Disturbed calcium-phosphate metabolism affects cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD), particularly in patients with ESRD.1 , 2 3 , 4 5 3 6 2 D3 ). Although in the past it was assumed that vitamin D therapy is “nephrotoxic,” probably as a result of vitamin D–induced hypercalcemia in patients with CKD,7 2 D3 and its analogues attenuate progression in various CKD models.3 , 8 , 9
We assessed various parameters of calcium-phosphate metabolism including c-terminal and biologically active intact FGF23 plasma concentrations in 227 patients who did not have diabetes and had primary CKD, 177 of whom were prospectively followed for a median of 53 mo. We examined the working hypothesis that FGF23 is a predictor of CKD progression.
RESULTS
Stages of CKD and Calcium-Phosphate Metabolism
Baseline clinical characteristics and laboratory data of 227 patients with CKD are reported in Table 1 . To elucidate the relationship between renal function and parameters of calcium-phosphate metabolism, we stratified renal patients into four groups according to National Kidney Foundation (NKF) criteria for CKD: GFR ≥90 ml/min per 1.73 m2 , GFR 60 to 89 ml/min per 1.73 m2 , GFR 30 to 59 ml/min per 1.73 m2 , and GFR <30 ml/min per 1.73 m2 (Table 1 ). We found a continuous and significant increase of Ca × P, PTH, and both c-terminal and intact FGF23 concentration across the various NKF stages of renal dysfunction. In addition, serum phosphate was significantly higher in patients with more advanced renal failure. We found significant correlations between c-terminal FGF23 as well as intact FGF23 with GFR, phosphate, and PTH (Figure 1 ). When we analyzed the data of patients with only CKD stages 2 through 5, we found similar results as for the entire population (data not shown).
Calcium-Phosphate Metabolism and Progression of CKD
Clinical characteristics and laboratory data of patients with follow-up are reported in Table 2 . The median follow-up after completion of the baseline investigation was 53 mo (range 3 to 84 mo), and during this follow-up, 65 patients had progressed to a renal end point (33 patients had doubled their serum creatinine, and 29 had reached terminal renal failure necessitating renal replacement therapy; the two groups differed with respect to baseline serum creatinine, GFR, c-terminal FGF23, Ca × P, and the use of phosphate binders). Patients who had reached a progression end point were significantly older and had higher protein excretion rates and lower GFR. In addition, they had significantly higher phosphate, PTH, and FGF23 levels and Ca × P. There were no differences for surrogate parameters of nutritional (body mass index, albumin) and inflammatory status (high-sensitivity C-reactive protein [hsCRP]).
Age- and gender-adjusted Cox regression analysis revealed that GFR, c-terminal and intact FGF23, phosphate, Ca × P, and PTH all showed a strong association with progression-free survival (all P < 0.001; Table 3 , model 1), and proteinuria showed a weaker association (P = 0.03). In a multivariable analysis adjusting for all variables in addition to age and gender, only baseline GFR (P < 0.001), c-terminal FGF23 (P = 0.002), and intact FGF23 (P = 0.005) remained significant predictors for progression, and serum calcium, phosphate, and PTH concentrations were not independently associated with disease progression (Table 3 , model 2). When we used only parameters of the calcium-phosphate metabolism without FGF23 levels in a third model, we observed PTH and Ca × P besides baseline GFR to predict CKD progression. Phosphate concentrations were of borderline significance (P = 0.063), and calcium was not associated with CKD progression (Table 3 , model 3). To test the goodness of fit of the models to the observed data, we also analyzed models with c-terminal and intact FGF23 concentrations that were transformed using the log base 2 logarithm (log2 c-terminal FGF23 or log2 intact FGF23). We found that both log-transformed parameters remained significant, and results were comparable to models interpreting hazard ratios for an increment of 10 units in FGF23 concentration (data not shown).
