Short-term administration of the bisphosphonate ibandronate increases bone volume and prevents hyperparathyroid bone changes in mild experimental renal failure

BACKGROUND: Bisphosphonates (BP) are potent antiresorptive agents that have been used successfully in several bone diseases associated with hyperresorption. Hyperresorption, hypercalcemia, and osteoporosis are frequent findings in patients with renal failure or after renal transplantation. The present study was carried out to determine the effects of a new BP, ibandronate, on bone in a state of normal vs. moderately impaired renal function. MATERIAL AND METHODS: Forty 90-day-old female rats were either 2/3 nephrectomized (Nx, n = 20) or sham-operated (Sham, n = 20). Half of the Nx and Sham rats received either ibandronate (1.25 microg/rat s.c.) or vehicle once weekly for three weeks. Before euthanasia, blood drawings were performed and 24-hr urine was collected. Femurs were analyzed by bone histomorphometry. RESULTS: Serum creatinine, parathyroid hormone, and osteocalcin levels were equally higher in Nx rats given ibandronate or vehicle than in Sham rats. There was no difference in serum calcium, phosphorus, alkaline phosphatase, and urinary creatinine among the groups. Ibandronate-treated rats had lower urinary calcium and deoxypyridinoline crosslink levels than their Sham counterparts. Ibandronate-treated rats had higher bone volume than vehicle-treated animals. Ibandronate prevented the increase in erosion depth and bone turnover in Nx rats. CONCLUSIONS: BPs such as ibandronate represent potentially useful tools in the treatment of certain facets of renal bone disease. Indications for BP therapy may include treatment of osteoporosis, hypercalcemia, and/or extraosseous calcifications. Optimal dose and frequency of BP administration need to be determined in these patients.     

Lifelong administration of high doses of ibandronate increases bone mass and maintains bone quality of lumbar vertebrae in rats

As part of a long-term safety study the bisphosphonate ibandronate was investigated for its effects on bone quality in lumbar vertebrae in rats. Bone area, bone density and mechanical properties were assessed by peripheral quantitative computed tomography (pQCT), dual-energy X-ray absorptiometry (DXA) and compression tests. Female and male groups of Wistar rats received either vehicle or 3, 7 or 15 mg/kg per day of ibandronate over 104 weeks orally by gavage. Compared with the control group, bone mineral density, compressive strength and stiffness were significantly higher in ibandronate-treated animals, whereas no changes occurred in strain or modulus of elasticity. The increase in vertebral body stress was significant in some of the ibandronate-treated groups. The changes in mechanical properties appear to be due mainly to an increase in bone mass. A highly significant correlation was found between bone mineral density measured either by DXA (r=0.86) or pQCT (r=0.85) and maximal strength in vertebral bodies (p<0.0001 each). In conclusion, we demonstrated that lifelong administration of doses of ibandronate far in excess of any therapeutically intended dose not only increases bone mass and apparent density, but also maintains or even slightly improves bone quality. Bone mineral density measured either by pQCT or DXA can be used as a predictor for ultimate strength in rat lumbar vertebral bodies after treatment with ibandronate.     

Comparison of peripheral bone and body axis skeleton in a rat model of mild-to-moderate renal failure in the presence of physiological serum levels of calcitropic hormones

The skeleton is characterized by anatomic heterogeneity of metabolic turnover. Site-dependent differences in hormonal effects seem likely. Hyporesponsiveness of osteoclasts to parathyroid hormone (PTH) and probably calcitriol was recently demonstrated in the fifth lumbar vertebra of a rat model with moderate renal failure. We compared histomorphometric findings of the tibial head to these data. Histomorphometric measurements were carried out in sections of the right tibial head of pair-fed male Sprague-Dawley rats. Subtotally nephrectomized (SNx), parathyroidectomized (PTx), rats, which received either solvent or rat PTH(1-34) (100 ng/kg per hour) + calcitriol (5 pmol/kg per hour) via osmotic minipumps were compared with sham-operated controls. Results were compared with data from the fifth lumbar vertebra reported recently. Osteoclast numerical density and osteoclast surface density were lower in the tibial head and the lumbar vertebra of solvent-treated SNxPTx rats than in sham-operated controls (p < 0.05), and could not be returned to normal by the substitution of PTH + calcitriol (p < 0.05). On the other hand, there were differences between interventional effects on the tibial head and on the lumbar vertebra concerning parameters describing osteoblasts and trabecular bone volume. In the tibial head, osteoblast surface density was nearly unchanged in both interventions. Nevertheless, bone volume increased after SNxPTx without substitution of PTH + calcitriol (p < 0.05), and no further changes occurred after hormonal replacement. In contrast, osteoblast surface density in the lumbar vertebra was decreased slightly compared with values in sham-operated rats; a clear but nonsignificant increase occurred after the administration of calcitropic hormones. This was accompanied by unchanged trabecular bone volume after SNxPTx. Hormonal replacement, however, caused an increase in trabecular bone volume (p < 0.05), which represents an anabolic effect, which contrasts with findings from the tibial head. The different interventional effects on the lumbar spine and on peripheral bone, regarding the parameters reflecting the condition of osteoblasts, may be intrinsic to the uremic syndrome itself as well as to dissimilar growth manner, function, and mechanical requirements. The findings substantiate the site dependence of bone surface cell metabolism in renal failure and should be the subject of further study. Corresponding results with regard to bone resorption argue for a bone-site-independent, diminished response of osteoclasts to calcitropic hormones.     

