A higher incidence of coronary artery and cardiac valve calcification occurs in dialysis patients compared to the general population1,2,3,4. Ectopic or vascular calcification in chronic renal failure (CRF) is associated with elevated levels of serum phosphorus and calcium-phosphate (Ca 
P) product and is likely an important contributor to increased cardiovascular morbidity and mortality5,6,7,8.
Vascular calcification in renal failure patients was previously considered a passive process resulting from deposition of calcium-phosphate crystals in the setting of an elevated Ca x P product and secondary hyperparathyroidism. Recent studies, however, suggest that vascular calcification is instead an active, cell-mediated process in which vascular smooth muscle cells develop bone-forming functions9,10,11,12. The mechanisms mediating and the treatment of this ectopic calcification remain incompletely defined6. In vitro studies by Jono et al13 have demonstrated that high inorganic phosphate levels, greater than 1.4 mmol/L, increase human smooth muscle cell calcification rates and increase the expression of the osteoblastic differentiation markers, osteocalcin and Osf2/Cbfa-1, via a sodium-phosphate cotransporter called Pit-1. Additional in vitro studies by Chen et al14 have shown that phosphorus and other factors in uremic serum increase osteopontin expression and calcification in bovine vascular smooth muscle cells14. Finally, a recent clinical trial published by Chertow, Burke, and Raggi15 revealed that sevelamer, when compared to calcium-based phosphate binders, attenuated the progression of coronary and aortic calcification in hemodialysis patients as identified by electron beam computed tomography. Thus, the control of hyperphosphatemia with phosphate binders in patients with CRF may not only prevent a worsening Ca P product and secondary hyperparathyroidism, but also influence the development of the active, cell-mediated process of vascular calcification.
In previous years, the most commonly used phosphate binders contained either aluminum or calcium salts, such as calcium carbonate (CaCO3) or acetate. Whereas aluminum exposure may cause neurologic, skeletal, and hematologic toxicity16,17, calcium-based phosphate binders can lead to hypercalcemia and potentially worsen the risk of soft tissue and cardiovascular calcifications18,19,20. To avoid these side effects, an aluminum- and calcium-free phosphate binder, sevelamer hydrochloride, has been developed. Sevelamer is effective in controlling hyperphosphatemia and reducing serum Ca P product levels in dialysis patients21,22,23.
Our group recently published the results of a study comparing the effects of the two phosphate binders CaCO3 and sevelamer in a 3-month experimental model of uremia. Despite an equal control of hyperphosphatemia and secondary hyperparathyroidism, uremic rats on a high phosphate diet and treated with sevelamer were noted to have less nephrocalcinosis compared to uremic rats treated with CaCO3. Uremic control rats fed a high phosphate diet without a phosphate binder had the greatest degree of kidney calcification. Furthermore, uremic rats treated with either of these phosphate binders were found to have decreased calcification in the myocardium and liver compared to uremic control rats. Uremic rats on sevelamer also had better renal function after 3 months compared to uremic controls and those rats on CaCO3. There was no evidence of aortic vascular calcification after 3 months of uremia in either uremic controls or those rats treated with phosphate binders24.
Based on these findings, the present studies were conducted in uremic rats fed a high phosphate diet to address any differences between 6-month treatment with sevelamer and CaCO3 on vascular calcification, kidney calcification, and renal function. Our findings indicate that sevelamer attenuated kidney and cardiovascular calcification more adequately than CaCO3, despite a similar control of serum phosphorus, Ca P product, parathyroid hormone (PTH), and parathyroid gland enlargement.