To evaluate the operating characteristics of FGF23 as a prognostic tool for the progression of kidney disease, we performed a receiver operating characteristic (ROC) analysis for c-terminal and intact FGF23 in comparison with GFR (Figure 2 ). GFR obtained the highest area under the curve (AUC; AUC = 0.84; 95% confidence interval [CI] 0.78 to 0.90; P < 0.001), followed by c-terminal FGF23 (AUC = 0.81; 95% CI 0.74 to 0.88; P < 0.001) and intact FGF23 (AUC = 0.72; 95% CI 0.64 to 0.80; P < 0.001). We further constructed Kaplan-Meier curves of progression-free survival comparing patients with c-terminal FGF23 values above and below the optimal cutoff level of 104 rU/ml and for intact FGF23 values above and below the median of 35 pg/ml (a value very close to the optimal cutoff derived by ROC analysis). Patients who had c-terminal and intact FGF23 levels above these threshold values had a worse prognosis and statistically significant shorter progression times compared with patients with values below the threshold (Figure 3 ): Mean time to progression 46.9 mo (95% CI 40.2 to 53.6) compared with 72.5 mo (95% CI 67.7 to 77.3; P < 0.0001), respectively, for c-terminal FGF23 and 54.6 mo (95% CI 48.2 to 61.0) compared with 69.8 mo (95% CI 64.8 to 74.8; P = 0.0004), respectively, for intact FGF23. We observed similar separation of survival curves when we used the median of c-terminal FGF23 (85 rU/ml) instead of the ROC-derived cutoff of 104 rU/ml (data not shown).
DISCUSSION
The results of this prospective long-term study in a sizable cohort of white patients with nondiabetic CKD have identified FGF23 as a novel risk marker for the progression of CKD. Remarkably, apart from baseline GFR, FGF23 was the only independent predictor of progression among several parameters of the calcium-phosphate metabolism assessed. Moreover, both the c-terminal fragment and the biologically active intact FGF23 were independent predictors of progression. This finding is of particular interest because it points to a role of FGF23 in CKD progression apart from the problem of accumulation as a result of reduced renal clearance, which could be one potential explanation for elevated concentrations of c-terminal FGF23.
FGF23 is a recently identified “phosphatonin” that is thought to be implicated in the systemic balance of phosphate maintained by the interaction of intestine, bone, and kidneys.9 – 11 2 D3 levels, and osteomalacia.12 , 13 in vivo produced similar derangements of calcium-phosphate metabolism, whereas inactivation of this gene caused hyperphosphatemia and high circulating 1,25-OH2 D3 levels.14 – 16 17 9 , 18 – 21 Table 1 , Figure 1 ). However, because we measured only baseline serum phosphate, calcium, PTH, and FGF23 levels in our cross-sectional study, we cannot distinguish whether the hormone levels are the cause of abnormal serum calcium and phosphate levels or their consequence.
In experimental studies, the increase of FGF23 levels preceded the decrease of serum 1,25-OH2 D3 concentrations, suggesting an important role of FGF23 in the development of secondary hyperparathyroidism of patients with CKD. This point is further corroborated by results of recent experimental22 21 et al. 21 10 , 11 23 et al. 24 25 26 27 28 post hoc analysis of the Cholesterol and Recurrent Events (CARE) study in 4127 patients, Tonelli et al. 29 29
This study points to an additional potential consequence, namely that serum phosphate levels have an impact on progression of renal disease. The experimental work of Haut and Alfrey and colleagues30 , 31
The cohort investigated in our study comprised relatively young patients with mostly mild to moderate impairment of renal function or even normal GFR at the baseline examination. The remarkable power of baseline GFR as a predictor of progression highlights the importance of impaired renal function as a key determinant of progressive renal damage. In addition, this finding may explain why more patients in the “progressor” subgroup received vitamin D and phosphate binders, because derangements of calcium-phosphate metabolism become more evident with advanced CKD. In our patients, FGF23 levels increased with decreasing GFR, but it is important to point out that the impressive prediction of progression by both c-terminal and intact FGF23 is already adjusted for GFR. We have to admit that the measurement of GFR even with iohexol clearance may not be perfect and also does not reflect all aspects of kidney dysfunction. Therefore, we cannot exclude the possibility that the prediction of progression by intact FGF23 might also result from commutation of biologically inert degradation products reflecting residual confounding by severity of kidney function. However, the estimates of CKD progression by FGF23 were still highly significant after adjustment for GFR. This finding suggests that FGF23 is not simply a surrogate marker for baseline GFR and that FGF23 is a marker on its own providing prediction beyond the information obtained by the measurement of GFR.
In our cohort, systolic and diastolic BP at baseline were comparable in patients who progressed to a renal end point during follow-up and those who did not. The former needed more aggressive antihypertensive treatment, however, to achieve the same level of BP control. We emphasize that the use of angiotensin-converting enzyme inhibitors was comparable between groups. It is likely that identification of further progression predictors and potentially progression promoters such as FGF23 was facilitated by the almost equal BP control in progressors and nonprogressors.
