Posttransplantation osteopenia leading to osteoporosis is being recognized as a severe complication because it is a crucial cause of spontaneous fracture and lowers the quality of life (1–3). Cyclosporine (CsA) is a clinically established immunosuppressant in the prevention of allograft rejection but unfortunately does cause osteopenia not only in experimental animals (4–7) but also in human (8–10).
FK506, a fungal macrolide produced by Streptomycestsukubaensis (11, 12), is a newly developed and more potent immunosuppressant than CsA. FK506 binds to “FK-binding protein,” and then FK506-FK-binding protein complex as well as CsA-cyclophilin complex inhibit calcineurin, a calcium-dependent phosphatase (13–16), resulting in inhibition of cytokine production including interleukin-2, interleukin-3, and γ-interferon from T lymphocytes (17–21). FK506 has been evaluated in a wide variety of experimental animal models such as heart, liver, small bowel, and kidney allograft transplantations, in which its immunosuppressive activity is represented 3–10-fold more potent than CsA (22–25). Based on these data, FK506 is currently being utilized for the management of rejection in patients after transplantation (26–28). Additionally, attempts are recently being made to use FK506 in the treatment of various autoimmune diseases including atopic dermatitis (29) and uveitis (30).
On the other hand, FK506 therapy has been shown to cause bone mineral loss as severe as that by CsA in some clinical reports (31, 32). In most cases, however, FK506 is being used in combination with steroids including prednisolone, and accordingly the effect of FK506 alone on bone mineral metabolisms could not be accurately elucidated. A recent prospective study has indicated that the bone loss by FK506 regimen after organ transplantation appeared to be less than that in CsA (33). Thus, the clinical evaluation of FK506 on bone metabolisms still remains controversial.
Our recent study has shown that FK506 has the potential to elevate plasma and hepatic levels of insulin-like growth factor (IGF)-I in rats, but CsA does not.2 IGFs are mitogenic and anabolic peptide hormones that play an important role in the growth and differentiation of tissue of mesenchymal origin in vitro and in vivo (34). IGF-I is the most potent of the IGFs and has potent osteogenetic properties on bone metabolisms (35–38). Taken together, it evoked the idea that FK506, unlike CsA, might have beneficial effects on bone metabolisms Thus, in this article, we investigated the effects of the treatment with FK506 on bone mineral metabolisms in rats in comparison with CsA.
MATERIALS AND METHODS
Animals.
Sixty 8-week-old male Sprague-Dawley rats were obtained from Charles River Japan, Inc. (Shiga, Japan). All rats were housed on a 12-hr light/dark cycle (lights on at 7:00 a.m.) with room temperature set at 22°C and maintained on a laboratory diet (Oriental Yeast, Tokyo, Japan) containing 0.91% phosphorus and 1.17% calcium, and tap water ad libitum.
Drugs.
Both FK506 and CsA were synthesized in the laboratory of Fujisawa Pharmaceutical Co. Ltd. FK506 in a 20% (w/w) solid dispersion formulation was suspended in distilled water to obtain final concentrations of 0.2 or 0.64 mg/ml. CsA was dissolved in olive oil (Nacalai tesque, Kyoto, Japan) to obtain final concentrations of 2 or 6.4 mg/ml.
Experimental protocols.
After 2-week acclimatization, and when the rats were 10 weeks old, they were weighed, divided into six groups (10 animals per group), and treated as follows; vehicle containing FK506-placebo, 1.0 mg/kg FK506, 3.2 mg/kg FK506, olive oil (CsA vehicle), 10 mg/kg CsA, and 32 mg/kg CsA. The drugs were administered by daily oral gavage to individual animals in a volume of 5 ml/kg for 28 days. All rats were weighed every 7 days. All the animal experiments in this study were approved by our institutional animal care committee.
Urine sample preparation and analysis.
All rats were placed individually in a stainless cage for 5-hr urine collection on days 0, 7, 14, 21, and 28. Urine samples were centrifuged and stored at −80°C until assayed. Urinary deoxypyridinoline was determined by enzyme-linked immunosorbent assay with a commercially available kit (Pyrilinks-D assay, Metra Biosystems Inc., Mountain View, CA). Creatinine was determined colorimetrically with a diagnostics kit (Wako Pure Chemicals, Osaka, Japan). Level of urinary deoxypyridinoline of each sample was represented as the value corrected with creatinine concentration.
