Chronic renal failure (CRF) is associated with the development of secondary hyperparathyroidism and vascular calcifications. We evaluated the efficacy of PA21, a new iron-based noncalcium phosphate binder, in controlling phosphocalcic disorders and preventing vascular calcifications in uremic rats. Rats with adenine-diet-induced CRF were randomized to receive either PA21 0.5, 1.5, or 5% or CaCO3 3% in the diet for 4 weeks, and were compared with uremic and nonuremic control groups. After 4 weeks of phosphate binder treatment, serum calcium, creatinine, and body weight were similar between all CRF groups. Serum phosphorus was reduced with CaCO3 3% (2.06 mM; P ≤ 0.001), PA21 1.5% (2.29 mM; P < 0.05), and PA21 5% (2.21 mM; P ≤ 0.001) versus CRF controls (2.91 mM). Intact parathyroid hormone was strongly reduced in the PA21 5% and CaCO3 3% CRF groups to a similar extent (1138 and 1299 pg/ml, respectively) versus CRF controls (3261 pg/ml; both P ≤ 0.001). A lower serum fibroblast growth factor 23 concentration was observed in the PA21 5%, compared with CaCO3 3% and CRF, control groups. PA21 5% CRF rats had a lower vascular calcification score compared with CaCO3 3% CRF rats and CRF controls. In conclusion, PA21 was as effective as CaCO3 at controlling phosphocalcic disorders but superior in preventing the development of vascular calcifications in uremic rats. Thus, PA21 represents a possible alternative to calcium-based phosphate binders in CRF patients.
Accelerated vascular calcification is associated with increased incidence of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD) (Foley et al., 1998). Arterial layers are more frequently and more intensively calcified in uremic patients than in nonuremic patients. Several metabolic abnormalities associated with CKD are believed to contribute to calcification, including the development of hyperphosphatemia, hypercalcemia, secondary hyperparathyroidism, inflammation, and oxidative stress (Goodman et al., 2000). Although the mechanisms whereby smooth muscle cells calcify in chronic renal failure (CRF) are not completely understood, the process appears to result from a complex interplay between factors that activate and inhibit tissue calcification (Haller et al., 1995; Hughes, 1995; Moe et al., 2002; Chen et al., 2010; Hruska et al., 2011). Matrix vesicles initiate mineral nucleation during skeletogenesis; similar vesicular structures are deposited at sites of pathologic vascular calcification. In vitro studies have shown that elevated levels of extracellular calcium and phosphorus can induce mineralization of vascular smooth muscle cells (Shanahan et al., 1999).
Recognition that hyperphosphatemia is a predictor of cardiac mortality has highlighted the importance of controlling hyperphosphatemia in patients with CKD using phosphate-lowering treatments (Raggi et al., 2002). Even though decreased consumption of phosphate-rich food is generally prescribed in CKD patients, use of phosphate binders such as calcium-based phosphate binders and, more recently, calcium-free compounds, such as sevelamer carbonate and lanthanum carbonate, is required to control serum phosphate (Chertow et al., 2002; Cozzolino et al., 2002; Phan et al., 2005; Persy et al., 2006b; Spasovski et al., 2006; Damment et al., 2011).
Fibroblast growth factor 23 (FGF23), a phosphaturic hormone that plays an important role in phosphate homeostasis, was recently identified as a causative factor in phosphate-wasting disorders, such as tumor-induced osteomalacia and autosomal dominant hypophosphatemic rickets (Gutierrez et al., 2009). FGF23 secretion from bone increases as renal function declines. Phosphate retention occurs early in the course of renal failure, and there are data in favor of the hypothesis that this is the principal abnormality of secondary hyperparathyroidism. FGF23 increases urinary phosphate excretion and can be viewed as an adaptive response to elevated phosphate load (Gutierrez et al., 2009). Klotho, an obligate coreceptor for FGF23, is a transmembrane protein mainly expressed in renal tubular cells, the parathyroid gland, and the choroid plexus. High FGF23 levels are associated with death and cardiovascular events in subjects with chronic kidney disease, even before the initiation of dialysis (Gutierrez et al., 2009).
