Abstract
Background. We recently reported that administration of high doses of lanthanum carbonate (1000 mg/kg/day) to chronic renal failure (CRF) rats can result in a mineralization defect. Our results suggested, however, that the impaired mineralization was not due to a direct toxic action of lanthanum on the bone, but rather was an indirect consequence of a phosphate depletion resulting from the compound's high phosphate-binding capacity. To further substantiate these results, in the present study, the effects of lanthanum carbonate on bone were compared to the effects of sevelamer, a nonabsorbed, non-metal-containing polymeric phosphate-binding agent. Methods. Male Wistar rats underwent a 5/6th nephrectomy to induce chronic renal failure, after which they were treated with either sevelamer (500 or 1000 mg/kg/day) or lanthanum carbonate (1000 mg/kg/day) by oral gavage for 12 weeks. Results. CRF animals treated with either sevelamer (500 or 1000 mg/kg/day) or lanthanum carbonate (1000 mg/kg/day) developed a phosphate depletion after 4 weeks of treatment, as evidenced by a marked reduction in phosphaturia. At sacrifice after 12 weeks of treatment, bone histomorphometry showed that a mineralization defect had developed in two out of six animals in the lanthanum-carbonate-treated group, in four out of seven animals in the 1000 mg/kg/day sevelamer group, and in one out of nine animals in the 500 mg/kg/day sevelamer group. Conclusions. These results corroborate our previous findings that the administration of a powerful phosphate-binding agent to CRF rats can induce phosphate depletion, resulting in a mineralization defect.
Introduction
Control of phosphatemia is still an important issue in the management of patients with renal failure. Due to renal insufficiency, urinary excretion of phosphorus is impaired, resulting in phosphate accumulation in these patients. This, in turn, can lead to various clinical conditions, such as secondary hyperparathyroidism, renal osteodystrophy, and extra-osseous calcifications. Since dialysis and dietary restriction of phosphate intake are often insufficient to control phosphatemia, additional phosphate-binding treatment is necessary to reduce gastrointestinal uptake of dietary phosphate.
Recently, lanthanum carbonate [La2(CO3)3, Fosrenol®] was introduced as a new phosphate-binding agent, with a very low gastrointestinal absorption, a high phosphate-binding capacity, and a good safety profile.Citation[1-3]
In chronic renal failure (CRF) rats given lanthanum carbonate during 12 weeks at a dose of 1000 mg/kg/day, we previously demonstrated the development of a mineralization defect, as indicated by an increased amount of osteoid and a decreased bone formation rate.Citation[4] No such lesions were seen in animals with normal renal function receiving the same lanthanum carbonate doses. Our results suggested that this mineralization defect occurred secondary to a phosphate depletion caused by the compound's high phosphate-binding capacity, in combination with reduced 25-(OH) vitamin D3 levels inherent to the CRF, rather than being the consequence of a direct effect of lanthanum on bone.
In order to further substantiate these results, the effects of lanthanum carbonate on bone were compared with those of another recently introduced phosphate-binding agent: sevelamer hydrochloride. Sevelamer is a calcium-free, non-metal-containing polyallylamine polymer that is not gastrointestinally absorbed.Citation[5] Hence, it is not readily expected to exert any direct effect on bone.
This study was set up to investigate whether the effects of lanthanum carbonate on bone could be reproduced in CRF rats receiving similar doses of sevelamer. This would provide additional evidence supporting the hypothesis that the previously observed effects of lanthanum carbonate on bone of CRF rats occurred secondary to phosphate depletion and were not due to a direct effect of lanthanum on bone.
Material and Methods
Experimental design
Thirty-one male Wistar rats (14 weeks old at the start of the study) underwent a 5/6th nephrectomy by ligation of two of the three renal arteries of the left kidney (week − 3), followed by a right nephrectomy 1 week later. Treatment was started after a 2-week stabilization period and consisted of daily oral gavages with lanthanum carbonate (1000 mg/kg/day, n = 6), sevelamer (500 and 1000 mg/kg/day, n = 9 and n = 7) or vehicle (2% carboxymethylcellulose, n = 9) during 12 weeks. A constant dose volume of 15 mL/kg was used, and individual treatment doses were adjusted to the most recently recorded body weight (at weekly intervals). All animals had free access to food and water throughout the study period. The diet used contained 1.00% Ca, 0.75% P, and 2000 IU/kg vitamin D3.
