C-Terminal to Intact Fibroblast Growth Factor 23 Ratio in Relation to Estimated Glomerular Filtration Rate in Elderly Population

Fibroblast growth factor 23 (FGF23) is a key bone-derived regulator of phosphate and vitamin D metabolism. FGF23 promotes phosphaturia by down regulating the expression of luminal sodium dependent phosphate transporters in the proximal renal tubule, and reduce synthesis of the active form of vitamin D by down regulating 1α-hydroxylase and up regulating 24-hydroxylase activity [1,2,3]. FGF23 is a protein consisting of 251 amino acids comprising a unique C-terminal sequence that mediates high affinity binding to the FGFR/Klotho receptor complex [3]. This hormone is secreted into extracellular fluid in a 32.5-kDa mature beta-trefoil form. FGF23 is degraded by intracellular pro-protein convertases (PC) before secretion by osteoblasts/osteocytes and furin, a proteolytical enzyme that recognizes the Arg176-X-X-Arg179 motif [4,5]. This cleavage abolishes phosphaturic FGF23 action [6]. The intracellular cleavage of FGF23 is considered the most important mechanism of FGF23 regulation. In predialysis patients, the FGF23 cleavage was decreased. The mechanism(s) of further biodegradation (proteolysis) of the 14 kDa C-terminal fragment of FGF23 are still unknown. Regardless of that FGF23 in CKD is considered to exert direct cardiotoxic effect [7].

Two-site enzyme-linked immunosorbent assays (ELISA) recognize the 32.5-kDa N-terminal beta-treofil structure and short cleaved 10-kDa C-terminal unique to human FGF23. Specific recognition of intact FGF23 protein (iFGF23) is only possible using the combination of two antibodies [8]. While the C-terminal ELISA assay recognize iFGF23 as well as C-terminal fragments derived from FGF23 proteolysis.

Several authors looked for analytical equivalence between iFGF23 and C-terminal FGF23 (cFGF23) assays. In regression analysis a slope between Immutopics FGF23 assays [cFGF-23 (RU/ml) = 2.1 x FGF23 (pg/ml) - 14.0] in a cohort of healthy subjects was shown [9]. A strong correlation between both was expected. However, in numerous studies, a striking lack of strong association between the measures was demonstrated [10]. Analytical disagreement appeared particularly at physiologic and moderately elevated levels of FGF-23, and can be explained by lower stability of iFGF23 after blood collection without the addition of protease inhibitor [11], or random errors in measurements in individual specimens. Regardless, when blood samples were stabilized with protease cocktail inhibitors, an unexpectedly weak correlation between intact and cFGF23 was demonstrated (R²= 0.428) in patients with mild to moderate chronic kidney disease (CKD). The explanation of this phenomenon is still elusive.

We have recently assessed both iFGF23 and cFGF23 levels in plasma samples coming from a large population from the PolSenior Study including 45.6% subject with CKD [12]. We have demonstrated that the increase of cFGF23 levels slightly preceded the increase of iFGF23 with eGFR decline. However, we did not analyze the ratio between both assays. Therefore, the aim of this study was to analyze the ratio between C-terminal and intact FGF23 levels in relation to estimated glomerular filtration rate (eGFR) in an elderly population.

Plasma samples stored at -70°C used in the presented cross-sectional study were obtained from participants of PolSenior study - a large, multicenter, interdisciplinary, publicly funded research project focused on elderly subjects, conducted in Poland from 2007 to 2012, during the morning after overnight fasting [13]. The PolSenior study, as well as additional analyses, stored in plasma samples had approval of the Bioethics Committee of Medical University of Silesia (KNW/0022/KB1/38/II/08/10; KNW-650138/I/08). Additionally, each subject gave written consent.

We measured plasma iFGF23 and cFGF23 concentrations by ELISA using commercially available kits from Immutopics (San Clemente, CA, U.S.). The mean intra- and inter-assay coefficients were 4.4% and 6.1% for iFGF-23 and 2.4% and 4.7% for cFGF23, respectively.

Other measurements: serum phosphorus, calcium, iron, uric acid, albumin (also in urine), creatinine, and urea concentrations were previously assessed by automated system (Modular PPE, Roche Diagnostics GmbH, Mannheim, Germany) in a single certified laboratory with inter-assay coefficients of variability below 1.4%, 1.5%, 1.8 %, 1.7%, 1.7%, 2.3%, 1.7%, respectively. Serum intact parathyroid hormone (iPTH) level was assessed by the electrochemiluminescence method (ECLIA) using commercially available kits on Cobas E411 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) with inter-assay coefficients of variability below 6.5%. We measured serum vitamin 25-OH-D₃ by the radioimmunoassay method (DIAsource ImmunoAssays, Nivelles, Belgium), with inter-assay coefficients of variability <5.3%.