There is considerable interest in identifying novel therapeutic strategies to control serum phosphorus without concomitant calcium overload. The new iron-based, calcium-free oral phosphate binder PA21 may offer an advantageous safety profile and an alternative to calcium-based phosphate binders. PA21 is a mixture of polynuclear iron(III)-oxyhydroxide (pn-FeOOH), sucrose, and starches. It contains approximately 33% (mass/mass) of pn-FeOOH, approximately 30% (mass/mass) of sucrose, approximately 28% (mass/mass) of starches and ≤ 10% (mass/mass) of water. The iron content in PA21 is approximately 21% (mass/mass). Addition of carbohydrates prevents iron oxyhydroxide from aging and maintains its phosphate binding capacity (Geisser and Philipp, 2010). Recent clinical studies have demonstrated that PA21 is an effective phosphate binder (Hergesell and Ritz, 1999; Geisser and Philipp, 2010; Wüthrich et al., 2013); however, whether PA21 has a favorable effect on vascular calcifications has not been investigated to date.
In rats, severe hyperparathyroidism occurs when uremia is chemically induced by addition of adenine to the diet (Tamagaki et al., 2006). Adenine is metabolized to 2,8-dihydroxyadenine, which precipitates as crystals in the microvilli and apical region of the proximal tubular epithelia, thereby inducing severe CRF (Yokozawa et al., 1986). The resulting CRF is characterized by increased levels of serum creatinine and phosphorus (Neven et al., 2009). This model, therefore, provides a rapid and reliable method of inducing severe secondary hyperparathyroidism (Tamagaki et al., 2006).
The aim of the present study was to evaluate the effects of PA21 compared with CaCO3 on serum phosphorus, calcium, intact parathyroid hormone (iPTH), and FGF23 levels, and to examine a potential effect on the development of vascular calcifications in an adenine-induced rat model of CRF.
Materials and Methods
All experiments were conducted in accordance with local guidelines for the care and use of experimental animals (National Institutes of Health publication No. 85–23; http://grants.nih.gov/grants/olaw/references/laba94.htm), and the protocol was accepted by the veterinary authorities. Male Wistar rats (Charles River Laboratories, L'Arbresle, France) were housed in polycarbonate cages in a pathogen-free, temperature-controlled (25°C) facility with a strict 12-hour light/dark cycle. The rats had free access to chow diet and water. To induce CRF (Yokozawa et al., 1986), 10-week-old rats were fed with a diet containing 0.75% adenine and a high phosphorus content (1.3% phosphorus, 1.06% calcium, 1000 IU/kg vitamin D3, and 23% protein) (Kliba Nagaf 3200, supplemented with phosphorus and calcium; Provimi Kliba AG, Kaiseraugst, Switzerland) for 4 weeks. After 4 weeks, adenine was withdrawn from the high-phosphate diet (Supplemental Fig. 1). Blood sampling was performed at the tail vein, and rats were randomly assigned to one of five treatment groups for an additional 4 weeks: three groups of CRF animals were treated with PA21 mixed into the diet at three different concentrations [0.5% (CRF PA21 0.5%; n = 9), 1.5% (CRF PA21 1.5%; n = 9), or 5% (CRF PA21 5%; n = 20)]; one CRF group was treated with CaCO3 3% mixed into the diet (CRF CaCO3 3%; n = 20); and one CRF control group did not receive any treatment (CRF control group; n = 20).Three groups of non-CRF animals comprising non-CRF control, non-CRF CaCO3 3%, and non-CRF PA21 5% (n = 8 per group) were constituted and fed with the same diet without adenine for the same duration of 4 weeks. In previous uremic rat studies, a concentration of 3% CaCO3 was commonly used (Cozzolino et al., 2002). The percentages of active moieties for calcium carbonate (calcium) and PA21 (iron) are similar if the concentrations are 3% for CaCO3 and 5% for PA21. Three percent of CaCO3 corresponds to 1.2% of calcium, and 5% of PA21 corresponds to 1% of iron. After 4 weeks of treatment, rats were sacrificed. Twenty-four hours before sacrifice, an arterial catheter was inserted under light isoflurane anesthesia to measure intra-arterial blood pressure and heart rate in awake conditions. At sacrifice, blood was taken through the catheter after anesthesia with 60 mg/kg body weight sodium pentobarbital via intraperitoneal injection.
PA21, lot 9550000A11 was provided by Vifor (International) Inc. (St. Gallen, Switzerland). CaCO3 Ph Eur (www.safcglobal.com/catalog/product/sial/12010?null), lot 2009.02.0547 was purchased from Hänseler AG (Herisau, Switzerland).
