Human Parathyroid Hormone 1–34 Prevents Bone Loss in Experimental Biliary Cirrhosis in Rats
2007; Elsevier BV; Volume: 134; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.10.025
ISSN1528-0012
AutoresRivka Dresner–Pollak, Yankel Gabet, Arza Steimatzky, Gilad Hamdani, Itai Bab, Zvi Ackerman, Miron Weinreb,
Tópico(s)Liver Diseases and Immunity
ResumoBackground & Aims: Reduced bone mass and increased fracture rate are complications of primary biliary cirrhosis (PBC). The effect of intermittent administration of human parathyroid hormone (hPTH) 1–34 on bone mass and architecture in bile duct-ligated (BDL) rats was studied. Methods: Six-month-old male rats were subjected to BDL or sham operation (SO) and were treated from the second postoperative week intermittently with either hPTH 1–34 40 μg/kg per day, 80 μg/kg per day, or a vehicle for 4 weeks. Femoral and tibial bones were evaluated ex vivo by dual x-ray absorptiometry, microcomputed tomography, and histomorphometry. Serum osteocalcin and urinary deoxypyridinoline cross-links (DPD) were determined. Results: BDL rats had decreased bone mass compared with SO rats as indicated by a 6% decrease in femoral and tibial bone mineral density (BMD), 18% reduction in femoral trabecular bone volume (bone volume/total volume [BV/TV]), 17% decrease in trabecular thickness, and 10% decrease in tibial cortical thickness. The administration of hPTH 1–34 at 40 μg/kg per day increased femoral and tibial BMD (9% and 9%), femoral trabecular BV/TV (50%), trabecular thickness (50%), tibial cortical thickness (17%), and serum osteocalcin (82%). On the other hand, hPTH 1–34 80 μg/kg per day had no effect on BMD and tibial cortical thickness, was associated with a smaller increase in trabecular BV/TV (24%), and had a higher osteoclast number and DPD compared with untreated BDL rats and the lower hPTH 1–34 dose treatment group. Conclusions: BDL rats exhibit loss of bone mass and structure, which can be prevented by the intermittent administration of hPTH 1–34, a potential therapy for osteoporosis in PBC. Background & Aims: Reduced bone mass and increased fracture rate are complications of primary biliary cirrhosis (PBC). The effect of intermittent administration of human parathyroid hormone (hPTH) 1–34 on bone mass and architecture in bile duct-ligated (BDL) rats was studied. Methods: Six-month-old male rats were subjected to BDL or sham operation (SO) and were treated from the second postoperative week intermittently with either hPTH 1–34 40 μg/kg per day, 80 μg/kg per day, or a vehicle for 4 weeks. Femoral and tibial bones were evaluated ex vivo by dual x-ray absorptiometry, microcomputed tomography, and histomorphometry. Serum osteocalcin and urinary deoxypyridinoline cross-links (DPD) were determined. Results: BDL rats had decreased bone mass compared with SO rats as indicated by a 6% decrease in femoral and tibial bone mineral density (BMD), 18% reduction in femoral trabecular bone volume (bone volume/total volume [BV/TV]), 17% decrease in trabecular thickness, and 10% decrease in tibial cortical thickness. The administration of hPTH 1–34 at 40 μg/kg per day increased femoral and tibial BMD (9% and 9%), femoral trabecular BV/TV (50%), trabecular thickness (50%), tibial cortical thickness (17%), and serum osteocalcin (82%). On the other hand, hPTH 1–34 80 μg/kg per day had no effect on BMD and tibial cortical thickness, was associated with a smaller increase in trabecular BV/TV (24%), and had a higher osteoclast number and DPD compared with untreated BDL rats and the lower hPTH 1–34 dose treatment group. Conclusions: BDL rats exhibit loss of bone mass and structure, which can be prevented by the intermittent administration of hPTH 1–34, a potential therapy for osteoporosis in PBC. Reduced bone mass and increased fracture risk are serious complications of human primary biliary cirrhosis (PBC).1Solaymani-Dodaran M. Card T.R. Aithal G.P. et al.Fracture risk in people with primary biliary cirrhosis: a population-based cohort study.Gastroenterology. 