METHODS
Experimental design
Renal insufficiency was induced by 5/6 nephrectomy in 5 to 6 weeks-old female Sprague-Dawley rats, weighing 200 to 225 g. In this procedure, several branches of the left renal artery were ligated and the right kidney excised. After 7 days of uremia, blood was taken to determine serum creatinine, calcium, and phosphorus. Rats were subsequently divided into three experimental groups, each with similar serum creatinine and serum Ca P product values. The three groups of uremic rats were fed a high phosphorus diet (0.9% phosphorus; 0.6% calcium, and 20% protein) for 6 months. Group 1 (38 rats), uremic 5/6 nephrectomized rats (U) fed a high phosphorus diet (HP) (U-HP), received no phosphate binder and served as the uremic control for the deleterious effects of prolonged high phosphorus intake. Two phosphate binders were compared. Group 2 (30 rats), U-HP + CaCO3 (U-HP + C), received 3% CaCO3 as the phosphate binder, whereas group 3 (29 rats), U-HP + sevelamer (U-HP + S), received 3% sevelamer. An additional group of rats with normal kidney function was fed a similar high phosphorus diet and served as a nonuremic control group. Powdered diets were purchased from Dyets, Inc. (Bethlehem, PA, USA). CaCO3 (Sigma Chemical Co., St. Louis, MO, USA) and sevelamer hydrochloride (RenaGel®) (GelTex Pharmaceuticals, Inc., Waltham, MA, USA) were added to the powdered, high phosphorus diet. The dose (3%) of sevelamer and CaCO3 was defined in pilot studies which demonstrated that 3% sevelamer and CaCO3 were equally effective in reducing serum phosphorus in uremic rats fed the same high phosphorus diet utilized in the present studies. On average, rats consumed approximately 20 g of powdered diet per day.
Following 5/6 nephrectomy, rats were weighed monthly and then placed into metabolic cages during the last 5 days of the 6-month treatment. Twenty-four–hour urine samples were collected and daily dietary intake monitored. Average 24-hour urinary excretion of calcium, phosphorus, and protein was measured during the last 3 days of treatment. After 6 months, rats were killed and blood was drawn from the dorsal aorta for analytic determinations. Parathyroid glands were microsurgically removed and weighed using a microbalance (CAHN-31) (Orion Instruments, Inc., Boston, MA, USA).
The remnant kidney and the entire myocardium were removed from rats at the time of the sacrifice, weighed on a microbalance, and cut into three pieces perpendicular to the longitudinal axis. One piece from each tissue was rinsed in phosphate buffered saline (PBS) and embedded in paraffin. The two remaining pieces were carefully snap-frozen for calcium deposition analysis.
Aortas were dissected and three (0.5 cm) pieces from the descending thoracic aorta to the iliac branch were removed per rat (at approximately the same anatomic level). Each sample was then cut into two pieces. One piece was weighed on a microbalance and retained for measurements of calcium deposition; the other was rinsed in PBS and embedded in paraffin.
All experimental protocols were approved by the Animal Studies Committee at Washington University School of Medicine.
Analytic determinations
Urine samples were acidified and analyzed for 24-hour excretion of creatinine, total protein, calcium, and phosphorus. Serum and urinary levels of phosphate and creatinine, as well as urinary protein, were determined using an autoanalyzer (COBAS-MIRA Plus, Branchburg, NJ, USA). Total serum and urinary calcium were measured by atomic absorption spectrophotometry using a Perkin-Elmer 1100B spectrophotometer (Perkin-Elmer, Norwalk, CT, USA). Creatinine clearances were calculated using the standard formula:
where CCr is creatinine clearance, UCr is urinary clearance, Vu is urinary volume, and SCr is serum creatinine. Urinary calcium, phosphorus, and protein were expressed as milligrams excreted in 24 hours. Intact PTH levels were measured by an immunoradiometric assay specific for intact rat PTH (Immutopics, San Clemente, CA, USA).
Quantification of calcium deposition in kidney, myocardium, and aorta
Calcium content in remnant kidney, myocardium, and aorta was measured as previously described by Jono et al13. Tissue (three samples from each remnant kidney, myocardium, and aorta) was weighed and decalcified with 0.6 N HCl for 24 hours. Calcium content in HCl supernatants was determined by atomic absorption spectrophotometry. Results were corrected by wet tissue weight and expressed as micrograms calcium/gram wet tissue.
