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From the Departments of Pediatrics (C.P.S., B.D.K., I.B.S.) and Medicine (P.A.A., W.G.G.), UCLA School of Medicine, Los Angeles, California 90095; and the Endocrine Unit (H.J.), Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: William G. Goodman, M.D., Division of Nephrology, 7–155 Factor Building, UCLA Medical Center, 10833 Le Conte Avenue, Los Angeles, California 90095. E-mail: wgoodman{at}ucla.edu
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Abstract |
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Introduction |
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The long-term consequences of adynamic renal osteodystrophy have yet to be determined. Hypercalcemia is more common, however, in patients with adynamic bone than in those with secondary hyperparathyroidism (3, 4). Preliminary work also suggests that the risk of fracture is greater in adult hemodialysis patients with adynamic lesions (5, 6). In the pediatric age group, linear growth has recently been reported to decline in prepubertal children who develop adynamic renal osteodystrophy during the treatment of secondary hyperparathyroidism with large intermittent doses of calcitriol (7). Such findings suggest that adynamic bone is associated with abnormalities in chondrocyte proliferation and/or differentiation within the epiphyseal growth plate at the ends of long bones in the developing skeleton. Whether mediated through direct or indirect mechanisms, disturbances in chondrocyte function may aggravate the growth retardation that characterizes children with renal failure (8, 9).
Factors associated with low serum PTH levels and adynamic renal osteodystrophy in adults include previous parathyroidectomy, age-related or postmenopausal osteoporosis, diabetes mellitus, and the use of large doses of calcitriol to treat secondary hyperparathyroidism (10, 11). In most patients, however, including children, the ingestion of large amounts of calcium to diminish intestinal phosphorus absorption and the use of high dialysate calcium concentrations are major contributors (10). The current study was undertaken, therefore, to test the hypothesis that a high dietary calcium intake lowers serum PTH levels and modifies chondrocyte proliferation and/or differentiation in the epiphyseal growth plate of growing animals with renal failure and to determine whether these changes are associated with impaired linear bone growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The day after completing the second set of surgical procedures, all animals were weighed, and body length was determined by measuring the distance from the tip of the nose to the end of the tail while animals were sedated. This marked the start of the four week study.
Nine sham-nephrectomized control (C, n = 9) animals and seven nephrectomized control (Nx-C, n = 7) animals continued to ingest standard rat chow containing 0.6% calcium and 0.68% phosphorus as previously described (13). In contrast, ten subtotally nephrectomized rats were placed on a diet containing 2.5% calcium and 0.68% phosphorus (Nx-Ca, n = 10) to induce biochemical changes consistent with adynamic bone. Nine other nephrectomized rats were placed on a diet containing 0.8% calcium and 1.2% phosphorus (Nx-P, n = 9) to promote the development of secondary hyperparathyroidism. Animals from each experimental group were maintained on the diets specified for the full four weeks of study.
Control rats with normal renal function were pair-fed with animals that had undergone subtotal nephrectomy by providing to controls the same amount of food that had been consumed the previous day by their nephrectomized counterparts (12, 13). Animals were weighed each week throughout the experiment, and body length was determined weekly.
After 4 weeks, rats were anesthetized with ketamine and xylazine, and the animals were killed by exsanguination by cardiac puncture. Blood was saved for subsequent biochemical determinations. Immediately after death, tissues were fixed by trans-cardiac perfusion using 4% paraformaldehyde in PBS (PFA/PBS) (13). Tibiae were excised and freed of adherent soft tissue (13). Tibial length was determined by measuring the distance between the proximal and distal articular surfaces using a metric ruler. Triplicate measurements were obtained for each bone, and the average of these three determinations was taken to represent overall tibial length. Bones were then immersed in 4% PFA/PBS for 48 h. Decalcification was completed using 0.1 M ethylenediamine tetra-acetic acid (EDTA, free acid) in PBS, pH 7.0, at 4 C for 2 weeks (13).
