S. Epstein

The Role of Testosterone in Cyclosporine‐Induced Osteopenia

Our laboratory has demonstrated that the immunosuppressants Cyclosporin A (CsA) and tacrolimus (FK506), in vivo in the rat, produce a high‐turnover osteopenia. CsA is known to decrease serum testosterone (Test) levels both in the rat and in human transplant patients. Less is known of FK506s effect on androgens. CsA‐induced hypogonadism may contribute to the aforementioned bone loss because hypogonadism itself is a risk factor for osteoporosis and fracture. The aim of this study was to assess serum androgen levels following CsA and FK506 therapy and to see whether Test replacement therapy, in the form of 28‐day controlled release subcutaneous pellet implants, could prevent CsA‐induced osteopenia. Two experiments were conducted. In experiment I, four groups of 6‐month‐old male Sprague‐Dawley rats received the following: (A) CsA vehicle and placebo pellet, (B) Test 15 mg pellet and CsA vehicle, (C) CsA 10 mg/kg and placebo pellet, (D) Test 15 mg pellet and CsA 10 mg/kg. In experiment II, two groups of rats received (E) FK506 vehicle and (F) FK506 4 mg/kg. CsA, FK506, and vehicles were given for 28 days by daily oral gavage. The rats were weighed and bled on days 0, 14, and 28. All rats received double fluorescent labeling, and on day 28 the tibiae were removed for histomorphometry. Whole blood was assayed for CsA and FK506 levels. Serum was assayed for total and free Test as well as for osteocalcin (BGP), blood urea nitrogen (BUN), creatinine, and calcium. Whole blood monoclonal CsA levels measured by fluorescent immunoassay were in the therapeutic range, while a drug concentration profile showed good absorption of FK506. Those rats receiving Test and FK506 lost weight, while those receiving CsA remained constant. BUN was only marginally elevated in the CsA‐treated groups on day 28 (p < 0.05), while creatinine was unchanged. On day 28, total and free Test was significantly reduced in the CsA‐treated rats versus control (p < 0.05), while Test replacement therapy maintained total Test levels above vehicle (p < 0.01) and free Test levels similar to vehicle on day 28. FK506 did not lower total or free Test levels. BGP levels were significantly increased in the CsA (p < 0.01) and FK506 (p < 0.001) groups on day 28. BGP in the groups receiving Test alone and in combination with CsA remained similar to vehicle. Histomorphometry confirmed CsA‐ and FK506‐induced high‐turnover osteopenia. The Test alone group marginally increased bone formation. Test replacement failed to prevent the CsA‐induced bone loss. In conclusion, immunosuppressive doses of CsA, but not FK506, lowers serum total and free Test. Hypoandrogenemia does not seem to be a major factor in CsA‐induced osteopenia because bone loss occurs despite Test replacement.

Alendronate prevents cyclosporin A-induced osteopenia in the rat

Post-transplantation bone disease is an increasingly recognized clinical entity whose etiology is multifactorial. The immunosuppressant agent cyclosporine-A (CsA) has repeatedly been shown experimentally to induce a high-turnover osteopenic state. Alendronate (Alen.) is a new generation bisphosphonate having far greater antiresorptive potency than previous bisphosphonates. It inhibits osteoclast resorption in vitro and in vivo without adversely affecting bone mineralization. This study was designed to investigate whether alendronate could prevent CsA-induced osteopenia in the rat. Forty-eight 8-month-old male Sprague Dawley rats were randomized into four groups to receive the following for 28 days: (1) CsA vehicle (veh.) p.o. daily and alendronate vehicle subcutaneously (s.c.) twice/week, (2) CsA 15 mg/kg p.o. daily and Alen. veh. s.c. twice/week, (3) Alen. 70 micrograms/kg s.c. twice/ week and CsA veh. p.o. daily, and (4) CsA 15 mg/kg p.o. daily and Alen. 70 micrograms/kg s.c. twice/week. Rats were weighed and bled and serum was assayed serially for calcium, PTH, 1,25(OH)2vit.D, and osteocalcin. Tibiae were removed following sacrifice on day 28, after double demeclocycline and calcein labeling, for histomorphometric analysis. Treated groups were compared to the vehicle-treated control. We confirmed previous findings that CsA produces elevated 1,25(OH)2 vitamin D and serum osteocalcin levels. Alendronate treatment by itself decreased osteocalcin by day 28 and resulted in a marginal decrease in serum total calcium on day 14. The histomorphometry findings reconfirmed that the administration of CsA induces a state of high-turnover osteopenia. Alendronate prevented CsAs adverse effects, particularly in maintaining trabecular bone volume, presumably by decreasing bone remodeling. Alendronate would seem to hold therapeutic promise in post-transplantation bone disease.

