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Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism
Kidney International, Vol. 65 (2004), pp. 1740–1748
Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism CHERYL P. SANCHEZ, YU-ZHU HE, ELLEN LEIFERMAN, and NORMAN J. WILSMAN Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin; and Department of Comparative Biological Sciences, University of Wisconsin School of Veterinary Medicine, Madison, Wisconsin
Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism. Background. Impairment of growth in children with chronic renal failure may be due, in part to the insensitivity to the actions of growth hormone by insulin-like growth factor-I (IGF-I) because of accumulations of IGF binding proteins. There are a few studies describing the changes that occur in the growth plate in renal failure. None of these studies has simultaneously compared the modifications in the expression of selected markers of endochondral bone formation in renal failure with mild or advanced secondary hyperparathyroidism. Methods. Forty-six rats that underwent 5/6 nephrectomy (Nx) were fed either standard rodent diet (Nx-control) or high phosphorus diet to induce advanced secondary hyperparathyroidism (Nx-phosphorus) for 4 weeks. Sections of the tibia were obtained for growth plate histomorphometry, immunohistochemistry studies, and in situ hybridization experiments for selected markers of endochondral bone formation. Results. Weight gain, gain in length, and tibial length were less in Nx animals. Serum parathyroid hormone (PTH) and phosphorus levels were higher and serum calcium levels were lower in the Nx-phosphorus group. The width of the growth plate was much shorter in the Nx-phosphorus group due to a decrease in both proliferative and hypertrophic zones. IGF-I protein and IGF binding protein-3 staining were diminished in both Nx groups without changes in the IGF-I receptor expression; the decline in IGF-I protein expression was much lower in the Nx-phosphorus group. PTH/PTH receptor protein (PTHrP) receptor mRNA transcripts decline and tartrate-resistant acid phosphastase (TRAP) staining increased only in the Nxphosphorus group. Conclusion. The growth impairment in renal failure may be worsened by the severity of secondary hyperparathyroidism.
The growth plate cartilage is important in bone elongation and longitudinal growth. Bone elongation is achieved by the complex interplay of chondrocyte proliferation,
Key words: growth plate, secondary hyperparathyroidism, endochondral bone formation. Received for publication September 5, 2003 and in revised form November 11, 2003 Accepted for publication December 10, 2003 C
2004 by the International Society of Nephrology
matrix synthesis, chondrocyte maturation and hypertrophy, matrix degradation, angiogenesis, and mineralization. In the presence of renal failure, abnormalities in the histology of the growth plate cartilage have been reported in animals, and these alterations may contribute to the impairment of linear growth reported in young uremic children. Cobo et al [1] have shown that the width of the growth plate was increased in nephrectomized rats compared to animals with normal renal function due to the widening of the hypertrophic zone. Furthermore, there was low chondrocyte turnover and impairment of cartilage resorption in the chondro-osseous junction [1]; however, the degree of secondary hyperparathyroidism in these experiments were unknown. In contrast, we have shown that the growth plate width was decreased and the microarchitecture of the growth plate was disorganized in rats with renal failure and severe secondary hyperparathyroidism accompanied by a decline in the expression of the parathyroid hormone/parathyroid hormone receptor protein (PTH/PTHrP) receptor mRNA in the proliferating and maturing chondrocytes [2]. Similar findings were reported by Urena ˜ et al [3] in rats that were given high phosphorus diet to induce severe secondary hyperparathyroidism. Currently, there are no concurrent studies available that compare changes in the growth plate cartilage in renal failure with mild or advanced secondary hyperparathyroidism. Our previous study was done mainly to evaluate the effects of growth hormone and calcitriol on the growth plate in renal failure, but the experiments were not done simultaneously [2]. Although serum insulin-like growth factor-I (IGF-I) levels have been reported to be within normal limits in rats with renal failure, there was a significant decline in the expression of IGF-I mRNA, growth hormone receptor transcripts, defective mineralization, and alterations in collagen metabolism [4–6]. As in previous reports, serum PTH levels were not obtained in these studies so the severity of secondary hyperparathyroidism cannot be evaluated. The current set of experiments was performed to compare the effects of the varying degrees of secondary hyperparathyroidism on chondrocyte proliferation, 1740
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chondrocyte hypertrophy, matrix degradation, and initiation of mineralization in young rats with renal failure. METHODS Forty-six male weanling Sprague-Dawley rats (Harlan Laboratories, Madison, WI, USA) with a mean weight of 57 ± 3 g were housed in individual cages at constant temperature with a 12-hour light and 12-hour dark cycle. The animals had free access to drinking water and standard rodent diet containing 23.4% protein, 0.6% calcium, and 0.6% phosphorus (Purina Mills, Indianapolis, IN, USA). After 24 hours of acclimatization, 16 rats underwent the first stage of a two-stage 5/6 nephrectomy (Nx), and 30 rats underwent sham nephrectomy (intact) using surgical procedures described elsewhere [2]. The second-stage nephrectomy was completed 7 days later. The 4-week study period began 24 hours after completion of the second surgery. All procedures were reviewed and approved by the Research Animal Resource Center at the University of Wisconsin, Madison, Wisconsin. Measurements of body weight and body length were obtained weekly. Fifteen sham-nephrectomized animals (intact-control), and six nephrectomized animals (Nxcontrol) continued to ingest standard rodent diet. To induce advanced secondary hyperparathyroidism, ten Nx rats were given high phosphorus diet (Nx-phosphorus) (1.2% phosphorus and 0.6% calcium) (Purina Mills). To ensure equal caloric intake, all intact-control (N = 15) animals were pair-fed with Nx animals by providing the amount of food each day to intact-control rats that had been consumed the previous day by Nx animals. A group of sham-Nx animals (intact-ad libitum) (N = 15) were fed ad libitum with the standard rodent chow. Another group of animals with normal renal function given high phosphorus diet was not included in the current experiment. To evaluate longitudinal bone growth, the animals were given intraperitoneal injections of oxytetracycline hydrochloride, 1 mg/kg/dose, 3 days and 1 day prior to sacrifice. Four weeks after the second-stage nephrectomy, the animals were randomly divided into two groups. In group I, animals from the Nx-control (N = 3), Nx-phosphorus (N = 5), intact-control (N = 10), and intact-ad libitum (N = 10) groups were anesthetized, killed by exsanguination by cardiac puncture, and underwent transcardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). The tibiae were decalcified for 2 weeks and embedded in paraffin as previously described [2]. Five micrometer sections of bone were obtained for morphometric analysis, in situ hybridization, and immunohistochemistry experiments. In group II, the remaining animals, Nx-control (N = 3), Nx-phosphorus (N = 5), intact-control (N = 5), and intact-ad libitum (N = 5), were given lethal doses of
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pentobarbital (300 mg/kg intraperitoneally). The growth plate in the proximal tibia of the left leg was quickly collected to measure the rate of elongation. For each of the growth plate, two thin slabs were cut and were observed under epifluorescence microscopy with an excitation filter of 330 to 380 nm and a barrier filter of 420 nm. To estimate bone elongation over 48 hours, the distance between the leading edges of the two fluorescent bands was measured four times using an ocular reticle and the average measurement was converted to elongation for 24 hours [7]. When there is only one label discernable, the measured distance between the visible label and the chondro-osseous junction is then used for calculation of bone elongation. For optimal fixation, the remaining growth plate was fixed in 2% glutaraldehyde in 0.05 mol/L cacodylate buffer (pH 7.35) with 0.7% ruthenium hexamine trichloride and embedded in Epon-araldite [8, 9], and sections were obtained for histology. The right tibia was also collected, freed of adherent tissue, fixed in 4% parafolmaldehyde in PBS for 24 hours, decalcified in 15% ethylenediaminetetraacetic acid (EDTA) in paraformaldehyde for approximately 2 weeks and processed together with group I as described above. All animals were included in all in situ hybridization and immunohistochemistry experiments, Nx-control (N = 6), Nx-phosphorus (N = 10), intact-control (N = 15), and intact-ad libitum (N = 15). Serum biochemical determinations Serum was obtained by centrifugation, and samples were stored at −70◦ C. Serum urea nitrogen, creatinine, calcium, and phosphorus levels were measured using standard laboratory methods. Serum intact PTH levels were measured using the Rat Intact PTH enzyme-linked immunosorbent assay (ELISA) Kit (Immutopics, Inc., San Clemente, CA, USA). Growth plate morphometry For morphometric analysis, three 5 lm sections of bone were obtained from each tibia, stained with hematoxylin and eosin, viewed by light microscopy at 20× and images were captured onto a computer monitor. The total width of the growth plate and the widths of the proliferative and hypertrophic zones were measured using an image analysis software (Elektronik 200) (Kontron Instruments, Ltd., Hallbergmoos, Germany) as previously described [2]. In situ hybridization (radioactive and nonradioactive) In situ hybridization was performed using methods described elsewhere [2, 10]. 35 S-labeled sense and antisense riboprobes encoding mouse matrix metalloproteinase9 (MMP-9)/gelatinase B and rat vascular endothelial growth factor were labeled to a specific activity of 1 to 2
