The Amino Bisphosphonate Ibandronate Prevents Calciphylaxis in the Rat at Doses that Inhibit Bone Resorption

The present experiments were carried out to test the hypothesis that there could be a common underlying biochemical mechanism that accounts for the different kinds of soft tissue calcification observed in animals that are treated with toxic doses of vitamin D. In previous studies we showed that lethal doses of vitamin D cause extensive calcification of arteries, lungs, kidneys, and cartilage, and that doses of ibandronate that inhibit bone resorption completely inhibit each of these soft tissue calcifications and prevent death [1, 2]. In the present experiments we have examined the effect of ibandronate on an entirely different type of calcification, the calciphylaxis induced by administration of a challenger to rats previously treated with sub-lethal doses of vitamin D.

The calciphylaxis response in the rat requires two treatments, an initial treatment with a sensitizer, such as a high dose of vitamin D or PTH, followed after an interval of a day or more by treatment with a calciphylactic challenger [3]. In the present study we have employed the same 3-day vitamin D injection schedule used in the prior ibandronate studies [1, 2], but have reduced the dosage of vitamin D to nonlethal levels. Two completely different kinds of calciphylactic challengers were used, subcutaneous injection of FeCl₃ and intrascapular epilation, and both challengers were administered 24 hours after the last of the three vitamin D doses. In previous studies of these calciphylactic responses in rats sensitized by prior treatment with toxic doses of vitamin D, calcification induced by epilation (hair plucking) started in the fibrous tissue surrounding the hair follicles and subsequently spread throughout the dermis, and calcification induced by FeCl₃ was accompanied by an inflammatory response in the ventral fascia and culminated in the formation of a highly calcified tissue button [3, 4].

Vitamin D₃ (cholecalciferol) was purchased from ICN (Costa Mesa, CA) and Ibandronate (Bondronat, Boehringer Mannheim) was purchased from Idis World Medicines (Surrey, UK). Ibandronate was diluted with 0.15 mol/l NaCl and stored at 4°C. Stock solutions of vitamin D were prepared fresh for each 3 day subcutaneous injection cycle at a concentration of 4.3 mmol/l in 7% emulphor (alkamuls EL-620, Rodia, Inc.) and then placed in foil-wrapped containers and stored at 4°C, as described previously [5]. Simonsen albino rats (Sprague-Dawley derived) were purchased from Simonsen labs (Gilroy, Ca).

Male Sprague Dawley rats were fed ad libitum with rodent diet 5001 (Purina Mills Inc., St. Louis, MO), a diet that is 0.67% phosphorus and 0.95% calcium by weight. In all experiments, rats were killed by exsanguination while under ether anesthetic. In the calciphylaxis experiments, 7-week-old male rats were first sensitized by 3 subcutaneous injections of 400,000 IU of vitamin D₃/kg body weight made at t = 0, 24, and 48 hours. Calciphylaxis was then initiated at t = 72 hours either by subcutaneous injection with 300 mg of FeCl₃ at each of two ventral sites in the thorax (see Fig. 1) or by mechanically removing all of the hair in a 9 cm² area of skin in the intrascapular region of the anesthesized rat (epilation; see Fig. 4). To assess the effect of ibandronate on ectopic calcifications, a subset of the rats also received subcutaneous injections of ibandronate at a dose of 0.25 mg/kg/day beginning 4 days prior to the first vitamin D injection and continuing until the animals were killed. Animals were killed by exsanguination 7 days after administration of the calciphylactic challenger and the appropriate tissues were removed within 30 min of death and either immediately frozen at −20°C until chemical analysis or fixed in formalin. The effect of vitamin D dose on the level of calcification in the aorta and at the site of calciphylactic challenge was determined in rats that received subcutaneous injections of 100,000, 200,000 or 300,000 IU of vitamin D₃/kg body weight at t = 0, 24, and 48 hours and of 300 mg of FeCl₃ at t = 72 hours. The UCSD Animal Subjects Committee approved all animal experiments.

For measurement of mineral accumulation in the aorta, the abdominal aorta section beginning 1 cm above the renal branch and ending at the femoral bifurcation was demineralized in 150 mmol/l HCl overnight at room temperature. For analysis of mineral accumulation at sites of calciphylaxis, the well-defined tissue button that formed at the subcutaneous sites of FeCl₃ injection (see Fig. 1) and the region of skin that underwent epilation (see Fig. 4) were removed and demineralized in 10% formic acid. Calcium levels in serum and in the acid extract of tissues were determined colorimetrically using cresolphthalein complexone (Sigma) and phosphate levels in serum and in the acid extract of tissues were determined colorimetrically as described [6]. Serum samples were analyzed to determine the level of cross-linked N-teleopeptides (OSTEOMARK NTx) by Ostex, Inc. (Seattle, WA) using a specific ELISA assay [7].