Bone metabolism in renal transplant patients treated with cyclosporine or sirolimus

Sirolimus is a new immunosuppressive agent used as treatment to prevent acute renal allograft rejection. One of the complications of renal transplantation and subsequent long‐term immunosuppression is bone loss associated with osteoporosis and consequent fracture. Two open‐label, randomized, phase 2 studies comparing sirolimus versus cyclosporine (CsA) included indices of bone metabolism as secondary end‐points. Markers of bone turnover, serum osteocalcin and urinary N‐telopeptides, were measured over a 1‐year period in 115 patients receiving either CsA or sirolimus as a primary therapy in combination with azathioprine and glucocorticoids (study A) or mycophenolate mofetil (MMF) and glucocorticoids (study B). Urinary excretion of N‐telopeptides and the concentrations of serum osteocalcin were consistently higher in the CsA‐treated patients and significantly different at week 24 for N‐telopeptides and at weeks 12, 24, and 52 for osteocalcin. In conclusion, future trials are warranted to test whether a sirolimus‐based regimen conserves bone mineral density compared with a CsA‐based regimen.

Amylin increases bone volume but cannot ameliorate diabetic osteopenia

Amylin is normally secreted in a regulated fashion by the pancreatic β-cells in parallel with insulin and has been reported to have bone-conserving properties. Type I diabetes mellitus results in a low-turnover osteopenia in the presence of decreased amylin, which is in contrast to type II diabetes where less bone loss, in the presence of high amylin levels, occurs. We investigated the effects of amylin on bone mineral metabolism in normal and dibetic (streptozotocin-induced) rats, in order to ascertain whether amylin would modify the streptozotocin-induced diabetic osteopenia. Tenweek-old male Sprague-Dawley rats were randomized as follows: group A (n=18) received normal saline; group B (n=18) received amylin; group C, diabetic rats (n=23), received normal saline; and group D, diabetic rats (n=23), received amylin. Amylin (100 pmol/100 g b. w.) was administered by a daily subcutaneus injection. Double calceinlabeled tibiae were removed for histomorphometric analysis followed sacrifice on day 19. Results showed no difference in blood ionized calcium between groups. Blood glucose remained above 600 mg/dl in the diabetic animals and was not affected by the administration of amylin. Serum osteocalcin, insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), and 1,25 dihydroxyvitamin D [1,25(OH)2D] were significantly lower in the diabetic rats compared with control group A by day 19. Amylin produced higher levels of serum osteocalcin in group B on day 9 (P<0.05) compared with controls but returned to control values (group A) by day 19; no such change occurred in the diabetic group. Amylin administration did not influence IGF-1, 1,25(OH)2D or PTH levels compared with the untreated animals. Analysis of the bone histomorphometry showed a low-turnover osteopenia in the diabetic animals. Amylin administration resulted in a significant increase in bone volume in the normal rats, group B (P<0.05), but was unable to significantly alter this parameter in the diabetic animals. In conclusion, amylin has a beneficial effect on the bone metabolism of the rat in vivo by increasing bone volume. It is, however, unable to overcome the osteopenia caused by streptozotocin-induced diabetes mellitus at the doses used in this study.

Immunosuppressive therapy and the skeleton.

