Time-evolution and reversibility of strontium-induced osteomalacia in chronic renal failure rats

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1 Kidney Interntionl, Vol. 7 (25), pp Time-evolution nd reversiility of strontium-induced osteomlci in chronic renl filure rts LINEOSTE, AN R. BERVOETS, GEERT J. BEHETS, GEERT DAMS, RITAL. MARIJNISSEN, HILDE GERYL, LUDWIG V. LAMBERTS, STEVEN C. VERBERCKMOES, VIVIANE O. VAN HOOF, MARC E. DE BROE, nd PATRICK C. D HAESE Deprtment of Nephrology-Hypertension, University of Antwerp, Antwerp, Belgium; Deprtment of Biochemistry, Antwerp University Hospitl, Antwerp, Belgium Time-evolution nd reversiility of strontium-induced osteomlci in chronic renl filure rts. Bckground. Ptients with impired renl function cn ccumulte strontium in the one, which hs een ssocited with the development of osteomlci. A cusl role for strontium in the development of the disese ws presented in chronic renl filure (CRF) rts. Strontium-rnelte hs een put forwrd s therpeutic gent in the tretment of osteoporosis. Since the trget popultion for strontium tretment consists minly in postmenopusl osteoporotic women, who my hve reduced renl function, the risk for osteomlci should e considered. Methods. To determine the time evolution nd reversiility of the strontium-induced minerliztion defect, CRF rts were loded with strontium (2 g/l) (± 2 mg/kg/dy) during 2,, nd 12 weeks, followed y wshout period of, 2, 4, or 8 weeks. Results. Histologic exmintion of the one of the nimls treted with strontium reveled signs of osteomlci lredy fter 2 weeks. Animls tht received strontium during nd 12 weeks hd significntly higher osteoid perimeter, re nd thickness s compred to CRF controls. After 12 weeks, the minerliztion ws significntly ffected, s evidenced y lower doule-leled surfce, minerl pposition nd one formtion rte in comintion with n incresed osteoid mturtion time nd minerliztion lg time. The osteolst perimeter ws significntly lower in the strontium-treted nimls. After the wshout periods, these effects were reversed nd the one lesions evolved to the vlues of CRF controls. This went long with n 18% reduction of the one strontium content. A significnt rise in serum lkline phosphtse (ALP) ctivity ws pprent in the strontium-treted nimls s compred to CRF controls. This ws not only due to higher levels of the one ALP ut lso to those of the liver nd the intestinl isoenzymes. Serum prthyroid hormone (PTH) levels decresed during strontium tretment. After cesstion of the tretment, the serum ALP ctivity nd PTH concentrtion reversed to control levels. Keywords: osteomlci, strontium, remnnt kidney, one minerliztion. Received for puliction June 25, 24 nd in revised form August 24, 24 Accepted for puliction Septemer 22, 24 C 25 y the Interntionl Society of Nephrology Conclusion. In this study evidence is provided for the rpid development of minerliztion defect in strontium-loded CRF rts, ccompnied y reduced osteolst numer, reduced PTH synthesis or secretion, nd incresed serum ALP levels. These effects cn e rpidly reversed fter withdrwl of the compound. Experimentl studies in nimls with norml renl function hve demonstrted strontium to exert eneficil s well s dverse effects on one, depending on the dose dministered [1]. Dt in literture indicte tht high dietry intke of strontium my distur one minerliztion nd induce one normlities [2 9]. In rts with intct renl function, the development of rickets ws induced fter dding the element to the nimls drinking wter t concentrtion of 4 g/l (± 4 mg/kg/dy, s extrpolted from the weight nd dily fluid intke of dult rts) [3, 1]. In contrst, there is experimentl evidence tht strontium dministrtion t low doses reduces one resorption nd increses one formtion, resulting in incresed one mss in norml nimls. Doses lower thn 4 g/l of orl stle strontium hve