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Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin 53706
Address all correspondence and requests for reprints to: Cheryl P. Sanchez, M.D., 3590 Medical Science Center/Pediatrics, University of Wisconsin Medical School, 1300 University Avenue, Madison, Wisconsin 53706. E-mail: cpsanchez{at}wisc.edu.
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Abstract |
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Introduction |
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GH and calcitriol are potent modifiers of longitudinal bone growth in children with chronic renal failure. In general, GH therapy produces a mitogenic response in the growth plate cartilage and bone including the stimulation of chondrocyte and osteoblastic proliferation, raising collagen production and increasing bone resorption by directly binding to the GH receptor (GHR) leading to stimulation of local IGF-I and IGF binding protein (IGFBP) production. When administered to children with chronic renal failure, GH therapy increases linear growth; however, the growth response is much less in children who are undergoing dialysis therapy compared with those who are on conservative medical management (2). Several factors may contribute to the blunted growth response in children maintained on chronic dialysis including the use of high doses of calcitriol or other vitamin D analogs to control secondary hyperparathyroidism, presence of low turnover bone disease and higher insensitivity to GH therapy. Our previous studies have shown that serum IGF-I levels did not increase in transplanted prepubertal children with adynamic bone after 12 months of GH therapy compared with those patients with normal bone formation (3). Calcitriol, on the other hand, has dose-dependent antiproliferative effects on chondrocytes and osteoblasts. Children with chronic renal failure are frequently treated with GH to enhance linear growth and calcitriol to control secondary hyperparathyroidism.
The objective of the current study, therefore, is to evaluate growth and the expression of selected molecular markers of chondrocyte proliferation and chondrocyte differentiation in calcium-loaded young rats with renal failure treated with GH.
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Materials and Methods |
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All animals were weighed, and body length measurements were determined in sedated animals by measuring the distance from the tip of the nose to the end of the tail. The 6-wk study period began 24 h after completion of the second surgery. Seven sham-nephrectomized (Intact-Control) rats, and five Nx animals continued to ingest standard rodent diet. To induce adynamic bone, a group of seven Intact-Ca2+ and the remaining Nx-Ca2+ animals (n = 29) were given a high-calcium diet (2.0% calcium, 0.68% phosphorus, 23.4% protein) (Purina Mills) for the duration of the study period (1). To ensure equivalent caloric intake in all animals, the Intact animals were pair-fed with animals that have undergone subtotal Nx (Nx-Control) by providing the amount of food each day to intact rats that had been consumed the previous day by Nx animals. Body weight and body length were obtained weekly.
At the end of 4 wk, a group of eight Nx animals received GH (Nx-Ca2+ + GH, n = 8) (Genentech, San Francisco, CA) at a dose of 10 IU/kg·d, calcitriol (Nx-Ca2+ + D, n = 8) (Abbot Laboratories, North Chicago, IL) at a dose of 50 ng/kg·d, combination of GH (10 IU/kg·d) and calcitriol (50 ng/kg·d) (Nx-Ca2+ + GH + D, n = 6). The remaining groups, Nx-Control (n = 5), Nx-Ca2+ (n = 7), Intact-Control (n = 7), and Intact-Ca2+ (n = 7), were all given saline injections. All injections were given daily at the same volume and administered at the same time by ip injection 2 wk before animals were killed. The dose and duration of GH and calcitriol therapy was based on an earlier study that showed changes in the growth plate only after 10 d of treatment (4). A control group with normal renal function treated with GH was not included in the current experiments. Earlier studies have shown that GH administered to rats with normal renal function increased body weight, body length, and growth plate width (5, 6).
After 6 wk of the study period, the rats were anesthetized, killed by exsanguination by cardiac puncture, and underwent transcardiac perfusion with 4%PFA in PBS. Blood was obtained for biochemical determinations for calcium, creatinine, phosphorus, urea nitrogen, and PTH and IGF-I. The proximal tibiae were excised, and tibial lengths were measured as previously described (4). Bones were decalcified in 15% ethylenediamine tetra-acetic acid in PBS (pH 7.0), at 4 C for approximately 2 wk and embedded in paraffin. Five-micrometer sections of bone for morphometric analysis, in situ hybridization, and immunohistochemistry were obtained using the Leica rotary microtome 2165 (Leica Microsystems, Nussloch, Germany).