Growth hormone receptor abundance in tibial growth plates of uremic rats: GH/IGF-I treatment

BACKGROUND: Children with chronic renal failure (CRF) exhibit growth retardation and a disturbed growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. Treatment of children with CRF with GH or GH/IGF-I can partially restore linear growth. The molecular basis for decreased longitudinal growth is not known but may involve an impaired action of GH. METHODS: We used the growth-retarded uremic rat model to determine the abundance and distribution of GH receptors (GHRs) in the tibial epiphyseal growth plate and the influence of GH, IGF-I, or combined GH/IGF-I treatment. Pair-fed rats were used as the control. RESULTS: While all treatment regimes increased body length and weight in both rat groups, only GH/IGF-I treatment increased the total growth plate width. This involved an increase in cell number in the hypertrophic zone, which could also be induced by IGF-I alone. Immunohistochemical analysis showed that uremic rats had decreased abundance of GHRs in the proliferative zone, and only GH/IGF-I therapy could overcome this decrease. These data thus suggest that growth retardation in uremic rats is, at least in part, due to a decrease in GHR abundance in chondrocytes of the proliferative zone of the tibial growth plate. This decreased GHR abundance can be overcome by combined GH/IGF-I therapy, thus enhancing generation and proliferation of hypertrophic zone chondrocytes and increasing growth-plate width. CONCLUSION: These studies point to a mechanism for the growth retardation seen in children with CRF, and suggest that combined GH/IGF-I treatment may provide more effective therapy for these patients than GH alone.     

Administration of ghrelin to young uraemic rats increases food intake transiently, stimulates growth hormone secretion and does not improve longitudinal growth.

BACKGROUND: Ghrelin administration stimulates appetite and growth hormone (GH) secretion. Whether these effects are preserved in young individuals with chronic renal failure (CRF) and their potential benefit on growth is questioned. METHODS: Three experiments were performed in subtotally nephrectomized young rats (Nx). (i) Food intake was monitored in CRF rats receiving saline intraperitoneally or a ghrelin dose (30 nmol) shown to increase food intake over 2 and 24 h in rats with normal renal function. (ii) Plasma levels of GH were measured after a dose of intravenous ghrelin (3 nmol) was given to three groups of five rats each: Nx, sham-operated fed ad libitum (SAL) and sham-operated pair-fed with Nx (SPF). (iii) Growth of Nx rats treated with intraperitoneal ghrelin (3 nmol) for 7 days (Nx-Ghr) was compared with that of SAL and Nx groups receiving saline (n=8-10 per group). RESULTS: In CRF rats, the dose of 30 nmol of ghrelin increased food consumption for 2 h (1.3+/-0.2 g vs 0.5+/-0.2 g, P<0.05) but not 24-h food intake (12.5+/-0.6 g vs 12.2+/-0.5 g). Ghrelin (3 nmol) increased plasma levels of GH, which were maximal 10 min after injection, no differences being observed among groups (SAL: 666.2+/-104.6 ng/ml; Nx: 691.6+/-90.7 ng/ml; SPF: 577.8+/-125.4 ng/ml). Return to basal GH levels was delayed in Nx. Ghrelin did not improve body length and weight gains, longitudinal bone growth rate or food intake in the Nx-Ghr group. CONCLUSIONS: In young uraemic rats, ghrelin increases appetite but not 24-h food intake, stimulates GH secretion and does not improve growth.     

Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats

BACKGROUND: Extensive renal ablation is associated with progressive sclerosis of the remnant kidney. Because lymphocytes and monocytes accumulate in the remnant kidney, it is likely that they play a role in the renal scarring. Therefore, we treated rats with 5/6 nephrectomy (5/6Nx) with mycophenolate mofetil (MMF), a drug that has an antiproliferative effect and that suppresses the expression of intercellular adhesion molecules. METHODS: Sprague-Dawley rats with 5/6Nx received MMF (30 mg. kg-1. day-1 by daily gastric gavage, N = 15) or vehicle (N = 16). Ten additional rats were sham operated. All rats were fed a 30% protein diet. Body weight, serum creatinine, and urinary protein excretion were determined weekly. Lipid peroxidation, as a measure of oxidative stress observed by urinary malondialdehyde determinations, was performed every two weeks. Histologic studies were done in the remnant kidney four weeks (9 rats from the vehicle-treated group, 7 rats from the MMF group, and 5 sham-operated rats) and eight weeks after surgery (the remaining rats). Glomerular volume, sclerosis in glomeruli (segmental and global) and interstitium (semiquantitative scale), infiltrating lymphocytes and macrophages (CD43- and ED1-positive cells), and expression of adhesion molecules (CD54, CD18, and CD11b) were analyzed. RESULTS: MMF treatment prevented the progressive increment in serum creatinine and the proteinuria observed in the 5/6 nephrectomized rats during the eight weeks of observation (P < 0.01). Weight gain was comparable in the MMF-treated and sham-operated rats, whereas weight gain was decreased in untreated 5/6 nephrectomized rats. Excretion of malondialdehyde increased after surgery but returned sooner to control levels in the MMF-treated rats. Increments in glomerular size and mean arterial blood pressure induced by renal ablation were not modified by MMF treatment. Eight weeks after surgery, segmental sclerosis was present in 48.4 +/- 8.35% (+/- sd) glomeruli in the vehicle-treated group versus 25 +/- 10.5% in the MMF-treated group (P < 0.001). Interstitial fibrosis was reduced significantly with MMF treatment (P < 0.001). Infiltration with CD43- and ED1-positive cells in glomeruli and interstitium was two to five times lower in MMF-treated rats (P < 0.01). Expression of adhesion molecules CD18 and CD11b was similarly reduced. CONCLUSION: MMF ameliorates the progressive renal damage in the remnant kidney after 5/6Nx. This effect is associated with a reduction in the infiltration of lymphocytes and monocytes, whereas glomerular hypertrophy and systemic hypertension are unchanged.     

The effect of growth hormone on the growth failure of chronic renal failure

To investigate the effects of growth hormone (GH) on the reversal of growth failure in uremia, recombinant human GH (rhGH) was administered to rats with chronic renal failure (CRF). The dosage of rhGH was 3 IU/day (i.p.) for 13 days after the induction of CRF by 5/6 nephrectomy. Animals were classified into four groups: untreated nephrectomized rats (NX,n=40), GH-treated nephrectomized rats (NX+GH,n=18), sham-operated rats fed ad libitum (SHAMAL,n=27), and sham-operated rats pair-fed with 10 NX rats (SHAMPF,n=10). NX and NX+GH rats developed a similar and moderate degree of CRF, serum urea nitrogen being (mean±SEM) 49±3 and 54±4 mg/dl, respectively, compared with 16±4 and 19±0 mg/dl in SHAMAL and SHAMPF groups. Weight (56.0±3.3 g) and length (3.5±0.1 cm) gains of NX rats were lower than those of SHAMAL rats (94.2±4.0 g,P<-0.0001 and 4.1±0.2 cm,P<-0.01). Growth of the SHAMPF group and the matched NX rats was not significantly different. Weight (56.2±5.0 g) and length (3.4±0.2 cm) gains of NX+GH and NX rats were similar, the beneficial effect of GH therapy on growth being observed in only those animals with more severe degrees of uremia. This growth-promoting action resulted from greater food efficiency and not from stimulated food intake. The hypercholesterolemia seen in NX rats, 81±2 mg/dl versus 55±3 mg/dl in SHAMAL (P0.0001), was not increased in the NX+GH group, 87±3 mg/dl. There was a positive and significant correlation between serum cholesterol and serum urea nitrogen values in NX and NX+GH animals. This study suggests that growth impairment of mild CRF is mainly due to malnutrition and is refractory to GH administration. GH therapy improves the growth rate of animals with advanced CRF without aggravating their lipid abnormalities.     