The exclusion criteria in this study yielded a selected group of patients. Further studies must show whether the association between FGF23 and progression can be found also in other types of CKD, such as diabetic nephropathy, or nephrotic forms of kidney disease. The study had a follow-up time of adequate duration, and the primary end points were reached by one third of the participants. Therefore, the data are sufficiently solid to draw firm conclusions for the entire cohort, despite a considerable loss during follow-up of participants who had superior kidney function at baseline when compared with the followed patients. An additional limitation is the lack of data on phosphate intake in our patients.
In this prospective study comprising a cohort of patients with primary CKD, we identified intact FGF23 and, additionally, the-easier-to-measure c-terminal FGF23 fragment as novel predictors of CKD progression. Given the physiologic role of FGF23 in calcium-phosphate metabolism, it may also serve as a clinically useful indicator of early alterations of calcium-phosphate metabolism, correction of which might modify progression of CKD31 29
CONCISE METHODS
Patients and Baseline Investigations
We examined at baseline 227 white patients who were between 18 and 65 yr of age and had nondiabetic CKD and various degrees of renal impairment. These patients were recruited from eight nephrology departments in Germany, Austria, and South Tyrol as described previously.32 2 per d); organ transplantation; allergy to ionic contrast media; and pregnancy. According to the NKF classification of CKD, our study cohort showed the following stages of CKD: GFR ≥90 ml/min per 1.73 m2 (stage 1) in 72 (31.7%) patients, GFR 60 to 89 ml/min per 1.73 m2 (stage 2) in 49 (21.6%) patients, GFR 30 to 59 ml/min per 1.73 m2 (stage 3) in 63 (27.8%) patients, and GFR <30 ml/min per 1.73 m2 (stages 4 and 5) in 43 (18.9%) patients. The primary cause of kidney disease was glomerulonephritis in 97 (biopsy-confirmed in 90) patients, adult polycystic kidney disease in 37 patients, interstitial nephritis in 24 patients, other types of kidney disease in 43 patients, and unknown in 26 patients.
For avoiding interobserver differences, all patients were recruited by one physician who visited all participating centers. Patient history, including smoking habits and antihypertensive treatment at baseline, was recorded by interview and confirmed by checking patient records. This was complemented by clinical examination, including assessment of body mass index and BP. Hypertension was defined as BP >140/90 mmHg and/or the use of antihypertensive medication. Antihypertensive medication was withheld on the day of the study to minimize interference with measurements of the GFR. Antihypertensive drugs were taken by 179 (79%) patients: Diuretics (n = 83; 37%), angiotensin-converting enzyme inhibitors (n = 123; 54%), calcium channel blockers (n = 78; 34%), β receptor blockers (n = 67; 30%), and α-1 receptor blockers (n = 36; 16%).
Prospective Follow-Up
After the baseline investigation, patients were followed pro-spectively until the primary study end point or the end of the observation period was reached. The primary end point was defined as doubling of baseline serum creatinine and/or terminal renal failure necessitating renal replacement therapy. A total of 177 (78%) patients from the baseline cohort could be assessed during the follow-up. Patients who were lost to follow-up (n = 50) had at baseline a significantly better renal function than patients who were not lost for follow-up (i.e. , a higher mean GFR [91 ± 44 versus 64 ± 39 ml/min per 1.73 m2 ; P < 0.01]). However, both groups did not differ significantly with respect to age and gender. Patients who were lost to follow-up had moved away or were not referred by their physicians for follow-up visits in the renal units.
