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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-beta (TGF-beta) 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-beta1 in humans, which, while improving immunosuppression, may also contribute to renal toxicity. This study was performed determine whether exogenously administered TGF-beta would modify the bone effects of CsA. Male Sprague-Dawley rats, 6 months of age, were randomized to receive: TGF-beta and CsA vehicle (group A); TGF-beta 5 microg/kg three times per week and CsA vehicle (group B); TGF-beta vehicle and CsA 10 mg/kg (group C); or TGF-beta 5 microg/kg three times per week and CsA 10 mg/kg (group D). These were compared with control over 28 days. CsA, but not TGF-beta, increased serum 1,25(OH)(2)D levels throughout the study. CsA increased osteocalcin (BGP), but TGF-beta negated this effect. Histomorphometry confirmed the known effects of CsA, whereas TGF-beta alone had no effect. However, in combination, TGF-beta 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-beta administration may have potential in modulating the deleterious bone effects of CsA.

Cyclosporine A (CsA) induces high turnover osteopenia in the rat and there is evidence for this in humans. Recent studies suggest that increases in parathyroid hormone (PTH) may be involved in posttransplantation bone loss. However, human studies are difficult to interpret since transplant patients usually receive a cocktail of immunosuppressants and have underlying disease. Our aim was to try to resolve the influence of the absence or presence of PTH on CsA-induced bone disease. Male Sprague Dawley rats aged 7-9 months, either sham operated or parathyroidectomized (PTX), were randomly divided into vehicle and CsA groups. All PTX rats were given oral calcium supplementation ad libitum. The rats were divided into groups: basal, sham/vehicle, sham/CsA, PTX/vehicle, and PTX/CsA. Serial biochemistry was performed 0, 14, and 28 days after the start of the experimental period; bone histomorphometry was performed 28 days after the start of the experimental period. Statistical analysis consisted of group comparisons and factorial analyses. The results showed that CsA alone produced a high turnover osteopenia consistent with previous studies. In the PTX animals there was an increase in bone mass. PTX also decreased osteoblast activity and recruitment, and serum 1,25OH2D levels. Serum levels of osteocalcin (BGP) were unaffected by PTX. The combination group (PTX/CsA) did not differ statistically from the controls in most of the histomorphometric parameters measured, with the exception of reduced mineral apposition and bone formation rates, reflecting the effects of PTX. Serum BGP and 1,25OH2D levels did not differ, but PTH was reduced from the control. Explanations for these results are (1) CsA and PTX exert their effects via separate mechanisms, negating each other; (2) in the absence of PTH, CsA managed to cause bone loss, and thus PTH may not be essential for CsA-induced bone loss; or (3) the profound accelerated bone loss produced by CsA in normal rats requires PTH. These findings may help explain the discrepancies found in clinical studies where bone loss occurs with either elevated or normal PTH levels.

Cyclosporine A (CsA) is associated with posttransplantation bone disease. Immunosuppressant drugs such as sirolimus (SRL), which are more potent and less deleterious than CsA, are being developed. Previous experiments have shown that SRL although immunosuppressive, is relatively bone sparing. The use of low doses of CsA and SRL in combination has displayed in vivo synergism. This study was initiated to examine the effect of low-dose CsA and SRL on bone metabolism, thereby hopefully providing a bone sparing immunosuppressive regimen for transplant recipients. One hundred and nineteen rats were divided into groups: basal, vehicle, CsA high dose, CsA low dose, SRL low dose, and combination low-dose CsA and SRL. The basal group was killed on day 0 for histomorphometry. The experimental groups were weighed and bled on days 0, 28, 56, and 84 and were killed on day 84 for histomorphometry. Serial assays for blood urea nitrogen (BUN), creatinine, and osteocalcin were performed. Osteocalcin was raised on days 28 and 56 in the high dose CsA group. Histomorphometry showed osteopenia with high-dose CsA. Low-dose CsA was relatively bone sparing, while low-dose SRL and combined low-dose CsA did not cause bone loss. In conclusion, the synergistic combination of low-dose CsA and SRL has the potential of providing both bone sparing and immunosuppressive benefits.