een shown to stimulte one formtion without ltering one resorption in the rt with norml renl function. This oservtion ws found to e due to strontium-induced stimultion of the osteolstic ctivity evidenced y n increse in the mount of osteoid tht ws oserved in the sence of ny minerliztion defect or ny chnge in osteoclstic surfce or numer [3, 1 13]. Strontium, s the rnelte compound, hs een shown to ct s n uncoupling gent therey incresing one formtion nd reducing one resorption, leding to gin of one mss nd improving one mechnicl properties in ovriectomized rts (77 to 38 mg/kg/dy during dys) [12], immoilized rts (8 mg/kg/dy during 2 weeks), mice (2 to 18 mg/kg/dy during 14 weeks) [14] nd monkeys (75 mg/kg/dy during months) [15]. In view of these findings, strontium-rnelte hs een proposed s 92

2 Oste et l: Development nd reversiility of strontium-induced osteomlci 921 potentil gent in the tretment of one diseses tht re chrcterized y desequilirium of the one remodeling process, such s osteoporosis [1 21]. In recent phse 3 clinicl tril, 3 yers of strontium-rnelte tretment (2 g/dy) (± 3 mg/kg/dy) hs een shown to produce n erly nd sustined reduction in the risk of verterl frctures in women with postmenopusl osteoporosis [22, 23]. However, s the ove studies were performed in nimls nd humns with norml renl function nd ecuse the min route of elimintion of strontium is the kidney [24], the risk for side effects due to n incresed ccumultion of the element in one should e considered in elderly people, who lredy hve modertely decresed renl function nd constitute the trget popultion for the tretment of osteoporosis nd osteorthritis. In dilysis ptients, we previously showed n ssocition etween incresed one strontium levels nd the presence of osteomlci [25 27]. Evidence for cusl role of the element in the development of the disese ws presented in chronic renl filure (CRF) rts [28, 29]. Indeed, orl dministrtion of strontium to CRF rts over 12-week period y dding the element to the drinking wter t concentrtion of 3.4 g/l (± 34 mg/kg/dy), resulted in the development of distinct osteomlcic lesions in contrst to the nimls tht hd not received the compound. These effects ppered to e dose-dependent. Animls tht were given strontium in their drinking wter t the lowest dose (.3 g/l) (± 3 mg/kg/dy) exhiited chrcteristics of low turn-over osteodystrophy (dynmic one disese) chrcterized y the sence of osteolsts, drmticlly reduced one formtion rte nd decresed mount of osteoid tissue. Rts tht received.75 g/l (± 75 mg/kg/dy) dose showed no significnt normlities. Rts receiving the highest strontium dose (1.5 g/l) (15 mg/kg/dy), however, developed minerliztion defect, reflecting the histologic fetures of osteomlci [29]. This multiphsic effect of strontium on one could e reproduced in n in vitro setup of primry rt osteolsts. Here, it ws lso shown tht strontium t low concentrtions impirs the in vitro differentition of osteoprogenitor cells towrd mture osteolsts nd disturs minerliztion t high concentrtions [3]. The moderte renl filure tht ws induced in these rts using the remnnt kidney procedure is comprle with the remining renl function of some elderly people. Dt in literture indicte tht in these ptients the therpeutic dministrtion of the compound t reltively high doses (up to 2 g/dy s proposed in clinicl trils) my led to distinct ccumultion of the element in one up to vlues tht re in the sme order of mgnitude s those seen in uremic rts of our previous studies nd plsm levels (up to >1 mg/l [23]) tht could clerly e ssocited with the development of osteomlci in CRF rts. To further elucidte the mechnism underlying the strontium-induced effect on one, we studied the tempo- Arrivl nd conditioning of mle Wistr rts, 8 weeks old Instlltion of CRF (5/ nephrectomy Week Numer of rts Sr loding 2 g/l in drinking wter