Serum biochemical determinations
Serum was obtained by centrifugation and samples were stored at –70 C until biochemical assays are done. Serum urea nitrogen, creatinine, calcium, and phosphorus levels were measured using standard laboratory methods. PTH levels were measured using the Rat Bioactive Intact PTH ELISA kit (Immutopics Inc., San Clemente, CA), and IGF-I levels were measured using the rat IGF-I ELISA kit (Diagnostic Systems Laboratories, Inc., Webster, TX).
Growth plate morphometry
For morphometric analysis, three 5-µm sections of bone were obtained from each tibia and stained with hematoxylin and eosin. Sections were viewed by light microscopy at x30, and images were captured onto a computer monitor using a video camera control unit (Hitachi Denshi Ltd., Tokyo, Japan). The total width of the growth plate at the proximal end of each tibia was measured at equally spaced intervals using an image analysis software (Elektronik 200; Kontron Instruments Ltd, Hallbergmoos, Germany) (4). The widths of the zones occupied by hypertrophic chondrocytes and proliferative chondrocytes were also measured by the same method, and the values are expressed as ratios of the selected zone to the total width of the growth plate.
In situ hybridization (radioactive and nonradioactive)
In situ hybridization was performed using methods described elsewhere (4). Briefly, 35S-labeled sense and antisense riboprobes were generated encoding mouse MMP-9/gelatinase B (provided by Drs. G. V. Segre and K. Lee, Massachusetts General Hospital, Boston, MA) and rat vascular endothelial growth factor (provided by Dr. Zena Werb, University of California, San Francisco, CA) and labeled to a specific activity of 1–2 x 109 cpm/µg using the Gemini transcription kit (Promega Corp., Madison, WI). After hybridization and posthybridization washing, the slides were exposed to x-ray film (Kodak Scientific Imaging Systems, Rochester, NY) overnight, and emulsion autoradiography was done using NTB-2 (Eastman Kodak, Rochester, NY) at 4 C.
For nonradioactive in situ hybridization, probes encoding Histone-4 kindly provided by Dr. Gary Stein (University of Massachusetts, Worcester, MA), IGF-I receptor provided by Dr. Derek LeRoith (NIH, Bethesda, MD), PTH/PTHrP receptor, type II collagen, and type X collagen given by Drs. G. V. Segre and K. Lee were linearized, purified, labeled with Digoxigenin-uridine triphosphate in the nucleotide triphosphate labeling mixture (Roche Diagnostics, Mannheim, Germany), and mixed in a hybridization mixture as described previously (7). The specimens were hybridized with specific riboprobes at 100 µl/specimen in a hybridization solution mixture (200 ng riboprobe/ml hybridization mixture) at 55–60 C in a humidified chamber overnight. After the overnight hybridization, the specimens were washed, incubated with the secondary antibody and color was developed (7).
Quantification of in situ hybridization signals
Slides were viewed at x100 under bright-field microscopy and the number of silver grains overlying each chondrocyte profile was counted using an image analysis system (Elektornik 200, Kontron Instruments Ltd.) (4). Data are expressed as the number of silver grains/1000 µm2 of cell profile.
For the nonradioactive in situ hybridization, the number of cells expressing the specific mRNA 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. 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.