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× 109 cpm/lg. After hybridization and posthybridization washing, the slides were exposed to x-ray film overnight, and emulsion autoradiography was done using NTB-2 (Eastman Kodak, Rochester, New York) at 4◦ C. For nonradioactive in situ hybridization, probes encoding histone-4, mouse type X collagen, rat PTH/PTHrP receptor, and IGF-I receptor were linearized, purified, labeled with digoxigenin-uridine triphosphate (UTP) in the nucleotide-triphosphate (NTP) labeling mixture (Roche Diagnostics, Mannheim, Germany), and mixed in a hybridization mixture [11]. The specimens were hybridized with specific riboprobes at 100 lL/specimen at 55◦ C to 60◦ C in a humidified chamber overnight. Following the overnight hybridization, the specimens were washed, incubated with the secondary antibody, and color was developed [11]. Quantification of in situ hybridization signals For the specimens hybridized with 35 S, the slides were viewed at 100× by bright field microscopy and the number of silver grains overlying each chondrocyte profile was counted using an image analysis system [2]. Data are expressed as the number of silver grains/1000 lm2 of cell profile. For the nonradioactive in situ hybridization and immunohistochemistry experiments, the number of cells expressing the specific mRNA or protein was counted and expressed as percentage of the number of positive cells to the total number of cells in the appropriate growth plate zone where the mRNA expression is localized [11]. The mRNA expression for MMP-9/gelatinase B was quantified by measuring the area with positive staining and results are expressed as labeled area over the total tissue area in the chondro-osseous junction [11]. Immunohistochemistry [Bcl-2, Bax, IGF-I protein, IGF binding protein-3, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) assay, tartrate-resistant acid phosphatase (TRAP)] Immunohistochemistry was performed using methods described previously using the following primary antibodies: monoclonal Bcl-2 antibody (Santa Cruz Biotechnology, San Diego, CA, USA), monoclonal Bax antibody (Santa Cruz Biotechnology), monoclonal IGF-I protein antibody (Upstate Biotechnology, Lake Placid, NY, USA), and polyclonal IGF binding protein-3 antibody (Santa Cruz Biotechnology) [11]. For PCNA, the tissues were immunostained with the mouse anti-PCNA antibody (Zymed Laboratories, South San Francisco, CA, USA). For the TUNEL assay, the specimens were labeled using the kit from Intergen (Gaithersburg, MD, USA). The number of TUNELpositive cells were counted and expressed as percent-
Table 1. Anthropomorphic and tibial length measurements in all groups Nxcontrol N=6 Gain in weighta g 100 ± 29c Gain in lengtha cm 11 ± 1.7c Tibial lengthb cm 3.5 ± 0.1c
Nxphosphorus N = 10
Intactcontrol Nx = 15
Intact-ad libitum N = 15
100 ± 34c 10 ± 2.2c 3.4 ± 0.1c
126 ± 21 13 ± 1.3 3.8 ± 0.1
217 ± 25d 17 ± 0.9d 4.0 ± 0.1d
Nx is nephrectomized. a Difference in weight or length between baseline and at the end of the 4-week study period; b Obtained at the end of the study period; c P < 0.0001 vs. intact-control; d P < 0.0001 vs. intact-control, Nx-control, and Nx-phosphorus.
age of the total number of terminal chondrocytes in each section. Histochemical staining for TRAP was done using methods reported previously [2]. Image analysis was done in tissue sections viewed at 50× and the number of TRAP-positive cells in the chondro-osseous junction was quantified and expressed as number of cells per area measured in the chondro-osseous junction and in the nearby primary spongiosa. Statistical analysis All results are expressed as mean values ± 1 SD. Data were evaluated by one-way analysis fo variance (ANOVA) and comparisons among groups were done using Bonferroni/Dunn post hoc tests using the StatView statistical software (SAS Institute, Cary, NC, USA). For all statistical tests, probability values less than 5% were considered to be significant. RESULTS Compared to our previous experiments, there was a significant increase in mortality rate from 5% to 10% in our earlier studies [2, 12] to approximately 25% in the current set of experiments. The rats used in this study were much younger with body weights between 50 to 60 g compared to 60 to 80 g in our previous studies. Most of the mortality occurred within 7 days after the secondstage surgery. Anthromorphic measurements As expected, the mean gain in body weight was twofold greater in the intact-ad libitum animals compared to the intact-control, Nx-phosphorus, and Nx-control groups (Table 1). There was a 35% and 15% increase in total body length at the end of the study period in the intactad libitum and intact-control groups (Table 1). Similarly, tibial length measurements were much longer by approximately 11% to 15% in the animals with normal renal function compared to the Nx animals (Table 1). Tibial length and gain in body length at the end of the study period did not differ between the two Nx groups; such
Sanchez et al: Growth plate alterations in renal failure
Table 2. Serum biochemical parameters in all groups obtained at the end of the study period
Parathyroid hormone pg/mL Calcium mg/dL Phosphorus mg/dL Urea nitrogen mg/dL Creatinine mg/dL
Nxcontrol N=6
Nxphosphorus N = 10
Intactcontrol N = 15
Intact-ad libitum N = 15
323 ± 206b
1038 ± 633a
120 ± 53
75 ± 46
9.2 ± 1.6
7.3 ± 0.7a
10 ± 1.3
10 ± 1.4
11 ± 1.3
16 ± 5.0a
10 ± 2.3
11 ± 1.5
100 ± 25b
81 ± 11b
16 ± 2
22 ± 3
1.0 ± 0.4b
0.7 ± 0.1b
0.3 ± 0.08
0.3 ± 0.07
Nx is nephrectomized. a < P 0.001 vs. Nx-control, intact-control, and intact-ad libitum; b P < 0.05 vs. intact-control and intact-ad libitum.
findings may be due in part to the mildly elevated mean serum creatinine level in the Nx-control animals (1.0 ± 0.1 mg/dL) compared to the phosphorus-loaded group (0.7 ± 0.1 mg/dL). Serum creatinine levels moderately correlated with tibial length and body length measurements in the current study (R= −0.7, P < 0.001). Serum urea nitrogen and serum creatinine levels were higher in the Nx animals (Table 2). Serum phosphorus levels rose and serum calcium levels declined in the Nx animals given high phosphorus diet (Nx-phosphorus) (Table 2). Intact PTH levels increased eightfold in the Nx-phosphorus animals and were modestly elevated in the Nx-control animals compared to the groups with intact renal function (Table 2). Serum PTH levels correlated with measurements of tibial length (R = −0.7, P < 0.001), gain in body length (R = −0.6, P < 0.001), and TRAP staining (R = 0.6, P < 0.05). The total thickness of the growth plate was wider by approximately 35% in the intact-ad libitum animals when compared to the intact-control and Nx-control groups (369 ± 29 lm versus 323 ± 50 lm and 335 ± 57 lm, respectively) (P < 0.007) (Fig. 1). In contrast, there was a significant reduction in the width of the growth plate in the Nx-phosphorus animals (288 ± 42 lm) (P < 0.04) (Fig. 1). As previously demonstrated in our earlier experiments, alterations in the architecture of the growth plate cartilage were evident mainly in the animals given high phosphorus diet with the loss of the columnar organization of the proliferating chondrocytes [2] (Fig. 1). The area occupied by the proliferating chondrocytes in the Nx animals decreased by approximately 20% compared to the groups with normal renal function (Fig. 1) (0.5 ± 0.04 versus 0.6 ± 0.04, respectively) (P < 0.02). In addition, the area occupied by the hypertrophic chondrocytes decreased by 11% in the phosphorus-loaded animals (0.40 ± 0.03 versus 0.46 ± 0.03, 0.45 ± 0.02, and 0.45 ± 0.03,
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respectively) (P < 0.001), in Nx-control, intact-control, and intact-ad libitum groups (Fig. 1). There was a mild reduction in double tetracycline labeling in the phosphorus-loaded nephrectomized animals (Nx-phosphorus), 122 ± 22 lm/day, compared to Nxcontrol, intact-control, and intact-ad libitum groups, respectively (212 ± 73, 178 ± 42, and 196 ± 25 lm/day), but these values did not reach statistical significance (P < 0.06). These findings reflect the lack of difference in tibial length and body length measurements at the end of the study period between the two Nx groups. It is unclear why the tetracycline label did not differ between the Nx animals and those with normal renal function. A possible explanation may be the limitation of the technique used in the current study, particularly the method of tetracycline administration and the system of measurement between the two labels. There may be a wide variability in the amount of tetracycline absorbed using the intraperitoneal method of injection such that some animals may not demonstrate a clear separation between the two tetracycline labels. IGF-I protein staining significantly declined by 24% in the Nx-phosphorus animals (P < 0.01), compared to intact-control and intact-ad libitum groups. In the Nxcontrol group, IGF-I protein expression decreased by 15% (P = 0.02 (Fig. 2). IGF-binding proteins (IGFBP)3 protein expression declined by approximately 39% in the Nx animals compared to the groups with normal renal function (P < 0.04) (Fig. 3). In contrast, IGF-I receptor mRNA expression did not differ among groups. IGF-I protein expression correlated with the measurements of body length (R = 0.6, P < 0.01) and with PTH/PTHrP receptor (R = 0.8, P < 0.01). There was a 30% reduction in PTH/PTHrP receptor mRNA transcripts (Fig. 4) and at least a twofold increase in the histochemical staining for TRAP in the phosphorus-loaded animals (0.005 ± 0.002 cells per area in Nx-phosphorus group compared to 0.002 ± 0.001 cells per area in Nx-control, intact-control and intact-ad libitum animals) (P < 0.02). Despite changes in PTH/PTHrP receptor, the protein expression for Indian hedgehog did not change. The PTH/PTHrP receptor expression correlated with serum PTH levels measured using the intact assay (R = −0.8, P < 0.001). Markers used to evaluate chondrocyte proliferation, including PCNA and histone-4 mRNA expression declined in both Nx groups; PCNA staining was 0.4 ± 0.1 and 0.5 ± 0.07 in Nx-phosphorus and Nx-control groups versus 0.61 ± 0.06 in intact-control and intact-ad libitum animals (P < 0.001), and histone-4 mRNA expression, 0.60 ± 0.04 in both Nx groups and 0.74 ± 0.07 in animals with normal renal function (P < 0.02). On the other hand, the expression of type II collagen, type X collagen, gelatinase B, Bcl-2 protein, Bax protein, and vascular endothelial growth factor did not differ in all groups.