After decalcification, tibiae were washed in 10 mM PBS for 24 h, dehydrated in increasing concentrations of ethanol, and embedded in paraffin. Paraffin blocks were carefully positioned in the microtome specimen holder to obtain frontal sections of the proximal tibia with the plane of section oriented parallel to the longitudinal axis of the bone. Five micrometer sections of bone for either morphometric analysis or in situ hybridization were obtained using a Jung-Reichert Model 1140 microtome (Warner Lambert, Buffalo, NY); these were mounted on Superfrost Plus slides (Fisher Scientific Co., Springfield, NJ) (13).
Serum biochemical determinations
After blood samples were obtained, serum was separated by
centrifugation, and samples were stored at -70 C until biochemical or
hormonal assays were begun. Blood ionized calcium levels were
determined at the time the rats were killed using a calcium-sensitive
electrode (ICA Radiometer, Copenhagen, Denmark) (13, 14). Serum
phosphorus, creatinine, and urea nitrogen levels were measured by
standard laboratory methods (12, 13). Serum PTH levels were measured
using an immunoradiometric assay for rat PTH (Immutopics Inc., San
Clemente, CA) (13).
Morphometric assessment of growth plate cartilage
For morphometric analysis, four 5-µm sections of bone were
obtained from each tibia as reported previously (13); these were
mounted on glass slides, stained with hematoxylin, and eosin, and
counter-stained with azure A. Sections were viewed by light microscopy
at 150x using a Leitz Dialux microscope equipped with a
drawing tube attachment (Leitz, Wezlar, Germany (15, 16).
The total width of the growth plate cartilage at the proximal end of
each tibia was measured at equally spaced intervals along an axis
oriented 90 degrees to the transverse plane of the growth plate and
parallel to the longitudinal axis of the bone using a digitizer tablet
(Summagraphics, Seymour, CT) interfaced with a microcomputer (13). At
least four width measurements were obtained from each epiphyseal growth
plate, and final width determinations in individual animals represent
the average of these values (13). The width of the zone occupied by
hypertrophic chondrocytes was measured by the same method. In addition
to the average width of the hypertrophic zone, the percentage of the
total width of the growth plate comprised of hypertrophic chondrocytes
was calculated (13).
Technique of in situ hybridization
In situ hybridization in sections of bone was
performed using methods described in detail elsewhere (13, 17, 18, 19, 20).
Individual sections of bone obtained from rats in each study group were
mounted together on individual glass microscope slides to permit valid
side-by-side comparisons among samples from each group and to minimize
differences that could be attributed to slide-to-slide variation during
specimen processing and development (13). Thus, each slide contained
four tissue sections, one from each group of experimental animals.
Approximately 80 slides were included in each hybridization procedure using 35S-labeled sense and antisense riboprobes encoding rat osteocalcin, rat type II collagen, mouse type X collagen, rat PTH/PTHrP receptor and mouse MMP-9/gelatinase B (kindly provided by Drs. K. Lee and G. V. Segre). Riboprobes were labeled to a specific activity of 1–2 x 109 cpm/µg using the Gemini transcription kit (Promega Corp., Madison, WI) and 35S-UTP (NEN Life Science Products, Boston, MA) (13). Probes were purified by ethanol/chloroform extraction and acetate/ethanol precipitation. Sense messenger RNA (mRNA) probes were used as negative controls.
Before hybridization, paraffin was removed from slides using xylene, and tissues were then rehydrated and digested with proteinase K, 4 µg/ml. Samples were postfixed in 4% paraformaldehyde, denatured in 0.2 N HCl, acetylated in 0.25% acetic anhydride/0.1 triethanolamine, pH 8.0, and dehydrated in ethanol. In situ hybridization was completed using 5–10 x 106 cpm of 35S-labeled riboprobe for selected mRNAs at 55 C overnight in buffer containing 50% formamide, 10 mM Tris-HCl, 200 µg/ml transfer RNA, 1x Denhardt’s solution, 10% dextran sulfate, 0.6 M NaCl, 0.25% SDS, 1 mM EDTA, and 50 mM DTT. After hybridization, sections were washed with 5 x SSC at 50 C, with 50% formamide and 2 x SSC at 50 C, with 10 µg/ml ribonuclease A/10 mM Tris-HCl, 500 mM NaCl, and 1 mM EDTA at 37 C, with 2 x SSC, and with 0.2 x SSC at 50 C together with increasing concentrations of ethanol in 0.3 M ammonium acetate (13). Slides were exposed to x-ray film overnight (Fujifilm, Fujo Photo Film Co.,Tokyo, Japan), and emulsion autoradiography was done at 4 C using NTB-2 (Eastman Kodak Co., New Haven, CT). The length of exposure for emulsion autoradiography was estimated from the intensity of images on developed x-ray films. Slides were developed and stained with hematoxylin and eosin.