Serum and urine creatinine, calcium, and phosphorus concentrations were measured using a standard colorimetric method (Cobas Mira; Roche, Basel, Switzerland). Serum iPTH was measured using a rat bioactive iPTH enzyme-linked immunosorbent assay kit (Alpco Diagnostics, Salem, NH) that was specific for the full-length intact 1–84 form of the molecule. Serum C-reactive protein (CRP), FGF23, and interleukin (IL)-1 and -6 were assessed using commercial enzyme-linked immunosorbent assay kits specific for rat CRP (Alpco Diagnostics), rat FGF23 (Millipore, Billerica, MA), and IL-6 or IL-1β (both IL kits were obtained from Invitrogen/Life Technologies Corporation, Camarillo, CA). Hematocrit was measured in triplicate immediately at the time of sacrifice by collecting a drop of blood in glass capillaries and spinning in a microfuge for 3 minutes at 12,000 rpm.
Assessment of Vascular Calcifications.
Vascular calcifications were evaluated by von Kossa staining as previously described (Phan et al., 2005). The proximal aorta was removed, fixed in neutral buffered formalin, and embedded upright in the same paraffin block, such that four different levels of proximal aorta (eight cross-sections) were analyzed per animal; 4-μm sections were stained with von Kossa’s method by the same operator. Vascular calcifications were then evaluated histomorphometrically with a magnification of 40×. Double-blind assessments of vascular calcifications were performed on random sections of aorta by separate investigators. The degree of von Kossa positivity was scored semiquantitatively, with scores ranging from 0 to 3 depending on the surface of von Kossa positivity. Score 0 indicated no von Kossa positivity; score 1, focal von Kossa positivity, larger than or not overlying a cell nucleus; score 2, partially circumferential von Kossa positivity in the tunica media of the vessel; and score 3, von Kossa positivity in the tunica media spanning the complete circumference of the vessel (Persy et al., 2006a; Neven et al., 2010).
Quantification of Nitrotyrosine Infiltration in Aortic Rings.
Nitrotyrosine and CD68 infiltrations were assessed as described previously (Phan et al., 2005). The sections of aorta rings were preincubated in peroxidase blocking solution before incubation with biotinylated nitrotyrosine monoclonal mouse antibody (nitrotyrosine: Millipore MAB5404; CD68: Serotec MCA341GA). Sections were treated with peroxidase-labeled streptavidin (Dako, Carpinteria, CA), followed by reaction with diaminobenzidine/hydrogen peroxidase. The aortic rings visible on a 40× magnification were captured on a microcomputer equipped with Leica software (Leica Microsystems, Buffalo Grove, IL) and analyzed by a computerized image analysis program. Semiquantitative assessments of nitrotyrosine and CD68 expressions were done by semiautomatic quantification of brown-field expression specifically inside aorta media, as described previously (Katsumata et al., 2003; Persy et al., 2006a). Data were expressed as the relative proportion (percentage) of the positive staining area of the aorta media to total surface area of each ring.
Blood Pressure and Heart Rate.
Twenty-four hours before sacrifice, surgical anesthesia was induced by placing rats in a chamber through which 5% isoflurane in oxygen was flushed at 1 l/min for 90 seconds (Whitesall et al., 2004). Isoflurane was adjusted (0.75–1.5%) to maintain a surgical plane of anesthesia. Eyes were coated with ophthalmic lubricant. The measurement of blood pressure required the surgical implantation of a catheter into the right femoral artery. The remaining procedures were all performed with the aid of a dissecting stereomicroscope. Catheters were placed in the femoral artery for the measurement of arterial pressure. The arterial catheters were filled with 500 U of heparin in saline, sealed, and opened only when they were used for recording. On the day of sacrifice, the rats were placed in large Plexiglas tubes without noise or visual stimulation for 2 hours. The catheter was attached to a combination pressure transducer, and arterial pressure data were collected using computerized data acquisition software, as previously described (Mattson et al., 1994; Mattson, 1998). Invasive arterial blood pressure was measured through a low compliance pressure line connected to an electronic pressure transducer. The invasive arterial pressure values were registered and stored. Histograms and normality plots were used to confirm that the data met the distributional assumptions. The device automatically adjusted cuff deflation rate and gain for parametric analysis.