Blood collection and analysis.
On day 28, 1 hr after drug administration, all rats were subjected to blood collection by drawing into heparinized glass capillary from the caudal vein. Plasma samples were obtained by centrifugation and stored at −80°C. IGF-I was determined by radioimmunoassay with a commercially available kit (Diagnostic Systems Laboratories Inc., Webster, TX). To remove IGF-I-binding proteins, each serum was extracted with acidic ethanol solution and centrifuged before applying to the assay system.
On day 29, rats were anesthetized with intraperitoneally injection of sodium pentobarbital (50 mg/kg) and killed by exsanguination via aortic puncture. Each blood was allowed to clot at room temperature. Sera were obtained by centrifugation and stored at −80°C. Total bile acid and total bilirubin were determined colorimetrically with diagnostics kits (Wako Pure Chemicals).
Measurement of bone mineral density.
When bone remodeling is impaired, the reduction in bone mineral density is detectable more clearly in the trabecular region. Measurement by peripheral quantitative computed tomography (pQCT) is available to evaluate the mineral density of trabecular bone separate from that of cortical bone (39). In a preliminary study, a distal section equal to 18% of the femur length was found to be an appropriate position for detecting the reduction in both trabecular and cortical bone mineral density. Thus, the effects of FK506 or CsA on bone mineral density were evaluated at that point of the femur by pQCT.
The right femurs were removed and preserved in 70% ethanol. Length of the isolated femurs was measured with a slide caliper and assessed by pQCT with a Stratec XCT960A (Norland, White Plains, NY). An 18% distal section described above was scanned with a voxel size of “E” (0.148 mm) and analyzed using contmode/1, peelmode/20, cortmode/1 for the calculation of trabecular, cortical, and total bone mineral densities.
Statistical methods.
Data in this article are represented as mean±SEM. Statistical evaluations of the effects of FK506 or CsA on the various parameters measured were made by one-way analysis of variance with Dunnett t test. Significance was ascribed at P ≦0.05.
RESULTS
Changes in body weight.
Body weight was increased in all rats during the treatment period of 28 days, although FK506 did not affect body weight gain whereas CsA-treated rats tended to gain less weight, and there were statistically significant differences between 32 mg/kg CsA-treated groups and its vehicle (Fig. 1).
;)
Figure 1: Time course of body weight. Rats were treated with vehicle (•), 1.0 mg/kg FK506 (▴), and 3.2 mg/kg FK506 (▪) (a) or with vehicle (○), 10 mg/kg CsA (▵), and 32 mg/kg CsA (squlo]) (b) by daily oral gavage and weighed every 7 days. Each point represents mean±SEM of 10 rats. ##, P <0.01 compared with CsA-vehicle group (Dunnett test).
Effects of FK506 and CsA on bone mineral density.
To investigate whether FK506 induces bone loss in rats as well as CsA, bone mineral density of the femur was measured by pQCT. The results are shown in Figure 2. The trabecular bone mineral densities in the FK506-treated groups were 90% and 81% of the value observed in its vehicle group at doses of 1 and 3.2 mg/kg, respectively, but these changes were not significant. The trabecular bone mineral densities in the CsA-treated groups were significantly decreased to 61% and 52% of the value observed in its vehicle at doses of 10 and 32 mg/kg, respectively. Similarly, treatment with FK506 slightly decreased both cortical and total bone mineral densities, whereas CsA caused a significant reduction in these parameters. There were no differences in femur length between all groups (data not shown).
Figure 2: Effects of FK506 (a, c, and e) and CsA (b, d, and f) on trabecular (a and b), cortical (c and d), and total (e and f) bone mineral density of femur in the rat. Bone mineral density was measured by pQCT. Each column represents mean±SEM of 10 femurs. ##, P <0.01 compared with CsA-vehicle group (Dunnett test).
Effects of FK506 and CsA on levels of plasma IGF-I.