Quantification of in situ hybridization signals
Slides were viewed at 100x by bright field microscopy using a
Jenalumar microscope (13); images were captured using a CCD video
camera control unit (Hamamatsu Photonics, Hamamatsu-City, Japan), and
these were displayed on a computer monitor. The number of silver grains
overlying each chondrocyte profile was counted using an image analysis
system (Kontron Instruments Ltd. Elektronik 200,
Hallbergmoos, Germany) (13). Twenty to 30 cell profiles were
assessed in each bone specimen, and the results for individual bone
samples represent the average of these measurements. Data are expressed
as the number of silver grains/1000 µm2 of cell
profile.
Bone histochemistry and immunohistochemistry
Histochemical staining for tartrate-resistant acid phosphatase
(TRAP) was done using methods reported previously on sections of bone
prepared and mounted in the same manner as for in situ
hybridization (21). Once again, this approach was taken to limit the
influence of technical variations in slide preparation on comparisons
among groups. Image analysis was done in tissue sections viewed at
250x to quantify histochemical staining for TRAP and mRNA expression
for MMP-9/gelatinase B (15, 16). Results are expressed as the
percentage of the total tissue area with positive staining (15, 16).
Statistical analysis
All results are expressed as mean values ± 1
SD. Data were evaluated by one-way ANOVA; comparisons among
groups were done using Scheffé’s multiple range test (22). For
all statistical tests, probability values less than 5% were considered
to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As expected, blood
ionized calcium concentrations were higher and serum PTH values were
lower in Nx-Ca than in either Nx-C or C (Table 1). In contrast, the
levels of blood ionized calcium were lower, whereas serum PTH values
were higher, in Nx-P than in Nx-C or C (Table 1). Serum phosphorus
levels did not differ between Nx-C and C, but values were lower in
nephrectomized rats ingesting a high-calcium diet, a finding consistent
with the known effect of calcium to diminish intestinal phosphorus
absorption (Table 1). In nephrectomized rats maintained on a
high-phosphorus diet, serum phosphorus levels exceeded those in either
Nx-C or C (Table 1).
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). The weight of animals at the end of
study and the amount of weight gained were less, however, in Nx-C than
in C (Table 2). Further reductions in these two measures were seen in
Nx-Ca and Nx-P (Table 2). Although the length of animals at the end of
the experiment did not differ between C and Nx-C, both total body
length and the increase in body length during study were less in Nx-Ca
and Nx-P than in either Nx-C or C (Table 2). Tibial length was also
less in Nx-Ca and Nx-P than in Nx-C or C (Table 2).
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).
The proportion of the growth plate occupied by either proliferating or
hypertrophic chondrocytes was also similar in these three groups of
animals (Fig. 1). In contrast, the epiphyseal growth plate was
substantially wider in Nx-Ca than in C, Nx-C, or Nx-P (Fig. 1). This
difference was largely due to an increase in the width of the zone of
hypertrophic chondrocytes immediately adjacent to and extending into
the primary spongiosa (Fig. 1).
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). These cells
were difficult to visualize by image analysis because of their severely
flattened profiles. As such, quantitative results for osteocalcin mRNA
expression could not be obtained, but overt differences in message
expression were not apparent between Nx-C and C (Fig. 2). In contrast,
osteocalcin mRNA expression was markedly lower in Nx-Ca than in either
control group, whereas the level of message expression was
substantially higher in Nx-P (Fig. 2). These qualitative differences in
mRNA expression correspond to the changes expected based upon marked
differences in serum PTH levels between Nx-Ca and Nx-P (Table 1
).