Kidney, myocardial, and aortic morphology
Five-micrometer tissue sections were stained with hematoxylin-eosin and processed for light microscopy. Sections were examined for calcium deposition using von Kossa staining. Briefly, slides were deparaffinized, hydrated with water, incubated in 5% silver nitrate solution (Sigma S-01334) (Sigma Chemical Co.) for 1 hour, rinsed four times with distilled water, followed by a thiosulphate solution for 5 minutes, and counterstained with nuclear fast red solution for 5 minutes. Slides were then rinsed in tap water, dehydrated, cleared in 95% ethyl alcohol, 100% ethyl alcohol, and xylene, and cover slips were mounted. Semiquantitative assessment of calcification was performed as follows. The entire kidney, myocardium, and aortic sections were examined and all foci of calcification were counted (four sections per animal and a total of six rats per group). Precise localization of calcium deposits within the myocardium was aided by immunohistochemical staining of endothelial cells with antibody to factor VIII. Briefly, slides were deparaffinized, hydrated, immersed in citrate buffer (pH = 6.0) for 10 minutes (2), and stained with polyclonal antibody to human factor VIII (1:1000) overnight (Sigma Chemical Co.). Slides were then rinsed, incubated with universal Link antibody (Biocare, CA, USA), and developed with diabenzidine (DAB). Endothelial cells lining arterioles and capillaries were highlighted with this stain. Histologic features were quantified by three different individuals blinded to treatment.
Statistical analyses
Analysis of variance (ANOVA) was employed to assess statistical differences between all experimental groups, except for mortality rates, in which a chi-square test was performed. Multiple comparisons using the stringent Bonferroni test measured the statistical significance of the differences between every possible two-group comparison. An unpaired, two-tailed t test was used to compare time points at baseline and 6 months within experimental groups.
RESULTS
Serum biochemistries (i.e., calcium, phosphorus, Ca P product, creatinine, and cholesterol), renal function, markers of secondary hyperparathyroidism, and calcium deposition in the kidney, myocardium, and aorta were compared between the three groups of rats (U-HP, U-HP + C, and U-HP + S).
Effects of sevelamer and CaCO3 on serum biochemistries
After 6 months of uremia, serum creatinine increased from baseline in U-HP and U-HP + C rats, but not in U-HP + S rats Table 1. Serum phosphorus and Ca P product were significantly lower in both U-HP + S and U-HP + C rats compared to U-HP control rats. Although there was a trend toward lower levels in U-HP + S rats, no statistical difference was found in serum phosphorus and Ca P product between U-HP + S and U-HP + C rats (P = 0.29 comparing phosphorus levels). Serum calcium levels were also similar among the three uremic groups after 6 months of uremia. Acid-base balance (serum bicarbonate and pH) and cholesterol levels were not statistically different among the three uremic groups.
Effects of sevelamer and CaCO3 on body weight
Baseline mean body weights for U-HP, U-HP + S, and U-HP + C groups, respectively, were 217 
21 g, 215 18 g, and 209 9 g. Those rats surviving to 6 months of uremia in U-HP, U-HP + S, and U-HP + C groups had the following mean body weights: 298 10 g, 332 11 g, and 294 9 g, respectively. U-HP + S rats had significantly higher body weights compared to U-HP and U-HP + C rats after 6 months of uremia.
Effects of sevalemer and CaCO3 on mortality
Mortality rates at six months were high among all uremic groups: U-HP 84%, U-HP + S 79%, and U-HP + C 80%. These values did not differ significantly between groups (P = 0.64).
Effects of sevelamer and CaCO3 on creatinine clearance, urinary protein, calcium, and phosphorus excretion
As shown in Table 2, creatinine clearance was equally impaired in CaCO3-treated rats and uremic controls. Sevelamer-treated rats had higher creatinine clearances and lower urinary protein excretion compared to the other two groups of uremic rats. Treatment with CaCO3 was associated with higher urinary calcium excretion compared to uremic controls, but did not differ with that of sevelamer treatment.