Immunohistochemistry [IGF-I, IGFBP-3, GHR, Indian hedgehog (Ihh), bone morphogenetic protein (BMP)-7, TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) assay, and tartrate-resistant acid phosphatase (TRAP)]
Immunohistochemistry experiments were performed using methods described previously (7) with the following primary antibodies: IGF-I (monoclonal antibody, Upstate Biotechnology, Lake Placid, NY) at a concentration of 10 µg/ml, IGFBP-3 at a concentration of 4 µg/ml (polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA), GHR at a concentration of 20 µg/ml (polyclonal antibody, American Diagnostica Inc., Stamford, CT), Ihh protein at a concentration of 10 µg/ml (polyclonal antibody, Santa Cruz Biotechnology), and BMP-7 at a concentration of 5 µg/ml (polyclonal antibody, Santa Cruz Biotechnology) incubated at 4 C overnight in a humidified chamber. For quantification, the number of cells expressing IGF-I, IGFBP-3, GHR, Ihh, and BMP-7 were counted and expressed as percentage of the labeled cells over the total number of cells in the appropriate zone in the growth plate. For the TUNEL assay, the specimens were labeled using the kit from Intergen (Gaithersburg, MD). The number of TUNEL-positive cells were counted and expressed as percentage of the labeled cells over the total number of terminal chondrocytes in each section.
Histochemical staining for TRAP was performed using methods reported previously (4). Image analysis was done in tissue sections viewed at x30, projected onto the computer screen and the number of TRAP-positive cells in the chondro-osseous junction was quantified and expressed as number of cells per area of the growth plate in the chondro-osseous junction.
Statistical analysis
All results are expressed as mean values ± 1 SD. Data were evaluated by one-way ANOVA and comparisons among groups were done using Bonferroni/DUNN post hoc tests using the StatView statistical software (SAS Institute, Cary, NC). The Pearson product moment correlation coefficient was performed to evaluate the relationship between two numerical variables. For all statistical tests, probability values less than 5% were considered to be significant.
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Results |
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). Body weight, body length, and tibial length were less in the Nx-Control group compared with the animals with normal renal function (Intact-Control), but the values did not reach statistical significance (Table 1). There was a significant correlation between the changes in body length obtained at the beginning and end of the study period and the measurements of tibial length at the end of the study period, R = 0.8, P < 0.0001. No correlation was demonstrated between the gain in length or tibial length to the serum intact PTH levels, R = –0.3, P = not significant. There were no differences in the average daily food intake and the food utilization ratio for each experimental group (Table 1).
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). In addition, serum intact PTH levels remained unchanged after 2 wk of GH and calcitriol therapy (Table 2). Serum calcium levels were higher and serum phosphorus levels were mildly lower in the Nx animals that received exogenous calcium (Table 2). As expected, serum creatinine and urea nitrogen levels were elevated in Nx animals compared with Intact-Control (Table 2). Serum IGF-I levels obtained at the end of the study period did not differ in all groups (Table 2).
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). After 2 wk of therapy, a reduction in the growth plate width was evident in the Nx animals treated with calcitriol alone and the combination of calcitriol and GH; the growth plate width was comparable to the Nx-Control group and to the animals with normal renal function (Intact-Control) (Fig. 1). There was a decline in the area occupied by the proliferating chondrocytes (calculated as percent area of the proliferative zone compared with the total width of the growth plate) in the Nx-Control animals compared with the Intact groups, 0.5 ± 0.05 vs. 0.6 ± 0.06, P < 0.02 (Fig. 1). There was further reduction in the width of the proliferative zone in Nx-Ca2+ rats; however, GH and calcitriol therapy increased the thickness of the proliferating chondrocytes comparable to the Nx-Control animals, 0.4 ± 0.02 vs. 0.5 ± 0.05, 0.53 ± 0.01, 0.54 ± 0.04, respectively, P < 0.05 (Fig. 1). The area occupied by the hypertrophic chondrocytes was much wider in the Nx-Ca2+ animals compared with Nx-Control, Intact-Control, and Intact-Ca2+ groups, 0.5 ± 0.05 vs. 0.45 ± 0.02, 0.44 ± 0.07, 0.45 ± 0.04, P < 0.002.
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). Type II collagen and PTH/PTHrP receptor mRNA expression, however, did not differ in all groups. There was no correlation between serum intact PTH levels and PTH/PTHrP receptor mRNA expression in the growth plate.
