Human Parathyroid Hormone 1–34 Prevents Bone Loss in Experimental Biliary Cirrhosis in Rats

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Human Parathyroid Hormone 1–34 Prevents Bone Loss in Experimental Biliary Cirrhosis in Rats RIVKA DRESNER–POLLAK,* YANKEL GABET,‡ ARZA STEIMATZKY,§ GILAD HAMDANI,§ ITAI BAB,‡ ZVI ACKERMAN,储 and MIRON WEINREB¶

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 ductligated (BDL) rats was studied. Methods: Six-monthold 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 ␮g/kg per day, 80 ␮g/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 ␮g/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 ␮g/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.

R

educed bone mass and increased fracture risk are serious complications of human primary biliary cirrhosis (PBC).1– 4 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.1 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.5–7 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 PBCrelated bone loss and fractures.8,9 Hormone replacement therapy has favorable effects on bone mass in PBC but can worsen cholestasis.10 Studies in humans with PBC are difficult to interpret because of patients’ heterogeneity with respect to disease stage and menopausal status. 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.11,12 Osteopenia in BDL rats was characterized by decreased bone formation and osteoblastsogenesis.12 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.13–21 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 (␮CT), Abbreviations used in this paper: BDL, bile duct-ligated; DPD, deoxypyridinoline cross-links; hPTH, human parathyroid hormone; ␮CT, microcomputerized tomography; PBC, primary biliary cirrhosis; SO, sham operation. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2007.10.025

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*Endocrinology and Metabolism Service, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; ‡Bone Laboratory, the Hebrew University of Jerusalem, Jerusalem; §Hadassah-Hebrew University Medical School, Jerusalem; 储Department of Medicine, Hadassah-Hebrew University Hospital Mount Scopus, Jerusalem; and ¶Department of Oral Biology, Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv, Israel

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and histomorphometry, the present study shows, for the first time, that hPTH 1–34 prevents cholestatic liver disease-induced bone loss.

Materials and Methods Animals and Experimental Protocol

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Six-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.11 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 ␮g/kg per day or 80 ␮g/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, ␮CT, and histomorphometric analyses. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Hebrew University-Hadassah Medical Center.

Liver Histology The liver of each animal was fixed in formaldehyde, and 5 ␮m 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 Analysis Serum 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).

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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/␮mol/L creatinine.

BMD Measurements Ex 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 Analysis Femurs 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 al22: 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-␮m sections of each bone were deplastified and subjected to tartrate-resistant acid phosphatase histochemical staining (Sigma Chemical Co). Stained os-

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Table 1. Biochemical Indices in Sham-Operated, BDL, and hPTH 1–34-Treated Rats Parameter

SO

BDL

BDL⫹PTH 40 ␮g/kg per day

BDL⫹PTH 80 ␮g/kg per day

P Value

Serum calcium (mmol/L/L) Serum phosphorus (nmol/L/L) Serum albumin (g/L) Serum ALT (IU) Serum total ALP (IU) Serum total bilirubin (␮mol/L) Plasma creatinine (␮mol/L)

2.48 ⫾ 0.05 2.63 ⫾ 0.18 33.4 ⫾ 1.2 105.8 ⫾ 19.8 142.7 ⫾ 9.4 8.3 ⫾ 2.7 55 ⫾ 4.4

2.39 ⫾ 0.09 2.42 ⫾ 0.27 25 ⫾ 2.5a 224.6 ⫾ 71.2 371.7 ⫾ 70a 73.3 ⫾ 15.5a 66.9 ⫾ 6.1

2.41 ⫾ 0.1 3.1 ⫾ 0.2 23.3 ⫾ 3.1a 181.4 ⫾ 28.1 412.3 ⫾ 75.7a 122.2 ⫾ 13.9a 64.2 ⫾ 3.7

2.43 ⫾ 0.06 2.9 ⫾ 0.19 25 ⫾ 2.5a 251.5 ⫾ 59.3 430.5 ⫾ 75.5a 101.6 ⫾ 31.8a 68 ⫾ 10.6

.8 .2 .01b .04b .05b .001b .5

NOTE. 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. aP ⬍ .05 vs SO. bP ⬍ .05 (ANOVA), obtained in 8 rats per group.