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Sanchez et al: Growth plate alterations in renal failure
Growth plate
Proliferative zone
Hypertrophic zone
Micrometer
500 d
400 300
a b
200
b c
100 0 Nx-Control
Nx-Phos
Intact-Control
The number of apoptotic terminal chondrocytes in the chondro-osseous junction remained unchanged in both Nx groups and in animals with normal renal function. DISCUSSION Impairment of linear growth in renal failure has been attributed to several factors, including tissue resistance to the actions of growth hormone partly due to a decrease in growth hormone receptor expression, low hepatic expression of IGF-I mRNA, and diminished bioactivity of IGF-I–dependent growth processes [13, 14]. In our current study, the reduction in IGF-I protein expression was much greater in rats with advanced secondary hyperparathyroidism compared to the animals with mild elevations in PTH levels. Although the severity of growth impairment was equivalent in both groups of Nx animals, the width of the growth plate cartilage was much narrower in the phosphorus-loaded animals and this reduction may lead to further reduction in longitudinal growth as the animals matures. Serum IGF-I levels were not obtained in the current study, but Tonshoff et al [13] have reported that although the plasma levels of IGF-I did not differ between animals with renal failure and normal renal function, there was an associated reduction in IGF-I mRNA expression in the liver, lung, and muscle of the uremic animals. Thus, serum levels of IGF-I may not be an important determinant of IGF-I mRNA expression in the presence of renal failure. Despite equivalent caloric intake, the Nx animals had significantly less weight gain
Intact-Ad Lib
Fig. 1. Measurements of the total width of the growth plate () and the width of the area occupied by the proliferating and () and hypertrophic chondrocytes ( ) are demonstrated. (Upper panel) a P < 0.04 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups; b P < 0.007 vs. intact-control and intact-ad libitum animals; c P < 0.03 vs, Nx-control, intact-control, and intact-ad libitum; d P < 0.007 vs. Nx-control and intact-control. The middle panel denotes the morphology of the growth plate optimally fixed in Ruthenium Hexamine Trichloride (20×). Note that there is distortion of the growth plate architecture in the phosphorus loaded animals (second panel), including the loss of columnar organization of the chondrocytes and changes in the growth plate matrix. (Lower panel) The low magnification of the growth plate (14×), including parts of the primary spongiosa.
and shorter body and tibial lengths at the end of the 4week study period when compared to the control animals. There was no difference demonstrated between the tibial length and body length measurements between the Nxcontrol and Nx-phosphorus groups. These findings may suggest that the slightly higher serum creatinine in the Nx-control animals may have affected growth independent of the degree of secondary hyperparathyroidism. IGF-I participates in longitudinal growth by partly enhancing chondrocyte proliferation, amplifying chondrocyte hypertrophy and increasing matrix production [15]. In the IGF-I null mice, longitudinal growth declined by 35% primarily due to the smaller size of the chondrocytes occupying the hypertrophic zone without any associated impairment in the expression of specialized proteins, including type X collagen and alkaline phosphatase [15]. In our current and previous experiments, alterations in the histology of the growth plate in rats with renal failure and advanced secondary hyperparathyroidism were not associated with changes in the expression of type II collagen and type X collagen in the proliferating and hypertrophic chondrocytes. In various clinical and animal studies, the administration of growth hormone, IGF-I or both have led to an increase in linear growth in children with chronic renal failure, augmented bone growth, and even enhanced IGF-I expression in the growth plate of uremic rats [4, 16, 17]. To stimulate growth in children, supraphysiologic doses of recombinant human growth hormone (rhGH) are given in order to increase circulating IGF-I levels
Sanchez et al: Growth plate alterations in renal failure
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Labeled cells Total no. of cells
1.2 1 0.8
b
a
0.6 0.4 0.2 0
Nx-Control
Nx-Phos
Intact-Control
Intact-Ad Lib
Fig. 2. Immunohistochemical localization of insulin-like growth factor-I (IGF-I) protein staining (denoted by brown color) in the lower proliferative zone and in the hypertrophic chondrocytes (50×). a P < 0.01 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups; b P < 0.02 vs. animals with normal renal function.
Labeled cells Total no. of cells
1.2 1 0.8 0.6
a
a
Nx-Control
Nx-Phos
0.4 0.2 0
Intact-Control
Intact-Ad Lib
Fig. 3. Quantification of the insulin-like growth factor binding protein-3 (IGFBP-3) signals. (Upper panel) a P < 0.04 vs. intactcontrol and intact-ad ibitum animals. (Lower panel) The localization of IGFBP-3 in the growth plate (brown color) (50×).
and counteract the effects of the accumulated IGFBP, especially IGFBP-3. Previous studies have shown that the growth response to rhGH therapy was much better in children with chronic renal failure on conservative management compared to children undergoing dialysis [18]; such findings may be attributed to the possibility that there is a greater insensitivity to growth hormone therapy in dialysis patients mediated in part by the lower IGF-I protein expression in the growth plate cartilage. Our current findings show that IGF-I protein expression was much less in rats with advanced secondary hyperparathyroidism compared to animals with mild secondary hyperparathyroidism, and these changes were accompanied by a decrease in PTH/PTHrP receptor mRNA. The decline in IGF-I protein without any changes in IGF-I receptor expression as demonstrated in the current study
may suggest the presence of postreceptor signaling defect that occur in the presence of renal failure and secondary hyperparathyroidism. PTHrP is an autocrine/paracrine factor that has a variety of physiologic functions and expressed in various tissues including the skeleton. In the growth plate cartilage, PTHrP and Indian hedgehog are responsible, at least in part, for the control of chondrocyte proliferation and chondrocyte differentiation [19, 20]. The growth plate cartilage was much smaller in the rats with advanced secondary hyperparathyroidism mainly due to the smaller proportion of the chondrocytes in the proliferative and hypertrophic zones. In contrast to other reports, we did not demonstrate a decrease in the width of the hypertrophic zone in Nx animals with mild secondary hyperparathyroidism and there were no changes in the
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Labeled cells Total no. of cells
1.2 1 0.8
a
0.6 0.4 0.2 0
Nx-Control
Nx-Phos
Intact-Control
Intact-Ad Lib Fig. 4. Parathyroid hormone/parathyroid hormone receptor protein (PTH/PTHrP) receptor mRNA expression in the lower proliferative and upper hypertrophic zones (denoted by black color) in all groups, including the quantification of the mRNA transcripts. (Upper panel) a P < 0.001 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups (50×).