Effects of sevelamer and CaCO3 on secondary hyperparathyroidism
As noted in Figure 1a, the average serum PTH in untreated uremic rats fed a high phosphate diet was 2085 616 pg/mL with a 95% confidence interval (95% CI) ranging from 503 to 3667 pg/mL. The elevation in serum PTH induced by high dietary phosphorus in uremic controls was markedly attenuated by treatment either with sevelamer (528 158 pg/mL, P < 0.01; 95% CI 122 to 934) or CaCO3 (708 285 pg/mL, P < 0.01; 95% CI 0 to 1439). Mean serum PTH levels did not differ significantly according to treatment with either sevelamer or CaCO3 (P = 0.30). Mean serum PTH levels in nonuremic rats fed a high phosphorus diet were 27.1 7.0 pg/mL; 95% CI 9.2 to 44.8 (data not shown in Figure 1a). Figure 1b shows that untreated uremic rats developed a marked increase in parathyroid gland weight (6.33 1.52 
g/g body weight) compared to uremic rats treated with phosphate binders. Treatment with both sevelamer (2.77 0.27 g/g body weight, P < 0.01) and CaCO3 (2.99 0.39 g/g body weight, P < 0.01) was associated with reduced enlargement of parathyroid glands compared to uremic controls.
Figure 1.
Effects of sevelamer and calcium carbonate (CaCO3) on serum parathyroid hormone (PTH) and parathyroid gland growth. Serum PTH (A) and parathyroid gland weight (B) in uremic (5/6 nephrectomized) rats undergoing one of the following experimental protocols for 6 months: (
) uremic control + high phosphorus diet (U-HP); (
) uremic + high phosphorus diet + 3% sevelamer (U-HP + S); (
) uremic + high phosphorus diet + 3% CaCO3 (U-HP + C). Results represent the mean SEM from six rats per group. P values were obtained by analysis of variance (ANOVA) and Bonferroni tests.
Effects of sevelamer and CaCO3 on calcium deposition in kidney, myocardium, and aorta
Table 3 shows calcium content in kidney, myocardium, and aorta in all experimental groups. All three groups of uremic rats had markedly increased calcium deposition in the kidney compared to normal, nonuremic rats fed the same high phosphate diet. However, a dramatic reduction of renal calcium content was observed in the sevelamer group compared to both uremic controls and the CaCO3 group. Interestingly, myocardial calcium content was even higher in CaCO3 group than in uremic controls. In contrast, sevelamer-treated rats developed less myocardial calcium deposition compared to CaCO3-treated rats. Untreated uremic rats had increased calcium content in the aorta compared to normal rats. Sevelamer treatment greatly reduced aortic calcium deposition compared to both uremic controls and CaCO3-treated rats.
Histologic effects of sevelamer and CaCO3 on kidney calcifications
Hematoxylin-eosin and von Kossa staining of representative kidney sections from each experimental group are shown in Figure 2. Kidney calcification was greater in untreated uremic Figure 2 b and f and CaCO3-treated rats Figure 2 d and h compared to normal animals Figure 2 a and e fed the same high phosphate diet. Kidney calcification was markedly lower in uremic rats treated with sevelamer Figure 2 c and g. In addition, semiquantitative analysis of kidney calcification Figure 3a showed an association between chemical measurement of calcium content and the number of foci of calcification, being lower in the sevelamer group than in uremic controls and the CaCO3 group.
Figure 2.
Effects of sevelamer and calcium carbonate (CaCO3) on kidney calcification. Representative photomicrographs of hematoxylin-eosin (A to D) and von Kossa (E to H) staining in remnant kidney tissue of normal and 5/6 nephrectomized rats undergoing one of the following experimental protocols for 6 months: normal + high phosphorus diet (N-HP) (A and E), uremic control + high phosphorus diet (U-HP) (B and F), uremic + high phosphorus diet + 3% sevelamer (U-HP + S) (C and G), uremic + high phosphorus diet + 3% CaCO3 (U-HP + C) (D and H). Magnification 400.
Figure 3.
Semiquantitative analysis of foci of calcifications in kidney, myocardium, and aorta. Mean of foci of calcification in kidney (A), myocardium (B), and aorta (C) in uremic (5/6 nephrectomized) rats undergoing one of the following experimental protocols for 6 months: () uremic control + high phosphorus diet (U-HP), () uremic + high phosphorus diet + 3% sevelamer (U-HP + S), (
) uremic + high phosphorus diet + 3% calcium carbonate (CaCO3) (U-HP + C). Normal rats do not have any calcification. Results represent the Mean SEM for six rats per group. P values were obtained by analysis of variance (ANOVA) and Bonferroni tests.