Microcomputed Tomographic Analysis Whole femora were examined by ␮CT (Desktop ␮CT 40; Scanco Medical AG, Bassersdorf, Switzerland). The scans were performed in 3 spatial dimensions. Twodimensional (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 ␮m. 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 Analysis Data 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).

Results Liver Disease Histologic 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).

Effect of BDL and hPTH 1–34 on BMD Mean 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 ␮g/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 ␮g/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-␮g/kg per day treatment group were significantly lower compared with the 40-␮g/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, ␮CT, histomorphometry, and biochemical analyses were performed.

Effect of BDL and hPTH 1–34 on Trabecular and Cortical Bone Mass and Structure BDL rats exhibited a significant reduction in femoral trabecular bone mass compared with SO rats as indicated by 18% lower BV/TV as determined by ␮CT (Figures 2 and 3A). This decrease was accompanied by a

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teoclasts were counted, and their number was determined per millimeter of cancellous bone surface (Oc.N/BS).

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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-␮g/kg per day hPTH 1–34 dose only (Figure 3D). 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 ␮g/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-␮g/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-␮g/kg per day treatment group compared with the 40-␮g/kg per day treatment group (Table 3). Middiaphyseal ␮CT 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).

Effects of BDL and hPTH 1–34 on Bone Formation and Resorption

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Figure 1. hPTH 1–34 prevents femoral and tibial bone mineral density (BMD) loss in male bile duct-ligated (BDL) rats. Femoral (A) and tibial (B) BMD in sham-operated (SO) rats, BDL rats, and BDL rats treated for 4 weeks with human parathyroid hormone (hPTH 1–34) at 40 ␮g/kg per day or 80 ␮g/kg per day. Data are mean ⫾ SE obtained in 8 rats per group. *aDifferent from BDL rats, P ⬍ .05; *bdifferent from BDL⫹hPTH 1–34 80 ␮g/kg per day rats, P ⬍ .05.

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 ␮g/kg per day and 80 ␮g/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

To 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 ␮g/kg per day and 80 ␮g/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). 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 ␮g/kg per day (Table 4). However, the administration of hPTH

Table 2. Bone Mineral Content and Area of the Femur and Tibiae Determined by Dual-Energy X-ray Absorptiometry Parameter

SO

BDL

BDL⫹PTH 40 ␮g/kg per day

BDL⫹PTH 80 ␮g/kg per day

P Value

Femoral bone mineral content (mg) Femoral bone area (cm2) Tibial bone mineral content (mg) Tibial bone area (cm2)

0.319 ⫾ 0.009 1.26 ⫾ 0.03 0.23 ⫾ 0.006 1.18 ⫾ 0.02

0.297 ⫾ 0.010 1.25 ⫾ 0.03 0.21 ⫾ 0.006a 1.18 ⫾ 0.02

0.340 ⫾ 0.010a,b 1.30 ⫾ 0.04 0.24 ⫾ 0.008a,b 1.20 ⫾ 0.03

0.280 ⫾ 0.010c 1.20 ⫾ 0.04 0.20 ⫾ 0.008c 1.10 ⫾ 0.03

.01d .1 ⬍.01d .07

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. aP ⬍ .05 vs BDL. bP ⬍ .05 vs BDL⫹PTH 80 ␮g/kg per day. cP ⬍ .05 vs SO. dP ⬍ .05 (ANOVA).

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263

Figure 2. Three-dimensional ␮CT 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 ␮g/kg per day. (D) BDL rat treated with hPTH 1–34 at 80 ␮g/kg per day.