Urine and blood samples were taken prior to the installation of the renal failure (week − 4), at the start of treatment (week 0) and at monthly intervals thereafter. Urine samples were collected by housing the animals in individual metabolic cages for 24 h. Blood samples were collected from the tail-vein under ketamine/xylazine anesthesia at the end of the urine-collection period. Serum and urine samples were stored at –80°C until analysis.
At the end of the treatment period, animals were labeled with tetracycline (30 mg/kg) and demeclocycline (30 mg/kg) by intraperitoneal injection at, respectively, 7 and 3 days before sacrifice. Animals were sacrificed by exsanguination through the aorta abdominalis under ketamine/xylazine anesthesia, and bone was removed. The tibias were fixed in Burckhardt's fixative for 24 h and stored in 70% ethanol at 4°C until further processing for histomorphometric analysis.
Biochemical Analysis
Creatinine, calcium, and phosphorus were determined in the serum samples with an automated Vitros 750 XRC system. Parathyroid hormone (PTH) levels were measured using a rat PTH IRMA kit (Immutopics Inc, San Clemente, CA), performed according to the manufacturer's instructions. Alkaline phosphatase was determined according to IFCC 1983/4Citation[6] using 4-nitrophenol-phosphate as substrate and intestinal alkaline phosphatase as standard.
Urinary phosphorus was determined using an automated Vitros 250 system. Urinary creatinine was measured with a modified Jaffé method. Total protein was determined according to Bradford.Citation[7] Urinary calcium was measured by flame atomic absorption spectrophotometry using a Perkin-Elmer model 372 AAS.Citation[8] 25-(OH)-vitamin D and 1,25-(OH)2-vitamin D were assessed using methods previously described by others.Citation[9&10]
Bone Histomorphometry
The tibias were dehydrated in increasing ethanol concentrations and impregnated in methyl-methacrylate for 6 days. Afterwards, polymerization was allowed to proceed for 48 h under N2-atmosphere at 4°C. After polymerization, 5 µm sections were Goldner stained for visualization of osteoid and mineralized bone. Ten µm sections were mounted unstained in 100% glycerol for visualization of tetracycline labels.
The sections were analyzed using a computerized KS-400 image analysis system. Calibration of image pixel size was performed before each measurement cycle by means of a calibration grid (Graticules Ltd, Kent, UK). Bone area, osteoid area, osteoid perimeter, eroded perimeter, and quiescent perimeter were measured by manually tracing the mineralized and osteoid area, and marking erosion and osteoblast and osteoclast perimeters on the computer screen, after which the system calculated the absolute areas and perimeters. Double-labeled perimeter and total perimeter were measured in a similar way on unstained sections under UV illumination. Interlabel distance was measured by tracing the labels, after which the system measured the distances between the labels at regular spatial intervals, perpendicular to the labels. Out of the primary measurements, the following derived parameters were calculated according to standardized procedures:Citation[11] mineral apposition rate, bone formation rate, osteoid width, and mineralization lag time. Because in rats, nonspecific single labeling is frequently observed, only the double-labeled perimeter was included in the calculation of the histodynamic parameters. In the absence of published histomorphometric reference values for defining renal osteodystrophy in the rat, histological assessment of bone lesions was based on a comparison with historical values from previous experiments of our own group using animals of the same age and gender.Citation[4]
Statistical Analysis
Results are shown as mean ± SEM. For each time point and variable, a Kruskall–Wallis test was performed to test for differences between groups, followed by a Mann–Whitney U-test with Bonferroni correction when significant differences were found. Comparisons between time points within each treatment group were made with a Friedman test, followed by a Wilcoxon signed rank test when significant differences between time points were noted. Differences were considered significant when p < 0.05.