Alterations in the growth plate cartilage of rats with renal failure receiving corticosteroid therapy

Growth retardation is a complication often associated with corticosteroid therapy. Corticosteroids are frequently used in the treatment of children with chronic renal failure. To examine the effects of corticosteroids on the growth plate cartilage in renal failure, selected markers of chondrocyte function and phenotype were evaluated in the proximal tibia of subtotally nephrectomized rats treated with corticosteroid. Serum parathyroid hormone (PTH), urea nitrogen, and creatinine levels were higher in the nephrectomized animals. Weight gain was less in the corticosteroid-treated animals; however, linear growth and tibial length did not differ among the groups after 10 days of corticosteroid therapy. The total width of the growth plate and the width of the proliferative zone were much smaller in corticosteroid-treated nephrectomized (Nx-MP) animals. Type II collagen mRNA expression was lower in animals treated with corticosteroids, and proliferating-cell nuclear antigen staining, histone-4, and insulin-like growth factor-1 (IGF-1)-receptor mRNA expression were further decreased in the Nx-MP group. There was an increase in TUNEL-positive cells in the corticosteroid-treated rats with normal renal function (intact-MP), associated with an increase in Bax and a decrease in Bcl-2 protein expression. In the Nx-MP group, both Bax and Bcl-2 protein staining was much less frequent, and TUNEL-positive cells were lower in number compared with the intact-MP group. Vascular endothelial growth factor expression in the hypertrophic chondrocytes was lower in corticosteroid-treated animals. There was less gelatinase B/matrix metalloproteinase-9 expression in the Nx-MP group, which was not associated with a decrease in tartrate-resistant acid phosphatase (TRAP) staining in the chondro-osseous junction. Inhibition of chondrocyte proliferation, diminishing of apoptosis, and lower angiogenic activity may contribute to the alterations in growth plate architecture and the significant reduction in growth plate width in rats with renal failure receiving corticosteroid therapy.     

Orchiectomy upregulates free soluble RANKL in bone marrow of aged rats

The osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) axis is thought to be involved in the upregulation of bone turnover following sex steroid deficiency. Here, we investigated the effects of orchiectomy (ORX) on bone turnover and free soluble RANKL (sRANKL) in aged rats. Free, bioactive sRANKL is a critical determinant and key mediator for survival and activity of mature osteoclasts. Thirty-three 9-month-old male Fischer-344 rats were either ORX or sham-operated (SHAM). Following in vivo fluorochrome labeling, vehicle (ricinus oil/benzyl benzoate)-treated SHAM and vehicle- or testosterone undecanoate (T, 6 mg/kg s.c. once weekly)-treated ORX rats (n = 8–9 each) were killed 2 months after surgery. Vehicle-treated ORX rats showed lower seminal vesicle weight, loss of proximal tibial trabecular bone mineral density, and reduced cortical thickness at the tibial shaft as measured by peripheral quantitative computed tomography relative to SHAM controls. Bone loss in vehicle-treated ORX rats was associated with enhanced bone turnover as evidenced by increases in tibial cancellous bone formation rate, osteoclast numbers, urinary excretion of calcium and deoxypyridinoline, and serum osteocalcin. T treatment of ORX rats restored seminal vesicle weight to SHAM control levels, and completely protected against post-ORX bone loss by suppressing bone turnover. Free sRANKL concentrations in bone marrow supernatants harvested from the proximal femur were about 3-fold higher in vehicle-treated ORX relative to SHAM rats, and returned to SHAM control levels in T-treated ORX rats. mRNA abundance of matrix metalloproteinase-14 (MMP-14) in bone marrow was 4-fold higher in vehicle-treated ORX rats relative to SHAM rats. T treatment of ORX rats suppressed MMP-14 mRNA expression to SHAM control levels. We conclude that orchiectomy increases the concentration of free sRANKL in bone marrow of aged rats. In addition, increased shedding of membrane-bound RANKL by MMP-14 may be a pivotal mechanism resulting in augmented free sRANKL concentrations in the bone marrow environment after androgen withdrawal.     

The bisphosphonate ibandronate improves implant integration in osteopenic ovariectomized rats

Osteoporosis is known to impair the process of implant osseointegration. Bisphosphonates are drugs that inhibit osteoclast-mediated bone resorption and normalize the high rate of bone turnover that characterizes this disease. Consequently, there is a rationale for using bisphosphonates to enhance the early stabilization of implants in subjects with low bone mass. In this study, 84 rats received titanium-only or hydroxyapatite (HA)-coated titanium femoral implants, 3 months after being ovariectomized (OVX) or sham operated. They were then treated for 4 weeks. The OVX rats were randomly assigned to daily subcutaneous injections of either saline or the bisphosphonate ibandronate (at a dose of 1 microg/kg or 25 microg/kg), while the sham-operated animals received saline throughout. The 1 microg/kg or 25 microg/kg ibandronate doses are considered translatable to doses used to treat osteoporosis and metastatic bone disease (MBD), respectively, in rats, and roughly reflect those used in humans. At the end of the treatment period, bone mineral density (BMD) at the lumbar spine increased in both of the ibandronate-treated groups when compared with the OVX control animals and to a level similar to that of the sham-operated control group. Osseointegration, determined by histomorphometric analysis and expressed as percentage of osseointegration implant surface (OIS), did not differ between groups for the titanium-only implants. For the HA-coated implants, however, OIS was 113.5% and 185% higher in the groups receiving 1 microg/kg or 25 microg/kg ibandronate, respectively, relative to the OVX controls. In turn, the OIS of the HA-coated implants was 56.5% lower in the OVX control group than in the sham control group. These findings clearly demonstrate that OVX-induced osteopenia impairs the osseointegration of HA-coated titanium implants and that ibandronate, administered at doses analogous to those used to clinically treat osteoporosis and MBD, counters this harmful effect. Ibandronate may, therefore, have a role in improving the osseointegration of implants in patients with osteoporosis and MBD.     