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) (13).
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and 5). The extent of histochemical staining
for TRAP within the primary spongiosa was moderately greater in Nx-C
than in C, whereas TRAP staining was markedly increased in Nx-P (Fig. 6). In contrast, TRAP staining was lower
in Nx-Ca than in either Nx-P or Nx-C (Fig. 6). Expression of mRNA for
MMP-9/gelatinase B did not differ between Nx-C and C (Fig. 6), but the
level of expression was higher in Nx-P than in Nx-C (Fig. 6).
MMP-9-gelatinase B mRNA expression was substantially lower in Nx-Ca
than in either Nx-C or C (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The morphological changes in the epiphyseal growth plate of calcium-supplemented animals were unexpected. Based upon clinical evidence that linear growth decreases in children with renal failure who develop adynamic bone during treatment with large intermittent doses of calcitriol, reductions, rather than increases, in growth plate thickness were expected. We sought, therefore, to determine whether widening of the growth plate in this experimental model of adynamic renal osteodystrophy was due to enhanced chondrogenesis or to reductions in the resorption of newly formed calcified cartilage within the primary spongiosa immediately adjacent to the growth plate.
As anticipated, the expression of mRNA for osteocalcin was lower in nephrectomized rats given supplemental calcium than in nephrectomized control animals, whereas osteocalcin mRNA expression in rats with secondary hyperparathyroidism exceeded that of nephrectomized controls. Such findings are consistent with the expected variation in osteoblastic activity in animals with markedly different serum PTH levels, and they most probably reflect PTH-mediated differences in the rates of bone remodeling in the primary and secondary spongiosa of the proximal tibial metaphysis.
Despite reductions in osteocalcin mRNA expression, the levels of mRNA for several key markers of differentiated chondrocyte function were no different in calcium-supplemented rats than in nephrectomized control or intact control rats. Thus, mRNA levels for type II collagen, type X collagen, and the PTH/PTHrP receptor did not differ among Nx-Ca, Nx-C, and C. The level of PTH/PTHrP receptor mRNA was less, however, in Nx-P, confirming data reported previously in rats with severe secondary hyperparathyroidism (13). The lack of change in markers of differentiated chondrocyte function in calcium-supplemented rats strongly suggests that widening of the epiphyseal growth plate in this rodent model of adynamic renal osteodystrophy is not due to enhanced chondrogenesis.
In contrast to these findings, expression of specific markers of osteoclastic/chondroclastic activity within the primary spongiosa of the proximal tibia was markedly reduced in nephrectomized rats maintained on a high calcium diet. Histochemical staining for tartrate-resistant acid phosphatase (TRAP) was lower in calcium-supplemented animals than in either nephrectomized controls or nephrectomized rats given supplemental dietary phosphorus, and similar reductions were seen in the expression of mRNA for MMP-9/gelatinase B. Such findings suggest that dietary calcium supplementation in rats with renal failure affects growth plate morphology by retarding the resorption of calcifying cartilage within the primary spongiosa of long bones.
Alternative explanations for the histological changes in growth plate morphology in the proximal tibia of calcium-supplemented rats were not readily apparent. Renal failure per se, which often leads to high serum PTH levels, is generally associated with decreases, rather than increases, in the width of epiphyseal growth plate cartilage (13). Persistent hypophosphatemia can cause osteomalacia, and widening of the epiphyseal growth plate cartilage is a characteristic feature of growing animals with rickets (24, 25). Although serum phosphorus levels were lower in nephrectomized, calcium-supplemented animals than in the other three experimental groups, this finding is unlikely to account for changes in growth plate morphology. In a companion study, subtotally nephrectomized rats ingesting the same diet used in the current experiment had no evidence of osteomalacia as judged by quantitative bone histomorphometry. Indeed, values for osteoid area and for double tetracycline-labeled trabecular bone perimeter in the proximal tibial metaphysis were lower in nephrectomized, calcium-supplemented animals than in nephrectomized controls, findings consistent with adynamic renal osteodystrophy.