Results are expressed as the mean ± S.E.M. Data obtained in the different experimental groups were compared using one-way analysis of variance with the Bonferroni post-hoc test. The only exception is for FGF23 data that do not follow a Gaussian distribution, and where only the median was reported with confidence intervals indicated in the legend to Fig. 2. Nonparametric analysis (i.e., Kruskal-Wallis test followed by Mann-Whitney post-hoc test) was performed on this set of data. χ2 analysis was performed to compare the vascular calcifications. A value of P < 0.05 was considered significant.
Of all the animals used in the study, only eight died because of bleeding when catheterized during the last day of the experiment. These animals were distributed in groups: 4/38 PA21 (with two rats lost in PA21 5%, one in PA21 1.5%, and one in PA21 0.5%), 1/20 CaCO3; 3/20 CRF control. Nevertheless, blood samples could be collected from some of these rats. Therefore, the number of rats listed in Tables 1 and 2 exactly reflects the number used in the statistical analyses.
Blood Pressure and Heart Rate.
No significant differences in systemic blood pressure or heart rate were observed when uremic rats were compared with nonuremic animals. Moreover, no differences in blood pressure and heart rate were observed between uremic rats treated with CaCO3 3% or PA21 at any dose (unpublished data).
After 4 weeks of adenine administration, serum creatinine concentrations in CRF rats were 4 times higher, and body weight was significantly lower, than in non-CRF animals (Supplemental Fig. 1; Table 1). Serum creatinine slightly decreased in all CRF treatment groups, including control animals, following cessation of adenine administration, and remained 4 times higher compared with the non-CRF group (Table 2). At sacrifice, after 4 weeks of phosphate binder treatment, there was no difference in serum creatinine between the groups receiving PA21 or CaCO3 3% versus the CRF control arm (Table 2). There was no observable difference in weight between the different PA21 and CaCO3 3% groups, and weight remained low in CRF animals even 4 weeks after adenine was withdrawn (Fig. 1; Table 2).
As expected, the development of CRF was associated with an increase in serum phosphorus, although serum calcium was still comparable in animals with or without CRF at the time of randomization (Table 1). PA21 induced a dose-dependent reduction in serum phosphorus, with PA21 5% resulting in a normalization of phosphorus levels (Table 2). Mean serum phosphorus concentration in the PA21 5% group was comparable to that obtained with CaCO3 3% (Table 2), despite a greater reduction in serum phosphorus from randomization to the end of the study with CaCO3 3%. Change in serum calcium from randomization to the end of the study was similar for all groups receiving phosphate binder. Phosphaturia was dramatically decreased in all uremic groups treated with phosphate binder, especially PA21 5% and CaCO3 3%, compared with the CRF control group (Table 3). CRF rats developed marked hyperparathyroidism, as reflected by elevated iPTH levels at sacrifice (Table 2). PA21 exerted a dose-dependent reduction in iPTH, with similar levels observed after 4 weeks of treatment with either PA21 5% or CaCO3 3%.
Uremia was associated with a higher serum FGF23 concentration; serum FGF23 was increased in the CaCO3 3% group and the CRF control group compared with CRF PA21 5%. No statistical difference was observed between CRF CaCO3 3% and CRF control (Fig. 2).
CRF animals developed anemia, as measured by hematocrit at sacrifice, the extent of which was similar in the CRF control group and across all treatment arms (Table 2). Serum ferritin concentration was increased in all CRF groups compared with non-CRF rats. In treated CRF groups, serum ferritin was increased, compared with the CRF control group, and reached statistical significance in the CRF CaCO3 3% arm (Table 2). No differences were observed between the different CRF groups for serum CRP, IL-1, and IL-6 or for the quantification of nitrotyrosine and CD68 staining in the thoracic aorta (data not shown).
Impact on Vascular Calcifications.
In total, 77 rats were evaluated. Due to a technical defect of von Kossa staining, 3 of 20 CRF control aortas, 2 of 20 CRF PA21 5%, and 1 of 20 CaCO3 3% could not be evaluated. CRF control animals developed marked vascular calcifications, with 13 of 17 (76%) showing severe vascular calcifications as defined by a score of 2–3 (Fig. 3; Table 4). PA21 5%, as well as 1.5%, was associated with fewer rats with a high vascular calcification score (Fig. 3; Table 4). The occurrence of calcifications in rats receiving the lowest dose of PA21 (0.5%) did not differ from that in control CRF rats. An increased score of vascular calcification was associated with a higher serum concentration of FGF23 and creatinine. A similar trend was observed for a high score of calcification and increased serum iPTH concentration (Fig. 4).