Effects of grwoth hormone therapy and malnutrition on the growth of rats with renal failure

We examined the effects of methionylhuman growth hormone (met-hGH) and malnutrition on the growth of 5/6 nephrectomized rats and sham-operated controls. One group of shamoperated rats (PFS) was pair-fed with a group of nephrectomized rats in renal failure (RF); another group of sham-operated rats was fed ad libitum (ALS), and a final group of rats with renal failure (RF-GH) was treated with 4 IU/day met-hGH. After 4 weeks, RF-GH rats gained 12.3±1.7 cm in length; this was more than the 10.2±1.2 cm gain of RF rats (P<0.05). Ingested food was converted into weight gain more efficiently by RF-GH rats than RF rats (267±26 vs 235±38 mg weight gain/g food intake,P<0.05). RF-GH rats also gained more weight (122±25 g) than RF rats (98±27 g), but this difference was not significant (0.05<P<0.1). Insulin-like growth factor (IGF)-I, glucose and insulin levels were not different between RF and RF-GH rats. Food intake of RF and PFS rats was 64% of ALS intake and was associated with poor gains in weight and length by the PFS and RF groups (relative weight and length gains were ALS>PFS>RF,P<0.05 for all comparisons); this suggests that the poor growth of RF rats when compared with PFS rats was due to factors other, than food intake. Serum IGF-I levels of 771±249 ng/ml in PFS rats were lower than levels of 1109±253 ng/ml found in the ALS group (P<0.05); this is consistent with the malnourished state of PFS rats. Serum IGF-I levels in RF rats (950±236 ng/ml) were not different from ALS or PFS levels despite the fact that RF rats gained less weight and length than either the ALS or PFS rats. We conclude that RF rats increase in length, use ingested calories more efficiently, and fail to develop marked insulin resistance when treated with met-hGH. We find that, in addition to poor food intake, other factors contribute to growth failure in this model of renal failure. Finally, we find that RF rats have normal levels of IGF-I, suggesting that low IGF-I levels are not a major cause of growth failure in rats with renal failure.     

Restoration of Bone Mineralization by Cinacalcet is Associated with a Significant Reduction in Calcitriol-Induced Vascular Calcification in Uremic Rats

The present study investigated to what extent normalization of bone turnover goes along with a reduction of high-dose calcitriol-induced vascular calcifications in uremic rats. Five groups of male Sprague–Dawley rats were studied: sham-operated controls (n?=?7), subtotally nephrectomized (SNX) uremic (CRF) animals (n?=?12), CRF?+?calcitriol (vitD) (0.25?μg/kg/day) (n?=?12), CRF?+?vitD?+?cinacalcet (CIN) (10?mg/kg/day) (n?=?12), and CRF?+?vitD?+?parathyroidectomy (PTX) (n?=?12). Treatment started 2?weeks after SNX and continued for the next 14?weeks. High-dose calcitriol treatment in hyperparathyroid rats went along with the development of distinct vascular calcification, which was significantly reduced by >50?%, in both CIN-treated and PTX animals. Compared to control animals and those of the CRF group, calcitriol treatment either in combination with CIN or PTX or not was associated with a significant increase in bone area comprising ±50?% of the total tissue area. However, whereas excessive woven bone accompanied by a dramatically increased osteoid width/area was seen in the CRF?+?vitD group, CIN treatment and PTX resulted in significantly reduced serum PTH level, which was accompanied by a distinct reduction of both the bone formation rate and the amount of osteoid. These data indicate that less efficient calcium and phosphorus incorporation in bone inherent to the severe hyperparathyroidism in vitamin D-treated uremic rats goes along with excessive vascular calcification, a process which is partially reversed by CIN treatment in combination with a more efficacious bone mineralization, thus restricting the availability of calcium and phosphate for being deposited in the vessel wall.     