expression of both type X collagen and type II collagen. The shorter proliferative zone in the rats with advanced secondary hyperparathyroidism may be partly explained by the low expression of PTH/PTHrP receptor mRNA. Several investigators have demonstrated that the layers of proliferating chondrocytes are mainly affected by PTHrP signaling such that the growth plate of PTHrP (−/−) null mice was 30% shorter than the wild-type and almost completely absent in the PTH/PTHrP receptor (−/−) mice [21–23]. Chondrocyte differentiation is controlled at various steps by both PTHrP and Ihh through PTH/PTHrP and Patched 1 receptors [24, 25]. In the current set of experiments, the expression of Ihh protein did not differ between the Nx groups and the animals with normal renal function despite a significant decline in PTH/PTHrP receptor expression in the phosphorusloaded animals; such findings may suggest a dysregulation in the PTH/PTHrP-Ihh signaling pathway in the presence of advanced secondary hyperparathyroidism. The expression of Patched 1 receptor was not evaluated in the current experiments. The determination of bone growth and bone elongation is partly dependent on the number of hypertrophic and proliferating chondrocytes available in the growth plate, and the interaction among multiple regulators and their receptors. The interactions of these regulators may be altered in the presence of renal failure and advanced secondary hyperparathyroidism. A dramatic increase in aggrecan synthesis in chondrocytes isolated from rib cages of rat embryos was demonstrated after treatment with PTH-(1–34) [26]. Proteoglycan synthesis was not evaluated in the current study; however, a decrease in the production of aggrecan may contribute to the shorter growth evident in rats with advanced secondary hyperparathyroidism. Alterations in the growth plate architecture are evident in the
current study, including changes in the matrix and the loss of columnar organization of the chondrocytes (see Fig. 1). Although, there was a significant elevation of the serum PTH levels in the current study particularly in the phosphorus-loaded rats, the increase may be due to the presence of the biologically inactive C-terminal component of PTH that may actually inhibit not only chondrocyte proliferative activity but also the synthesis of proteoglycans. The PTH assay used in our study measured both the intact PTH-(1–84) and the PTH Cterminal fragment (39–84). PTH C-terminal (7–84) fragments have been reported to inhibit the calcemic actions of hPTH-(1–34) or hPTH-(1–84) in parathyroidectomized rats and exert a direct anti-resorptive effect on neonatal mouse calvarial bone independent of the type I PTH receptor [27, 28]. Harvey et al [26] reported that only the administration of N-terminally intact PTH and PTHrP were effective in both promoting synthesis and incorporation of aggrecan into the cell and extracellular matrix in the rat costal chondrocytes. These findings may explain the lack of differences in the expression of type X collagen and type II collagen particularly in the hypertrophic chondrocytes of Nx animals in the current experiment. The PTH-stimulated aggrecan synthesis has been reported to be dependent on the IGF-I/IGF binding protein axis since the addition of a neutralizing antibody to IGF-I during PTH treatment resulted in the inhibition of aggrecan synthesis [26]. In addition, Bikle et al [29] have reported that the anabolic effects of PTH in bone did not occur in the absence of IGF-I. Since IGF-I has been postulated to mediate the anabolic actions of PTH on the bone, the significantly lower expression of IGF-I in the current set of experiments, may at least in part contribute to the impairment of growth in renal failure. The
Sanchez et al: Growth plate alterations in renal failure
down-regulation of PTH/PTHrP receptor transcripts and the increase staining for chondroclast/osteoclast activity in the chondro-osseous junction may further worsen the growth retardation in these animals by increasing cartilage/bone resorption without any increase in chondrocyte proliferation or chondrocyte turnover. IGFBP-3 protein expression decreased in both groups of rats with renal failure. The biologic activity of IGFBP-3 is complex. Under preincubation conditions with IGF-I, IGFBP-3 enhanced IGF-I responsiveness by facilitating IGF-I cell surface binding, whereas IGFBP-3 under coincubation conditions with IGF-I, exerted an antiproliferative and inhibitory effect [30]. In our study, the low IGFBP-3 protein expression was certainly associated with decreased IGF-I protein staining despite comparable expression of IGF-I receptor in the animals with intact renal function and rats with renal failure. Overall, the impairment of growth evident in rats with renal failure and mild secondary hyperparathyroidism may, in part be due to the mild decrease in IGF-I activity and lower IGFBP-3 expression. In rats with advanced secondary hyperparathyroidism, however, the decline in PTH/PTHrP receptor mRNA expression and further decrease in IGF-I protein staining may worsen bone growth in these animals despite higher levels of serum PTH and comparable caloric intake. These findings have important implications in the therapeutic armamentarium currently available to enhance linear growth in young children with chronic renal failure. Supraphysiologic doses of rhGH have been demonstrated to increase height in children with chronic renal failure [31], but the growth response in dialysis patients is suboptimal [18]. Possible explanations for the blunted growth response in dialysis patients include the use of higher doses of vitamin D especially calcitriol required to control secondary hyperparathyroidism. When administered in high doses, calcitriol inhibits chondrocyte proliferation probably through its actions on IGF-I and IGFBP-4 [32, 33]. Growth hormone therapy, however, is not without its problems since worsening of secondary hyperparathyroidism has been reported in children undergoing dialysis therapy [34][abstract; Kuizon BD et al, J Am Soc Nephrol 11:577A, 2000]. On the other hand, the administration of IGF-I has been shown to increase height velocity when given to children with growth hormone insensitivity syndrome and improved growth in uremic animals, but the growth response is not comparable to growth hormone [35]. In the long run, exogenous IGF-I administration may suppress endogenous growth hormone production and the reported metabolic complications associated with IGFI, particularly hypoglycemia may not be well tolerated in children [35]. Further studies are required to evaluate the safety and efficacy of IGF-I therapy in children with chronic renal failure. Future development of therapeutic agents that can selectively enhance local IGF-
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I expression and increase PTH/PTHrP receptor in the growth plate cartilage may be helpful in the improvement of growth in children with chronic renal failure and secondary hyperparathyroidism. ACKNOWLEDGMENTS This study was supported by NIH grant DK56688 and by Research funds from the Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin. We would like to thank Dr. G.V. Segre and Dr. K. Lee from Massachusetts General Hospital, Boston, Massachusetts, for providing us with the mouse type X collagen, type II collagen, mouse MMP-9/gelatinase B, and rat PTH/PTHrP receptor cDNA. The rat vascular endothelial growth factor was provided by Dr. Zena Werb, University of California, San Francisco, California, histone4 was provided by Dr. Gary Stein, University of Massachusetts, Worcester, Massachusetts, and IGF-I receptor was given by Dr. Derek LeRoith from NIH, Bethesda, Maryland. Reprint requests to Cheryl P. Sanchez, M.D., 3590 MSC/Pediatrics, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. E-mail: [email protected]
REFERENCES 1. COBO A, LOPEZ JM, CARBAJO E, et al: Growth plate cartilage formation and resorption are differentially expressed in growth retarded uremic rats. J Am Soc Nephrol 10:971–979, 1999 2. SANCHEZ CP, SALUSKY IB, KUIZON BD, et al: Growth of long bones in renal failure: Roles of hyperparathyroidism, growth hormone and calcitriol. Kidney Int 54:1879–1887, 1998 3. URENA P, FERREIRA A, MORIEUX C, et al: PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilage growth plate of uremic rats. Nephrol Dial Transplant 11:2008–2016, 1996 4. HANNA JD, SANTOS F, FOREMAN JW, et al: Insulin-like growth factorI gene expression in the tibial epiphyseal growth plate of growth hormone-treated uremic rats. Kidney Int 47:1374–1382, 1995 5. MEHLS O, RITZ E, GILLI G, et al: Skeletal changes and growth in experimental uremia. Nephron 18:288–300, 1977 6. ALVAREZ J, BALBIN M, FERNANDEZ M, et al: Collagen metabolism is markedly altered in the hypertrophic cartilage of growth plates from rats with growth impairment secondary to chronic renal failure. J Bone Miner Res 16:511–524, 2001 7. BREUR GJ, VANENKEVORT BA, FARNUM CE, et al: Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates. J Orthop Res 9:348–359, 1991 8. WILSMAN NJ, FARNUM CE, LEIFERMAN EM, et al: Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics. J Orthop Res 14:927–936, 1996 9. HUNZIKER EB: Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Techniq 28:505– 519, 1994 10. LEE K, DEEDS JD, BOND AT, et al: In situ localization of PTH/PTHrP receptor mRNA in the bone of fetal and young rats. Bone 14:341– 345, 1993 11. SANCHEZ CP, HE Y-Z: Alterations in the growth plate cartilage of rats with renal failure receiving corticosteroid therapy. Bone 30:692– 698, 2002 12. SANCHEZ CP, HE Y-Z: Effects of thyroparathyroidectomy, exogenous calcium, and short-term calcitriol therapy on the growth plate in renal failure. J Am Soc Nephrol 14:148–158, 2003 13. TONSHOFF B, POWELL DR, ZHAO D, et al: Decreased hepatic insulinlike growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia. Endocrinology 128:938– 946, 1997 14. BLUM WF, RANKE MB, KIETZMANN K, et al: Growth hormone resistance and inhibition of somatomedin activity by excess of insulinlike growth factor binding protein in uremia. Pediatr Nephrol 5: 539–544, 1991