Histologic effects of sevelamer and CaCO3 on myocardium and aortic calcifications
Figures 4 and 5 show representative myocardial and aortic sections, respectively, from each experimental group. Both hematoxylin-eosin and von Kossa staining demonstrate the efficacy of sevelamer in attenuating calcification of the myocardium and aorta. Myocardial Figure 4 and aortic Figure 5 sections from untreated Figure 4 b and f and Figure 5 b and f and CaCO3-treated Figure 4 d and h and Figure 5 d and h uremic rats showed more calcification than normal animals Figure 4 a and e and Figure 5 a and e and sevelamer-treated rats Figure 4 c and g and Figure 5 c and g. Calcifications in the myocardium were specifically localized within capillaries, as demonstrated in heart sections stained with an antibody to the endothelial marker factor VIII Figure 6. No calcifications were observed within the cardiac interstitium. Calcifications in the aorta were localized within the intimal and medial layers. Figure 3 b and c also depict a higher number of foci of calcification in the myocardium and aorta of uremic controls and CaCO3-treated rats, which parallels the higher calcium content in these tissues measured biochemically. Again, sevelamer-treated rats had markedly reduced foci of calcification in myocardium and aorta compared to the other two uremic groups.
Figure 4.
Effects of sevelamer and calcium carbonate (CaCO3) on myocardial calcification. Representative photomicrographs of hematoxylin-eosin (A to D) and von Kossa (E to H) staining in myocardial tissue of normal and 5/6 nephrectomized rats undergoing one of the following experimental protocols for 6 months: normal + high phosphorus diet (N-HP) (A and E), uremic control + high phosphorus diet (U-HP) (B and F), uremic + high phosphorus diet + 3% sevelamer (U-HP + S) (C and G), uremic + high phosphorus diet + 3% CaCO3 (U-HP + C) (D and H). Magnification 400.
Figure 5.
Effects of sevelamer and calcium carbonate (CaCO3) on aortic calcification. Representative photomicrographs of hematoxylin-eosin (A to D) and von Kossa (E to H) staining in aortic tissue of normal and 5/6 nephrectomized rats undergoing one of the following experimental protocols for 6 months: normal + high phosphorus diet (N-HP) (A and E), uremic control + high phosphorus diet (U-HP) (B and F), uremic + high phosphorus diet + 3% sevelamer (U-HP + S) (C and G), uremic + high phosphorus diet + 3% CaCO3 (U-HP + C) (D and H). Magnification 400.
Figure 6.
Intravascular localization of myocardial calcification. Representative photomicrograph demonstrating calcifications within myocardial capillaries. Calcifications and factor VIII (a specific endothelial cell marker) stain brown (magnification 800).
DISCUSSION
These studies provide a long-term, experimental model of the influence of uremia and the phosphate binders, sevelamer and CaCO3, on vascular and kidney calcification in rats with CRF. Aortic calcification was evident after 6 months of uremia, unlike our previous 3-month study24. Furthermore, despite a similar efficacy in controlling serum phosphorus, Ca P product, and secondary hyperparathyroidism, those uremic rats treated with sevelamer developed less vascular calcification in the myocardium and aorta and less nephrocalcinosis compared to uremic rats treated with CaCO3. Interestingly, in those rats surviving to 6 months, treatment with sevelamer also attenuated the deterioration in renal function seen in uremic controls and CaCO3-treated rats. Treatment with either phosphate binder had no influence on mortality compared to uremic controls.
In recent years, vascular calcification in patients with CRF and end-stage renal disease (ESRD) has been increasingly recognized as an important contributor to overall cardiovascular morbidity and mortality. The exact factors and mechanisms regulating vascular calcification remain incompletely defined, yet there is increasing evidence that this is an active, cell-mediated process10,11,13,14,25.
Many factors, such as high phosphorus, calcium, and vitamin D therapy, may contribute to the development of vascular calcification25. Various in vitro studies have demonstrated that phosphorus and unidentified factors in uremic serum play a potential role in the pathogenesis of vascular calcification13,14. Such studies have demonstrated an increased expression of osteoblast-specific proteins and calcification by vascular smooth muscle cells in response to high levels of serum phosphorus and uremic serum. Hyperphosphatemia itself has been identified as an independent risk factor for increased cardiovascular mortality in patients with ESRD25. Consequently, the control of hyperphosphatemia with phosphate binders in patients with CRF and ESRD has been appropriately emphasized.
Phosphate binders currently used to manage hyperphosphatemia in ESRD patients include sevelamer and the calcium-containing binders, CaCO3, and calcium acetate. Sevelamer is an aluminum- and calcium-free phosphate binder, which does not promote hypercalcemia and has favorable effects on the lipid profile, with an associated reduction in low-density lipoprotein (LDL) and increase in high-density lipoprotein (HDL) cholesterol21. A potential concern with the use of calcium-containing phosphate binders has been suggested over the past few years. A study of young hemodialysis patients by Goodman et al1 noted a correlation between coronary artery calcification detected by electron beam computed tomography and duration of dialysis, serum phosphorus levels, serum Ca P product, and daily intake of calcium. Another study in 200 hemodialysis patients by Chertow, Burke, and Raggi15 showed that sevelamer attenuated the progression of coronary and aortic calcification better than calcium-based phosphate binders after 1 year. Subjects treated with sevelamer had lower serum calcium, total cholesterol, and LDL levels compared to subjects treated with calcium-based phosphate binders. Thus, sevelamer's effects on vascular calcification may have been secondary to its lower calcium load versus lipid-lowering properties. Furthermore, the influence of type of the phosphate binder on cardiovascular morbidity and mortality has yet to be prospectively studied in humans.
Our study in long-term experimental uremia shows that treatment with sevelamer, when compared to CaCO3, is associated with less vascular calcification within the myocardium, aorta, and kidney. One potential explanation for the increased vascular calcification in CaCO3-treated rats could be the greater severity of renal dysfunction in this group compared to sevelamer-treated rats. A recent in vitro study by Chen et al14 demonstrated that there are unidentified factors in uremic serum that potentiate vascular calcification. In support of this concept, uremic control rats in our study also had worse renal function and excessive aortic calcification compared to sevelamer-treated rats.
Hypothetically, a difference in lipid metabolism is yet another potential contributing factor for the increased vascular calicification noted in CaCO3-treated rats compared to sevelamer-treated rats. In vitro studies have shown that acetylated LDL stimulates26 while HDL inhibits27 vascular smooth muscle cell calcification. In our study, total cholesterol levels of CaCO3-treated rats trended lower than sevelamer-treated rats, a finding which could be explained by reduced dietary intake in the setting of worse renal function. LDL and HDL levels were not evaluated in our study. In human studies, sevelamer has been shown to consistently reduce LDL and frequently increase HDL levels. Such an improved lipid profile could potentially play a role in the lower degree of vascular calcification seen in sevelamer-treated rats. However, as already noted, the CaCO3-treated rats had lower total cholesterol levels and LDL levels typically parallel such levels.
In addition to a lower degree of vascular calcification, treatment with sevelamer for 6 months was associated with less kidney calcification compared to CaCO3. These results are similar to those published by our group involving a 3-month experimental model of uremia. Interestingly, as noted above, renal function, as measured by serum creatinine, creatinine clearance, and proteinuria, was preserved in those rats treated with sevelamer and surviving to 6 months. The reason for this is unclear and remains speculative. Nephrocalcinosis may have been a contributing factor, as suggested by our previous studies24. Uremic rats in each treatment group were fed a diet with similar phosphorus and protein content, thus minimizing the effect of other potential confounding factors known to influence the progression of renal failure. It is important to note that, despite the preserved renal function and lower vascular calcification in sevelamer-treated rats, there were high mortality rates in all uremic groups, which did not differ significantly.
CONCLUSION
These studies of long-term experimental uremia suggest that, despite similar control of hyperphosphatemia and other markers of secondary hyperparathyroidism, sevelamer may be more effective than CaCO3 in attenuating cardiovascular and kidney calcification. Potential mechanisms mediating these differences are only in the early stages of elucidation.
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Acknowledgments
This research was supported in part by grants from Research in Renal Diseases, Washington University, and from Genzyme Pharmaceutical. Dr. Slatopolsky is a consultant for Geltex Pharmaceutical.