Laboratory Measurements
Blood samples for measurement of routine chemistry, hsCRP, PTH, and c-terminal and intact FGF23 were taken after an overnight fast of at least 12 h. The samples were immediately centrifuged at 1500 × g and 4°C for 10 min, and the supernatants were stored in aliquots at −80°C until further use. PTH was measured with an immunoradiometric assay, and FGF23 was measured using both the human c-terminal and the intact ELISA (Immutopics, San Clemente, CA).10 33
Statistical Analyses
Statistical analysis was performed with the SPSS for Windows 12.01 (SPSS, Chicago, IL). Univariate comparisons of continuous variables between various groups were performed using an unpaired t test or the nonparametric Wilcoxon rank sum test in case of non-normally distributed variables. Dichotomized variables were compared using Pearson χ2 test. All statistical tests were performed at the two-sided 0.05 level of significance. Data are presented as means ± SD and as median and 25th and 75th percentiles for skewed variables where appropriate. Univariate correlation analysis was performed by Spearman correlation analysis. For calculation of ROC curves for the predictor variables c-terminal and intact FGF23, patients were stratified into two groups (those who progressed and those who did not over the study period), and for each cutoff value c, sensitivity was defined as the proportion of progressors with predictor variables above c and specificity as the proportion of nonprogressors with values below c. The optimal cutoff value for the predictor variables was defined as the cutoff obtaining the highest sum of sensitivity and specificity. Calculation of the AUC and a test of the null hypothesis that it equals 50% (equivalent to a random 50:50 prognosis) were performed using the Mann-Whitney test. Kaplan-Meier time-to-event curves were generated for patients with c-terminal and intact FGF23 concentrations above and below optimal cutoff levels. Adjusted risk estimates for progression end points were calculated using multivariable Cox proportional hazards regression analysis. Because Ca × P is the product term of the variable calcium and phosphate, estimates of Ca × P were based on a model that did not include calcium and phosphate at the same time, to avoid multicollinearity. Similarly, intact and c-terminal FGF23 were not offered at the same time in a particular model. Exploratory graphical analysis and tests of specific violations indicated no departure from the assumption of proportional hazards.
DISCLOSURES
None.
Figure 1: Correlation analysis of c-terminal (A through C) as well as intact fibroblast growth factor 23 (FGF23; D through F) with GFR, serum phosphate, and parathormone. All parameters have been analyzed based on the log scale.
Figure 2: Receiver operating characteristics (ROC) curve of GFR and plasma c-terminal and intact FGF23 concentrations with progression of kidney disease as status variable. The area under the curve (AUC) for GFR, c-terminal, and intact FGF23 are 0.84, 0.81, and 0.72, respectively. The inserted tables present sensitivity and specificity of the four cutoffs derived from the quintile thresholds for c-terminal and intact FGF23, respectively. For c-terminal FGF23, the threshold between the third and fourth quintiles was equal to the ROC-derived optimal cutoff. For intact FGF23, the optimal cutoff is presented additionally.
Figure 3: Kaplan-Meier curves of renal end points in patients with below and above optimal cutoff of plasma c-terminal (A) FGF23 concentrations and for intact (B) FGF23 concentrations below and above the median. In patients with c-terminal FGF23 levels above the optimal cutoff (>104 rU/ml), progression was significantly faster (log-rank test, P < 0.0001); the same was observed in patients with intact FGF23 levels above the median (35 pg/ml; log-rank test, P = 0.0004). Numbers near the survival curves represent the number of patients at risk with FGF23 levels below and above the optimal cutoff at the times 0, 12, 24, 36, 48, 60, and 72 mo.
Table 1: Baseline clinical and laboratory data of 227 patients stratified according to GFR in NKF stagesa
Table 2: Baseline clinical and laboratory data of 177 patients with completed follow-up with further stratification of those with and without progression during the follow-up perioda
Table 3: Association of baseline variables with progression of kidney disease during the observation period using multiple Cox proportional hazards regression modelsa
Parts of this work were supported by the “Genomics of Lipid-associated Disorders—GOLD” of the Austrian Genome Research Programme GEN-AU and by grants from the Austrian Nationalbank (Project 9331) and the Austrian Heart Fund to F.K.
The following members of the Mild and Moderate Kidney Disease (MMKD) Study Group collaborated with the authors of this project: Erich Kuen, Division of Genetic Epidemiology, Innsbruck Medical University (Innsbruck, Austria); Paul Ko[Combining Diaeresis]nig, Innsbruck University Hospital (Innsbruck, Austria); Gu[Combining Diaeresis]nter Kraatz, Ernst Moritz Arndt University (Greifswald, Germany); Johannes F.E. Mann, Mu[Combining Diaeresis]nchen Schwabing Hospital (Munich, Germany); Gerhard A. Mu[Combining Diaeresis]ller, Georg August University (Go[Combining Diaeresis]ttingen, Germany); Ulrich Neyer, Feldkirch Hospital (Feldkirch, Austria); Hans Ko[Combining Diaeresis]hler, Medizinische Universita[Combining Diaeresis]tskliniken des Saarlandes (Homburg/Saar, Germany); and Peter Riegler, Bozen Hospital (Bozen, Italy).
Published online ahead of print. Publication date available at www.jasn.org
D.F. and B.K. contributed equally to this work.
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