Wsh out (WO) Norml feeding Strontium groups groups Fig. 1. Time scheme of the experimentl study setup. CRF is chronic renl filure; WO is wshout. rl development of the minerliztion defect nd investigted the reversiility of the one lesion fter cesstion of the strontium loding. METHODS Experimentl design CRF ws induced in mle Wistr rts weighing 15 to 2 g y pplying the remnnt kidney model (5/ nephrectomy) s previously descried [28]. Two weeks fter initition of the renl filure, oth control nimls (N = 19), receiving no strontium nd study nimls receiving 2 g/l (± 2 mg/kg/dy) strontium vi the drinking wter (N = ) were divided into different tretment groups s shown in Figure 1. nd strontium-loded nimls hd free ccess to food nd wter. Wter s well s food consumption were recorded every 2 weeks. Blood nd urine smples were tken every 2 weeks. For 24-hour urine collection the nimls were housed individully in metolic cges. Blood ws tken from the til vein during the study period nd y ortic puncture t scrifice. Serum nd urine smples were frozen t 8 C until nlysis. At 7 nd 3 dys efore scrifice, rts were leled with tetrcycline (3 mg/kg) nd demeclocycline (25 mg/kg), respectively. At scrifice, of ech rt, one tii ws removed for histomorphometric nlysis nd one femur ws used for the determintion of strontium nd clcium. Biochemicl nd chemicl nlyses Cretinine, clcium, nd phosphorus in the serum were mesured using Vitros 95 AT Autonlyser System

3 922 Oste et l: Development nd reversiility of strontium-induced osteomlci (Ortho Clinicl Dignostics, Johnson & Johnson, Rritn, NJ, USA). Cretinine in urine ws determined ccording to the Jffé method, while the method of Brdford ws used to mesure proteinuri nd urinry phosphorus concentrtion ws determined using photometric method (inorgnic phosphte kit) (Merck, Drmstdt, Germny). Intct prthyroid hormone (ipth) ws determined using rt PTH immunordiometric ssy (IRMA) kit (Immutopics, Inc., Sn Clemente, CA, USA). Alkline phosphtse (ALP) ctivity ws quntified spectrophotometriclly y mesurement of the relese of p-nitrofenol from p-nitrophenylphosphte, reflected y the chnge in sornce t 45 nm over 5-minute period. This procedure is sed on the kinetic method for mesuring totl ALP cknowledged y the Interntionl Federtion of Clinicl Chemistry (IFCC) [31]. The ALP isoenzymes were mesured using n optimized grose gel electrophoretic method, shown to hve high ccurcy, precision, nd reproduciility [32]. This method consists of n electrophoresis on commercilly ville grose gel (Beckmn, Anlis, Nmur, Belgium). The serum smples were pretreted with phospholipse C for etter seprtion of the liver nd one ALP isoenzyme. Visuliztion of the frctions is done y incution of the grose gel with 5-romo-4-chloro- 3-indolyl phosphte (BCIP) nd quntifiction is performed y scnning the gel in computerized densitometer (Apprise, Beckmn Instruments, Bre, CA, USA). Strontium in serum, urine, nd one ws nlyzed using Zeemn 33 tomic sorption spectrometer equipped with n HGA- grphite furnce, n AS- utosmpler, nd n Andex DP-95B silent scrie printer, ll from Perkin-Elmer (Norwlk, CT, USA). To determine strontium in serum, smples were diluted fourfold in solution contining.5 ml/l Triton X-1 nd 1 ml/l HNO 3 solution, wheres urine smples were diluted 1:2 in2ml/l HNO 3 solution. Strontium in one ws ssessed fter digestion with concentrted HNO 3 [33]. Clcium determintions in urine nd one were done y flme-aas (model 311) (Perkin-Elmer) fter diluting the smples 1:5 in reverse osmosis wter to which 1 g/l lnthnum nitrte ws dded to void chemicl nd ioniztion interferences. Bone histology At scrifice, left femur nd tii were freed from soft tissue. From ech rt, the femur ws weighed immeditely fter removl of skin nd muscle tissue nd stored t +4 C until chemicl nlysis. The tii ws used for histologic exmintion fter 24 