For calculation of the mean urinary phosphorus levels and further statistical analysis, results below the detection limit of the assay (4.4 mg/dL) were set at half the detection limit (2.2 mg/dL).
Complete biochemical data were available for four vehicle-treated animals, five and seven animals treated with, respectively, 500 and 1000 mg/kg/day of sevelamer, and five animals treated with lanthanum carbonate.
Results
Biochemical Analysis
Serum creatinine levels did not show any significant differences between treatment groups (). A statistically significant increase was found after the 5/6th nephrectomy (week 0) when compared with preoperative levels (week − 4), after which serum creatinine levels remained stable throughout the treatment period. Proteinuria showed a steady increase in all treatment groups from week 4 onwards, which was statistically significant from week 8 onwards. These results indicate that a stable renal insufficiency was created, which was comparable in all treatment groups.
Table 1 Serum and urine biochemistry (Mean ± SEM)
Serum alkaline phosphatase showed no significant differences, neither between treatment groups nor over time. Although not statistically significant, serum PTH levels tended to increase after installation of the chronic renal failure in all treatment groups (). Individual animals occasionally showed a significant increase over time.
Serum phosphate showed a transient decrease in all groups postsurgery (week 0), which was normalized after 4 weeks of treatment. No statistically significant differences were found between treatment groups (). Administration of the phosphate-binding agents led to a significant decrease in phosphaturia. Already after 4 weeks of treatment, phosphaturia was significantly reduced in the lanthanum carbonate and the high-dose sevelamer-treated animals when compared to vehicle-treated animals (p = 0.048 and p = 0.018), and this decrease persisted until the end of the treatment period (). In the low-dose sevelamer group, statistical significance was only reached after 8 weeks of treatment.
Figure 1 Serum phosphate (top) and urinary phosphorus excretion (bottom). Serum phosphate levels show a statistically significant reduction at week 0, possibly as a result of reduced food intake postsurgery. During the study period, serum phosphate levels returned to normal. No statistically significant differences were noted between treatment groups. Already after 4 weeks of treatment, a statistically significant decrease in phosphaturia was noted in the treated animals. °p < 0.05 versus baseline (week 4); *p < 0.05 versus start of treatment (week 0); a: p < 0.05 vehicle versus lanthanum carbonate and sevelamer 1000 mg/kg/day treated; b: p < 0.05 vehicle versus phosphate binder treated.
Serum calcium showed a significant rise after installation of CRF and remained stable from that time onwards. No statistically significant differences were found between treatment groups. Calciuria showed a statistically significant increase after the installation of the renal failure in all treatment groups (). Interestingly, at the end of the treatment period, urinary calcium excretion tended to be higher in the sevelamer-treated groups, almost reaching statistical significance in the high-dose sevelamer group (p = 0.051).
Figure 2 Serum calcium (top) and urinary calcium excretion (bottom). A statistically significant increase in serum calcium levels is seen after induction of the renal failure. During treatment, serum calcium levels remained stable. Urinary calcium excretion increased postsurgery. At the end of the treatment period, sevelamer-treated animals showed higher (but not statistically significant) calcium excretions, when compared to vehicle or lanthanum carbonate treated animals. °p < 0.05 versus baseline (week − 4); *p < 0.05 versus start of treatment (week 0).
At sacrifice, 25-(OH) vitamin D3 levels did not differ significantly between treatment groups (). In animals treated with 1000 mg/kg/day sevelamer, however, a significant decrease in 1,25-(OH)2 vitamin D3 levels was found when compared to control animals as well as lanthanum carbonate treated animals ().
Figure 3 1,25-(OH)2 vitamin D3 levels at sacrifice (week 12). Sevelamer treatment induced a dose-dependent, statistically significant decrease in 1,25-(OH)2 vitamin D3 levels when compared to vehicle or lanthanum carbonate treatment. (*p < 0.05.)