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). Likewise, IGF-I receptor mRNA expression considerably declined in the Nx-Ca2+ control animals and in those treated with calcitriol (Nx-Ca2+ + D) (Fig. 3B). GH alone or in combination with calcitriol increased IGF-I receptor expression comparable to Nx-Control and Intact-Control groups. There was no correlation between the serum IGF-I levels and the IGF-I protein expression in the chondrocytes.
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). Such decline was also evident in the Nx-Ca2+ rats and in those treated with GH alone (Nx-Ca2+ + GH) (Fig. 3C). Calcitriol therapy increased IGF-binding protein-3 comparable to the Intact-Control animals whether administered alone or in combination with GH (Fig. 3C).
GHR expression was lower in the Nx-Control and in rats given a high-calcium diet (Nx-Ca2+) (Fig. 3D). Both GH and calcitriol therapy administered alone or together increased GHR staining in the chondrocytes (Fig. 3D).
BMP-7 staining localized mostly in the zone occupied by the hypertrophic chondrocytes declined in all Nx animals (Fig. 3E). Although GH therapy given alone or with calcitriol mildly increased the expression for BMP-7 in the hypertrophic chondrocytes, such increase was not comparable to the Intact-Control group at the end of the study period (Fig. 3E). On the other hand, 2 wk treatment with calcitriol alone further decreased BMP-7 protein expression by approximately 25% when compared with the animals given GH alone or in combination with calcitriol (Fig. 3E). Type X collagen mRNA expression localized to the hypertrophic chondrocytes and Ihh protein staining did not differ in all groups.
As previously reported in our earlier experiments, gelatinase B mRNA expression was also lower only in the Nx group given a high-calcium diet (Nx-Ca2+) when compared with all other Nx animals and to rats with normal renal function (Intact-Control), 0.11 ± 0.037 vs. 0.22 ± 0.079 and 0.24 ± 0.026, P < 0.03, respectively (Fig. 4). Histochemical staining for TRAP was much less in all Nx animals fed a high-calcium diet including the group with normal renal function (Intact-Ca2+) (Fig. 5). Vascular endothelial growth factor mRNA expression in the terminal chondrocytes did not differ in all groups.
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Discussion |
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Chronic renal failure, per se, has been associated with derangements in GH, IGF, and IGFBP axis leading to relative resistance to the actions of GH and IGF-I. Tonshoff et al. (9) have demonstrated a 37% decline in hepatic IGF-I gene expression, 20–30% decrease in hepatic GHR and lower hepatic GH binding protein expression. Although the serum levels of IGF-I levels did not change in renal failure, there was an increase in the plasma levels of IGFBP-1, -2, and -4; no changes were demonstrated in IGFBP-3 (9). Hanna et al. (10) have reported a widening of the growth plate cartilage and an increase in the IGF-I mRNA expression in the chondrocytes after GH therapy in rats with renal failure. In contrast to previous studies, the current findings did not show any significant increase in the serum levels of IGF-I or an enhancement in IGF-I protein expression or IGFBP-3 staining in the chondrocytes after 2 wk of GH treatment. The high doses of extracellular calcium may up-regulate the expression of cytokine suppressors that participate in the hepatic and local production of IGF-I after treatment with GH.
Adynamic renal bone disease in patients with chronic renal failure is associated with low to normal bone formation rate and low osteoblast and osteoclast number. In addition, biochemical findings include low to normal parathyroid hormone levels, low alkaline phosphatase and problems with hypercalcemia. PTH is a potent anabolic agent that affects bone formation and bone resorption. It has been thought that adynamic bone is a relative state of hypoparathyroidism because clinical investigations have demonstrated that a higher serum intact PTH level is required to maintain normal bone formation in chronic renal failure. As demonstrated in the current study, exogenous calcium loading considerably lowered serum PTH levels and the administration of GH did not correct this problem. The lack of response to GH therapy may be due, in part to the low PTH levels demonstrated in the animals given a high-calcium diet with or without vitamin D. Although there were no changes in serum IGF-I levels in the animals that received GH, IGF-I receptor, and IGFBP-3 expression declined in these animals after calcium loading. The combination of low PTH levels and a decreased in IGF-I expression may be responsible for the blunted effects of GH in the growth plate cartilage. The stimulatory effects of PTH on bone have been reported to be directly affected by the presence of IGF-I. Exogenous PTH administration to IGF-I (minus/minus) null mice only increased bone formation parameters when the animals were given IGF-I at the same time (11, 12).