We estimated GFR according to the MDRD formula [14], and ensured G1-G4 CKD stages were properly scored, according to the guidelines [15]. Obesity was diagnosed according to the WHO criteria [16], and hypertension based on home measurements, during two visits, and the mean values from two measurements when systolic blood pressure was at least 140 mm Hg and/or mean diastolic blood pressure was at least 90 mm Hg, and/or the subject reported treatment with antihypertensive medications [17]. We established diagnosis of coronary artery disease (CAD) and diabetes based on medical history, medication use, and fasting serum glucose ≥126 mg/dL.

Statistical analysis was performed using STATISTICA 10.0 PL (StatSoft, Cracow, Poland), StataSE 12.0 (StataCorp LP, TX, U.S.). Statistical significance was set at a p value below 0.05. All tests were two-tailed. Imputations were not done for missing data. Nominal and ordinal data were expressed as percentages, whilst interval data were expressed as mean value ± standard deviation in the case of normal distribution or as median (lower - upper quartile) in the case of data with skewed distribution. For comparison of data between CKD groups, one-way ANOVA analysis was used with RIR Tukey post-hoc test. Categorical variables were compared using χ2 tests. Distribution of variables was evaluated by the Shapiro-Wilk test, and homogeneity of variances was assessed by the Levene test. In order to assess the relationship between c/i FGF23 ratio [RU/pg] and other variables, the least angle square regression was used.

We studied 3264 subjects, 1720 men and 1544 women (Table 1). Among them, 1152 (35.3%) had eGFR < 60 ml/min/1.73m², that was more prominent in women than men (40.0% vs. 31.0%; p < 0.001). Hyperphosphatemia (serum phosphate concentration ≥ 4.6 mg/dL) was diagnosed in 0.9% study population, and only secondary hyperparathyroidism met the criteria of serum iPTH level ≥ 65 pg/mL in 14.2%. Serum vitamin 25-OH-D₃ levels below 30 ng/mL were observed in 37.2 % of the study population (Table 1).

The least angle square regression model (LARS) enclosed: gender, age, eGFR values, and serum levels of hs-CRP and Fe. The coefficient values are yielded for the minimum estimation of prediction error C_p and in order from the most significant to the least ones. The LARS procedure revealed that the log₁₀(c/i FGF23) is diminished by eGFR values (β = -0.0014), then by serum levels of Fe (β = -0.0006) and CRP (β = -0.0183), increased with age (β = 0.0015) and in women (β = 0.0056).

For the log₁₀(c/i FGF23 ratio), one-way analysis of variance showed a significant effect for the CKD stages (G1, G2, G3a, G3b, G4) (F = 11.12, p < 0.001) - Figure 1. The log₁₀(c/i FGF23 ratio) was statistically lower in G1 group than in G3a (p < 0.05), G3b (p < 0.001) and G4 (p < 0.05) groups, lower in G2 group than in G3a (p < 0.01), G3b (p < 0.05) groups.

The results of our study demonstrate modestly increasing C-terminal fragments to iFGF ratio with the deterioration of kidney excretory function with kits developed by Immutopic in an elderly population. It suggests a decreased degradation of C-terminal fragment of FGF23 but does not exclude coexisting diminished iFGF23 cleavage.

Our recent study revealed that the prevalence of increased cFGF23 levels across CKD stages was greater than iFGF23 levels [12]. The observation supports the hypothesis that decreased biodegradation of C-terminal cFGF23 fragments by the kidneys is an additional mechanism to the increased production of FGF23, stimulated by the accumulation of phosphates in the circulation. However, certainly decreased biodegradation capacity of the failing kidney is not the main mechanism of the rise of plasma cFGF23 in CKD.

Our findings do not exclude diminished FGF23 cleavage in CKD shown by Smith et al. [9]. They demonstrated the decreased formation of 14 kDa C-terminal fragment of FGF23 recognized by polyclonal antibodies identifying epitops within the region of 186-206 amino acid residues. The epitope is inside the region recognized by the polyclonal antibodies (180-251 amino acids residues) utilized by the manufacturer (Immutopics). However, we cannot exclude that these antibodies may detect a different group of C-terminal peptides, especially those resulting from the further degradation of the 14 kDa C-terminal FGF23 fragments generated by furin cleavage. Most likely, without some kind of inhibited cleavage, the increase in c/iFGF23 ratio in CKD stages would be greater.