2006; 131: 1752-1757Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 2Guanabens N. Pares A. Marinoso L. et al.Factors influencing the development of metabolic bone disease in primary biliary cirrhosis.Am J Gastroenterol. 1990; 85: 1356-1362PubMed Google Scholar, 3Guanabens N. Pares A. Ros I. et al.Severity of cholestasis and advanced histological stage but not menopausal status are the major risk factors for osteoporosis in primary biliary cirrhosis.J Hepatol. 2005; 42: 573-577Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Menon K.V. Angulo P. Weston S. et al.Bone disease in primary biliary cirrhosis: independent indicators and rate of progression.J Hepatol. 2001; 35: 316-323Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar A recent study demonstrated an approximately 2-fold increased risk of any fracture, hip fracture, and wrist fracture among patients with PBC compared with the general population.1Solaymani-Dodaran M. Card T.R. Aithal G.P. et al.Fracture risk in people with primary biliary cirrhosis: a population-based cohort study.Gastroenterology. 2006; 131: 1752-1757Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The etiology of bone loss in PBC has not been completely elucidated. Both reduced bone formation because of decreased osteoblast function and increased bone resorption have been suggested as underlying mechanisms.5Hodgson S.F. Dickson E.R. Wahner H.W. et al.Bone loss and reduced osteoblast function in primary biliary cirrhosis.Ann Intern Med. 1985; 103: 855-860Crossref PubMed Scopus (204) Google Scholar, 6Janes C.H. Dickson E.R. Okazaki R. et al.Role of hyperbilirubinemia in the impairment of osteoblast proliferation associated with cholestatic jaundice.J Clin Invest. 1995; 95: 2581-2586Crossref PubMed Scopus (175) Google Scholar, 7Hodgson S.F. Dickson E.R. Eastell R. et al.Rates of cancellous bone remodeling and turnover in osteopenia associated with primary biliary cirrhosis.Bone. 1993; 14: 819-827Abstract Full Text PDF PubMed Scopus (93) Google Scholar As a result, no specific therapy for PBC-related bone loss has been definitely established. Agents such as 25-hydroxyvitamin D3, calcitonin, etidronate, alendronate, or sodium fluoride failed to demonstrate definite beneficial effects in preventing PBC-related bone loss and fractures.8Lindor K.D. Jorgensen R.A. 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Thus, an animal model is needed to understand better the mechanisms of bone loss and the efficacy of therapeutic interventions.We have previously shown that bile duct-ligated (BDL) male rats develop the clinical features of biliary cirrhosis accompanied by the hallmarks of osteoporosis: reduced bone mass and decreased mechanical strength.11Ackerman Z. Weinreb M. Amir G. et al.Bone mineral metabolism and histomorphometry in rats with cholestatic liver disease.Liver. 2002; 22: 166-172Crossref PubMed Scopus (24) Google Scholar, 12Weinreb M. Pollak R.D. Ackerman Z. Experimental cholestatic liver disease through bile-duct ligation in rats results in skeletal fragility and impaired osteoblastogenesis.J Hepatol. 2004; 40: 385-390Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Osteopenia in BDL rats was characterized by decreased bone formation and osteoblastsogenesis.12Weinreb M. Pollak R.D. Ackerman Z. Experimental cholestatic liver disease through bile-duct ligation in rats results in skeletal fragility and impaired osteoblastogenesis.J Hepatol. 2004; 40: 385-390Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The aim of this study was to investigate the effects of a bone anabolic agent in the prevention of BDL-induced bone loss in rats.Intermittent administration of parathyroid hormone (PTH) by daily subcutaneous injections effectively stimulates cancellous and often cortical bone formation and reverses the bone loss induced by estrogen deficiency, orchidectomy, and immobilization of limbs in rats, other animal species, and humans.13Hock J.M. Gera I. Fonseca J. et al.Human parathyroid hormone-(1–34) increases bone mass in ovariectomized and orchidectomized rats.Endocrinology. 