Calcium loading in rats with renal failure led to a reduction in histone-4 expression in the chondrocytes. Concurrent calcitriol administration in rats fed a high-calcium diet further decreased histone-4 staining and GH was not effective in counteracting the effects of both vitamin D and exogenous calcium. Calcitriol has been reported to have dose-dependent antiproliferative effects on chondrocytes and osteoblasts. Recent experiments by Eelen et al. (13) have shown that calcitriol when coincubated in high doses with mouse osteoblasts exerts its antiproliferative effect by inducing a G1 arrest in the cell cycle and significant down-regulation of the E2F family of transcription factors. The mechanisms by which high calcium lower chondrocyte proliferative activity in renal failure are currently unknown; however, the concomitant administration of vitamin D in children receiving high doses of calcium salts to control hyperphosphatemia may lead to further reduction in linear growth.
The changes in the growth plate cartilage in the current experiments cannot be all explained by renal failure because the changes in chondrocyte proliferation and chondrocyte differentiation in the Nx rats given high doses of exogenous calcium were more severe compared with the Nx-Control animals. In addition, the average daily food intake and the food utilization ratio were comparable in all experimental groups, so poor nutrition may not be able to explain all the changes in the growth plate. Currently, there is no information available on the direct or indirect effects of exogenous calcium loading on GH signal transduction or IGF-I activation. It is plausible that the high extracellular calcium concentration may directly impair the reported increase in locally produced IGF-I in chondrocytes after the administration of GH, or that the high calcium may adversely affect GH signaling in the chondrocytes. Exogenous calcium may also directly inhibit transcription factors necessary in stimulating the cyclin D proteins that are important positive regulators of chondrocyte proliferation, and this inhibitory effect may not be reversed by GH alone. In addition, cyclin-dependent kinase inhibitors may also be directly or indirectly up-regulated by exogenous calcium loading leading to the reduction in the proliferation of the chondrocytes in the growth plate cartilage in renal failure. The processes involved in cell proliferation have not been fully elucidated in uremic animals.
GH not only affects cell proliferation but also participates in chondrocyte differentiation. In the current study, rats with renal failure given exogenous calcium had a considerable widening of the zone occupied by the hypertrophic chondrocytes, and these alterations in the growth plate persisted even after GH administration. Interestingly, calcitriol therapy alone decreased the width of the hypertrophic zone comparable to the Nx and Intact-Control animals. In our earlier experiments, we have demonstrated that the decline in gelatinase B mRNA expression and the reduction in histochemical staining for TRAP may contribute to the delay in mineralization and a wider zone occupied by the hypertrophic chondrocytes (1). Although GH increased gelatinase B expression and TRAP activity in the chondro-osseous junction comparable to the Nx-Control group, GH therapy alone was not able to reverse the changes induced by exogenous calcium loading. Such findings may also be due to the low serum PTH levels demonstrated in the calcium-loaded animals because gelatinase B activity has been reported to be enhanced by PTH administration (14, 15).
Calcitriol increased gelatinase B mRNA expression and further decreased TRAP staining in the calcium-loaded animals, yet the growth plate width is more comparable with the Nx-Control or Intact-Control group. Although calcitriol has antiproliferative effects on chondrocytes, the increase in gelatinase B activity in the current study may have prevented the widening of the hypertrophic zone and restored the thickness of the growth plate as demonstrated in calcium-supplemented rats. In vitro experiments have also demonstrated that the coincubation of IGF-I and calcitriol with rat epiphyseal chondrocytes has a dose-dependent increase (especially with low doses) in IGF-I receptor expression, DNA synthesis, cell proliferation, and alkaline phosphatase activity (16). There were no changes demonstrated in the expression of the Ihh protein or in the PTH/PTHrP receptor mRNA transcripts in the current study. These findings may indicate in part that the transition between chondrocyte proliferation to chondrocyte maturation may not be significantly affected in this study.