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15. WANG J, ZHOU J, BONDY CA: IGF-I promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. FASEB J 13:1985–1990, 1999 16. FINE RN, ATTIE KM, KUNTZE J, et al: Recombinant human growth hormone in infants and young children with chronic renal insufficiency. Pediatr Nephrol 9:451–457, 1995 17. KOVACS GT, OH J, KOVACS J, et al: Growth promoting effects of growth hormone and IGF-I are additive in experimental uremia. Kidney Int 49:1413–1421, 1996 18. WUHL E, HAFFNER D, NISSEL R, et al: Short dialyzed children respond less to growth hormone than patients prior to dialysis. Pediatr Nephrol 10:294–298, 1996 19. VORTKAMP A, LEE K, LANKSE B, et al: Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273:613–622, 1996 20. LANSKE B, KARAPLIS AC, LEE K, et al: PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273:663–666, 1996 21. LANSKE B, AMLING M, NEFF L, et al: Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 104:399–407, 1999 22. AMIZUKA N, WARSHAWSKY H, HENDERSON JE, et al: Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol 126:1611–1623, 1994 23. CHUNG U-I, SCHIPANI E, MCMAHON AP, et al: Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 107:295–304, 2001 24. KOBAYASHI T, CHUNG U-I, SCHIPANI E, et al: PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development 129:2977–2986, 2002 25. FARQUHARSON C, JEFFERIES D, SEAWRIGHT E, et al: Regulation of chondrocyte terminal differentiation in the postembryonic growth plate: The role of the PTHrP-Indian hedgehog axis. Endocrinology 142:4131–4140, 2001 26. HARVEY AK, YU X-P, FROLIK CA, et al: Parathyroid hormone-(1–
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34) enhances aggrecan synthesis via an insulin-like growth factor-I pathway. J Biol Chem 274:23249–23255, 1999 DIVIETI P, JOHN MR, JUPPNER H, et al: Human PTH-(7–84) inhibits bone resorption in vitro actions independent of the type 1 PTH/PThrP receptor. Endocrinology 143:171–176, 2002 LANGUB MC, MONIER-FAUGERE M-C, WANG G, et al: Administration of PTH-(7–84) antagonizes the effects of PTH-(1–84) on bone in rats with moderate renal failure. Endocrinology 144:1135–1138, 2003 BIKLE DD, SAKATA T, LEARY C, et al: Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res 17:1570–1578, 2002 KIEPE D, ANDRESS DL, MOHAN S, et al: Intact IGF-binding protein4 and -5 and their respective fragments isolated from chronic renal failure serum differentially modulate IGF-I actions in cultured growth plate chondrocytes. J Am Soc Nephrol 12:2400–2410, 2001 FINE RN, KOHAUT EC, BROWN D, et al: Growth after recombinant human growth hormone treatment in children with chronic renal failure: Report of a multicenter randomized double-blind placebocontrolled study. J Pediatr 94:374–382, 1994 SCHARLA SH, STRONG DD, MOHAN S, et al: 1,25-dihydroxyvitamin D 3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts. Endocrinology 129:3139–3146, 1991 SILBERMANN M, MIRSKY N, LEVITAN S, et al: The effect of 1,25dihydroxyvitamin D 3 on cartilage growth in neonatal mice. Metab Bone Dis Rel Res 4:337–345, 1983 PANCZYK-TOMASZEWSKA M, ZIOLKOWSKA H, DEBINSKI A, et al: Vitamin D metabolite requirements in dialysed children receiving recombinant human growth hormone. Nephrol Dial Transplant 15:375–378, 2000 BACKELJAUW PF, UNDERWOOD LE: Therapy for 6.5–7.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: A clinical research center study. J Clin Endocrinol Metab 86:1504–1503, 2001
Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism CHERYL P. SANCHEZ, YU-ZHU HE, ELLEN LEIFERMAN, and NORMAN J. WILSMAN Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin; and Department of Comparative Biological Sciences, University of Wisconsin School of Veterinary Medicine, Madison, Wisconsin
Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism. Background. Impairment of growth in children with chronic renal failure may be due, in part to the insensitivity to the actions of growth hormone by insulin-like growth factor-I (IGF-I) because of accumulations of IGF binding proteins. There are a few studies describing the changes that occur in the growth plate in renal failure. None of these studies has simultaneously compared the modifications in the expression of selected markers of endochondral bone formation in renal failure with mild or advanced secondary hyperparathyroidism. Methods. Forty-six rats that underwent 5/6 nephrectomy (Nx) were fed either standard rodent diet (Nx-control) or high phosphorus diet to induce advanced secondary hyperparathyroidism (Nx-phosphorus) for 4 weeks. Sections of the tibia were obtained for growth plate histomorphometry, immunohistochemistry studies, and in situ hybridization experiments for selected markers of endochondral bone formation. Results. Weight gain, gain in length, and tibial length were less in Nx animals. Serum parathyroid hormone (PTH) and phosphorus levels were higher and serum calcium levels were lower in the Nx-phosphorus group. The width of the growth plate was much shorter in the Nx-phosphorus group due to a decrease in both proliferative and hypertrophic zones. IGF-I protein and IGF binding protein-3 staining were diminished in both Nx groups without changes in the IGF-I receptor expression; the decline in IGF-I protein expression was much lower in the Nx-phosphorus group. PTH/PTH receptor protein (PTHrP) receptor mRNA transcripts decline and tartrate-resistant acid phosphastase (TRAP) staining increased only in the Nxphosphorus group. Conclusion. The growth impairment in renal failure may be worsened by the severity of secondary hyperparathyroidism.
The growth plate cartilage is important in bone elongation and longitudinal growth. Bone elongation is achieved by the complex interplay of chondrocyte proliferation,
Key words: growth plate, secondary hyperparathyroidism, endochondral bone formation. Received for publication September 5, 2003 and in revised form November 11, 2003 Accepted for publication December 10, 2003 C
2004 by the International Society of Nephrology
matrix synthesis, chondrocyte maturation and hypertrophy, matrix degradation, angiogenesis, and mineralization. In the presence of renal failure, abnormalities in the histology of the growth plate cartilage have been reported in animals, and these alterations may contribute to the impairment of linear growth reported in young uremic children. Cobo et al [1] have shown that the width of the growth plate was increased in nephrectomized rats compared to animals with normal renal function due to the widening of the hypertrophic zone. Furthermore, there was low chondrocyte turnover and impairment of cartilage resorption in the chondro-osseous junction [1]; however, the degree of secondary hyperparathyroidism in these experiments were unknown. In contrast, we have shown that the growth plate width was decreased and the microarchitecture of the growth plate was disorganized in rats with renal failure and severe secondary hyperparathyroidism accompanied by a decline in the expression of the parathyroid hormone/parathyroid hormone receptor protein (PTH/PTHrP) receptor mRNA in the proliferating and maturing chondrocytes [2]. Similar findings were reported by Urena ˜ et al [3] in rats that were given high phosphorus diet to induce severe secondary hyperparathyroidism. Currently, there are no concurrent studies available that compare changes in the growth plate cartilage in renal failure with mild or advanced secondary hyperparathyroidism. Our previous study was done mainly to evaluate the effects of growth hormone and calcitriol on the growth plate in renal failure, but the experiments were not done simultaneously [2]. Although serum insulin-like growth factor-I (IGF-I) levels have been reported to be within normal limits in rats with renal failure, there was a significant decline in the expression of IGF-I mRNA, growth hormone receptor transcripts, defective mineralization, and alterations in collagen metabolism [4–6]. As in previous reports, serum PTH levels were not obtained in these studies so the severity of secondary hyperparathyroidism cannot be evaluated. The current set of experiments was performed to compare the effects of the varying degrees of secondary hyperparathyroidism on chondrocyte proliferation, 1740