Glucocorticoids and other immunosuppressive drugs, such as cytoclosporine A, are increasingly used today. One of the most common clinical situations in which they are prescribed is for immunosuppression after organ transplantation. These drugs have diverse effects on the skeleton, however, and one of the most common sequelae of organ transplantation is osteoporosis and fractures. Patients treated with immunosuppressive drugs should be carefully evaluated for osteoporosis, preferably prior to or at the time of initiation of immunosuppressive therapy. They should be followed carefully with sequential bone-density measurements and biochemical indices of bone turnover. Measures to prevent bone loss should be initiated early in the course of immunosuppressive therapy.

Interferon-α, unlike interferon-γ, does not cause bone loss in the rat

Abstract Interferons (IFN) are a group of related glycoproteins. IFN-γ, in vitro, has been shown to inhibit resorption; however, an in vivo experiment showed that it had the opposite effect, resulting in bone loss that was comparable to that caused by cyclosporine A. IFN-α has numerous clinical applications but is used most extensively in the treatment of chronic hepatitis B and chronic hepatitis C. Research into the effects of IFN-α on bone mineral metabolism has been very sparse, and the majority of studies reflect in vitro models. Like IFN-γ, there exists discordance between in vitro and in vivo studies on IFN-α. Both in vivo and in vitro studies demonstrate that IFN-α decreases bone resorption, whereas osteoblasts may or may not be affected in vivo. This study was designed to provide information on the in vivo effects of IFN-α in the rat model, because we feel that, given its widespread clinical use, this is an extremely important issue. Rats were given low dose IFN-α (1.6 × 10 6 IU/m 2 ), intermediate dose IFN-α (5.35 × 10 6 IU/m 2 ), and high dose IFN-α (30 × 10 6 IU/m 2 ) three times per week for 28 days. Serum osteocalcin (bone gla protein, or BGP) and parathyroid hormone (PTH) were measured serially and, after double labeling, the bones were examined histomorphometrically. IFN-α did not alter any of the histomorphometric parameters measured and did not affect PTH. However, it produced a disparate BGP response. Low dose IFN-α resulted in a statistically significant increase in serum BGP on days 14 and 28, whereas intermediate and high doses of IFN-α did not. Overall, these results provide no evidence of a deleterious effect of IFN-α on bone metabolism and confirm the limited clinical study.

The role of the T-lymphocyte in estrogen deficiency osteopenia.

Our laboratory has previously demonstrated that the T‐lymphocyte is critical in the development of cyclosporin A–induced osteopenia in the rat model. A similar state of osteopenia is induced by estrogen depletion in the ovariectomized (OVX) rat, which is the animal model of postmenopausal bone loss. However, the role of the immune system, and particularly the T‐lymphocyte, in estrogen deplete osteopenia has not been elucidated. We used the Rowett athymic nude rat as our model of T‐lymphocyte deficiency. In this study, the experimental rats were divided into four groups as follows: (1) sham‐operated Rowett heterozygous (rnu/+) euthymic rats (control group); (2) OVX Rowett heterozygous (rnu/+) euthymic rats; (3) sham‐operated Rowett homozygous (rnu/rnu) athymic nude rats, which are T‐lymphocyte deficient; and (4) ovariectomized Rowett homozygous (rnu/rnu) rats. Rats were weighed, and venous blood was taken in weeks 2, 4, and 6 for determination of serum osteocalcin. Serum 1,25‐dihydroxyvitamin D (1,25(OH)2D) was determined on the day of sacrifice. Following sacrifice, histomorphometry was performed on double‐labeled proximal tibial metaphyses. Flow cytometric analysis of splenic mononuclear cell isolates stained for OX19‐positive (CD5) T‐lymphocytes was performed. T‐lymphocyte analysis revealed significant reductions in both athymic nude groups, while OVX euthymic rats demonstrated a diminished number of T‐cells relative to their sham‐operated counterparts. Histomorphometric data indicated that both OVX groups exhibited a significant loss of trabecular volume, with associated increases in indices for bone formation and resorption, with resorption likely outstripping formation, resulting in osteopenia. Serum osteocalcin was significantly elevated in the ovariectomized euthymic group throughout the experimental period compared with the control group (p < 0.01); it was elevated in the ovariectomized athymic group on week 4 only (p < 0.01 vs. control). It appears that the T‐lymphocyte may not be an essential component in the pathogenesis of estrogen deficiency osteopenia. The contribution of circulating T‐lymphocytes as well as other T‐lymphocyte–rich organs needs to be explored further.