The Effects of Synthetic Conjugated Estrogens, A (Cenestin) on Trabecular Bone Structure and Strength in the Ovariectomized Rat Model

This study evaluated the effect of Cenestin, a synthetic conjugated estrogens product, on the maintenance of trabecular bone microarchitecture, bone strength, and of bone turnover in the ovariectomized (ovx) rat model. Two doses of Cenestin were chosen in an attempt to approximate the equivalent human oral doses of 0.3 mg and 0.625 mg. Forty-nine 6-month-old retired female breeder Sprague-Dawley rats were randomly assigned to one of four groups: (1) sham-operated + vehicle; (2) ovx + vehicle; (3) ovx + day 1 post-ovariectomy Cenestin (8.12 mg/kg); (4) ovx + day 1 post-ovariectomy Cenestin (16.24 mg/kg) for 8 weeks. Trabecular structure of the right proximal tibia of each rat was imaged noninvasively by microCT. A compression test to induce a tibial plateau fracture was performed to determine the mechanical properties of the proximal tibia. Urine was collected on days 0, 14, 28, 42 and 56 and serum on days 0, 28 and 56 to assess biochemical markers of bone turnover including deoxypyridinoline crosslinks and osteocalcin. Both biochemical markers of bone turnover were analyzed by ELISA. Trabecular bone mass, structure, and connectivity density in the Cenestin-treated groups did not differ statistically (p>0.05) from those of the sham-operated + vehicle-treated rats, but all were significantly higher (p<0.05) than in the ovx + vehicle-treated rats. Structure Model Index, a measure of trabecular plate morphometry, in Cenestin-treated rats maintained a more equal mix of plate- and rod-like structures similar to the sham group, whereas the ovx group had predominantly rod-like trabeculae. Fracture load in the Cenestin (16.24 mg/kg) treated group was 31% (p<0.01) higher than in the sham + vehicle-treated group and 61% (p<0.05) higher than in the ovx + vehicle-treated group. Both the sham-operated + vehicle-treated and Cenestin-treated groups showed significantly lower urinary deoxypyridinoline crosslink excretion at all timepoints and serum osteocalcin at day 56 compared with the ovx + vehicle-treated group. In summary, Cenestin maintained trabecular bone microarchitecture and strength in an ovariectomized rat model of estrogen deficiency. Received: 19 October 2001 / Accepted: 16 May 2002     

Impaired growth plate function in bmp-6 null mice

Bone morphogenetic protein 6 (BMP-6) is expressed by different skeletal cells including osteoblasts and growth plate chondrocytes, suggesting roles in bone formation and growth regulation. To address these possibilities, we examined whether cancellous and cortical bone parameters, or indices of growth plate function, are altered in bmp-6 null mice as assessed under basal conditions, and following stimulation of bone formation and suppression of growth by estrogen treatment. Ten-week-old female littermate bmp-6 null and wild-type (WT) mice were administered vehicle or E(2) 4, 40, 400 or 4,000 microg/kg/day by daily sc injection for 28 days (6-8 per group). Tibias were removed, and detailed histomorphometric analysis of the proximal metaphysis and growth plates, and tibial diaphysis were performed on longitudinal and transverse sections respectively. Long bone area as measured by DXA was reduced in vehicle-treated bmp-6 null mice compared with WT littermate controls. In addition, vehicle-treated bmp-6 null mice had a reduced cross-sectional area at the tibial mid-diaphysis as assessed by histomorphometry, whereas cancellous bone indices were unaffected. Histomorphometry of the proximal tibial metaphysis demonstrated a defect in bone formation immediately adjacent to the growth plate in bmp-6 null mice compared to WT mice following E(2) treatment. E(2) administration was also associated with a dose-responsive decrease in longitudinal growth rate, and proliferative and hypertrophic zone parameters of the growth plate (p<0.0001). Significantly greater reductions following E(2) treatment were observed in longitudinal growth rate (p<0.01), proliferating and hypertorphic zone widths (p<0.001), and proliferating (p<0.0002) and hypertrophic (p<0.002) cells per column of bmp-6 null mice compared to WT mice. Our observation that long bones are reduced in size compared to wild-type mice primarily through a decrease in cortical cross-sectional area, whilst cancellous bone mass is unaltered, suggests a non-redundant role for BMP-6 in periosteal but not trabecular bone formation. Moreover, growth plate function was reduced in bmp-6 null mice receiving estrogen, leading to an impaired cancellous bone response to estrogen at the highest dose, suggesting that BMP-6 also plays a physiological role in maintaining growth plate function.     