1–34 at 80 ␮g/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 ␮g/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 ␮g/kg per day compared with SO rats, BDL rats, and BDL rats treated with hPTH 1–34 40 ␮g/kg per day (Table 5), suggesting an increase in both bone resorption and formation with the 80 ␮g/kg per day dose.

Discussion The 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. hPTH 1–34 at 40 ␮g/kg per day restored bone mineral density, as well as trabecular bone mass and thickness. At a dose of 80 ␮g/kg per day, hPTH 1–34 increased indices of both bone formation and resorption, resulting in no net gain in BMD.

To our knowledge, this is the first study to evaluate the skeletal effects of a bone anabolic agent in an animal model of biliary cirrhosis. We have previously reported a reduction in bone formation and osteoblastogenesis in male BDL rats.11,12 Likewise, low bone mass in similar rats in the present study was associated with low bone turnover as indicated by serum osteocalcin level, urinary DPD, and osteoclast number. These findings are consistent with previous reports of reduced bone formation in PBC patients, as demonstrated in iliac crest biopsy specimens obtained in these patients.23,24 Furthermore, histomorphometric analysis of iliac crest bone biopsy specimens in patients with advanced PBC or primary sclerosing cholangitis revealed low bone formation rate with no significant increase in bone resorption in the male patients.24 hPTH 1–34 administration to BDL rats at 40 ␮g/kg per day increased trabecular bone mass by increasing trabecular bone volume and trabecular thickness with no significant effect on trabecular number. This anabolic effect resulted from stimulation of bone formation, primarily through an increase in the extent of mineralizing surfaces, which reflects increased osteoblast precursor proliferation and/or maturation. Similar effects were previously reported in PTH 1–34-treated ovariectomized (OVX) rats.15,25 The 20% PTH-induced increase in BV/TV

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Figure 3. Trabecular bone mass and architecture parameters determined by ␮CT 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 ␮g/kg per day or 80 ␮g/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.

in BDL rats in this study is somewhat lower compared with the effect in OVX rats treated for a similar time period (⬃45%15,25). This difference may result from the underlying mechanism of bone loss in estrogen deficiency, characterized by a high bone turnover and rapid bone loss, as opposed to the low turnover bone loss in male BDL rats. Studies of intermittent hPTH 1–34 administration to female BDL rats and OVX BDL rats are needed to evaluate fully the effects of hPTH 1–34 in this

model. Another possible explanation for the reduced response to PTH in our model is some degree of vitamin D deficiency, even though the rats in this study were supplemented with vitamin D, and we have previously demonstrated no histomorphometric evidence of osteomalacia in this BDL model.11 In addition, deleterious effects of cytokines and bilirubin on osteoblasts, as previously suggested by others and us,6,12 could have also diminished the response to PTH. PTH 1–34 at 40 ␮g/kg

Table 3. Histomorphometric Indices of Cortical Bone Obtained in the Tibial Diaphysis Parameter

SO

BDL

BDL⫹PTH 40 ␮g/kg per day

BDL⫹PTH 80 ␮g/kg per day

P Value

Cortical thickness (mm) Cortical area (mm2)

0.86 ⫾ 0.01 6.18 ⫾ 0.18

0.77 ⫾ 0.02a 5.55 ⫾ 0.2a

0.90 ⫾ 0.02b,c 6.32 ⫾ 0.23b,c

0.81 ⫾ 0.01 5.27 ⫾ 0.22a

⬍.001d ⬍.01d

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. aP ⬍ .05 vs SO. bP ⬍ .05 vs BDL. cP ⬍ .05 vs BDL⫹PTH 80 ␮g/kg per day. dP ⬍ .05 (ANOVA).