hours fixtion in Burkhrdt solution nd susequent trnsfer to 7% ethnol solution until further processing. Bone smples were then emedded in 1% methylmetcrylte. Longitudinl sections (5 lm) of the proximl tii were cut with Jung K microtome nd stined ccording to Goldner for descriptive histology nd unstined 1 lm sections were used for visuliztion of the tetrcycline lels. Bone histologic dt, s well s dynmic prmeters, re reported using stndrdized nomenclture nd definitions [34]. The prmeters mesured include (1) totl one re (%), the re of treculr one, including oth minerlized one nd osteoid, expressed s percentge of the totl one tissue re; (2) osteoid re (%), the mesured re of osteoid expressed s percentge of the totl one re; (3) osteoid width (lm), the men width of surfce osteoid sems, clculted y dividing the mesured osteoid re y the length of the osteoid sems; (4) osteoid perimeter (%), the treculr one perimeter occupied y osteoid s percentge of the totl one perimeter; (5) eroded perimeter (%), the percentge of treculr one perimeter with sclloped one resorptive lcune; () doule-leled perimeter (%), the percentge of totl treculr surfce exhiiting doule fluorescent tetrcycline lel; (7) minerl pposition rte (lm/dy), the rte y which one is minerlized, clculted s the verge distnce etween the midpoints of two consecutive tetrcycline lels, divided y the time intervl etween the leling periods; (8) minerliztion lg time (dys), men time intervl etween deposition nd minerliztion of ny infinitesiml volume of mtrix, verged over the entire life spn of the osteoid sem; (9) one formtion rte (lm 2 /mm 2 /dy), the volume of one formed per unit of time, clculted s the product of minerl pposition rte nd minerlising surfce; nd (1) osteolst perimeter (%), percentge of osteoid perimeter covered y cuoidl (ctive) osteolsts. Sttistics Sttisticl nlysis of the dt ws done using the Kruskl-Wllis test nd the Mnn-Whitney test for unpired comprisons, followed y Bonferroni correction when more thn two groups were compred. Dt re expressed s men ± SEM. A P vlue <.5 ws considered to e significnt t two-tiled level. RESULTS Biochemicl nd chemicl dt Dily food nd wter consumption were comprle etween the study groups. Body weights stedily incresed during the oservtion period nd did not differ etween the controls nd the strontium-loded nimls. Urinry output ws higher fter initition of renl filure, ut ws comprle for oth control nd strontium-loded nimls. Serum cretinine mesurements in control nimls efore nd fter initition of the 5/ nephrectomy indicte tht moderte degree of renl filure ws induced (Tle 1). Fourteen

4 Oste et l: Development nd reversiility of strontium-induced osteomlci 923 Tle 1. Biochemicl prmeters in serum nd urine of the vrious study groups. Strontium Pre- CRF Serum cretinine mg/dl.39 ± ± ± ± ± ±.35, 1.2 ±.34 Serum clcium mg/dl 9.95 ± ± ± ± ± ± ±.55 Serum phophorus mg/dl 7. ± ± ± ± ± 1.7,.7 ± 1.83, 4.93 ± 2.5 Cretinuri mg/24 hours 1.98 ± ± ± ± ± ± ± 3.77 Cretinine clernce.78 ± ±.8.27 ±.9.1 ± ±..2 ±.7.19 ±.7 ll/min/1 g Proteinuri mg/24 hours 8.14 ± ± ± ± ± 4.37, ± 45.54, 8.19 ± 49.3 CFR is chronic renl filure. P <.5 versus pre-crf; P <.5 versus control. weeks postsurgery, serum levels of cretinine were elevted 4.3-fold nd cretinine clernce ws 78% lower in the CRF rts s compred to the control nimls. Induction of renl filure cused rise in proteinuri in oth control nimls nd nimls loded with strontium (Tle 1). Serum clcium nd phosphorus concentrtions were within the norml rnge nd did not significntly chnge during the course of the experiment, neither etween groups. Strontium-loding did not ffect serum cretinine, clcium, or phosphte levels, neither did the urinry cretinine excretion chnge during the course of the experiment nor ws it ffected y tretment with strontium (Tle 1). Phosphturi tended to decrese