Bipedal stance exercise enhances antiresorption effects of estrogen and counteracts its inhibitory effect on bone formation in sham and ovariectomized rats

In this study we employed a raised cage model in combination with estrogen to observe their effects on the proximal tibial metaphysis (PTM) and tibial shaft (TX) in sham-operated or ovariectomized rats. A total of 105 6-month-old female Sprague-Dawley rats were used in the study. Bilateral sham ovariectomy or ovariectomy was performed at day 0 and the rats were housed in normal height or raised cages (RCs) and injected subcutaneously twice per week with 10 microg/kg of 17beta-estradiol (E2) or vehicle for 4 and 8 weeks. Because the time course of bone loss or bone gain distribution was not uniform in the metaphyses of the tibia, we subdivided the PTM into three zones (medial, central, and lateral) to observe the different bone loss or bone gain patterns after ovariectomy and/or raised cages. We found that: (1) E2 alone did not alter bone area or architecture in sham rats, whereas RC alone increased trabecular thickness and area of PTM, but had no effects on TX; (2) Ovx induced most bone loss from the central zone of the PTM and endocortical surface of TX, accompanied by decreased trabecular number and increased bone resorption; (3) E2 alone prevented ovx-induced bone loss by preserving trabecular number and depressing bone resorption; (4) RC alone partially compensated for bone loss following ovx by thickening the surviving trabeculae in lateral and medial zones, and tended to stimulate bone formation and decrease bone resorption; and (5) RC plus E2 increased trabecular bone area by having an additive effect on bone resorption and bone turnover. RCs helped to prevent the depressive effect of estrogen on periosteal bone formation. In conclusion, early and rapid bone loss occurred in the central zone of the metaphysis and endocortical surface after ovx. Estrogen replacement therapy prevented this loss. Raised cages partially compensated for bone loss following ovx by thickening the trabeculae in the lateral area of the metaphysis and decreased endocortical erosion. Combination treatment added bone to the PTM and prevented the decrease of periosteal bone formation after estrogen administration.     

A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate

The mechanisms that underlie growth plate chondrocyte volume increase and hence bone lengthening are poorly understood. Many cell types activate the Na‐K‐Cl cotransporter (NKCC) to bring about volume increase. We hypothesised that NKCC may be responsible for the volume expansion of hypertrophic chondrocytes. Metatarsals/metacarpals from 16 rat pups (P₇) were incubated in the presence/absence of the specific NKCC inhibitor bumetanide and measurement of whole‐bone lengths and histologic analysis of the growth plate were done after 24 hours. Fluorescent NKCC immunohistochemistry was visualised using a confocal laser scanning microscopy on seven rat tibial growth plates (P₇). Microarray analysis was performed on mRNA isolated from proliferative and hypertrophic zone cells of tibial growth plates from five rats of each of three ages (P_49/53/58). Exposure to bumetanide resulted in approximately 35% reduction (paired Student's t test, p < .05) of bone growth in a dose‐dependent manner; histologic analysis showed that a reduction in hypertrophic zone height was responsible. Quantification of fluorescence immunohistochemistry revealed a significant (paired Student's t test, p < .05) change in NKCC from the intracellular space of proliferative cells to the cytosolic membrane of hypertrophic zone cells. Further, microarray analysis illustrated an increase in NKCC1 mRNA between proliferative and hypertrophic cells. The increase in NKCC1 mRNA in hypertrophic zone cells, its cellular localization, and reduced bone growth in the presence of the NKCC inhibitor bumetanide implicate NKCC in growth plate hypertrophic chondrocyte volume increase. Further investigation is warranted to determine the regulatory control of NKCC in the mammalian growth plate and the possible detrimental effect on bone growth with chronic exposure to loop diuretics. © 2010 American Society for Bone and Mineral Research     

Beneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factors

In renal failure, parathyroid hormone (PTH) is not only involved in the genesis of disturbed calcium/phosphate metabolism and ostitis fibrosa; it is also a permissive factor in the genesis of hypertension, cardiovascular damage, and dyslipidemia. The allosteric activator of the calcium sensing receptor NPSR-568 (R-568) has been shown to reduce the serum intact PTH (iPTH) concentration in uremic rats. It was the purpose of this study in subtotally nephrectomized (SNX) rats to compare pharmacologic abrogation of secondary hyperparathyroidism by R-568 with parathyroidectomy (PTX). The effects on progression of renal failure, BP, and lipid and structural parameters of kidney and heart were studied. Four groups of male SD-rats were studied: (1) sham-operated + vehicle-treated rats (controls); (2) SNX + vehicle-treated rats (SNX); (3) parathyroidectomized SNX + vehicle-treated rats (SNX+PTX); and (4) SNX + calcimimetic R-568-treated rats (SNX+R-568). R-568 (50 micro mol/kg per d) was administered by gavage. Eight weeks after SNX, serum creatinine concentration, urinary albumin excretion, BP, and serum LDL-cholesterol concentration were significantly lower in both R-568-treated and parathyroidectomized SNX compared with vehicle-treated SNX. In addition, structural abnormalities of the kidney (glomerulosclerosis, tubulointerstitial changes) and the heart (interstitial fibrosis, capillary length density, arteriolar wall thickness) were significantly less pronounced than in vehicle-treated SNX. It is concluded that in experimental renal failure abrogation of hyperparathyroidism by administration of a calcimimetic or PTX similarly attenuates progression of renal failure. Furthermore, it interferes with the development of cardiovascular risk factors and cardiac remodeling.     