This is the first study demonstrating decreased vascular calcifications with PA21 versus uremic controls and CaCO3-treated rats. Importantly, CRF groups were similar in terms of weight and serum creatinine, calcium, and phosphorus concentrations before phosphate binder commencement. PA21 induced dose-dependent decreases in serum phosphorus and iPTH, with PA21 5% exhibiting similar efficacy to CaCO3 3%.
In our study, the weight of all CRF animals remained low even 4 weeks after adenine was withdrawn (Fig. 1). Such a decrease of the body weight in adenine-induced CRF animals has been well described in previous studies (Neven et al., 2009) and is probably due to the occurrence of rapid symptoms of uremia with an absence of appetite during the administration of adenine.
PA21 5% and CaCO3 3% resulted in normalization of serum phosphorus and significantly decreased iPTH levels. Hyperphosphatemia has an important role in vascular calcifications. A graded independent relationship between increasing levels of serum phosphorus and extent of coronary artery calcification in hemodialysis patients has been demonstrated (Goodman et al., 2000). Normalization of serum phosphorus in animal models can reduce or prevent arterial calcification (Moe and Chen, 2008). Studies in uremic animal models showed beneficial effects of noncalcium phosphate binders on hyperphosphatemia, secondary hyperparathyroidism, and prevention of vascular calcification; however, few studies have compared noncalcium phosphate binders with CaCO3 (Katsumata et al., 2003; Phan et al., 2005; Neven et al., 2009). We observed less progression of vascular calcifications in the CRF PA21 5% group versus the CRF CaCO3 3% group, despite comparable serum phosphorus, calcium, and iPTH. Cozzolino et al. (2002) assessed the severity of ectopic calcification in a 5/6 nephrectomy rat model treated with sevelamer or CaCO3 3% for 3 months. Hypercalcemia was observed in the CaCO3 3% group after 3 months. Both binders elicited similar efficacy in reducing uremia-induced myocardial and hepatic calcifications, but sevelamer reduced renal calcium deposition. In another study, the same authors demonstrated that sevelamer attenuated vascular and kidney calcification (Cozzolino et al., 2003). In our study, CaCO3 3% was not associated with worsening vascular calcification, but reduced vascular calcifications were observed versus CRF controls. This could be explained partly by the absence of excess hypercalcemia associated with strict control of phosphorus and iPTH, as shown previously (Phan et al., 2008). One hypothesis is that CaCO3 may, in contrast to the calcium-free phosphate binder PA21, have less of a preventive effect against vascular calcification due to calcium absorption. The clinical importance of maintaining plasma calcium and phosphorus within normal limits, and reducing transient hypercalcemia, for preventing vascular calcification has been confirmed (Kapustin et al., 2011). Moreover, rats with experimentally induced CKD treated with the less-calcemic vitamin D analog, 22-oxa-1α-25-dihydroxyvitamin D3, had reduced aorta medial vascular calcification versus calcitriol-treated rats, supporting a role for altered mineral metabolism in vascular calcification (Hirata et al., 2003). Exposure to calcium intake could result in a positive calcium balance in CKD leading to calcium loading, which may worsen vascular calcification. In a stage 3–4 CKD-study, increased calcium load, when moving from 800- to 2000-mg calcium intake, did not result in increased serum calcium concentration (Spiegel and Brady, 2012). The authors suggested that serum calcium cannot be used as a guide to evaluate calcium balance, and that normal serum calcium concentrations do not preclude calcium loading.
Oxidative stress and inflammation are believed to play roles in calcification and atherosclerosis in uremia (Massy et al., 2005; Mizobuchi et al., 2009; Sowers and Hayden, 2010). Guilgen et al. (2011) revealed increased nitrotyrosine production due to oxidative stress in arteries from CKD patients versus controls. In the present study, the high ferritin levels, as a potential inflammatory marker, were unexpected in the CRF CaCO3 3% group versus CRF controls. We also observed a similar, nonstatistically significant trend for the CRF PA21 5% group. Ferritin metabolism was not examined extensively in this rat model, and no interaction between calcium administration and serum ferritin concentration has been described (Bendich, 2001). Our study did not demonstrate whether PA21 interferes with these processes. No differences in nitrotyrosine, CD68, serum CRP, or IL-1 and IL-6 were observed as potential indicators of inflammatory state or oxidative stress.