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chondrocyte hypertrophy, matrix degradation, and initiation of mineralization in young rats with renal failure. METHODS Forty-six male weanling Sprague-Dawley rats (Harlan Laboratories, Madison, WI, USA) with a mean weight of 57 ± 3 g were housed in individual cages at constant temperature with a 12-hour light and 12-hour dark cycle. The animals had free access to drinking water and standard rodent diet containing 23.4% protein, 0.6% calcium, and 0.6% phosphorus (Purina Mills, Indianapolis, IN, USA). After 24 hours of acclimatization, 16 rats underwent the first stage of a two-stage 5/6 nephrectomy (Nx), and 30 rats underwent sham nephrectomy (intact) using surgical procedures described elsewhere [2]. The second-stage nephrectomy was completed 7 days later. The 4-week study period began 24 hours after completion of the second surgery. All procedures were reviewed and approved by the Research Animal Resource Center at the University of Wisconsin, Madison, Wisconsin. Measurements of body weight and body length were obtained weekly. Fifteen sham-nephrectomized animals (intact-control), and six nephrectomized animals (Nxcontrol) continued to ingest standard rodent diet. To induce advanced secondary hyperparathyroidism, ten Nx rats were given high phosphorus diet (Nx-phosphorus) (1.2% phosphorus and 0.6% calcium) (Purina Mills). To ensure equal caloric intake, all intact-control (N = 15) animals were pair-fed with Nx animals by providing the amount of food each day to intact-control rats that had been consumed the previous day by Nx animals. A group of sham-Nx animals (intact-ad libitum) (N = 15) were fed ad libitum with the standard rodent chow. Another group of animals with normal renal function given high phosphorus diet was not included in the current experiment. To evaluate longitudinal bone growth, the animals were given intraperitoneal injections of oxytetracycline hydrochloride, 1 mg/kg/dose, 3 days and 1 day prior to sacrifice. Four weeks after the second-stage nephrectomy, the animals were randomly divided into two groups. In group I, animals from the Nx-control (N = 3), Nx-phosphorus (N = 5), intact-control (N = 10), and intact-ad libitum (N = 10) groups were anesthetized, killed by exsanguination by cardiac puncture, and underwent transcardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). The tibiae were decalcified for 2 weeks and embedded in paraffin as previously described [2]. Five micrometer sections of bone were obtained for morphometric analysis, in situ hybridization, and immunohistochemistry experiments. In group II, the remaining animals, Nx-control (N = 3), Nx-phosphorus (N = 5), intact-control (N = 5), and intact-ad libitum (N = 5), were given lethal doses of
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pentobarbital (300 mg/kg intraperitoneally). The growth plate in the proximal tibia of the left leg was quickly collected to measure the rate of elongation. For each of the growth plate, two thin slabs were cut and were observed under epifluorescence microscopy with an excitation filter of 330 to 380 nm and a barrier filter of 420 nm. To estimate bone elongation over 48 hours, the distance between the leading edges of the two fluorescent bands was measured four times using an ocular reticle and the average measurement was converted to elongation for 24 hours [7]. When there is only one label discernable, the measured distance between the visible label and the chondro-osseous junction is then used for calculation of bone elongation. For optimal fixation, the remaining growth plate was fixed in 2% glutaraldehyde in 0.05 mol/L cacodylate buffer (pH 7.35) with 0.7% ruthenium hexamine trichloride and embedded in Epon-araldite [8, 9], and sections were obtained for histology. The right tibia was also collected, freed of adherent tissue, fixed in 4% parafolmaldehyde in PBS for 24 hours, decalcified in 15% ethylenediaminetetraacetic acid (EDTA) in paraformaldehyde for approximately 2 weeks and processed together with group I as described above. All animals were included in all in situ hybridization and immunohistochemistry experiments, Nx-control (N = 6), Nx-phosphorus (N = 10), intact-control (N = 15), and intact-ad libitum (N = 15). Serum biochemical determinations Serum was obtained by centrifugation, and samples were stored at −70◦ C. Serum urea nitrogen, creatinine, calcium, and phosphorus levels were measured using standard laboratory methods. Serum intact PTH levels were measured using the Rat Intact PTH enzyme-linked immunosorbent assay (ELISA) Kit (Immutopics, Inc., San Clemente, CA, USA). Growth plate morphometry For morphometric analysis, three 5 lm sections of bone were obtained from each tibia, stained with hematoxylin and eosin, viewed by light microscopy at 20× and images were captured onto a computer monitor. The total width of the growth plate and the widths of the proliferative and hypertrophic zones were measured using an image analysis software (Elektronik 200) (Kontron Instruments, Ltd., Hallbergmoos, Germany) as previously described [2]. In situ hybridization (radioactive and nonradioactive) In situ hybridization was performed using methods described elsewhere [2, 10]. 35 S-labeled sense and antisense riboprobes encoding mouse matrix metalloproteinase9 (MMP-9)/gelatinase B and rat vascular endothelial growth factor were labeled to a specific activity of 1 to 2
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× 109 cpm/lg. After hybridization and posthybridization washing, the slides were exposed to x-ray film overnight, and emulsion autoradiography was done using NTB-2 (Eastman Kodak, Rochester, New York) at 4◦ C. For nonradioactive in situ hybridization, probes encoding histone-4, mouse type X collagen, rat PTH/PTHrP receptor, and IGF-I receptor were linearized, purified, labeled with digoxigenin-uridine triphosphate (UTP) in the nucleotide-triphosphate (NTP) labeling mixture (Roche Diagnostics, Mannheim, Germany), and mixed in a hybridization mixture [11]. The specimens were hybridized with specific riboprobes at 100 lL/specimen at 55◦ C to 60◦ C in a humidified chamber overnight. Following the overnight hybridization, the specimens were washed, incubated with the secondary antibody, and color was developed [11]. Quantification of in situ hybridization signals For the specimens hybridized with 35 S, the slides were viewed at 100× by bright field microscopy and the number of silver grains overlying each chondrocyte profile was counted using an image analysis system [2]. Data are expressed as the number of silver grains/1000 lm2 of cell profile. For the nonradioactive in situ hybridization and immunohistochemistry experiments, the number of cells expressing the specific mRNA or protein was counted and expressed as percentage of the number of positive cells to the total number of cells in the appropriate growth plate zone where the mRNA expression is localized [11]. The mRNA expression for MMP-9/gelatinase B was quantified by measuring the area with positive staining and results are expressed as labeled area over the total tissue area in the chondro-osseous junction [11]. Immunohistochemistry [Bcl-2, Bax, IGF-I protein, IGF binding protein-3, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) assay, tartrate-resistant acid phosphatase (TRAP)] Immunohistochemistry was performed using methods described previously using the following primary antibodies: monoclonal Bcl-2 antibody (Santa Cruz Biotechnology, San Diego, CA, USA), monoclonal Bax antibody (Santa Cruz Biotechnology), monoclonal IGF-I protein antibody (Upstate Biotechnology, Lake Placid, NY, USA), and polyclonal IGF binding protein-3 antibody (Santa Cruz Biotechnology) [11]. For PCNA, the tissues were immunostained with the mouse anti-PCNA antibody (Zymed Laboratories, South San Francisco, CA, USA). For the TUNEL assay, the specimens were labeled using the kit from Intergen (Gaithersburg, MD, USA). The number of TUNELpositive cells were counted and expressed as percent-
Table 1. Anthropomorphic and tibial length measurements in all groups Nxcontrol N=6 Gain in weighta g 100 ± 29c Gain in lengtha cm 11 ± 1.7c Tibial lengthb cm 3.5 ± 0.1c
Nxphosphorus N = 10
Intactcontrol Nx = 15
Intact-ad libitum N = 15
100 ± 34c 10 ± 2.2c 3.4 ± 0.1c
126 ± 21 13 ± 1.3 3.8 ± 0.1
217 ± 25d 17 ± 0.9d 4.0 ± 0.1d
Nx is nephrectomized. a Difference in weight or length between baseline and at the end of the 4-week study period; b Obtained at the end of the study period; c P < 0.0001 vs. intact-control; d P < 0.0001 vs. intact-control, Nx-control, and Nx-phosphorus.
age of the total number of terminal chondrocytes in each section. Histochemical staining for TRAP was done using methods reported previously [2]. Image analysis was done in tissue sections viewed at 50× and the number of TRAP-positive cells in the chondro-osseous junction was quantified and expressed as number of cells per area measured in the chondro-osseous junction and in the nearby primary spongiosa. Statistical analysis All results are expressed as mean values ± 1 SD. Data were evaluated by one-way analysis fo variance (ANOVA) and comparisons among groups were done using Bonferroni/Dunn post hoc tests using the StatView statistical software (SAS Institute, Cary, NC, USA). For all statistical tests, probability values less than 5% were considered to be significant. RESULTS Compared to our previous experiments, there was a significant increase in mortality rate from 5% to 10% in our earlier studies [2, 12] to approximately 25% in the current set of experiments. The rats used in this study were much younger with body weights between 50 to 60 g compared to 60 to 80 g in our previous studies. Most of the mortality occurred within 7 days after the secondstage surgery. Anthromorphic measurements As expected, the mean gain in body weight was twofold greater in the intact-ad libitum animals compared to the intact-control, Nx-phosphorus, and Nx-control groups (Table 1). There was a 35% and 15% increase in total body length at the end of the study period in the intactad libitum and intact-control groups (Table 1). Similarly, tibial length measurements were much longer by approximately 11% to 15% in the animals with normal renal function compared to the Nx animals (Table 1). Tibial length and gain in body length at the end of the study period did not differ between the two Nx groups; such
Sanchez et al: Growth plate alterations in renal failure
Table 2. Serum biochemical parameters in all groups obtained at the end of the study period
Parathyroid hormone pg/mL Calcium mg/dL Phosphorus mg/dL Urea nitrogen mg/dL Creatinine mg/dL
Nxcontrol N=6
Nxphosphorus N = 10
Intactcontrol N = 15
Intact-ad libitum N = 15
323 ± 206b
1038 ± 633a
120 ± 53
75 ± 46
9.2 ± 1.6
7.3 ± 0.7a
10 ± 1.3
10 ± 1.4
11 ± 1.3
16 ± 5.0a
10 ± 2.3
11 ± 1.5
100 ± 25b
81 ± 11b
16 ± 2
22 ± 3
1.0 ± 0.4b
0.7 ± 0.1b
0.3 ± 0.08
0.3 ± 0.07
Nx is nephrectomized. a < P 0.001 vs. Nx-control, intact-control, and intact-ad libitum; b P < 0.05 vs. intact-control and intact-ad libitum.