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Table 4. Histomorphometric Measurements in Trabecular Bone Parameter

SO

BDL

BDL⫹PTH 40 ␮g/kg per day

BDL⫹PTH 80 ␮g/kg per day

P Value

Double-labeled surface (%) Mineralizing surface (%) Mineral apposition rate (␮m/day) Bone formation rate (mm3/mm2/day) Osteoclast number (mm⫺1)

34.3 ⫾ 3.7 48.8 ⫾ 4.3 2.63 ⫾ 0.2 129.7 ⫾ 14.4 1.79 ⫾ 0.16

16.5 ⫾ 35.8 ⫾ 5.0 2.29 ⫾ 0.3 110.7 ⫾ 23.5 1.21 ⫾ 0.14a

35 ⫾ 52.4 ⫾ 5.5 2.47 ⫾ 0.25 128.8 ⫾ 18.2 1.44 ⫾ 0.14

32.9 ⫾ 52.4 ⫾ 4.6 2.03 ⫾ 0.2 103.6 ⫾ 15.4 2.27 ⫾ 0.16b,d

.04c .08 .3 .5 .001

5.0a

4.7b

4.0b

per day prevented BDL-induced loss of tibial cortical bone thickness as indicated by histomorphometric analysis. ␮CT analysis did not reveal any changes in femoral cortical bone. These differences may be site or methodology related. The effects observed in the tibial cortical bone by histomorphometry are in agreement with the BMD measurements, which reflect the combined changes in trabecular and cortical bone. Dose-related differences in the efficacy of hPTH 1–34 were observed, with bone anabolic effects detected with the 40-␮g/kg per day dose. The skeletal effects of a wide range of PTH 1–34 doses were evaluated in OVX, aged OVX, orchiectomized, and hind-limb unloaded female and male rat models of osteoporosis.15–18 Turner et al have recently shown that a human therapeutic dose as low as 1 ␮g/kg per day was sufficient to prevent trabecular bone loss and maintain normal bone architecture in hind-limb unloaded rats, whereas the high dose of 80 ␮g/kg per day excessively increased bone formation and trabecular thickness beyond normal control animals.17 Elevated levels of PTH increase bone turnover leading to either anabolic or catabolic effects, depending on the pattern and duration of exposure. The anabolic effects require brief exposures to higher than average PTH concentrations, whereas the catabolic effects result from continuously sustained levels of the hormone as in primary or secondary hyperparathyroidism. Exogenous PTH 1–34 treatment was shown to stimulate bone formation by

increasing the remodeling rate and the amount of bone laid down in each remodeling unit, resulting in increased trabecular thickness and connectivity.25,26 At the cellular level, enhanced recruitment, proliferation, and differentiation as well as reduced apoptosis of osteoblasts have been reported.27 An additional mechanism whereby PTH stimulates bone formation involves uncoupling of bone formation from resorption by directly activating lining cells on previously quiescent bone surfaces.28 On the other hand, the catabolic effects of continuous PTH result from an increase in receptor activator of nuclear factor-␬B expression and inhibition of osteoprotegerin with consequent increased osteoclastogenesis.29,30 The pharmacokinetic profile of PTH 1–34 associated with either an anabolic or catabolic bone response was previously studied in male rats31 and was shown to be determined by the length of time that serum concentration of PTH 1–34 remained above baseline levels of endogenous PTH and only secondarily by the maximum serum concentrations of PTH 1–34 or the area under the curve. The pharmacokinetics and pharmacodynamics of intermittently administered PTH 1–34 in severe hepatic disease have not been studied, and its volume of distribution may be altered in the presence of ascites. The precise PTH 1–34 dose range necessary to prevent and treat the bone loss associated with cholestatic liver disease needs further investigation.

Table 5. Biochemical Markers of Bone Turnover in SO, BDL, and hPTH 1–34-Treated BDL Rats Biochemical marker

SO

BDL

Serum osteocalcin (ng/mL) Urinary DPD (nmol/L/␮mol/L creatinine)

13.9 ⫾ 0.7 94.6 ⫾ 8.6

11.9 ⫾ 1.1 91.7 ⫾ 10.6

BDL⫹PTH 40 ␮g/kg per day 21.7 ⫾ 83.8 ⫾ 3.9

3.5a,b

BDL⫹PTH 80 ␮g/kg per day

P Value

24.1 ⫾ 156.2 ⫾ 24.6a,b,d

.01c .02c

2.5a,b

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. aP ⬍ .05 vs SO. bP ⬍ .05 vs BDL. cP ⬍ .05 (ANOVA). dP ⬍ .05 vs BDL⫹PTH 40 ␮g/kg per day.