during 2 nd weeks of strontium loding, which ws not reversed during the withdrwl period (Fig. 2A). Strontium loding induced n increse in clciuri tht reversed to norml levels fter withdrwl of the compound (Fig. 2B). Loding with strontium during 2,, nd 12 weeks resulted in rise in the serum strontium concentrtion nd urinry strontium concentrtion, which decresed gin during the wshout period (Fig. 3A nd B). The rise of serum strontium concentrtion ws ccompnied y time-dependent ccumultion of the element in one, s indicted y the one strontium concentrtions (Fig. 3C). After withdrwl of strontium during 2, 4, or 8 weeks, limited ut sttisticlly significnt decrese of the one strontium content ws noted. From these dt, the hlftime for removl of strontium from the one ws estimted to e 9.7 ± 2.9 weeks. As shown in Figure 4, comprison of the ipth levels efore nd fter induction of renl filure reveled n increse of this prmeter, though not sttisticlly significnt. Strontium loding during 2,, or 12 weeks went long with decrese in serum PTH levels compred to control nimls, however, gin rose to control vlues during the wshout period. At scrifice, control nimls hd lower serum ALP ctivity, compred to vlues efore initition of renl filure. Loding with strontium, however, cused significnt increse in totl serum ALP ctivity (8% increse versus controls) fter 2 weeks of strontium loding, Phosphturi, mg/24h Clciuri, mg/24h A B Sr Sr + 2 w WO Sr + 8 w WO 4 w wo Sr loding time Fig. 2. Effect of strontium (Sr) loding nd susequent wshout (WO) period on (A) phosphturi nd (B) clciuri. Vlues re expressed s men ± SEM. P <.5 versus controls; P <.5 versus strontiumloding without wshout.

5 924 Oste et l: Development nd reversiility of strontium-induced osteomlci Bone Sr, mg/g Urinry Sr, mg/24h Serum Sr, µg/ml A B C Sr Sr + 2 w WO Sr + 8 w WO 4 w wo Sr loding time Serum PTH, pg/ml Pre- CRF Sr Sr + 2 w WO Sr + 8 w WO 4 w wo Sr loding time Fig. 4. Effect of renl filure, strontium (Sr) loding, nd wshout (WO) on prthyroid hormone (PTH) concentrtion. Vlues re expressed s men ± SEM. P <.5 versus controls; P <.5 versus strontium-loding without wshout; c P <.5 versus pre-chronic renl filure (CRF). 281% fter weeks, nd 77% fter 12 weeks, which decresed gin during the 2, 4, or 8 weeks wshout periods (Fig. 5A). It ws further demonstrted tht strontium induced n increse in ll three ALP isoenzymes, which ws sttisticlly significnt for liver nd one ALP (Fig. 5B to D). The one isoenzyme ws the mjor contriutor to the totl ALP mesured in serum. All three forms decresed gin fter withdrwl of strontium. Histologic dt Rts treted with strontium for nd 12 weeks displyed significntly incresed mounts of osteoid tissue s evidenced y the incresed osteoid perimeter, osteoid re, nd osteoid thickness (Fig. ). The minerl pposition rte, djusted pposition rte, doule-leled surfce nd one formtion rte decresed fter 2,, nd 12 weeks of strontium dministrtion, which ws ccompnied y n increse in minerliztion lg time nd osteoid mturtion time (Fig. ). These dt, together with the incresed mount of osteoid, provide evidence for the development of minerliztion defect reflected y the histologic picture of osteomlci in the strontiumloded nimls (Fig. 7). This ws ccompnied y drmtic decrese in osteolst perimeter during the vrious loding periods (Figs. H nd 7A to D). Administrtion of strontium ppered to hve no effect on osteoclst surfce or eroded perimeter, nor on the totl one re (dt not shown). Fig. 3. Strontium (Sr) concentrtion during 2,, nd 12 weeks of strontium loding nd serum strontium concentrtion fter wshout (WO) of 2, 4, or 8 weeks. (A)Men serum strontium concentrtion. (B) Men urinry strontium concentrtion. (C) Bulk strontium concentrtion in one. Vlues re expressed s men ± SEM. P <.5 versus controls; P <.5 versus strontium loding without wshout.