findings may be due in part to the mildly elevated mean serum creatinine level in the Nx-control animals (1.0 ± 0.1 mg/dL) compared to the phosphorus-loaded group (0.7 ± 0.1 mg/dL). Serum creatinine levels moderately correlated with tibial length and body length measurements in the current study (R= −0.7, P < 0.001). Serum urea nitrogen and serum creatinine levels were higher in the Nx animals (Table 2). Serum phosphorus levels rose and serum calcium levels declined in the Nx animals given high phosphorus diet (Nx-phosphorus) (Table 2). Intact PTH levels increased eightfold in the Nx-phosphorus animals and were modestly elevated in the Nx-control animals compared to the groups with intact renal function (Table 2). Serum PTH levels correlated with measurements of tibial length (R = −0.7, P < 0.001), gain in body length (R = −0.6, P < 0.001), and TRAP staining (R = 0.6, P < 0.05). The total thickness of the growth plate was wider by approximately 35% in the intact-ad libitum animals when compared to the intact-control and Nx-control groups (369 ± 29 lm versus 323 ± 50 lm and 335 ± 57 lm, respectively) (P < 0.007) (Fig. 1). In contrast, there was a significant reduction in the width of the growth plate in the Nx-phosphorus animals (288 ± 42 lm) (P < 0.04) (Fig. 1). As previously demonstrated in our earlier experiments, alterations in the architecture of the growth plate cartilage were evident mainly in the animals given high phosphorus diet with the loss of the columnar organization of the proliferating chondrocytes [2] (Fig. 1). The area occupied by the proliferating chondrocytes in the Nx animals decreased by approximately 20% compared to the groups with normal renal function (Fig. 1) (0.5 ± 0.04 versus 0.6 ± 0.04, respectively) (P < 0.02). In addition, the area occupied by the hypertrophic chondrocytes decreased by 11% in the phosphorus-loaded animals (0.40 ± 0.03 versus 0.46 ± 0.03, 0.45 ± 0.02, and 0.45 ± 0.03,
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respectively) (P < 0.001), in Nx-control, intact-control, and intact-ad libitum groups (Fig. 1). There was a mild reduction in double tetracycline labeling in the phosphorus-loaded nephrectomized animals (Nx-phosphorus), 122 ± 22 lm/day, compared to Nxcontrol, intact-control, and intact-ad libitum groups, respectively (212 ± 73, 178 ± 42, and 196 ± 25 lm/day), but these values did not reach statistical significance (P < 0.06). These findings reflect the lack of difference in tibial length and body length measurements at the end of the study period between the two Nx groups. It is unclear why the tetracycline label did not differ between the Nx animals and those with normal renal function. A possible explanation may be the limitation of the technique used in the current study, particularly the method of tetracycline administration and the system of measurement between the two labels. There may be a wide variability in the amount of tetracycline absorbed using the intraperitoneal method of injection such that some animals may not demonstrate a clear separation between the two tetracycline labels. IGF-I protein staining significantly declined by 24% in the Nx-phosphorus animals (P < 0.01), compared to intact-control and intact-ad libitum groups. In the Nxcontrol group, IGF-I protein expression decreased by 15% (P = 0.02 (Fig. 2). IGF-binding proteins (IGFBP)3 protein expression declined by approximately 39% in the Nx animals compared to the groups with normal renal function (P < 0.04) (Fig. 3). In contrast, IGF-I receptor mRNA expression did not differ among groups. IGF-I protein expression correlated with the measurements of body length (R = 0.6, P < 0.01) and with PTH/PTHrP receptor (R = 0.8, P < 0.01). There was a 30% reduction in PTH/PTHrP receptor mRNA transcripts (Fig. 4) and at least a twofold increase in the histochemical staining for TRAP in the phosphorus-loaded animals (0.005 ± 0.002 cells per area in Nx-phosphorus group compared to 0.002 ± 0.001 cells per area in Nx-control, intact-control and intact-ad libitum animals) (P < 0.02). Despite changes in PTH/PTHrP receptor, the protein expression for Indian hedgehog did not change. The PTH/PTHrP receptor expression correlated with serum PTH levels measured using the intact assay (R = −0.8, P < 0.001). Markers used to evaluate chondrocyte proliferation, including PCNA and histone-4 mRNA expression declined in both Nx groups; PCNA staining was 0.4 ± 0.1 and 0.5 ± 0.07 in Nx-phosphorus and Nx-control groups versus 0.61 ± 0.06 in intact-control and intact-ad libitum animals (P < 0.001), and histone-4 mRNA expression, 0.60 ± 0.04 in both Nx groups and 0.74 ± 0.07 in animals with normal renal function (P < 0.02). On the other hand, the expression of type II collagen, type X collagen, gelatinase B, Bcl-2 protein, Bax protein, and vascular endothelial growth factor did not differ in all groups.
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Growth plate
Proliferative zone
Hypertrophic zone
Micrometer
500 d
400 300
a b
200
b c
100 0 Nx-Control
Nx-Phos
Intact-Control
The number of apoptotic terminal chondrocytes in the chondro-osseous junction remained unchanged in both Nx groups and in animals with normal renal function. DISCUSSION Impairment of linear growth in renal failure has been attributed to several factors, including tissue resistance to the actions of growth hormone partly due to a decrease in growth hormone receptor expression, low hepatic expression of IGF-I mRNA, and diminished bioactivity of IGF-I–dependent growth processes [13, 14]. In our current study, the reduction in IGF-I protein expression was much greater in rats with advanced secondary hyperparathyroidism compared to the animals with mild elevations in PTH levels. Although the severity of growth impairment was equivalent in both groups of Nx animals, the width of the growth plate cartilage was much narrower in the phosphorus-loaded animals and this reduction may lead to further reduction in longitudinal growth as the animals matures. Serum IGF-I levels were not obtained in the current study, but Tonshoff et al [13] have reported that although the plasma levels of IGF-I did not differ between animals with renal failure and normal renal function, there was an associated reduction in IGF-I mRNA expression in the liver, lung, and muscle of the uremic animals. Thus, serum levels of IGF-I may not be an important determinant of IGF-I mRNA expression in the presence of renal failure. Despite equivalent caloric intake, the Nx animals had significantly less weight gain
Intact-Ad Lib
Fig. 1. Measurements of the total width of the growth plate () and the width of the area occupied by the proliferating and () and hypertrophic chondrocytes ( ) are demonstrated. (Upper panel) a P < 0.04 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups; b P < 0.007 vs. intact-control and intact-ad libitum animals; c P < 0.03 vs, Nx-control, intact-control, and intact-ad libitum; d P < 0.007 vs. Nx-control and intact-control. The middle panel denotes the morphology of the growth plate optimally fixed in Ruthenium Hexamine Trichloride (20×). Note that there is distortion of the growth plate architecture in the phosphorus loaded animals (second panel), including the loss of columnar organization of the chondrocytes and changes in the growth plate matrix. (Lower panel) The low magnification of the growth plate (14×), including parts of the primary spongiosa.
and shorter body and tibial lengths at the end of the 4week study period when compared to the control animals. There was no difference demonstrated between the tibial length and body length measurements between the Nxcontrol and Nx-phosphorus groups. These findings may suggest that the slightly higher serum creatinine in the Nx-control animals may have affected growth independent of the degree of secondary hyperparathyroidism. IGF-I participates in longitudinal growth by partly enhancing chondrocyte proliferation, amplifying chondrocyte hypertrophy and increasing matrix production [15]. In the IGF-I null mice, longitudinal growth declined by 35% primarily due to the smaller size of the chondrocytes occupying the hypertrophic zone without any associated impairment in the expression of specialized proteins, including type X collagen and alkaline phosphatase [15]. In our current and previous experiments, alterations in the histology of the growth plate in rats with renal failure and advanced secondary hyperparathyroidism were not associated with changes in the expression of type II collagen and type X collagen in the proliferating and hypertrophic chondrocytes. In various clinical and animal studies, the administration of growth hormone, IGF-I or both have led to an increase in linear growth in children with chronic renal failure, augmented bone growth, and even enhanced IGF-I expression in the growth plate of uremic rats [4, 16, 17]. To stimulate growth in children, supraphysiologic doses of recombinant human growth hormone (rhGH) are given in order to increase circulating IGF-I levels
Sanchez et al: Growth plate alterations in renal failure
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Labeled cells Total no. of cells
1.2 1 0.8
b
a
0.6 0.4 0.2 0
Nx-Control
Nx-Phos
Intact-Control
Intact-Ad Lib
Fig. 2. Immunohistochemical localization of insulin-like growth factor-I (IGF-I) protein staining (denoted by brown color) in the lower proliferative zone and in the hypertrophic chondrocytes (50×). a P < 0.01 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups; b P < 0.02 vs. animals with normal renal function.