1988; 122: 2899-2904Crossref PubMed Scopus (224) Google Scholar, 14Tada K. Yamamuro T. 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Miler M.A. Newman M.K. et al.Daily treatment of aged ovariectomized rats with human parathyroid hormone (1–84) for 12 months reverses bone loss and enhances trabecular and cortical bone strength.Calcif Tissue Int. 2006; 79: 262-272Crossref PubMed Scopus (49) Google Scholar, 19Alexander J.M. Bab I. Fish S. et al.Human parathyroid hormone 1–34 reverses bone loss in ovariectomized mice.J Bone Miner Res. 2001; 16: 1665-1673Crossref PubMed Scopus (136) Google Scholar, 20Finkelstein J.S. Klibanski A. Schaefer E.H. et al.Parathyroid hormone for the prevention of bone loss induced by estrogen deficiency.N Engl J Med. 1994; 331: 1618-1623Crossref PubMed Scopus (216) Google Scholar, 21Neer R.M. Arnaud C.D. Zanchetta J.R. et al.Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis.N Engl J Med. 2001; 344: 1434-1441Crossref PubMed Scopus (3820) Google Scholar Human PTH 1–34 (hPTH 1–34) is currently the only US Food and Drug Administration-approved anabolic agent for the therapy of osteoporosis. In this study, we investigated the effects of intermittent administration of hPTH 1–34 on bone mass, architecture, and turnover in BDL male rats. Using densitometry, microcomputerized tomography (μCT), and histomorphometry, the present study shows, for the first time, that hPTH 1–34 prevents cholestatic liver disease-induced bone loss.Materials and MethodsAnimals and Experimental ProtocolSix-month-old male Sprague-Dawley rats (Harlan, Israel) were used for this study. Males only were used to eliminate the influence of estrogen deficiency. Animals were maintained on standard rat chow containing 0.8%–1.2% calcium, 0.7%–0.9% phosphor, and ∼3000 U vitamin D per kg (Kofflok, Tel-Aviv, Israel) and were housed in regular cages at 24°C with a 12-hour light/darkness cycle. Four groups of 10 rats each were studied: group 1 underwent a sham operation (SO); groups 2, 3, and 4 underwent bile-duct ligation (BDL), as previously described by us.11Ackerman Z. Weinreb M. Amir G. et al.Bone mineral metabolism and histomorphometry in rats with cholestatic liver disease.Liver. 2002; 22: 166-172Crossref PubMed Scopus (24) Google Scholar A 4-week daily treatment (5 days a week) of subcutaneous injections of either a vehicle (saline containing 0.001 N HCL + 2% heat-inactivated rat serum) (groups 1 and 2) or hPTH 1–34 (Advanced ChemTech, Louisville, KY) at 40 μg/kg per day or 80 μg/kg per day (groups 3 and 4, respectively) was started 1 week after surgery. The SO rats and the PTH-treated BDL rats were pair fed to the BDL rats. Animals' weight was recorded twice weekly. To label bone-forming surfaces, all rats were injected subcutaneously with calcein (Sigma Chemical Co, St. Louis, MO) at 15 mg/kg, 8 and 2 days before death. All animals were killed by CO2 overdose on the 35th day postsurgery. A 24-hour urine collection was conducted 1 day prior to death. Upon death, blood samples were collected for biochemical analysis, the livers were removed for histology, and the femurs and tibiae were removed for bone mineral density (BMD) measurements, μCT, and histomorphometric analyses. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Hebrew University-Hadassah Medical Center.Liver HistologyThe liver of each animal was fixed in formaldehyde, and 5 μm sections were stained with H&E and evaluated by light microscopy. A histologic scoring of the liver damage induced by BDL was graded as follows: portal inflammation: none, 0; mild, 1; moderate, 2; marked, severe, 3; bile duct proliferation: none, 0; mild, 1; moderate, 2; marked, severe, 3; fibrosis: none, 0; portal expansion, 1; bridging fibrosis, 2; cirrhosis, 3.Biochemical AnalysisSerum calcium, phosphorus, albumin, creatinine, total alkaline phosphatase, total bilirubin, alanine aminotransferase (ALT), and urinary calcium and creatinine were measured by the dry chemistry method (VITROS 950/950AT Chemistry System; Ortho-Clinical Diagnostics, Rochester, NY).Serum total osteocalcin was measured with a rat-specific 2-site immunoradiometric assay (IRMA kit; Immutopics Inc, San Clemente, CA). The intra- and interassay coefficients of variation were 2.3% and 4.3%, respectively, and the assay sensitivity was 0.02 ng/mL. Urinary deoxypyridinoline cross-links (DPD) were measured by the Pyrilinks-D kit (Meta Biosystems Inc, Mountain View, CA). The intra- and interassay coefficients of variation were less than 10% and 15%, respectively, and the assay sensitivity was 1.1 nmol/L/liter. The results were corrected for urinary creatinine concentrations and are presented as nmol/L DPD/μmol/L creatinine.BMD MeasurementsEx vivo BMD measurements of whole femurs and tibiae were performed by dual-energy x-ray absorptiometry using a calibrated QDR 4500A densitometer (Hologic, Waltham, MA), equipped with the Small Animal Regional High Resolution software. BMD is expressed in milligrams per centimeter squared (mg/cm2). Bone mineral content (BMC) is expressed in milligrams and bone area in square centimeters.Histomorphometric AnalysisFemurs and tibiae were dehydrated in increasing concentrations of ethanol. Tibiae were embedded in methyl methacrylate, and 6-micron frontal sections were made from the proximal half. Some sections were deplastified using 2-methoxyethyl acetate (Merck, Darmstadt, Germany) and were stained with the Von-Kossa stain for mineralized tissue. Trabecular bone surface was measured in the tibial proximal metaphysis in an area extending 0.75 to 2.75 mm distal to the growth plate. In addition, unstained sections were used to determine the extent of bone surface with single (sL) or double (dL) calcein labels as the percentage of the total bone surface (BS) in an area located 0.75–2.25 mm distal to the growth plate. The mineralizing surface (MS) was calculated according to Parfitt et al22Parfitt A.M. Drezner M.K. Glorieux F.H. et al.Bone histomorphometry: standardization of nomenclature, symbols, and units Report of the ASBMR Histomorphometry Nomenclature Committee.J Bone Miner Res. 1987; 2: 595-610Crossref PubMed Scopus (4881) Google Scholar: MS = (dL+1/2 sL)*100/BS. The mean distance between 2 calcein labels was measured in all sections, and the cancellous mineral apposition rate (MAR) was derived accordingly. Bone formation rate (BFR) was calculated according to the following formula: BFR = (MS/100)*MAR. In addition, the tibiofibular junction zone was embedded in methyl methacrylate, cross sections were made, and cortical bone area and thickness were measured.To determine osteoclast number, tartrate-resistant acid phosphatase staining was used. Briefly, femurs were dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in polymethylmethacrylate (Technovit 9100; Heraeus Kulzer, Wehrheim, Germany). Longitudinal 5-μm sections of each bone were deplastified and subjected to tartrate-resistant acid phosphatase histochemical staining (Sigma Chemical Co). Stained osteoclasts were counted, and their number was determined per millimeter of cancellous bone surface (Oc.N/BS).Microcomputed Tomographic AnalysisWhole femora were examined by μCT (Desktop μCT 40; Scanco Medical AG, Bassersdorf, Switzerland). The scans were performed in 3 spatial dimensions. Two-dimensional (2D) CT images were reconstructed in 1024 × 1024 pixel matrices by using a standard convolution-back projection procedure with a Shepp and Logan filter. Images were stored in 3D arrays with an isotropic voxel size of 30 μm. Thresholds of 150 and 210 in permille of maximal image gray value were used for trabecular and cortical bone, respectively. All morphometric parameters were determined using a direct 3D approach. In the femur, 3 regions were analyzed: (1) whole