6 Oste et l: Development nd reversiility of strontium-induced osteomlci 925 Totl ALP ctivity, U/L A Sr Sr + 2 w WO Sr + 8 w WO 4 w wo Bone ALP ctivity, U/L B C 2 D 1 Liver ALP ctivity, U/L Intestinl ALP ctivity, U/L Sr loding time Sr loding time Fig. 5. Serum lkline phosphtse (ALP) isoenzymes. (A) Totl serum ALP ctivity. (B) Bone ALP ctivity. (C) Liver ALP ctivity. (D) Intestinl ALP ctivity. Vlues re expressed s men ± SEM. P <.5 versus controls; P <.5 versus strontium (Sr) loding without wshout (WO). The mount of osteoid decresed lredy fter 2 weeks wshout nd reched norml vlues 4 (in the 12 weeks loded nimls) or 8 (in the weeks loded nimls) weeks fter strontium withdrwl. Cesstion of strontium dministrtion went long with significnt increse in the osteolst perimeter fter 8 weeks of wshout in nimls tht received strontium during 2 nd weeks. As indicted y the histodynmic prmeters, strontium withdrwl lso restored minerliztion, n effect tht ecme significnt fter 2 nd 4 weeks of wshout in rts tht received strontium during 12-week period. DISCUSSION Previously, we showed n ssocition etween incresed one strontium levels nd osteomlci in one iopsies from 1 dilysis ptients [25, 2]. Administrtion of the element to CRF rts induced minerliztion defect tht ppered to e dose-dependent [28, 29]. The experiment descried in this pper ws designed to study the temporl evolution nd reversiility of the strontium-induced minerliztion defect previously oserved in CRF nimls. We used the remnnt kidney rt model, which is wellchrcterized model llowing the induction of mild, stle CRF, s reflected y the moderte increse in serum cretinine levels nd urinry protein excretion. This mild renl filure cn e compred to tht oserved in some elderly people, including osteoporotic postmenopusl women who constitute the trget popultion for tretment with strontium-rnelte, compound tht hs recently een introduced on the mrket

7 92 Oste et l: Development nd reversiility of strontium-induced osteomlci Osteoid perimeter 1% 8% % 4% 2% Sr Sr + 2 w WO Sr + 8 w WO 4 w wo Osteoid re 5% 4% 3% 2% 1% % % Osteoid width, µm Minerl pposition rte, µm/dy Doule leled perimeter 2% 15% 1% 5% % Bone fomtion rte µm 2 /mm 2 /dy Minerliztion lg time, dys Sr loding time Osteolst perimeter 4% 3% 2% 1% % Fig.. Histologic dt of the vrious study groups. Dt re s men ± SEM. P <.5 versus controls; P <.5 versus strontium Sr loding time (Sr) loding without wshout (WO). s n ntiosteoporotic gent. As shown in Figure 1, rts were dministered strontium vi the drinking wter t 2 g/l concentrtion, dose t which minerliztion defect hs een induced in previous studies [29]). Indeed, histomorphometric one nlysis of the nimls treted with strontium reveled cler signs of osteomlci ( defective minerliztion in comintion with n incresed osteoid volume) lredy fter 2 weeks, which ecme sttisticlly significnt fter nd 12 weeks. These dt confirm our previous results concerning the development of osteomlci in strontium-treted CRF rts [29]. Moreover, in the rts treted with strontium, the numer of cuoidl osteolsts lredy decresed fter 2 weeks nd further disppered fter nd 12 weeks.

8 Oste et l: Development nd reversiility of strontium-induced osteomlci 927 Fig. 7. Goldner-stined histologic one smples nd tetrcycline leling. (A) Norml one histology in 12-week control chronic renl filure (CRF) rt with lrge, cuoidl osteolsts lining the osteoid tissue. (B) Bone histology of CRF rt fter 12 weeks of strontium loding. There is n

9 928 Oste et l: Development nd reversiility of strontium-induced osteomlci To which extent this is (1) either the result of the lower ipth levels seen in these groups, (2) due to direct cell iologic effect of strontium t the level of the osteolst s demonstrted in vitro [3], or (3) the consequence of comintion effect of oth is uncler. In this context the ility of strontium to ct on the osteolst vi novel ction-sensing mechnism, s suggested recently y Pi nd Qurles [35] is worth mentioning. After 2 weeks of strontium loding, there ws lredy limited increse in the mount of osteoid tissue, despite sustntilly decresed numer of ctive, cuoidl osteolsts. At this time point, however, the effect of strontium on the vrious prmeters of one minerliztion is much more pronounced. This suggests tht direct effect of strontium on one minerliztion must hve preceded the element s effect on the osteolst. In other words, we hypothesize tht in the erly phse of strontium exposure, osteolsts secrete osteoid mtrix t reduced rte tht, however, is not minerlized in n optiml wy. Ultimtely, this results in the histologic picture of osteomlci, reflecting n imlnce etween osteoid deposition nd minerliztion. In contrst to other studies in oth rts nd mice with norml renl function, in which strontium tretment resulted in decresed osteoclst surfce/numer [11, 12], no significnt histologic effect of strontium on one resorption ws shown in the present study. Mesurement of the one strontium concentrtion reveled tht dministrtion of strontium ws ccompnied y time-dependent ccumultion of the element in the one. Even fter 12 weeks of tretment, there ws still incorportion of the element, indicting tht the one strontium content hd not yet reched plteu level. Serum strontium concentrtions, however, reched mximl vlues lredy fter 2 weeks of strontium dministrtion, nd remined stle during the whole loding period. In rts with norml renl function, Dhl et l [3] lso showed tht the plsm strontium level reches stedy stte within 1 dys. Withdrwl of strontium from the diet ws ccompnied y very rpid decrese of the serum strontium concentrtion, lmost to control levels fter 8 weeks wshout, nd sustntil decrese (± 18%) of one strontium. Bsed on the dt of the present study, the hlf-life of strontium in one ws clculted to e 9.7 ± 2.9 weeks. Becuse of the limited oservtion periods it ws not possile to check whether the elimintion of strontium out of one is liner over time or tkes plce vi multiphsic wy. In the 12 weeks control group, serum levels of PTH were elevted pproximtely twofold, indicting the development of mild secondry hyperprthyroidism nd reflecting the sitution in humns with moderte CRF. In nimls treted with strontium, t ll time points decrese of serum ipth ws found. Previously, it hs lredy een suggested tht strontium my exert clcimimetic effect t the level of the clcium-sensing receptor, resulting in decresed PTH secretion in rts with norml renl function [37] nd in ovine prthyroid cells in vitro [38]. Aside from the prthyroid glnd, the clcimimetic effect of strontium my lso e ctive t the level of the kidney. Indeed, the incresed clciuri might t lest in prt result from decresed tuulr clcium resorption in strontium-treted nimls. Moreover, the decresed phosphturi found fter strontium loding my e relted to the reduced PTH levels in these groups, since lowering PTH stimultes tuulr phosphte resorption [39]. The totl serum ALP ctivity ws significntly higher in the nimls treted with strontium. This is in greement with other studies in which higher serum totl ALP levels were lso found fter strontium dministrtion to ovriectomized [12] nd immoilized rts [4]. Higher one-specific ALP ws lso found fter strontium-rnelte tretment in osteoporotic women [23]. In this context, however, it should e mentioned tht the mesurement of totl serum ALP encompsses the codetermintion of multiple other isoenzymes derived from severl other tissues such s the smll intestine, liver, nd kidney. Therefore, the totl serum ALP does not lwys correlte with the one isoenzyme, neither with one histologic prmeters [41, 42]. In the present study, mesurement of the ALP isoenzymes indicted tht strontium loding is not only ssocited with n incresed concentrtion of the one ALP, ut of the intestinl nd liver ALP isoenzymes s well. The exct mechnism underlying this phenomenon is not yet cler nd could e ttriutle to n effect of strontium on the synthesis of ALP, its relese into the serum, its enzymtic ctivity or to decrese in the metolic clernce rte of this enzyme. Previously, we investigted the direct interction of strontium with the ALP enzymtic ctivity y incuting humn serum smple with different strontium concentrtions nd we found no difference in the electrophoretic moility or enzymtic ctivity [3]. This ws confirmed fter dding vrious mounts of strontium to rt serum in the present study (dt not shown). An effect of strontium on the metolic clernce rte of incresed mount of osteoid tissue without the presence of ctive osteolsts. (C) Bone histology of CRF rt fter 12 weeks of strontium loding, followed y wshout period of 2 weeks. Lrge mounts of osteoid tissue re still present ut osteolsts hve reppered. (D) Bone histology of CRF rt fter 12 weeks of strontium loding, followed y wshout period of 4 weeks. Active osteolsts re lining norml mount of osteoid tissue. (E) Fluorescent tetrcycline leling in CRF rt fter 12 weeks of strontium loding. No lels re incorported, which indictes the development of minerliztion defect. (F) Tetrcycline leling in 12-week control CRF rt. Norml doule lels re present.