Labeled cells Total no. of cells
1.2 1 0.8 0.6
a
a
Nx-Control
Nx-Phos
0.4 0.2 0
Intact-Control
Intact-Ad Lib
Fig. 3. Quantification of the insulin-like growth factor binding protein-3 (IGFBP-3) signals. (Upper panel) a P < 0.04 vs. intactcontrol and intact-ad ibitum animals. (Lower panel) The localization of IGFBP-3 in the growth plate (brown color) (50×).
and counteract the effects of the accumulated IGFBP, especially IGFBP-3. Previous studies have shown that the growth response to rhGH therapy was much better in children with chronic renal failure on conservative management compared to children undergoing dialysis [18]; such findings may be attributed to the possibility that there is a greater insensitivity to growth hormone therapy in dialysis patients mediated in part by the lower IGF-I protein expression in the growth plate cartilage. Our current findings show that IGF-I protein expression was much less in rats with advanced secondary hyperparathyroidism compared to animals with mild secondary hyperparathyroidism, and these changes were accompanied by a decrease in PTH/PTHrP receptor mRNA. The decline in IGF-I protein without any changes in IGF-I receptor expression as demonstrated in the current study
may suggest the presence of postreceptor signaling defect that occur in the presence of renal failure and secondary hyperparathyroidism. PTHrP is an autocrine/paracrine factor that has a variety of physiologic functions and expressed in various tissues including the skeleton. In the growth plate cartilage, PTHrP and Indian hedgehog are responsible, at least in part, for the control of chondrocyte proliferation and chondrocyte differentiation [19, 20]. The growth plate cartilage was much smaller in the rats with advanced secondary hyperparathyroidism mainly due to the smaller proportion of the chondrocytes in the proliferative and hypertrophic zones. In contrast to other reports, we did not demonstrate a decrease in the width of the hypertrophic zone in Nx animals with mild secondary hyperparathyroidism and there were no changes in the
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Sanchez et al: Growth plate alterations in renal failure
Labeled cells Total no. of cells
1.2 1 0.8
a
0.6 0.4 0.2 0
Nx-Control
Nx-Phos
Intact-Control
Intact-Ad Lib Fig. 4. Parathyroid hormone/parathyroid hormone receptor protein (PTH/PTHrP) receptor mRNA expression in the lower proliferative and upper hypertrophic zones (denoted by black color) in all groups, including the quantification of the mRNA transcripts. (Upper panel) a P < 0.001 vs. nephrectomized (Nx)-control, intact-control, and intact-ad libitum groups (50×).
expression of both type X collagen and type II collagen. The shorter proliferative zone in the rats with advanced secondary hyperparathyroidism may be partly explained by the low expression of PTH/PTHrP receptor mRNA. Several investigators have demonstrated that the layers of proliferating chondrocytes are mainly affected by PTHrP signaling such that the growth plate of PTHrP (−/−) null mice was 30% shorter than the wild-type and almost completely absent in the PTH/PTHrP receptor (−/−) mice [21–23]. Chondrocyte differentiation is controlled at various steps by both PTHrP and Ihh through PTH/PTHrP and Patched 1 receptors [24, 25]. In the current set of experiments, the expression of Ihh protein did not differ between the Nx groups and the animals with normal renal function despite a significant decline in PTH/PTHrP receptor expression in the phosphorusloaded animals; such findings may suggest a dysregulation in the PTH/PTHrP-Ihh signaling pathway in the presence of advanced secondary hyperparathyroidism. The expression of Patched 1 receptor was not evaluated in the current experiments. The determination of bone growth and bone elongation is partly dependent on the number of hypertrophic and proliferating chondrocytes available in the growth plate, and the interaction among multiple regulators and their receptors. The interactions of these regulators may be altered in the presence of renal failure and advanced secondary hyperparathyroidism. A dramatic increase in aggrecan synthesis in chondrocytes isolated from rib cages of rat embryos was demonstrated after treatment with PTH-(1–34) [26]. Proteoglycan synthesis was not evaluated in the current study; however, a decrease in the production of aggrecan may contribute to the shorter growth evident in rats with advanced secondary hyperparathyroidism. Alterations in the growth plate architecture are evident in the
current study, including changes in the matrix and the loss of columnar organization of the chondrocytes (see Fig. 1). Although, there was a significant elevation of the serum PTH levels in the current study particularly in the phosphorus-loaded rats, the increase may be due to the presence of the biologically inactive C-terminal component of PTH that may actually inhibit not only chondrocyte proliferative activity but also the synthesis of proteoglycans. The PTH assay used in our study measured both the intact PTH-(1–84) and the PTH Cterminal fragment (39–84). PTH C-terminal (7–84) fragments have been reported to inhibit the calcemic actions of hPTH-(1–34) or hPTH-(1–84) in parathyroidectomized rats and exert a direct anti-resorptive effect on neonatal mouse calvarial bone independent of the type I PTH receptor [27, 28]. Harvey et al [26] reported that only the administration of N-terminally intact PTH and PTHrP were effective in both promoting synthesis and incorporation of aggrecan into the cell and extracellular matrix in the rat costal chondrocytes. These findings may explain the lack of differences in the expression of type X collagen and type II collagen particularly in the hypertrophic chondrocytes of Nx animals in the current experiment. The PTH-stimulated aggrecan synthesis has been reported to be dependent on the IGF-I/IGF binding protein axis since the addition of a neutralizing antibody to IGF-I during PTH treatment resulted in the inhibition of aggrecan synthesis [26]. In addition, Bikle et al [29] have reported that the anabolic effects of PTH in bone did not occur in the absence of IGF-I. Since IGF-I has been postulated to mediate the anabolic actions of PTH on the bone, the significantly lower expression of IGF-I in the current set of experiments, may at least in part contribute to the impairment of growth in renal failure. The
Sanchez et al: Growth plate alterations in renal failure
down-regulation of PTH/PTHrP receptor transcripts and the increase staining for chondroclast/osteoclast activity in the chondro-osseous junction may further worsen the growth retardation in these animals by increasing cartilage/bone resorption without any increase in chondrocyte proliferation or chondrocyte turnover. IGFBP-3 protein expression decreased in both groups of rats with renal failure. The biologic activity of IGFBP-3 is complex. Under preincubation conditions with IGF-I, IGFBP-3 enhanced IGF-I responsiveness by facilitating IGF-I cell surface binding, whereas IGFBP-3 under coincubation conditions with IGF-I, exerted an antiproliferative and inhibitory effect [30]. In our study, the low IGFBP-3 protein expression was certainly associated with decreased IGF-I protein staining despite comparable expression of IGF-I receptor in the animals with intact renal function and rats with renal failure. Overall, the impairment of growth evident in rats with renal failure and mild secondary hyperparathyroidism may, in part be due to the mild decrease in IGF-I activity and lower IGFBP-3 expression. In rats with advanced secondary hyperparathyroidism, however, the decline in PTH/PTHrP receptor mRNA expression and further decrease in IGF-I protein staining may worsen bone growth in these animals despite higher levels of serum PTH and comparable caloric intake. These findings have important implications in the therapeutic armamentarium currently available to enhance linear growth in young children with chronic renal failure. Supraphysiologic doses of rhGH have been demonstrated to increase height in children with chronic renal failure [31], but the growth response in dialysis patients is suboptimal [18]. Possible explanations for the blunted growth response in dialysis patients include the use of higher doses of vitamin D especially calcitriol required to control secondary hyperparathyroidism. When administered in high doses, calcitriol inhibits chondrocyte proliferation probably through its actions on IGF-I and IGFBP-4 [32, 33]. Growth hormone therapy, however, is not without its problems since worsening of secondary hyperparathyroidism has been reported in children undergoing dialysis therapy [34][abstract; Kuizon BD et al, J Am Soc Nephrol 11:577A, 2000]. On the other hand, the administration of IGF-I has been shown to increase height velocity when given to children with growth hormone insensitivity syndrome and improved growth in uremic animals, but the growth response is not comparable to growth hormone [35]. In the long run, exogenous IGF-I administration may suppress endogenous growth hormone production and the reported metabolic complications associated with IGFI, particularly hypoglycemia may not be well tolerated in children [35]. Further studies are required to evaluate the safety and efficacy of IGF-I therapy in children with chronic renal failure. Future development of therapeutic agents that can selectively enhance local IGF-
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I expression and increase PTH/PTHrP receptor in the growth plate cartilage may be helpful in the improvement of growth in children with chronic renal failure and secondary hyperparathyroidism. ACKNOWLEDGMENTS This study was supported by NIH grant DK56688 and by Research funds from the Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin. We would like to thank Dr. G.V. Segre and Dr. K. Lee from Massachusetts General Hospital, Boston, Massachusetts, for providing us with the mouse type X collagen, type II collagen, mouse MMP-9/gelatinase B, and rat PTH/PTHrP receptor cDNA. The rat vascular endothelial growth factor was provided by Dr. Zena Werb, University of California, San Francisco, California, histone4 was provided by Dr. Gary Stein, University of Massachusetts, Worcester, Massachusetts, and IGF-I receptor was given by Dr. Derek LeRoith from NIH, Bethesda, Maryland. Reprint requests to Cheryl P. Sanchez, M.D., 3590 MSC/Pediatrics, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. E-mail: [email protected]
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