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NOTE. 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. aP ⬍ .05 vs SO. bP ⬍ .05 vs BDL. cP ⬍ .05 (ANOVA). dP ⬍ .05 vs BDL⫹PTH 40 ␮g/kg per day.

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The liver plays a major role in PTH metabolism and is the main site for clearance of the circulating hormone.32 Kupffer cells were shown to uptake the intact hormone and degrade it to generate various circulating fragments.33 Active fragments are generated because of the high content of the endopeptidase cathepsin D in Kupffer cells, which specifically cleaves PTH to generate 1–34, 1–37, 34 – 84, and 37– 84 fragments.33 The 1–34 and 1–37 N-terminal fragments, which are of high biologic activity, are degraded locally by Kupffer cells and do not reemerge from the liver into the circulation, whereas the C-terminal fragments are released to the bloodstream and are removed mainly by the kidneys.32 Studies in hepatectomized versus nephrectomized rats have shown that the liver is the principal source of circulating Cterminal PTH fragments.34,35 Hepatic production of Cterminal PTH fragments requires initial uptake of the intact hormone by recognizing determinants present in it (28 – 48) but not in PTH 1–34.36 Indeed, intact PTH 1– 84, but not PTH 1–34, binds to Kupffer cells.37 On the other hand, both PTH 1– 84 and PTH 1–34 bind to hepatocytes and sinusoidal cells.37 The physiologic significance of this binding is unclear. Although the amino-terminal region of PTH is both necessary and sufficient for mediating the bone anabolic actions of the hormone through activation of the PTH1 receptor, the biologic actions of the carboxyterminal fragments are still unclear. In vitro and in vivo data suggest the existence of discrete receptors for these fragments and a physiologic role for these determinants.32 Data regarding endogenous PTH metabolism in cirrhosis are sparse. Elevated serum levels of the midregion fragment (44 – 68) and the C-terminal (70 – 84) fragment of PTH were previously reported in patients with cirrhosis and PBC.38,39 The significance of these findings is yet unknown. Serum levels of endogenous PTH or its fragments were not determined in this study, but changes in uptake and clearance by the cirrhotic liver are likely and may in part explain the lack of effect observed with the higher dose of exogenously administered hPTH 1–34. In conclusion, hPTH 1–34 shows marked positive effects on bone mass and architecture when administered to male adult BDL rats, a rodent model of PBC-induced bone loss. Dose-related differences were observed with respect to the skeletal anabolic effects of hPTH 1–34, which may be related to the combined effects of exogenously administered hPTH 1–34 and the endogenous intact PTH and its fragments in liver disease. Further studies are needed to determine the effects of hPTH 1–34 and its optimal dose in females in this model. To the extent that rodent models of biliary cirrhosis can be applied to the human disease, hPTH 1–34 holds promise in the treatment of PBC-induced osteoporosis.

GASTROENTEROLOGY Vol. 134, No. 1

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Received May 16, 2007. Accepted September 27, 2007. Address requests for reprints to: Rivka Dresner-Pollak, MD, Endocrinology and Metabolism Service, Hadassah-Hebrew University Medical Center, POB 12000, Jerusalem 91120, Israel. e-mail: rivkap@ hadassah.org.il; fax: (972) 2-6437940. Supported in part by a grant from the Chief Scientist’s office of the Ministry of Health, Israel. Z.A and M.W contributed equally to this work, and should be considered joint last authors. Conflicts of interest: None of the authors has any conflict of interest.

BASIC–LIVER, PANCREAS, AND BILIARY TRACT

January 2008