bone, (2) trabecular bone in the distal metaphysis extending proximally 5 mm from the proximal tip of the primary spongiosa, and (3) cortical bone in a diaphyseal segment extending 1.32 mm distally from the midpoint between the femoral ends. Parameters determined in the metaphyseal trabecular bone included bone volume (BV/TV), trabecular thickness, trabecular number, and trabecular spacing. Cortical bone parameters included cortical thickness and the percent medullary cavity volume.Statistical AnalysisData are presented as mean ± standard error (SE). The significance of the differences between study groups was determined by analysis of variance (ANOVA). When significant differences were indicated by ANOVA, group means were compared using the Tukey test for pairwise comparisons. Level of significance was set at P < .05. Statistical analyses were performed using Statistix 8.0 (Analytical Software, Tallahassee, FL).ResultsLiver DiseaseHistologic analysis of sections from BDL rats demonstrated the presence of secondary biliary cirrhosis with portal fibrosis, bile duct obstruction with portal and lobular inflammation in all animals in groups 2–4. The livers of SO rats were normal. Serum levels of total bilirubin and alkaline phosphatase were markedly increased in BDL rats compared with SO rats, whereas albumin was markedly reduced (Table 1). In BDL rats, there was no association between serum levels of total bilirubin, alkaline phosphatase, or ALT and the bone parameters presented below. The administration of PTH at either dose had no effect on serum calcium, phosphorus, creatinine, or albumin (Table 1).Table 1Biochemical Indices in Sham-Operated, BDL, and hPTH 1–34-Treated RatsParameterSOBDLBDL+PTHBDL+PTHP Value40 μg/kg per day80 μg/kg per daySerum calcium (mmol/L/L)2.48 ± 0.052.39 ± 0.092.41 ± 0.12.43 ± 0.06.8Serum phosphorus (nmol/L/L)2.63 ± 0.182.42 ± 0.273.1 ± 0.22.9 ± 0.19.2Serum albumin (g/L)33.4 ± 1.225 ± 2.5aP < .05 vs SO.23.3 ± 3.1aP < .05 vs SO.25 ± 2.5aP < .05 vs SO..01bP < .05 (ANOVA), obtained in 8 rats per group.Serum ALT (IU)105.8 ± 19.8224.6 ± 71.2181.4 ± 28.1251.5 ± 59.3.04bP < .05 (ANOVA), obtained in 8 rats per group.Serum total ALP (IU)142.7 ± 9.4371.7 ± 70aP < .05 vs SO.412.3 ± 75.7aP < .05 vs SO.430.5 ± 75.5aP < .05 vs SO..05bP < .05 (ANOVA), obtained in 8 rats per group.Serum total bilirubin (μmol/L)8.3 ± 2.773.3 ± 15.5aP < .05 vs SO.122.2 ± 13.9aP < .05 vs SO.101.6 ± 31.8aP < .05 vs SO..001bP < .05 (ANOVA), obtained in 8 rats per group.Plasma creatinine (μmol/L)55 ± 4.466.9 ± 6.164.2 ± 3.768 ± 10.6.5NOTE. Data are mean ± SE.ALT, alanine aminotransferase; ALP, serum total alkaline phosphatase; BDL, bile duct-ligated rats; PTH, intermittently administered human parathyroid hormone 1–34; SO, sham-operated rats; IU, international units.a P < .05 vs SO.b P < .05 (ANOVA), obtained in 8 rats per group. Open table in a new tab Effect of BDL and hPTH 1–34 on BMDMean BMD of the femur and tibia was significantly lower (by 5.5% and 6.6%, respectively) in BDL rats compared with SO rats (Figure 1A and B). This reduction resulted from a decrease in BMC because there was no significant change in bone area (Table 2). The administration of hPTH 1–34 at 40 μg/kg per day increased femoral and tibial BMD in BDL rats by 8.7% and 9.2%, respectively (Figure 1A and B). This increase resulted from a higher BMC without a significant change in bone area (Table 2). In contrast, femoral and tibial BMD in the hPTH 1–34 80 μg/kg per day treatment group was not significantly different from untreated BDL rats (Figure 1A and B). Thus, femoral and tibial BMD and BMC in the hPTH 1–34 80-μg/kg per day treatment group were significantly lower compared with the 40-μg/kg per day treatment group (Figure 1A and B, Table 2). To evaluate further the specific effects of BDL and hPTH 1–34 administration on bone structure and turnover, μCT, histomorphometry, and biochemical analyses were performed.Table 2Bone Mineral Content and Area of the Femur and Tibiae Determined by Dual-Energy X-ray AbsorptiometryParameterSOBDLBDL+PTHBDL+PTHP Value40 μg/kg per day80 μg/kg per dayFemoral bone mineral content (mg)0.319 ± 0.0090.297 ± 0.0100.340 ± 0.010aP < .05 vs BDL., bP < .05 vs BDL+PTH 80 μg/kg per day.0.280 ± 0.010cP < .05 vs SO..01dP < .05 (ANOVA).Femoral bone area (cm2)1.26 ± 0.031.25 ± 0.031.30 ± 0.041.20 ± 0.04.1Tibial bone mineral content (mg)0.23 ± 0.0060.21 ± 0.006aP < .05 vs BDL.0.24 ± 0.008aP < .05 vs BDL., bP < .05 vs BDL+PTH 80 μg/kg per day.0.20 ± 0.008cP < .05 vs SO.