Transforming growth factor-β administration modifies cyclosporine A-induced bone loss

Abstract Cyclosporine A (CsA), a potent immunosuppressant used in transplantation, induces increased formation with excess resorption in the rat with resultant osteopenia. These findings are confirmed in the human model. Transforming growth factor-β (TGF-β) is reported to be involved in the coupling of bone formation with resorption and in vivo and in vitro stimulates osteoblasts, and in vitro inhibits osteoclasts. CsA stimulates secretion of TGF-β1 in humans, which, while improving immunosuppression, may also contribute to renal toxicity. This study was performed determine whether exogenously administered TGF-β would modify the bone effects of CsA. Male Sprague-Dawley rats, 6 months of age, were randomized to receive: TGF-β and CsA vehicle (group A); TGF-β 5 μg/kg three times per week and CsA vehicle (group B); TGF-β vehicle and CsA 10 mg/kg (group C); or TGF-β 5 μg/kg three times per week and CsA 10 mg/kg (group D). These were compared with control over 28 days. CsA, but not TGF-β, increased serum 1,25(OH)2D levels throughout the study. CsA increased osteocalcin (BGP), but TGF-β negated this effect. Histomorphometry confirmed the known effects of CsA, whereas TGF-β alone had no effect. However, in combination, TGF-β blocked CsA’s effect and increased osteoblast recruitment and activity, as reflected by increased percent mineralizing surface, percent osteoid perimeter, bone formation rate (bone volume referent), and activation frequency. Thus, it appears as if TGF-β administration may have potential in modulating the deleterious bone effects of CsA.

Cytokines and osteoporosis

Abstract Cytokines are polypeptide molecules which modulate the biologic actions of cells. They are recognized as local factors that regulate the various functions of bone cells and are implicated in various metabolic bone diseases including osteoporosis. Several interleukins (ILs) appear to be key players: IL-1 has potent bone-resorbing activities both in vitro and in vivo . It stimulates osteoclast formation which may depend on prostaglandins. In vivo , IL-1 produces high bone turnover with bone volume loss and hypercalcemia. The role of IL-1 in postmenopausal osteoporosis is unclear since current evidence provides conflicting results. IL-4 inhibits bone resorption in vitro whereas an in vivo study in transgenic mice reveals low turnover osteoporosis. IL-6 is another major cytokine which has been extensively studied in bone diseases especially estrogen-deficient osteoporosis. IL-6 is believed to induce bone resorption by activating osteoclastic stem cells rather than mature osteoclasts although the exact mechanism and its role in osteoporosis need to be clarified. IL-11 has been shown to enhance bone resorption and inhibit bone formation in vitro , however, its significance in bone metabolism in vivo is as yet undefined. Tumor necrosis factor (TNF), like IL-1, is a bone resorber both in vitro and in vivo and is one of the cytokines that may well play an important role in postmenopausal osteoporosis. Lymphotoxin (LT), similar to TNF, stimulates bone resorption in vitro but its role in osteoporosis has not been established. The effects of several colony-stimulating factors (CSFs) have also been investigated. Despite no bone-resorbing activity, granulocyte-CSF (G-CSF) is able to produce osteoporosis in transgenic mice and monocyte-CSF (M-CSF) administration is capable of correcting murine osteopetrosis. Interferon gamma (IFNγ) has been recognized as an inhibitor of bone resorption in vitro until recently when in vivo studies demonstrated its bone-resorbing effect by producing loss of bone volume in rats and in osteopetrotic patients. As another bone resorber in vitro , leukemia inhibitory factor (LIF) is also a candidate in the pathogenesis of osteoporosis although further studies are needed. The problem of defining roles for these cytokines may rest in the different biological systems, that is, in vitro versus in vivo studies, and interaction of systemic hormones and a stepwise cascade series of regulatory events that make one particular cytokines role difficult to evaluate in isolation.