For histological analysis of mineral accumulation, the appropriate tissues were fixed in formalin for at least 24 hours at room temperature. Sectioning and histological staining (Hematoxylin & Eosin and von Kossa) of formalin-fixed tissues were carried out by San Diego Pathology (San Diego, Ca). Alizarin red staining of formalin-fixed tissues was carried out as described [8, 9]. Photographs of freshly dissected and Alizarin red-stained tissues were made by the OLR Photo Lab at UCSD.

All data are presented as means ± standard deviation. Differences between groups were analyzed by the Student's t-test. Differences of P < 0.05 were accepted as significant.

The first experiments were carried out to determine the effects of ibandronate on the subcutaneous calciphylaxis induced by the injection of the FeCl₃ challenger into rats sensitized by the prior injection of vitamin D. As seen in Figure 1, the calciphylactic response at the two sites of FeCl₃ injection forms a whitish button about 0.8 cm in diameter that is intensely stained for calcification with Alizarin red. This calciphylactic response is identical to that previously reported for calciphylaxis produced by FeCl₃ injection into rats sensitized by prior vitamin D administration [3]. In agreement with these earlier studies, no calciphylactic button was found at the site of FeCl₃ injection in animals that had not been sensitized by prior vitamin D treatment (data not shown).

The effect of ibandronate on FeCl₃-induced calciphylaxis was examined using the ibandronate dose and injection schedule previously shown to completely inhibit artery calcification in rats treated with vitamin D [1, 2]. As seen in Figure 1, ibandronate treatment markedly reduced the size of the button formed at the site of FeCl₃ injection and largely eliminated the Alizarin red staining of the button. Figure 2 shows that the buttons contained high levels of calcium and phosphate and that ibandronate treatment dramatically lowered the concentration of both mineral components. Arteries were removed from each experimental animal at the end of the calciphylaxis experiment and stained for calcification with Alizarin red in order to confirm the effects of vitamin D and ibandronate on artery calcification. As seen in Figure 3, vitamin D treatment caused extensive calcification of the artery similar to that reported previously, and ibandronate treatment completely inhibited this effect.

Previous studies have shown that the timing of the calciphylactic challenge with agents such as FeCl₃ has a critical bearing on the strength of the calcification response [3]. Additional measurements were therefore undertaken to establish serum chemistry at the time of FeCl₃ injection, which was 72 hours after the first vitamin D injection. Table 1 shows that treatment with vitamin D alone increased serum levels of cross-linked N-teleopeptides, a marker for bone resorption activity, by 82%. Serum levels of cross-linked N-teleopeptides were slightly below control values in the rats treated with vitamin D plus ibandronate, which shows that ibandronate treatment completely inhibited the increased level of bone resorption activity produced by vitamin D treatment. Serum calcium levels were elevated by 30% in all animals treated with vitamin D, but there was no significant difference between the elevated serum calcium levels seen in animals treated with vitamin D alone and those with vitamin D plus ibandronate (P > 0.15) (Table 1).

The second experiments were carried out to determine the effects of ibandronate on the skin calciphylaxis induced by epilation in rats sensitized by the prior injection of vitamin D. The vitamin D injection schedule used to sensitize rats for the calciphylaxis response in these experiments is identical to the injection schedule used in the FeCl₃-induced calciphylaxis studies above, and to that used in previous studies to induce artery calcification [2, 10]. The timing of calciphylactic challenge by epilation was again 24 hours after the last vitamin D injection, and the epilation was achieved by mechanically plucking the hair from a 9 cm² area of skin above the shoulders of the anesthetized rat. As seen in Figure 4, the calciphylactic response at the site of the epilation challenge is seen as raised areas of skin discoloration that are intensely stained for calcification with Alizarin red. These raised areas of skin are palpably hard. This calciphylactic response to epilation is identical to that previously reported in rats sensitized by prior vitamin D administration [3, 4]. In agreement with these earlier studies, no skin calcification was found at the site of epilation in animals that had not been sensitized by prior vitamin D treatment, and no skin calcification was seen in animals treated with vitamin D at skin sites where hair had not been removed (data not shown).

The effect of ibandronate on epilation-induced skin calciphylaxis was examined with the ibandronate dose and injection schedule used in the FeCl₃-induced calciphylaxis experiments and in previous studies on the effect of ibandronate on vitamin D-induced artery calcification. As seen in Figure 4, ibandronate treatment eliminated all visible signs of calciphylaxis at the site of epilation, and completely eliminated the Alizarin red staining of the skin. Figure 5 shows that skin at the epilation site contained high levels of calcium and phosphate and that ibandronate treatment reduced the level of each mineral component to that seen in rats that were not subject to epilation. The level of calcium and phosphate in the skin from untreated control rats was not significantly different from that seen in vitamin D-treated rats that were not subjected to epilation (data not shown).