As was mentioned above, several authors described the significant correlation between measurements performed with iFGF23 and cFGF23 assays to indicate the analytical equivalence of these two methods [18,19,20,21,22]. Certainly, a strong correlation between cFGF23 and iFGF23 measurements was anticipated, as both kits detect the same hormone or products of its degradation (C-terminal peptides). Surprisingly, numerous studies in which simultaneous measurements of FGF23 has been performed using iFGF23 and cFGF23 assays demonstrated a lack of association between these assessments [23,24,25,26,27,28].

Poor analytical compatibility was shown, particularly at near-physiologic and modestly elevated concentrations. This was claimed to be the consequence of not only the calibration differences, but also the detection of C-terminal fragments using the cFGF23 assay, normally present in plasma, that do not reflect physiological regulatory processes [9].

In the context of CKD, it was initially hypothesized, that high concentration of circulating FGF23 is the result of reduced renal clearance and accumulation of C-terminal fragments. Other supposition was that with renal function deterioration the increasing stimuli for FGF23 production could possibly overwhelm the capacity of the physiological inactivation mechanism that ordinarily controls bioactive FGF23 concentrations. In addition, the proteolytic mechanism may be down-regulated by uremic milieu, i.e. accumulation of multiple glycation and nitration adducts, oxidized proteins, acidosis, and systemic inflammation [29,30].

The mechanism postulated to explain the decreased degradation rate of cFGF23 in CKD remains unknown. Potentially, proteolytic machinery may be down-regulated by uremic milieu [31]. The impaired generation of the 14 kDa C-terminal fragment of FGF23 was shown by Smith et al. [9]. Perhaps the proteolysis of smaller C-terminal FGF23 fragments, detected in our study is also decreased?

Furthermore, our analysis shows that systemic inflammation and low serum iron concentration increase the c/i FGF23 ratio, previously suggested by other authors [32,33]. This data supplements our previous analysis showing parallel, linear increase in plasma iFGF23 and cFGF23 levels in subjects with serum iron level below 60 µg/dL [34]. Calculation of the c/i FGF23 ratio is just a more sensitive method to demonstrate subtle changes in the greater accumulation of C-terminal FGF23 fragments. It is worth notice that the c/i FGF23 ratio increases with age independently to the GFR deterioration. This observation may suggest impairment of proteolysis capacity in very old subjects.

In addition, our study demonstrates high variability of the c/i FGF23 ratio not related to the kidney excretory function.

However, it should be noted that cFGF23 levels show smaller than iFGF23 circadian cycling [9,35,36], and week-to-week intra individual variability [9,37,38].

Other important factors that influence FGF23 measurements are sample type and pre-analytical stability of this protein, one of the major factors that caused the differentiation of FGF23 assays. Smith et al. indicate that in case of Immutopic assays, iFGF23 is significantly more stable in lithium heparin or EDTA anticoagulated plasma than in serum. These observations were also confirmed by Fassbender [39]. Additionally, losing immunoreactivity of FGF23 due to degradation [9,11,40] may be attenuated by addition of protease inhibitors. However, assays manufacturers do not provide any guidance on what antiprotease protection should be employed. It should be stressed that we used EDTA plasma samples, as recommended by the manufactures, but did not use other protease inhibitors.

Our study has some limitations as there are factors influencing FGF23 measurements, which we cannot entirely detect and eliminate. We have no complete information on the macro and micronutrient intake of our subjects. As was indicated above, suspected low dietary phosphate intake in our elderly population precludes the application of the data to younger populations with greater dietary phosphate intake. Additionally, as was mentioned before, we did not collect plasma samples using protease inhibitors, which might raise the pre-analytical bias. Furthermore, we did not use the recently developed CKD-EPI formula to estimate eGFR since that technique was not calibrated to an isotope dilution mass spectrometry (IDMS) method. Finally, the cross-sectional nature of the study does not allow us to observe the changes in the c/i FGF23 ratio with the progression of chronic kidney disease. On the other hand, the size of the study population is the strength of our study, enabling detection of small changes in c/i FGF23 ratio.

In summary, our results suggest impairment in the cleavage of the C-terminal FGF23 fragments and their accumulation in the circulation with the deterioration of kidney excretory function and age in the elderly population. Inflammation and low serum iron level seems to diminish degradation capacity of FGF23 fragments.

None of the authors declares conflict of interest

FGF23 assessments were covered by grant from National Centre of Science (No DEC-2011/03/B/NZ5/05837) and grant from Medical University of Silesia (KNW-1-022/D/2/0).

We thank Dr Mike Smertka for his invaluable editorial help during the preparation of this manuscript.