As there were no changes in blood pressure in CRF rats, hemodynamic changes do not explain observed differences in vascular calcifications. Six et al. (2012) investigated the effects of phosphate loading and sevelamer hydrochloride (HCl) on vascular function, and demonstrated that sevelamer HCl improved endothelial dysfunction, aortic systolic expansion rate, and pulse wave velocity in CKD mice on a normal diet (0.65% phosphate). Vascular calcifications were not observed in this uremic C57/BL6 mice model. In our study, we used a different model, a higher-phosphorus (1.3%) diet, and shorter treatment duration (4 versus 8 weeks).
High FGF23 levels are associated with death and cardiovascular events in subjects with chronic kidney disease (Gutierrez et al., 2008). In early CKD stages, circulating FGF23 levels increase with declining renal function (Gutierrez et al., 2008), as a physiologic compensation to stabilize serum phosphorus levels. Arterial calcification is an early characteristic of vascular injury in CKD. Some clinical studies suggest an association between elevated FGF23 and vascular calcification (Jean et al., 2009). In our study, we observed a positive association between high calcification score and FGF23. Whether the FGF23 can directly inhibit calcification or whether the effect is indirect due to reduced availability of calcification promoting mineral ions is still being debated. Preliminary clinical attempts have been made to suppress FGF23 upregulation using phosphate binders (Bleskestad et al., 2012; Shigematsu and Negi, 2012; Soriano et al., 2013). In our study, serum FGF23 concentration was higher in the CaCO3 3% group versus the PA21 5% group, despite similar levels of serum calcium, phosphorus, and iPTH, and urinary phosphorus excretion. Data related to the influence of CaCO3 or calcium loading in chronic kidney disease on FGF23 (Isakova et al., 2011) are few and sometimes conflicting; however, it was shown recently that calcium deficiency reduced FGF23 levels in nonuremic rats (Rodriguez-Ortiz et al., 2012; Yasin et al., 2013). Yasin et al. (2013) showed that calcium correlated positively with FGF23 in adolescents and young adults with CKD.Studies have examined the relationship between FGF23 and vascular calcification in CKD (Srivaths et al., 2011) in an attempt to establish whether FGF23 has a direct role in vascular calcification or whether it modulates calcification indirectly through complex interactions with other factors involved in mineral metabolism (Jean et al., 2009). However, such a link conflicts with recent results (Scialla et al., 2013); in this study, the unadjusted prevalence of vascular calcification was greater in the higher quartiles of FGF23, but the effect was abolished when further adjustments were made for cardiovascular risk factors, and FGF23 appeared to have no direct effect on calcification of cultured mouse aortic rings (Scialla et al., 2013). A 6-week dose-titration study evaluated effects of calcium acetate or sevelamer HCl on PTH and FGF23 levels in normo-phosphoremic patients with stage 3–4 CKD. Reduced FGF23 levels were not observed in patients receiving calcium acetate (Oliveira et al., 2010). Despite similar efficacy in terms of intestinal phosphate binding, phosphate binders exert disparate effects on serum FGF23 concentrations (Oliveira et al., 2010; Yilmaz et al., 2012). These results indicate that complex associations between different factors contribute to a high prevalence of cardiovascular events in CKD patients. Additional studies should evaluate how noncalcium phosphate binders, such as PA21, affect interplay between FGF23 and vascular calcification.
In conclusion, the present study indicates that, if phosphorus and iPTH are well controlled, PA21 can inhibit the development of uremia-related vascular media calcification. These results need to be confirmed in humans. Regarding the impact of calcium-containing phosphate binders on the progression of arterial calcification, the debate is ongoing, but it may be advisable to consider noncalcium alternatives.
Participated in research design: Phan, Maillard, Funk, Burnier.
Conducted experiments: Phan, Maillard, Peregaux, Mordasini, Stehle.
Performed data analysis: Phan, Maillard, Funk, Burnier.
Wrote or contributed to the writing of the manuscript: Phan, Maillard, Funk, Burnier.
- Received March 15, 2013.
- Accepted May 21, 2013.
The work was funded by Vifor (International) Ltd., St. Gallen, Switzerland. O.P. was recipient of a travel congress grant, M.B. is a member of the Board of Galenica, and F.F. is an employee of Vifor (International) Ltd.
This work was previously presented in a poster session at Renal Week 2010: American Society of Nephrology 43rd Annual Meeting; 2010 November 16–21; Denver Colorado.
- chronic kidney disease
- chronic renal failure
- C-reactive protein
- fibroblast growth factor 23
- intact parathyroid hormone
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics