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*Renal Division, Department of Internal Medicine and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri; 
Renal Division, Hospital San Paolo, Milan, Italy; and GelTex Pharmaceuticals, Inc, Waltham, Massachusetts.
Correspondence to Dr. Eduardo Slatopolsky, Renal Division, Box 8126, Department of Internal Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: 314-362-7208; Fax: 314-362-7875; E-mail: eslatopo{at}im.wustl.edu
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Abstract |
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Introduction |
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Dietary P restriction, dialysis treatment, and administration of phosphate-binders are the current therapies for hyperphosphatemia in chronic renal failure. The most commonly used phosphate-binders contain aluminum salts, calcium carbonate (CaCO3), or calcium acetate. Calcium salts increase serum Ca and could worsen soft tissue calcifications, especially in patients on vitamin D therapy. Administration of 1,25(OH)2D3, while suppressing PTH synthesis, increases intestinal Ca absorption and calcium-phosphate mobilization from bone (9–10).
The role of P in the progression of renal failure (11) and the protective effects of P restriction on renal function (12) have been known for more than 20 yr. Chronic renal failure causes a reduction in nephron mass and in P excretion. Phosphate retention not only induces secondary hyperparathyroidism but also accelerates renal failure by promoting renal calcification (13). Gimenez et al. (14) showed a correlation between renal Ca deposition, hyperphosphatemia, and the progression of renal failure in 246 renal biopsies. Patients with serum creatinine levels above 1.5 mg/dl had higher serum Ca x P product, renal Ca content, and histologic Ca deposition (14).
Ibels et al. (15) showed that dietary restriction of P prevents the progression of renal failure in nephrectomized rats. In contrast, high dietary P induced a rapid deterioration of renal function (16). Phosphorus toxicity associates with renal calcium-phosphate precipitation and tubulointerstitial damage, resulting in acceleration of nephrocalcinosis (17).
In 1980, Walser (18) described the association between CaCO3 administration in uremic patients and increases in serum creatinine concentration after 2 to 4 wk of treatment. At the time, he concluded that "... an increase in the serum Ca x P product may accelerate progression of renal failure and suggest caution in the use of calcium supplements for this reason."
To reduce the side effects of the commonly used calcium salts, a new aluminum- and calcium-free phosphate-binder was developed. Poly-allylamine hydrochloride (sevelamer hydrochloride, RenaGel; GelTex Pharmaceuticals, Inc, Waltham, MA) controls serum P levels with no hypercalcemia. Furthermore, sevelamer reduces LDL cholesterol by 30% and increases HDL cholesterol by 18% (19).
The present studies compare sevelamer and CaCO3 in the control of serum P, prevention of secondary hyperparathyroidism, and reduction of renal calcifications in an experimental model of chronic renal failure. Sevelamer and CaCO3 are equally effective in reducing serum P levels and in preventing secondary hyperparathyroidism. Importantly, sevelamer is more effective than CaCO3 in preventing increases in serum Ca x P product and in reducing renal Ca deposition, including better preservation of renal function.
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Materials and Methods |
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All experimental protocols were approved by the Animal Study Committee at Washington University School of Medicine.
Analytical Determinations
Rats were weighed monthly, and blood was drawn (tail vein) at 1, 4, and 8 wk to monitor serum creatinine, Ca, P, and Ca x P product. On the last 5 d of treatment, rats were placed in metabolic cages. Twenty four–hour urine were collected, and daily dietary intake was monitored. Results were taken from the last 3 d of treatment. After 12 wk, rats were anesthetized and sacrificed by exsanguination. Arterial blood (aortic puncture) was drawn for analytical determinations. Urine samples were acidified, and each 24-h urine sample was analyzed for creatinine, calcium, and phosphorus. Plasma and urinary phosphate, and serum and urinary creatinine were determined using an autoanalyzer (COBAS-MIRA Plus, Branchburg, NJ). Total serum and urinary calcium were measured by atomic absorption spectrophotometry using a Perkin-Elmer 1100B spectrophotometer (Perkin-Elmer, Norwalk, CT). Creatinine clearance measurements were calculated using the standard formula: CCr = (UCr x Vu)/SCr. Urinary excretion is expressed as milligrams of total calcium or phosphorus excreted in 24 h. Intact PTH levels were measured by an immunoradiometric assay specific for intact rat PTH (Immunotopics, San Clemente, CA). Parathyroid glands were surgically removed and weighed on a CAHN-31 microbalance (Cahn Instruments, Inc. Cerritos, CA). 1,25(OH)2D3 levels were measured in plasma samples using the solid phase extraction procedure and radioreceptor assay by Hollis et al. (20).
Immunohistochemical Analyses of Parathyroid Glands
Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and transforming growth factor-
(TGF-) was performed on sections of 10% neutral buffered formalin-fixed overnight at 4°C and switched to 70% ethanol, paraffin embedded parathyroid glands following protocols described in previous studies (21). Specificity of the primary antibodies was tested by immunohistochemical staining of rat parathyroid tissue replacing the primary antibody with mouse IgG1. For TGF- immunostaining, parathyroid tissue was pretreated with 0.05% saponin for 30 min at room temperature. Tissue was then blocked with 10% preimmune goat serum and incubated with primary antibody (1.13 µg/ml for PCNA; 10 µg/ml for TGF-) for 12 h at room temperature. Twenty-four consecutive sections of tissue were cut for each parathyroid gland. Immunohistochemical staining was evaluated independently by three different blinded individuals. Ten different tissue sections were analyzed per rat for each experimental condition.
Immunohistochemical staining of PCNA protein was quantitated using a Nikon Diaphot-TMD microscope coupled to a camera and an image analysis system. Images of stained tissue sections were acquired using a DAGE-330 color camera and captured with a Pentium P-166 IBM compatible computer. The digitized images were converted to a gray scale and analyzed using Image-Pro plus software (Media Cybernetics) according to Mize’s study (22) as described before (21). To eliminate variation, the microscope light source intensity used during image capture was kept constant for all sections stained on a given day.
Quantification of Calcium Deposition in Kidney, Myocardium, and Liver
Calcium content in kidney, myocardium, and liver was measured as described by Jono et al. (23). Tissue (three samples for each remnant kidney, myocardium, or liver) was weighed on a CAHN-31 microbalance (Cahn Instruments, Inc) and decalcified with 0.6 N HCl for 24 h. The calcium content of HCl supernatants was determined by atomic absorption spectrophotometry using a Perkin-Elmer 1100B spectrophotometer. Calcium content in each sample was corrected by wet tissue weight and expressed as µg Ca/g wet tissue.
Morphologic Analysis of Kidney Calcification
After sacrifice, the remnant kidney was removed and cleaned of fascia and adipose tissue. Sagittal sections of renal tissue were fixed in buffered formalin. Five-micrometer sections were stained with hematoxylin-eosin and with periodic acid-Schiff (PAS) and then processed for light microscopic evaluation.
The entire tissue section was evaluated for calcium deposition by von Kossa and Alizarin red S stains as follows. For von Kossa stain, slides were deparaffinized and hydrated to water. Five-percent silver nitrate solution (S-01334, Sigma) was placed on the slides and incubated for 1 h. Slides were rinsed four times in distilled water, placed in thiosulfate solution for 5 min, and counterstained in nuclear fast red solution for 5 min. Slides were then rinsed in tap water, dehydrated, cleared in 95% ethylalcohol, 100% ethylalcohol, and xylene, and cover slips were mounted. For Alizarin red S stain, slides were deparaffinized, hydrated, and placed in Alizarin red S solution (Alizarin sodium monosulfonate from A-3757, Sigma). When red-orange color appeared, excess stain was taken off. Slides were counterstained, cleared, and mounted as previously reported (24). For Alizarin red S stain, the tissue was viewed under polarized light. Semiquantitative counts of calcifications were performed as follows. The entire kidney section was examined, and all the foci of calcification were counted (four kidney sections per animal, for a total of five rats per group). Histologic features were quantified by three different individuals blinded to treatment of the rats.
Statistical Analyses
ANOVA was employed to assess statistical differences between all experimental groups tested. Multiple comparisons using the stringent Bonferroni test measured the statistical significance of the differences between every possible two-group comparison. Unpaired two-tailed t test was used to compare baseline and uremia 3-mo time points within experimental groups.
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Results |
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Serum Chemistry
Table 1 shows serum chemistries in uremic rats for all the experimental conditions tested at the beginning of the study and after the 3 mo of treatment. Serum creatinine increased in uremic control animals fed the high-P diet from a basal of 1.5 ± 0.1 to 2.3 ± 0.2 mg/dl (P < 0.01) after 3 mo of uremia. The increase in serum creatinine levels was prevented in uremic rats fed the same high-P diet by treatment with either sevelamer (1.4 ± 0.2 mg/dl; P < 0.05) or CaCO3 (1.7 ± 0.2 mg/dl; P < 0.05).
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As expected, serum total Ca was higher in the uremic rats treated with CaCO3 (10.6 ± 0.1 mg/dl) compared with those receiving sevelamer (9.5 ± 0.1 mg/dl; P < 0.05) or uremic controls (8.6 ± 0.5 mg/dl; P < 0.01).
Serum Ca x P product was reduced in both sevelamer-treated uremic rats (110 ± 6.8 to 61 ± 8.3 mg2/dl2; P < 0.01) and the CaCO3 group (109 ± 3.8 to 80 ± 5.3 mg2/dl2; P < 0.01). Importantly, only in the sevelamer-treated group, the serum Ca x P product differed significantly from that in uremic-untreated animals (61 ± 8.3 mg2/dl2 versus 101 ± 4.5 mg2/dl2; P < 0.05).
Although serum pH decreased with the progression of renal failure in all experimental groups, both sevelamer and CaCO3 prevented the drop in pH below the physiologic range that occurred in uremic controls.
Serum 1,25(OH)2D3 levels did not differ significantly between uremic controls (20.2 ± 3.8 pg/ml) and sevelamer-treated animals (17.0 ± 4.5 pg/ml) but were reduced in the CaCO3 group (10.3 ± 4.3 pg/ml).
Creatinine Clearance, Urinary Calcium, and Urinary Phosphorus
As shown in Table 2, the reduction in creatinine clearance in the U-HP (0.30 ± 0.05 ml/min) group was prevented only by sevelamer treatment (0.60 ± 0.14 ml/min; P < 0.01), whereas CaCO3 had no effect (0.36 ± 0.04 ml/min).
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Table 2 shows that 24-h urinary phosphorus decreased from 201 ± 13 mg/24 h in the uremic control group to 150 ± 15 mg/24 h with sevelamer treatment and to 137 ± 16 mg/24 h with CaCO3. As with serum phosphorus levels, the decrease in urinary phosphate was not different between sevelamer and CaCO3-treated rats.
Effects of Sevelamer and CaCO3 on Serum PTH and Parathyroid Gland Growth
Figure 1A depicts serum PTH levels in each experimental condition. In the untreated uremic rats fed the high-P diet, serum PTH (1808 ± 150 pg/ml) levels were much higher than in the CaCO3- or sevelamer-treated rats fed the same diet. Both sevelamer (387 ± 48 pg/ml; P < 0.01) and CaCO3 (356 ± 50 pg/ml; P < 0.01) prevented the increase in serum PTH induced by high dietary P.
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, a marker associated with uremia- and high P–induced parathyroid hyperplasia. Figure 2 (upper panels) shows higher levels of PCNA and TGF- in a parathyroid gland from uremic rats fed a high-P diet compared with CaCO3- (middle panels) or sevelamer-treated (lower panels) groups. Parathyroid PCNA expression after 3 mo of uremia was lower (40% reduction) in rats fed the high-P diet when treated with either sevelamer or CaCO3. These findings demonstrate that sevelamer is as effective as CaCO3 in reducing both parathyroid hormone secretion and parathyroid-cell growth induced by uremia and high dietary phosphorus.
Effects of Sevelamer and CaCO3 on Calcium Deposition in Myocardium and Liver
Chronic renal failure increased Ca content in rat myocardium and liver compared with animals with normal renal function fed the same high-P diet (13.1 ± 8.5 versus 3.5 ± 1.1 µg/g wet myocardial tissue; 6.1 ± 2.8 versus 2.9 ± 0.6 µg/g wet liver tissue). Both sevelamer and CaCO3 reduced Ca deposition in myocardium and liver. The decrease in Ca content at 3 mo did not differ with either phosphate binder.
Effects of Sevelamer and CaCO3 on Renal Calcium Deposition
Figure 3 shows kidney Ca content in all experimental groups. Uremia markedly increased kidney Ca content compared with rats with normal renal function fed the same high-P diet (175.5 ± 45.7 versus 5.8 ± 0.8 µg/g wet tissue; P < 0.01). Most importantly, a dramatic reduction of renal Ca deposition was observed in the sevelamer group (29.8 ± 8.6 µg/g wet tissue) compared with both uremic controls (175.5 ± 45.7 µg/g wet tissue; P < 0.01) and the CaCO3 group (58.9 ± 13.7 µg/g wet tissue; P < 0.04).
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Discussion |
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Hyperphosphatemia due to decreased P excretion (25,26) worsens secondary hyperparathyroidism, which is commonly present in patients with chronic renal failure. High serum P directly enhances parathyroid cell proliferation and PTH synthesis and secretion (6,7). High P also enhances parathyroid function indirectly by decreasing calcitriol synthesis and serum ionized Ca levels, which further elevates circulating PTH (27,28). High serum PTH induces osteitis fibrosa and bone loss, thus increasing serum Ca x P product (29,30) and ectopic calcification (3,31). In addition to the described effects regarding bone resorption, high PTH may also cause metastatic microcalcifications through elevations in cytosolic Ca (9,10). In rats, Borle et al. (16) showed that high P-induced hyperparathyroidism caused nephrocalcinosis. Elevated levels of serum PTH induced intracellular Ca accumulation and Ca-P deposition in renal tissue (16).
