [41] and thus prevent uraemia-induced catabolism.

In summary, CIN does not impact on epiphyseal chondrocyte function and longitudinal growth in healthy and uraemic rats, but exerts anabolic action on uraemic animals. The latter deserves further experimental analyses to elucidate the underlying molecular mechanisms; the first should prompt clinical trials in children with CKD to provide the scientific base for routine clinical administration of these highly efficient compounds in this patient group.



   Acknowledgments
 
We thank V. Loredo and L. Mallada for excellent technical support. C.P.S. received financial support from Amgen, TO, USA, for experimental studies. The study was in part supported by FIS PI 02-1050 and by Fundacion Nutricion y Crecimiento funds.

Conflict of interest statement. FS has been adviser to AMGEN. CPS has received financial support from AMGEN for experimental studies, for lecturing and to attend conferences.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 2. 7.07
Accepted in revised form: 21. 2.08


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