To elucidate the effects of FK506 and CsA on induction of IGF-I, levels of plasma IGF-I on day 28 were measured. FK506 induced significant increases in plasma IGF-I level by 24% and 25% at doses of 1.0 and 3.2 mg/kg, respectively, over its vehicle, whereas CsA had no effect on plasma IGF-I (Fig. 3).
Figure 3: Effects of FK506 (a) and CsA (b) on plasma levels of IGF-I in the rat. IGF-I was determined by radioimmunoassay. Each column represents mean±SEM of 10 samples. *, P <0.05 compared with FK506-vehicle group (Dunnett test).
Effects of FK506 and CsA on levels of serum bile acid and bilirubin.
To investigate the effects of FK506 and CsA on hepatic function, serum levels of bile acid and bilirubin were measured. Data are shown in Table 1. Treatment with FK506 caused a slight decrease in both parameters, whereas CsA significantly elevated serum level of bile acid and slightly elevated that of bilirubin over its vehicle group.
Table 1: Serum levels of biochemical parameters after 28-day treatment with FK506 or CsAa
Effects of FK506 and CsA on levels of urinary deoxypyridinoline excretion.
To investigate the effects of FK506 and CsA on the activity of bone resorption, levels of urinary deoxypyridinoline were measured. Figure 4 represents urinary deoxypyridinoline levels corrected with creatinine concentration. There were no differences between the FK506-treated groups including its vehicle at either point of measurement. By contrast, urinary deoxypyridinoline levels were elevated in the CsA-treated groups, and especially at a dose of 32 mg/kg a significant elevation was observed by 50% and 95% compared with initial value and vehicle, respectively, at day 14.
Figure 4: Effects of FK506 (a) and CsA (b) on urinary excretion of deoxypyridinoline in the rat. Rats were treated with vehicle (•), 1.0 mg/kg FK506 (▴), and 3.2 mg/kg FK506 (▪) (a) or with vehicle (○), 10 mg/kg CsA (▵), and 32 mg/kg CsA (□) (b). Urine samples were collected every 7 days. Urinary deoxypyridinoline was measured by enzyme-linked immunosorbent assay. Each point represents mean±SEM of 10 samples. ##, P <0.01 compared with CsA-vehicle group (Dunnett test).
DISCUSSION
Posttransplantation osteopenia is one of complications caused by immunosuppressants including CsA (1–3, 8–10). This leads to spontaneous fracturing and immobility, and thus contributes to the lowering of the quality of life. Currently, FK506, as well as CsA, is being used worldwide for the prevention of rejection in most orthotopic transplantations, and has been reported to have less adverse effects than CsA (26). However, the clinical evaluation of FK506 on bone metabolisms remains controversial. Thus, in this study, to investigate whether FK506 could cause severe osteopenia similar to CsA or not, we measured the bone mineral density of femur in rats treated with FK506 and compared with that of CsA.
Interestingly, FK506 slightly, but not significantly, reduced the bone mineral density of rat femur, whereas CsA markedly and significantly reduced it. FK506 did not affect the excretion of urinary deoxypyridinoline, a marker of bone resorption, whereas CsA significantly increased it. These results clearly demonstrate that, in comparison with CsA, FK506 did not cause severe bone loss. The effects of CsA on bone metabolism observed in this study were in good accordance with the previous findings that CsA caused severe bone loss characterized by high-turnover bone metabolism in rats (4–7). On the other hand, the effects of FK506 on bone metabolism we described here were not concomitant with the previous findings mainly by Epstain and colleagues (40, 41), in which FK506 has been shown to cause severe bone loss as well as CsA in rats. Although they evaluated the effect of FK506 by histomorphometry instead of densitometry, this difference of maneuvers should not be discussed because trabecular bone mineral density by densitometry with a pQCT device has been reported to reflect trabecular bone volume by histomorphometry (39). Rather, the dose of FK506 in their studies should be taken into account. The 5 mg/kg dose of FK506 used in their studies seemed to be a slight overdose because 1.0 or 3.2 mg/kg has been described as the effective dose on FK506 allograft transplantation in rats (23, 24). As an excess dose might blunt the essential effects of FK506 on bone metabolism, we chose 1.0 and 3.2 mg/kg for the doses in this study. As shown in Figure 2, FK506 at the doses of 1.0 and 3.2 mg/kg did not cause a severe reduction in bone mineral density in rats without influencing bone resorption in our study. This finding is likely to accord with a recent prospective study in which the bone loss in FK506 regimen after organ transplantation appeared to be less than that in CsA (33). As the reduction in bone mineral density is known to be associated with the increase in bone fragility, the bone mineral density can be regarded as a significant parameter in predicting spontaneous fracture. Spontaneous fracturing caused by osteopenia in patients receiving immunosuppressants including CsA is an important clinical problem (8–10). Thus, from this finding that FK506 caused less reduction in bone mineral density compared with CsA in rats, it can be expected that the FK506-treated patients might show a lower incidence of fracturing compared with CsA.