Conversely, P restriction counteracts the mitogenic signals for parathyroid hyperplasia triggered by renal failure, thus preventing parathyroid gland enlargement (7). Furthermore, in an experimental model of established secondary hyperparathyroidism, the switch from high P intake to P restriction normalized serum PTH levels within 1 wk (32). The molecular mechanisms by which phosphate restrictions effectively suppress hyperparathyroidism are incompletely understood. However, it is clear that the control of serum P is critical for effective treatment in renal failure patients. Because of difficulties with patients’ compliance to a P restricted diet, the current treatment of hyperphosphatemia demands phosphate binders. One obvious limitation of calcium-based phosphate binders, increased Ca load and serum Ca in patients with end-stage renal disease (33–35), led to the development of a new phosphate binder, sevelamer. In dialysis patients, this calcium- and aluminum-free compound reduces serum phosphorus and PTH levels with no hypercalcemia (19,36,37).
In the present studies of chronic renal failure in rats, sevelamer treatment reduced serum P independently of increases in serum Ca levels, leading to a lower serum Ca x P product when compared with uremic controls. Sevelamer reduction of serum P appears to mediate its efficacy to control both parathyroid hyperplasia and PTH secretion; serum 1,25(OH)2D3 levels were similar between uremic controls and the sevelamer-treated rats.
Although sevelamer and CaCO3 were equally effective in controlling serum P levels and secondary hyperparathyroidism, no difference in Ca x P product was evident between CaCO3-treated rats and uremic controls. It is clear that an additional factor, such as sevelamer’s improved control of the Ca x P product, mediated the higher efficacy of sevelamer in reducing renal Ca deposition. In fact, Ahmed et al. (38) showed an association between hyperphosphatemia, elevated serum Ca x P product, and calciphylaxis in dialysis patients. Although nephrocalcinosis is not a common factor in the progression of renal failure, Gimenez et al. (17) reported a significant positive correlation between renal Ca content and serum creatinine in patients with impaired renal failure. Biopsied patients with serum creatinine higher than 1.5 mg/dl had higher levels of serum P, serum Ca x P product, and renal Ca content (17). Recent studies by Goodman (2) and Guerin (34) have implicated the dose of calcium-based P binders as a risk for coronary artery calcification in end-stage renal failure patients.
In our uremic rat model, there was no evidence of high P–induced aortic calcifications 3 mo after the onset of renal failure. However, Ca content in rat myocardium and liver was higher than in normal controls. Both sevelamer and CaCO3 were equally effective in reducing Ca deposition in these tissues.
Importantly, despite the similarities of sevelamer and CaCO3 in controlling myocardial Ca deposition in rats after 3 mo of uremia and high-Pe diet, differences were evident when Ca deposition was measured in the kidneys. These findings suggest a tissue-specific and time-dependent sensitivity for Ca x P product induction of calcification (Figure 4). In fact, there was a significant reduction of renal Ca content in sevelamer-treated rats compared with the CaCO3 group, also evident in histologic studies using von Kossa and Alizarin red S staining of kidney sections. Further validation came from the demonstration that sevelamer was more effective than CaCO3 in preventing elevations in the number of foci of calcification compared with uremic controls (Figure 5). These findings suggest that the significant reduction of renal Ca deposition found in the uremic rats treated with sevelamer may be in association with the lower serum Ca x P product compared with uremic controls and CaCO3-treated animals. Moreover, renal function deterioration, assessed by measurements of creatinine clearance, was prevented only in sevelamer-treated rats. No differences in creatinine clearance were observed between the uremic and the CaCO3-treated animals. In fact, the severe tubulointerstitial fibrosis, present in remnant kidneys of uremic rats fed high dietary P (50% of the kidney surface area), was reduced to 30% of the kidney surface area by treatment with CaCO3 and almost abolished in sevelamer-treated rats (5% of the kidney surface area) (Figure 6). These data support the existing evidence on the role of hyperphosphatemia in the deterioration of renal failure in rats and the efficacy of sevelamer in better ameliorating its progression compared with CaCO3.
In 5/6-nephrectomized rats, low dietary P prevented increases in serum creatinine levels, improved kidney histology, and decreased renal Ca content (14). Moreover, in uremic rats fed a normal-P diet (0.5% P), renal histology presented extensive tubulointerstitial lesions and nephrocalcinosis compared with a low-P diet (0.2% P) (39). Furthermore, 5/6-nephrectomized rats placed on high-P (1.0% or 2.0%) diets developed higher renal Ca content and more histologic damage compared with animals on a normal-P (0.5%) diet (11). The demonstration in our experimental model in the rat that both P binders were equally effective in controlling serum P indicates that the lower Ca x P product in the sevelamer group may be the main determinant of its advantage in a better preservation of renal function.
In conclusion, sevelamer is an effective agent in reducing Ca x P product, preventing kidney calcification, and preserving renal function in uremic rats. These findings cannot be extrapolated to human disease, and further studies in patients are necessary to determine the benefits of either (CaCO3 versus sevelamer) P binder.
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Acknowledgments |
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