The changes in growth plate morphology in calcium-supplemented nephrectomized rats resemble qualitatively those described in mice with targeted deletions of both alleles of the gene for MMP-9/gelatinase B, but they are much less extensive (26). MMP-9/gelatinase B is a matrix metallo-proteinase (MMP) capable of cleaving extracellular matrix proteins including certain collagens. It is highly expressed during embryogenesis at sites of active tissue remodeling and in osteoclasts (27, 28, 29), and it may participate with other MMPs in cartilage degradation and in the regulation of angiogenesis near the epiphyseal growth plate (26). By 2 months of age, mice with deletions of both alleles for MMP-9/gelatinase B exhibit 6- to 8-fold increases in the length of the zone of hypertrophic chondrocytes at the ends of long bones (26). Cartilaginous septae within the metaphysis extend much further from the epiphysis into the diaphysis of long bones, whereas the overall length of long bones is approximately 10% less than in wild-type controls (26). These structural alterations appear to arise from diminished vascular invasion at the interface between epiphyseal cartilage and bone marrow, and they are associated with a delay in apoptosis of hypertrophic chondrocytes (26). In contrast, chondrocyte proliferation and differentiation within epiphyseal cartilage proceeds normally (26).
In the current experiment, markers of differentiated chondrocyte function were unaffected in nephrectomized, calcium- supplemented rats as judged by the levels of expression of mRNAs for type II collagen, type X collagen and the PTH/PTHrP receptor. Resorption activity immediately adjacent to the epiphyseal growth plate cartilage was reduced, however, as reflected by decreases in TRAP staining and by reductions in MMP-9/gelatinase B mRNA expression, changes similar to those seen in MMP-9/gelatinase B knock-out mice with the same degree of growth retardation.
Angiogenesis and vascular invasion are thought to be important regulators of chondrocyte apoptosis during endochondral bone formation (30, 31). Other factors such as vascular endothelial growth factor (VEGF) also contribute (30, 31), and it has been shown that MMP-9/gelatinase B plays a key role in these two processes (26). The results of the current investigation indicate that a high dietary calcium intake in rats with renal failure modifies MMP-9/gelatinase B expression immediately adjacent to epiphyseal growth plate cartilage, leading to morphological changes consistent with impaired cartilage degradation and resorption. Decreases in MMP-9/gelatinase B expression were demonstrated only in calcium-supplemented, nephrectomized rats, whereas the level of mRNA expression for MMP-9/gelatinase B was unchanged both in nephrectomized control animals and in rats with overt secondary hyperparathyroidism in whom the morphology of the epiphysis was largely preserved. Accordingly, neither renal failure per se nor differences in serum PTH levels adequately explain the reductions in MMP-9/gelatinase B expression in rats with biochemical features of adynamic renal osteodystrophy.
Whether the changes in growth plate morphology documented in calcium-supplemented, nephrectomized animals occur in other clinical or experimental forms of adynamic renal osteodystrophy such as that arising from the use of large doses of 1,25-dihydroxyvitamin D remains to be determined (7). Little is known about the impact of vitamin D administration on the morphology of growth plate cartilage in animals with renal failure. Calcitriol has potent antiproliferative effects on chondrocytes, on osteoblasts, and on many other cells (32, 33); these actions would be expected to diminish chondrocyte proliferation and differentiation and to reduce, rather than increase, the width of the epiphyseal growth plate. As such, the mechanism of impaired longitudinal bone growth in rats with renal failure and adynamic renal osteodystrophy may differ according to the specific pathogenic factor responsible for the development of this type of renal bone disease.
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Acknowledgments |
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Footnotes |
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2 Current address: Room 3590 MSC, 1300 University Avenue, University
of Wisconsin School of Medicine, Madison, Wisconsin 53706.
Received September 17, 1999.
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