Resistance to GH therapy can also be attributed to the increase in IGFBPs that prevents the binding of IGF-I to its receptor. The biological action of IGF-I is mediated via the type I IGF-I receptor; however, the IGFBPs bind IGF-I with affinities similar or higher to those of type I IGF-I receptor. In the current study, there was a mild increase in IGF-I receptor and GHR in the chondrocytes of GH-treated animals without any increase in IGF-I and IGFBP-3 expression. In the presence of renal failure, IGFBPs, especially IGFBP-1 and IGFBP-2, have been identified as inhibitors of IGF-I bioactivity and may play a significant role in the growth failure demonstrated in uremic children (17). Studies that evaluated the relationship of IGF-I and IGFBP-3 in children with chronic renal failure, however, are quite variable. Intact IGFBP-3 in chronic renal failure may have a growth-promoting action via the formation of the 150-kDa ternary complex, whereas the smaller molecular fragments may act as IGF-I inhibitors by competing with the IGF receptors for IGF binding (18).
GH was not able to increase the serum PTH levels previously demonstrated in earlier experiments (4). When administered to children with chronic renal failure to improve linear growth, worsening of secondary hyperparathyroidism have been reported in these children (19). These findings may be due in part to the inhibitory effects of calcium on the parathyroid gland. The mitogenic response to GH may also be mediated by an increase in serum PTH levels, but these changes were not shown in the current study because serum PTH levels remained low in rats that were given exogenous calcium despite GH treatment. Thus, the dose and frequency of GH therapy in the presence of adynamic bone or low turnover bone disease induced by exogenous calcium loading as in the current study may not be able to counteract the inhibitory effects of calcium on the growth plate cartilage.
BMP-7 or osteogenic protein-1, a member of the TGF-ß family of proteins participates in chondrocyte differentiation and mineralization. In rats with renal failure, the expression of BMP-7 in the growth plate cartilage was down-regulated when compared with the Intact-Control group. There was also a considerable decline in BMP-7 staining in all Nx animals loaded with exogenous calcium that was not reversed by either GH or calcitriol therapy. The reduction in BMP-7 expression may play a significant role in the widening of the growth plate cartilage, maintenance of the chondrocytes in the hypertrophic stage, and delay in mineralization in the calcium-loaded rats with renal failure as demonstrated in the current study.
The abnormalities reported in endochondral bone formation in rats with renal failure given exogenous calcium persisted despite GH therapy. The lack of anabolic response to GH therapy in the current study may not be fully explained by the high doses of calcium and calcitriol because the doses and the mode of administration of GH may not be optimal in these animals. Further studies are required to evaluate whether there is greater resistance to GH therapy in uremic animals given exogenous calcium loading.
The findings in the current study, however, may explain in part the fair to poor growth response in some children with adynamic bone who are undergoing chronic dialysis therapy or those who have undergone renal transplantation. We have reported that serum IGF-I levels did not increase in prepubertal children with functioning renal allograft after 12 months of treatment with GH (3). The concurrent treatment with calcitriol and administration of high doses of exogenous calcium should be followed very closely in growth-retarded children that may require GH therapy; the combination of these agents may contribute to the suboptimal growth response to GH therapy in renal failure. Other anabolic agents that may increase PTH levels such as exogenous PTH administration may play a role in improving the abnormalities demonstrated in the growth plate of calcium-loaded animals with renal failure.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMP, Bone morphogenetic protein; GHR, GH receptor; IGFBP, IGF binding protein; Ihh, Indian hedgehog; Nx, 5/6 nephrectomy/nephrectomized; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling.
Received September 30, 2003.
Accepted for publication March 10, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25 (OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes. Kidney Int 53:1152–1161[CrossRef][Medline]
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), proliferative zone (
), and hypertrophic zone (