<.01dP < .05 (ANOVA).Tibial bone area (cm2)1.18 ± 0.021.18 ± 0.021.20 ± 0.031.10 ± 0.03.07NOTE. Data are mean ± SE obtained in 8 rats per group.BDL, bile duct-ligated rats; PTH, intermittently administered human parathyroid hormone 1–34; SO, sham-operated rats.a P < .05 vs BDL.b P < .05 vs BDL+PTH 80 μg/kg per day.c P < .05 vs SO.d P < .05 (ANOVA). Open table in a new tab Effect of BDL and hPTH 1–34 on Trabecular and Cortical Bone Mass and StructureBDL rats exhibited a significant reduction in femoral trabecular bone mass compared with SO rats as indicated by 18% lower BV/TV as determined by μCT (Figures 2 and 3A). This decrease was accompanied by a reduction in trabecular thickness of 17% with no significant change in trabecular number (Figure 3B and C). In both hPTH 1–34 treatment groups (40 μg/kg per day and 80 μg/kg per day), trabecular bone volume (BV/TV) was restored to SO level (Figure 3A). The increase in BV/TV was accompanied by an increase in trabecular thickness with both hPTH 1–34 doses (50% and 40%, respectively, Figure 3B). There was no significant change in trabecular number with PTH administration (Figure 3C). As expected, trabecular spacing was significantly reduced (by 24%) with the 40-μg/kg per day hPTH 1–34 dose only (Figure 3D).Figure 2Three-dimensional μCT images of distal femoral metaphyseal trabecular bone obtained from animals with median trabecular bone density values. (A) Sham-operated rat. (B) Bile duct-ligated (BDL) rat. (C) BDL rat treated with hPTH 1–34 at 40 μg/kg per day. (D) BDL rat treated with hPTH 1–34 at 80 μg/kg per day.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Trabecular bone mass and architecture parameters determined by μCT in the distal femoral metaphysis of sham-operated (SO) rats, bile duct-ligated (BDL) rats, and BDL rats treated for 4 weeks with hPTH 1–34 at 40 μg/kg per day or 80 μg/kg per day. (A) Trabecular bone volume. (B) Trabecular thickness. (C) Trabecular number. (D) Trabecular spacing. Data are mean ± SE obtained in 8 rats per group. *Different from BDL rats, P < .05; **different from BDL rats, P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tibial cortical thickness and area, determined by histomorphometry, were lower in BDL rats compared with SO rats (by 10% and 10%, respectively, Table 3). The administration of hPTH 1–34 at 40 μg/kg per day increased tibial cortical thickness and area by 17% and 14%, respectively (Table 3). On the other hand, in the hPTH 1–34 80-μg/kg per day treatment group, cortical thickness was not significantly different than in BDL rats (Table 3). Both indices were significantly lower in the 80-μg/kg per day treatment group compared with the 40-μg/kg per day treatment group (Table 3). Middiaphyseal μCT measurements of cortical bone in the femur revealed no significant changes in untreated BDL compared with SO rats or in the PTH-treated rats with either hPTH 1–34 dose (data not shown).Table 3Histomorphometric Indices of Cortical Bone Obtained in the Tibial DiaphysisParameterSOBDLBDL+PTHBDL+PTHP Value40 μg/kg per day80 μg/kg per dayCortical thickness (mm)0.86 ± 0.010.77 ± 0.02aP < .05 vs SO.0.90 ± 0.02bP < .05 vs BDL., cP < .05 vs BDL+PTH 80 μg/kg per day.0.81 ± 0.01<.001dP < .05 (ANOVA).Cortical area (mm2)6.18 ± 0.185.55 ± 0.2aP < .05 vs SO.6.32 ± 0.23bP < .05 vs BDL., cP < .05 vs BDL+PTH 80 μg/kg per day.5.27 ± 0.22aP < .05 vs SO.