Bone Histomorphometry
Bone histomorphometry did not show any statistically significant differences between treatment groups, which most likely must be ascribed to the relatively high biological variability within treatment groups (). In order to allow us to interpret the results more accurately, individual histomorphometric results were compared with historical control valuesCitation[4] [impaired mineralization, expressed as either osteomalacia or mixed lesion: osteoid area > 1.86% and bone formation rate (BFR) < 570 µm2/mm2/day (osteomalacia) or BFR < 3147 µm2/mm2/day (mixed lesion); increased bone turnover: BFR > 3147 µm2/mm2/day, increased serum PTH], which allowed us to classify the individual animals within a particular category of renal osteodystrophy (). Using this classification, four out of seven animals treated with the 1000 mg/kg/day sevelamer dose and two out of six animals treated with lanthanum carbonate (1000 mg/kg/day) showed an impaired mineralization (low bone formation rate, in combination with an increased amount of osteoid). The effects of sevelamer on the bone histomorphometry appeared to be dose-dependent, because only one out of nine animals showed a mineralization defect in the low-dose (500 mg/kg/day) group. Animals receiving vehicle either showed normal bone histology (four out of nine animals) or had developed hyperparathyroidism (five out of nine animals), as indicated by an increased bone formation rate and normal to slightly increased osteoid area.
Figure 4 Bone histomorphometric results. Dots represent individual animals, the horizontal bar indicates median values. Normal values are indicated by the gray background. No statistically significant differences were noted between treatment groups. However, a trend toward increased mineralization lag time and osteoid maturation time, combined with higher osteoid area and lower bone formation rate is visible in the phosphate-binder-treated groups. Based on the histomorphometric and biochemical data, each individual animal was also classified according to the type of renal osteodystrophy (see ). Treatment groups: 1: Vehicle; 2: Sevelamer 500 mg/kg/day; 3: Sevelamer 1000 mg/kg/day; 4: Lanthanum carbonate 1000 mg/kg/day.
Figure 5 Distribution of different types of renal osteodystrophy seen after the 12-week treatment period. A mineralization defect could be induced by either phosphate-binding agent. (HPTH: hyperparathyroid bone disease; ABD: adynamic bone disease; Min. Def: Mineralization defect.)
Discussion
Serum creatinine measurements and proteinuria reflected the successful induction of renal failure in all study animals, the degree of which was comparable among all treatment groups. Furthermore, the installation of chronic renal failure went along with a varying degree of hyperparathyroidism, as evidenced by the increased serum PTH levels in some animals, and the induction of hyperparathyroid bone disease in a substantial proportion of the vehicle-treated animals.
Bone histology showed an impaired mineralization in combination with an increased osteoid area in two out of six animals treated with 1000 mg/kg/day lanthanum carbonate and in four out of seven animals treated with 1000 mg/kg/day sevelamer, and one out of nine animals treated with 500 mg/kg/day sevelamer.
We previously showed that treatment with lanthanum carbonate at doses of 1000 mg/kg/day in CRF animals induced a mineralization defect.Citation[4] We hypothesized that the effects of lanthanum carbonate occurred secondary to a phosphate depletion due to the powerful phosphate-binding capacity of lanthanum carbonate, in combination with reduced 25-(OH) vitamin D3 levels inherent to the CRF, rather than being the consequence of a direct effect of the element on bone. The main objective of the present study was to further substantiate this hypothesis. To this end, the effects of lanthanum carbonate on bone were compared with those of sevelamer. Sevelamer hydrochloride is a non-metal-containing, powerful polymeric phosphate binder that is virtually not gastrointestinally absorbed,Citation[5] hence, a direct effect of the compound on bone mineralization is not expected.
Serum phosphorus levels showed a slight reduction versus baseline at the start of the treatment when compared with baseline values, most likely caused by the reduced food intake during the days following surgery. Urinary phosphate excretion showed a significant decrease following the use of either phosphate-binding agent after 4 weeks of treatment. This decrease was sustained until sacrifice.
Excessive phosphate binding in the gut may lead to a state of phosphate depletion, which is not necessarily evidenced by serum phosphate levels. Lotz et al.Citation[12] already in 1968 showed a rapid, dramatic decrease in urinary phosphorus excretion with undetectable phosphaturia, after 6 days of treatment with magnesium-aluminium hydroxide, this without any significant effect on phosphatemia. Recently, Nagano et al.Citation[13] also showed a significant decrease in phosphaturia in rats fed a diet containing 1% sevelamer during only 8 days, in the absence of any change in phosphatemia. They also showed a significant increase in the expression of the type II Na/Pi cotransporter protein, which most likely reflects the need for increased phosphate reabsorption by the kidney, resulting in a decreased phosphaturia. Phosphate status is critical for bone mineralization, and deficiency is known to induce mineralization defects, as reported in dietary phosphate deficiency,Citation[14] hereditary hypophosphatasemia,Citation[15] tumor-associated hypophosphatasemia,Citation[16] and drug-induced hypophosphatasemia associated with excessive use of phosphate-binding antacids.Citation[17] Hence, the reduced phosphate availability (as evidenced by the marked decrease in phosphaturia) may thus result in either a reduced incorporation into or increased mobilization of phosphate out of bone reflected by an impaired mineralization, seen in animals treated with either lanthanum carbonate or sevelamer.
Urinary calcium excretion did not differ significantly between treatment groups. However, by the end of the treatment period, the animals treated with sevelamer showed a trend toward higher calciuria than those treated with vehicle or lanthanum carbonate, which almost reached statistical significance (p = 0.051) in the highest-dose sevelamer group. This is in line with a decreased incorporation into, or increased efflux of calcium out of bone concomitant with the fluxes of phosphorus at the level of the bone. The reason why such an increase in calciuria is not seen in the lanthanum carbonate treated animals may be due to the ability of lanthanum to block the calcium channels in the gut, when present at high doses,Citation[18] although such effects have yet to be demonstrated in vivo.
Interestingly, in the animals treated with the high dose of sevelamer, a statistically significant decrease in 1,25-(OH)2 vitamin D3 levels was noted. This effect was much less pronounced in the sevelamer 500 mg/kg group. These data are in agreement with the findings of Nagano et al. who also reported reduced vitamin D levels in sevelamer-treated animals treated for 84 days,Citation[19] but not in animals treated for 8 days.Citation[13] Although vitamin D deficiency is also well known as a causal factor for osteomalacia, in the current study, 1,25-(OH)2 vitamin D3 levels dropped only to approximately one half of normal. This decrease in 1,25-(OH)2 vitamin D3 is unlikely to be the only factor involved in the development of the mineralization defect, but it could have aggravated the bone lesions observed in the 1000 mg/kg sevelamer group by further limiting the phosphate/calcium absorption in the gut.
In conclusion, treatment of CRF rats with sevelamer or lanthanum carbonate at doses of 1000 mg/kg/day resulted in a mineralization defect, characterized by an increased osteoid volume and thickness, and reduced bone formation rate, in some animals. In addition, an important reduction in phosphaturia (but not phosphatemia) was noted. Both phosphate-binding agents, having completely different metabolisms, induced phosphate depletion and a similar mineralization defect. These results further support the hypothesis that the previously observed mineralization defect, seen in lanthanum carbonate treated CRF ratsCitation[4] occurs secondary to phosphate depletion, induced by the pharmacological actions of the phosphate binder, rather than being due to a direct effect of lanthanum on bone.
Acknowledgments
La2(CO3)3 was provided by Shire Pharmaceutical Development Ltd. The authors would also like to thank Prof. V. Van Hoof of the Department of Biochemistry of the University Hospital of Antwerp, Belgium, for the biochemical analysis using the Vitros systems, and Prof. R. Bouillon of the Laboratory for Experimental Medicine and Endocrinology of the Catholic University of Leuven, Belgium, for the vitamin D measurements.
Parts of this work have been presented as posters at the 2001 ASN meeting (J. Am. Soc. Nephrol. 12, 740A) and the 2002 ASBMR meeting (J. Bone. Miner. Res. 17, M415).
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