Estrogen deficiency following ovariectomy or menopause results in bone loss. Although evidence strongly suggests that the immune system is involved in the pathogenesis of estrogen-deficient osteoporosis, it is not clear what role, if any, the T-lymphocyte plays in this process. Therefore, we examined the distribution of T-cell subsets in lymphoid organs and tissues, under varying estrogenic states in the rat. Six-month-old female Sprague-Dawley rats, ovariectomized (Ovx) and sham-operated, were randomized 5 d post-surgery into six groups to receive the following treatments: (A) sham/placebo; (B) sham/low-dose E2; (C) sham/high-dose E2; (D) Ovx/placebo; (E) Ovx/low-dose E2; (F) Ovx/high-dose E2. Half of the treated rats (groups A-F) were sacrificed on d 14; the remainder on d 28. Following euthanasia, mononuclear cells were isolated from the thymus, peripheral blood, spleen, lymph node and bone marrow, and were labeled for flow cytometric analysis using mouse anti-rat monoclonal antibodies directed against CD5, CD4, and CD8 antigenic markers. In the thymus, ovariectomy caused a dramatic increase and E2 treatment caused a dose-dependent decrease in weight that was proportional to the number of thymocytes. In the bone marrow, ovariectomy caused a significant reduction in the percentage of all T-cell subsets examined and this effect persisted throughout the duration of the study. Estrogen replacement therapy at the low-dose reversed the effects of ovariectomy and high-dose E2 treatment caused an increase in T-cell subsets in both the sham and Ovx groups, an effect that was more pronounced at d 14 compared with d 28. Although the percentages of some T-cell subsets in the other lymphoid organs/tissues were altered by ovariectomy or E2 treatment at d 0 and 14, all these changes had normalized by d 28 except for CD5 and CD4 cells in peripheral blood. In summary, with the exception of T-lymphocytes in the bone marrow, the effects of varying estrogenic states on T-cells were variable and transient. The influence of estrogen status on bone marrow T-lymphocytes suggests that these cells may play a role in mediating the effects of estrogen on bone turnover and warrant additional studies focusing on the functional role of T-cells in the bone marrow compartment.

Interferons (IFN) are a group of related glycoproteins. IFN-gamma, 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-alpha 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-alpha on bone mineral metabolism has been very sparse, and the majority of studies reflect in vitro models. Like IFN-gamma, there exists discordance between in vitro and in vivo studies on IFN-alpha. Both in vivo and in vitro studies demonstrate that IFN-alpha 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-alpha 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-alpha (1.6 x 10(6) IU/m2), intermediate dose IFN-alpha (5.35 x 10(6) IU/m2), and high dose IFN-alpha (30 x 10(6) IU/m2) 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-alpha did not alter any of the histomorphometric parameters measured and did not affect PTH. However, it produced a disparate BGP response. Low dose IFN-alpha resulted in a statistically significant increase in serum BGP on days 14 and 28, whereas intermediate and high doses of IFN-alpha did not. Overall, these results provide no evidence of a deleterious effect of IFN-alpha on bone metabolism and confirm the limited clinical study.

Post-renal-transplantation bone disease is a well known entity. Immunosuppressive agents and persistence of hyperparathyroidism have primarily been implicated in its etiology. Renal transplantation patients are unique in that the bone changes occur on a background of pre-existing renal osteodystrophy. This review focuses on post-renal-transplantation bone disease. Unfortunately, the existing data in the review period, besides being scanty, provide conflicting information. This is due to the diversity of immunosuppressive regimens employed, the patient populations studied, diagnostic tools and criteria used by different centers, and the lack of formal trials.

Background: Posttransplantation bone disease is a well-described phenomenon; among its etiology is immunosuppressant-induced bone disease. Mycophenolate mofetil (MMF) has emerged as a promising new immunosuppressant. Our study was designed to investigate the effect of MMF on in vivo bone mineral metabolism., Methods: Twenty-four 6-month-old male Sprague-Dawley rats were randomized into two groups to receive either MMF vehicle daily for 28 days or 30 mg/kg MMF daily for 28 days. The serum was assayed for osteocalcin and 1,25-dihydroxy vitamin D3. Subsequent to double-labeling, the right tibiae were removed on day 28 for histomorphometry., Results: MMF suppressed bone gla protein (osteocalcin) levels on days 14 and 28 (P < 0.05). Except for percentage osteoid perimeter, there was no difference in bone histomorphometry between the two groups., Conclusion: In this relatively short-term study, MMF did not cause osteopenia in the rat model, but the suppressed bone gla protein merits further study.

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 FK506's 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 wether 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 weighted 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 marignally 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.