<.01dP < .05 (ANOVA).NOTE. Data are mean ± SE obtained in 8 rats per group.BDL, bile-duct ligated rats; PTH, intermittently administered human parathyroid hormone 1–34; SO, sham-operated rats.a P < .05 vs SO.b P < .05 vs BDL.c P < .05 vs BDL+PTH 80 μg/kg per day.d P < .05 (ANOVA). Open table in a new tab Effects of BDL and hPTH 1–34 on Bone Formation and ResorptionTo gain further insight into the mechanism leading to the different responses to the higher vs the lower hPTH 1–34 doses, we analyzed bone formation and resorption in the various treatment groups using calcein labeling, osteoclast count, and biochemical markers of bone turnover. A 52% reduction in the percentage double calcein labels' surface was found in BDL compared with SO rats (Table 4). Mineral apposition rate, an index of osteoblast activity, was not significantly different in BDL and SO rats (Table 3). The administration of hPTH 1–34 at 40 μg/kg per day and 80 μg/kg per day increased the percentage double calcein labels' surface by 99% and 112%, respectively, whereas the mineralizing surfaces were increased by 46% with both doses (Table 4).Table 4Histomorphometric Measurements in Trabecular BoneParameterSOBDLBDL+PTHBDL+PTHP Value40 μg/kg per day80 μg/kg per dayDouble-labeled surface (%)34.3 ± 3.716.5 ± 5.0aP < .05 vs SO.35 ± 4.7bP < .05 vs BDL.32.9 ± 4.0bP < .05 vs BDL..04cP < .05 (ANOVA).Mineralizing surface (%)48.8 ± 4.335.8 ± 5.052.4 ± 5.552.4 ± 4.6.08Mineral apposition rate (μm/day)2.63 ± 0.22.29 ± 0.32.47 ± 0.252.03 ± 0.2.3Bone formation rate (mm3/mm2/day)129.7 ± 14.4110.7 ± 23.5128.8 ± 18.2103.6 ± 15.4.5Osteoclast number (mm−1)1.79 ± 0.161.21 ± 0.14aP < .05 vs SO.1.44 ± 0.142.27 ± 0.16bP < .05 vs BDL., dP < .05 vs BDL+PTH 40 μg/kg per day..001NOTE. Data are mean ± SE obtained in 5–8 rats per group.BDL, bile-duct ligated rats; PTH, intermittently administered human parathyroid hormone 1–34; SO, sham-operated rats.a P < .05 vs SO.b P < .05 vs BDL.c P < .05 (ANOVA).d P < .05 vs BDL+PTH 40 μg/kg per day. Open table in a new tab On the other hand, osteoclast number was lower in BDL rats compared with SO rats but not significantly different in BDL rats treated with hPTH 1–34 40 μg/kg per day (Table 4). However, the administration of hPTH 1–34 at 80 μg/kg per day was associated with an almost 2-fold increase in osteoclast number compared with BDL rats and BDL rats treated with hPTH 1–34 40 μg/kg per day (Table 4).The histologic evidence of increased bone turnover with the higher hPTH 1–34 dose was further supported by the level of bone turnover markers. Serum osteocalcin was significantly higher in BDL rats treated with either hPTH 1–34 dose beyond SO and BDL levels (Table 5). On the other hand, urinary DPD was significantly higher in BDL rats treated with hPTH 1–34 at 80 μg/kg per day compared with SO rats, BDL rats, and BDL rats treated with hPTH 1–34 40 μg/kg per day (Table 5), suggesting an increase in both bone resorption and formation with the 80 μg/kg per day dose.Table 5Biochemical Markers of Bone Turnover in SO, BDL, and hPTH 1–34-Treated BDL RatsBiochemical markerSOBDLBDL+PTHBDL+PTHP Value40 μg/kg per day80 μg/kg per daySerum osteocalcin (ng/mL)13.9 ± 0.711.9 ± 1.121.7 ± 3.5aP < .05 vs SO., bP < .05 vs BDL.24.1 ± 2.5aP < .05 vs SO., bP < .05 vs BDL..01cP < .05 (ANOVA).Urinary DPD (nmol/L/μmol/L creatinine)94.6 ± 8.691.7 ± 10.683.8 ± 3.9156.2 ± 24.6aP < .05 vs SO., bP < .05 vs BDL., dP < .05 vs BDL+PTH 40 μg/kg per day..02cP < .05 (ANOVA).NOTE. Data are mean ± SE.BDL, bile-duct ligated rats; DPD, deoxypyridinoline cross-links; PTH, intermittently administered human parathyroid hormone 1–34; SO, sham-operated rats.a P < .05 vs SO.b P < .05 vs BDL.c P < .05 (ANOVA).d P < .05 vs BDL+PTH 40 μg/kg per day. Open table in a new tab DiscussionThe present study demonstrates that bile duct ligation in male rats induces bone loss and microarchitectural changes in the femur and tibia, which can be prevented by the intermittent administration of hPTH 1–34
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