Additional experiments were carried out to determine whether the dose of vitamin D needed to sensitize rats for calciphylaxis is comparable to the dose needed to induce artery calcification and to increase bone resorption activity. Animals were first treated with different vitamin D doses according to the same 3-day injection protocol used in the above experiments, and the calciphylaxis response to the FeCl₃ challenge and the level of artery calcification were evaluated in each animal. As can be seen in Table 2, the 100,000 IU/kg dose of vitamin D produced barely detectable calcification in the artery and at the FeCl₃ calciphylaxis site and no significant increase in bone resorption activity, whereas higher doses of vitamin D dramatically increased bone resorption activity and calcification at both sites. Serum calcium was elevated at all vitamin D doses and did not correlate with bone resorption activity or with the extent of either type of ectopic calcification (Table 2). The dissociation of serum calcium and bone resorption activity is further supported by the observation that serum calcium levels remained elevated at 10 days after the first vitamin D injection, whereas the serum NTx measure of bone resorption activity had returned to normal by this time (data not shown).

The present studies show that the calcification of arteries and calcification at two different kinds of calciphylaxis sites are all comparably inhibited by doses of the amino bisphosphonate ibandronate that inhibit bone resorption, and that calcification at the calciphylactic site and in the artery have a similar dependence on vitamin D dose. These observations strongly support the hypothesis that there is a common underlying biochemical mechanism in the vitamin D-treated rat that is responsible for the calcification of arteries and other soft tissues as well as the calcification at calciphylaxis sites.

We speculate that the underlying biochemical mechanism that is responsible for the diverse array of soft tissue calcifications and calciphylactic responses in rats treated with toxic doses of vitamin D arises from bone resorption activity. This hypothesis is supported by the observation that doses of vitamin D that promote these types of ectopic calcification also potently stimulate bone resorption activity (Tables 1 and 2), and by the observation that treatment with the bone resorption inhibitor ibandronate normalizes bone resorption activity and prevents all vitamin D-induced calcifications [1, 2]. This hypothesis is also consistent with the finding that vitamin D-induced calcification of arteries, lungs, kidneys, tracheal cartilage, stomach, and intestine are each prevented by the inhibition of bone resorption using the cytokine osteoprotegerin [10].

The nature of the biochemical mechanism that is responsible for the putative linkage between bone resorption and soft tissue calcification in the vitamin D-treated rat is presently unclear. One possibility is that soft tissue calcification could be a direct physicochemical consequence of the effect of the observed vitamin D-induced hypercalcemia on the nucleation and growth of calcium phosphate mineral in soft tissues. This hypothesis is not, however, supported by the observations that serum calcium levels in animals treated with vitamin D plus ibandronate are not significantly different from those seen in animals treated with vitamin D alone in this (Table 1) or in previous experiments [1, 2]. This hypothesis is also not consistent with the present observation that increasing the vitamin D dose from 200,000 IU to 300,000 IU/kg caused a 2.5-fold increase in the accumulation of calcium and phosphate in the abdominal aorta but did not further increase the level of hypercalcemia (Table 2). Another possibility is that soft tissue calcification is promoted by crystal nuclei generated at sites of bone resorption which travel in blood and occasionally lodge in soft tissue structures [1]. This hypothesis is supported by the observation that, under some circumstances, a complex of a calcium phosphate mineral phase and the proteins fetuin and matrix Gla protein is released from bone and can be detected in blood [11], and by the observation that the release of this complex from bone is inhibited by inhibitors of bone resorption [12].

The term calciphylaxis has also been used to refer to the syndrome of ischemic ulceration of skin due to metastatic calcification of subcutaneous tissue and small arteries that occurs in uremic patients [13, 14, 15]. While there is as yet no biochemical evidence that the ectopic calcifications seen in uremic patients are caused by the same basic biochemical mechanisms that appear to be responsible for the diverse types of soft tissue calcification seen in rats treated with toxic doses of vitamin D, there are similarities that should be noted. Uremic calciphylaxis in human subjects and vitamin D-induced calciphylaxis in the rat are both characterized by massive calcification of the elastic lamellae in the media of arteries. Rats treated with toxic doses of vitamin D and human patients with end-stage renal disease are also both prone to extra-vascular calcification at a number of ectopic sites, suggesting that both could involve a systemic increase in the tendency of soft tissues to calcify. Finally, early studies by Selye [3] showed that uremia acts as a calciphylactic sensitizer in the rat with an activity similar to that of exogenous vitamin D or PTH, and that parathyroidectomy abolishes this ability of uremia to promote calciphylaxis [3]. We speculate that the calcification of arteries and other soft tissues found in uremic patients is linked to the increase in bone resorption that arises as a consequence of the secondary hyperparathyroidism often found in these patients. This observation is supported by the observation that uremic calciphylaxis is, in some patients, ameliorated by parathyroidectomy [16, 17]. An important future test of the hypothesis that soft tissue calcification is linked to bone resorption in the uremic patient will be to determine whether inhibitors of bone resorption will prevent soft tissue calcification induced by uremia.