Growth plate cartilage formation and resorption are differentially depressed in growth retarded uremic rats

To characterize the modifications of growth plate in individuals with growth impairment secondary to chronic renal failure, young rats were made uremic by subtotal nephrectomy (NX) and, after 14 d, their tibial growth plates were studied and compared with those of sham-operated rats fed ad libitum (SAL) or pair-fed with NX (SPF). NX rats were growth retarded and severely uremic. Growth plate height (mean +/- SD) was much greater (P<0.05) in NX (868.4+/-85.4 microm) than SAL (570.1+/-93.5 microm) and SPF (551.9+/-99.7 microm) rats as a result of a higher (P<0.05) hypertrophic zone (661.0+/-89.7 versus 362.8+/-71.6 and 353.0+/-93.9 microm, respectively). The increased size of the growth plate was associated with a greater number of chondrocytes and modifications in their structure, particularly in the hypertrophic zone adjacent to bone. In this zone, chondrocytes of NX animals were significantly (P<0.05) smaller (12080.4+/-1158.3 microm3) and shorter (34.1+/-2.5 microm) than those of SAL (16302.8+/-1483.4 microm3 and 37.8+/-2.0 microm) and SPF (14465.8+/-1521.0 microm3 and 36.3+/-1.8 microm). The interface between the growth plate cartilage and the metaphyseal bone appeared markedly irregular in NX rats. Kinetics of chondrocytes was also modified (P<0.05) in the NX rats, which had lower cell turnover per column per day (5.4+/-0.9), longer duration of hypertrophic phase (89.0+/-15.2 h), and reduced cellular advance velocity (7.4+/-2.2 microm/h) compared with SAL (8.0+/-1.6, 32.1+/-6.7 h, and 11.3+/-2.7 microm/h) and SPF (7.2+/-1.1, 34.8+/-5.1 h, and 10.1+/-2.5 microm/h). Cell proliferation was no different among the three groups. Because the growth plates of SPF and SAL rats were substantially not different, modifications observed in the NX rats cannot be attributed to the nutritional deficit associated with renal failure. These findings indicate that chronic renal failure depresses both the activity of the growth plate cartilage by altering chondrocyte hypertrophy and the replacement of cartilage by bone at the metaphyseal end. The two processes are differentially depressed since cartilage resorption is more severely lowered than cartilage enlargement and this leads to an accumulation of cartilage at the hypertrophic zone.     

Assessment of effects of lanthanum carbonate with and without phosphate supplementation on bone mineralization in uremic rats

AIMS: Previous studies have indicated that impaired bone mineralization in 5/6 th nephrectomized rats given high doses of lanthanum carbonate is due to phosphorus depletion caused by excessive binding to, and reduced absorption of, dietary phosphate. This study aimed to test this hypothesis by: 1) directly comparing the effects of a supratherapeutic dose of lanthanum carbonate or dietary phosphorus restriction on bone mineralization in a rodent model of chronic renal failure (CRF); and 2) investigating whether phosphorus supplementation would prevent the bone mineralization defect associated with lanthanum carbonate treatment. METHODS AND MATERIALS: Male Sprague-Dawley rats were subjected to sham surgery or a two-step 5/6th nephrectomy to induce CRF and randomized across five treatment groups: sham, CRF, CRF + dietary phosphorus deficiency, CRF + lanthanum carbonate (1000 mg/kg/ day), and CRF + lanthanum carbonate + parenteral phosphorus repletion. RESULTS: Rats with 5/6th nephrectomy had elevated serum creatinine, blood urea concentration, and urine volume and protein, consistent with impaired renal function, and increased urinary phosphorus and serum parathyroid hormone, consistent with hyperparathyroidism. Lanthanum carbonate and dietary phosphate insufficiency induced parallel changes in serum and urine markers of phosphate homeostasis and increased osteoid formation. These changes induced by lanthanum carbonate were normalized by systemic phosphate supplementation. CONCLUSIONS: These findings provide further support for the concept that supratherapeutic doses of lanthanum carbonate induce effects on bone mineralization in uremic rats via an indirect pharmacological mechanism (phosphate depletion) and not via direct bone toxicity.