10 Oste et l: Development nd reversiility of strontium-induced osteomlci 929 ALP is lso highly unlikely, ecuse ll three isoforms re ffected in the sme wy y strontium dministrtion. Whether strontium my specificlly promote hydrolysis of the memrne nchoring of ALP deserves further investigtion. These dt indicte, however, tht incresed concentrtions of one-specific ALP in serum re not necessrily reflection of n incresed osteolst numer or ctivity. After cesstion of strontium tretment, the effects of strontium on ALP nd PTH were reversed nd the histologic nd histodynmic prmeters evolved to control levels during the susequent wshout periods of 2, 4, or 8 weeks. To certin extent, these findings re in line with those of Johnson, Armstrong, nd Singer [43], otined in young rts with norml renl function, showing tht during wshout period of 35 dys, following 28-dy dietry strontium tretment (.45% strontium), the excess osteoid ws ctolized nd the one trecule evolved to more norml ppernce. Dt of the present study do not llow us to define the exct mechnism y which strontium induces one minerliztion defect. The rpid development of this defect, however, is comptile with the ility of strontium to compete with clcium to form complexes with orgnic components of the mture osteoid mtrix such s phospholipids, glycosminoglycns, nd phosphoproteins tht my ct s nucletion points for crystl development nd growth []. Evidence for such mechnism is further provided y the finding tht, once circulting strontium levels re lowered y withdrwl of the element, reminerliztion rpidly occurs, despite the elevted levels t which strontium is still present in one. Aprt from this, other mechnisms y which strontium my interfere with the one minerl hve to e considered. During (impired) ptite formtion, strontium my still e incorported in the crystl lttice y heteroionic exchnge with clcium s indicted y previous oservtions demonstrting the element to e minly present in proximity of the minerliztion front [28] (i.e., in newly formed one [44]). Once entrpped in the crystl lttice, removl of the element will resonly e slow nd will minly occur vi osteoclstic resorption; notion eing in line with the high one strontium levels tht re still present fter wshout. To which extent the incorported strontium, in the long run, my ffect the crystl lttice stility or one strength deserves further investigtion. In this context it is worth mentioning tht, given the ntomic heterogeneity of one, locl strontium concentrtions my e reched t which, using X-ry diffrction (XRD) nd Fourier-trnsformed infrred spectroscopy (FTIR) nlysis, we recently showed reduced crystllinity nd ltered crystl lttice [45]. Finlly, the rpid ccumultion nd rpid prtil removl (± 18%) of the element fter withdrwl is indictive for heteroionic exchnge with clcium t the level of the hydrtion shell surrounding the ptite crystl. This hydrtion shell is considered the mjor site for fst ionic exchnge etween the extrcellulr fluid nd the minerlized one comprtment y which influx/efflux of strontium my occur t oth the minerlizing nd quiescent one surfces [4]. Loclistion of strontium t this site most proly hs no deleterious effect on one. CONCLUSION In this study evidence is provided for the rpid development of minerliztion defect in strontium-loded CRF rts, ccompnied y reduced osteolst numer, reduced PTH synthesis or secretion, nd incresed serum ALP levels. These effects cn e rpidly reversed fter withdrwl of the compound. ACKNOWLEDGMENTS The uthors re indeted to Croline Verstrete of the lortory of Biochemistry, Antwerp University Hospitl, for the serum mesurements of clcium, phosphorus, nd cretinine. 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