CsA has been reported to change the bone mineral metabolism into high-turnover “uncoupling” state and to cause bone loss dose-dependently in the rat (9). It has also been reported the bone loss caused by CsA is mediated by T lymphocytes because it was not observed in the T lymphocyte-deficient Rawett athymic nude rat (42), suggesting the possible relationship between bone loss and T lymphocyte-mediated immunosuppression by CsA. Inasmuch as FK506 as well as CsA exert their immunosuppressive activities by the suppression of T lymphocytes, it is expected that the drug might also reduce bone mineral density essentially. As shown in Figure 2, however, FK506 did not cause a severe bone loss in rats. Therefore, mechanisms apart from the T lymphocyte-related one appear to be involved in lesser bone loss by FK506. Recently, we have found that FK506 stimulated an increase in both plasma and hepatic IGF-I in rats after 1-week oral treatment. IGF-I in vitro activates osteogenetic functions such as increase in the replication of osteoblastic lineage, enhancement of osteoblastic collagen synthesis, and decrease in collagen degradation (34–36), and in vivo ameliorates bone loss in the ovariectomized rat (37) and the unloaded rat (38). Therefore, in this study, plasma levels of IGF-I were measured, and FK506 was confirmed to elevate the plasma levels of IGF-I, whereas CsA did not. Although the plasma level of IGF-I in the FK506-vehicle group was lower than that in the CsA-vehicle group, which could not be explained clearly, there was no significant difference in bone mineral densities between the two groups. Moreover there were significant reductions in bone mineral density in the rats treated with CsA despite the fact that plasma levels of IGF-I were sustained. Hence, although the achieved levels of IGF-I by FK506 were comparable to those with CsA, it was confirmed that IGF-I induced by FK506 exerted some effectiveness on bone mineral density. Additionally, the serum level of bile acid was reduced in the FK506-treated groups, suggesting that plasma IGF-I induced by FK506 had physiological significance. Therefore, little reduction in the bone mineral density by FK506 may be in part accounted for endogenously elevated IGF-I. Indeed, the mechanisms for the osteogenic activity of FK506 independent of the IGF-I mediated pathway can not be excluded. Recently, Akahane et al. (43) reported that FK506 could promote osteoblastic differentiation of bone marrow stromal cells.
In the present study, we evaluated the effect of FK506 on bone mineral density using normal rats. However, further investigations in animals with bone disorder are required to clarify whether FK506 causes less bone loss than CsA, considering that both drugs are being clinically used for the prevention of allograft rejection. For example, the ovariectomized rat may be available to achieve this purpose, inasmuch as, in this animal model, rapid osteopenia is observed similarly to that in patients after transplantation and CsA has been reported to cause bone loss additively (44–46). Rats with liver cirrhosis may be also useful, as it has been reported that bone loss observed in rats with CCl4-induced liver cirrhosis was partially improved by treatment with IGF-I (47). Patients with liver cirrhosis, who can be objects of liver transplantation, have lower levels of IGF-I (48) and suffer from osteoporosis in significantly higher incidence compared with age-matched normal subjects (49, 50). Thus, rats with ovariectomy or with liver cirrhosis are considered to be useful animal models for evaluating the effect of FK506 on bone metabolism and further clarifying its clinical usefulness.
In conclusion, FK506 caused lesser bone loss than CsA in the rat, which might be in part a result of the induction of IGF-I. The results suggest that FK506 may exert favorable effects on bone metabolism in patients with organ transplantation compared with CsA. To assess this idea, further clinical investigations focused on bone metabolism will be required.
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