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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1606
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 3 959-963
Copyright © 2006 by The Endocrine Society

BRIEF REPORT

Fibroblast Growth Factor 23 Is Increased in Calcium Nephrolithiasis with Hypophosphatemia and Renal Phosphate Leak

Domenico Rendina, Giuseppe Mossetti, Gianpaolo De Filippo, Michele Cioffi and Pasquale Strazzullo

Department of Clinical and Experimental Medicine (D.R., G.M., P.S.), Federico II University Medical School, 80131 Naples, Italy; Unit of Pediatric Endocrinology (G.D.F.), Azienda Ospedaliera di Rilievo Nazionale "G. Rummo," 82100 Benevento, Italy; and Department of General Pathology (M.C.), Second University of Naples, 80121 Naples, Italy

Address all correspondence and requests for reprints to: Giuseppe Mossetti, Dipartimento di Medicina Clinica e Sperimentale, Università Federico II, Via S. Pansini, 5-80131 Naples, Italy. E-mail: giumosse{at}unina.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Nephrolithiasis affects about 10% of the population in industrialized countries, with calcium salts composing more than 80% of renal stones. A significant percentage of patients with calcium nephrolithiasis and normal parathyroid function show hypophosphatemia and reduced renal phosphate reabsorption (i.e. a renal phosphate leak).

Objectives: The objective of the study was to compare serum levels of fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, in 110 recurrent stone formers with or without renal phosphate leak, six patients affected by X-linked hypophosphatemic rickets, five patients affected by oncogenic osteomalacia, and 60 unrelated healthy controls.

Design: This was a prospective interventional study.

Methods: Renal phosphate leak was identified based on the occurrence of idiopathic hypophosphatemia [serum phosphate concentration < 2.50 mg/dl (<0.80 mmol/liter)] and reduced renal threshold phosphate concentration [<2.2 mg/liter (<0.70 mmol/liter)].

Results: In 22 stone formers with renal phosphate leak, serum FGF23 concentration was significantly higher as compared with 88 stone formers without renal phosphate leak and with controls [83.3 (65.6–101.1) vs. 32.1 (26.8–37.4) and 24.5 (19.8–29.1) reference units (RU)/ml, respectively]. Stone formers with renal phosphate leak showed lower FGF23, compared with patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets [572.3 (235.9–908.7) RU/ml]. Among stone formers and controls, serum FGF23 concentration displayed a strong inverse association with serum phosphate (r = –0.784, P = 0.009) and the rate of tubular phosphate reabsorption (r = –0.791, P = 0.008).

Conclusions: In our study population, renal phosphate leak affected 20% of stone formers and was strongly associated with increased serum FGF23 concentration.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
NEPHROLITHIASIS IS A common disorder, affecting about 10% of the population in Europe and the United States. It results from the interaction between environmental influences and hormonal and genetic factors (1). Epidemiological studies indicate that calcium salts compose more than 80% of renal stones and that approximately 20% of patients with calcium nephrolithiasis and normal parathyroid function show idiopathic hypophosphatemia, reduced renal phosphate reabsorption, and urinary phosphate excretion inappropriately normal relative to the prevailing hypophosphatemia (i.e. renal phosphate leak) (2). Because urinary phosphate excretion influences the calcium salt urinary saturation, it has been suggested that this phenomenon might contribute to the pathogenesis of nephrolithiasis (2, 3, 4).

The cause for the relatively common occurrence of renal phosphate leak is not yet clear. Very recently experimental studies have suggested a role for fibroblast growth factor 23 (FGF23) in the regulation of phosphate homeostasis in physiological as well as pathological conditions other than nephrolithiasis (5, 6, 7, 8, 9, 10). The aim of our study was to evaluate the serum FGF23 levels in calcium stone-forming patients with or without a renal phosphate leak as well as healthy control subjects. Serum FGF23 concentration was also measured in patients affected by X-linked hypophosphatemic rickets and oncogenic osteomalacia, two hypophosphatemic disorders previously associated with elevated serum FGF23 concentrations (10).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We enrolled 110 recurrent stone-forming patients [62 males and 48 females; mean age 40.5 (36.2–44.7) yr; body mass index (BMI) 26.6 (25.5–27.7) kg/m2] between January 2002 and September 2003. All patients reported the elimination or removal of two or more calcium-oxalate (86, 78.2%) or calcium-phosphate (24, 21.8%) stones in different occasions over the preceding 4 yr. Sixty unrelated control subjects, without a history of nephrolithiasis [37 males and 23 females, mean age, 40.4 (35.4–45.4) yr; BMI, 26.4 (25.1–27.6) kg/m2] were enrolled among research fellows and employees of "Federico II" Medical School, family reference panel (one per family), and patients’ spouses.

Exclusion criteria for stone formers and controls were urinary tract infections; primary or secondary hyperparathyroidism; cystinuria greater than 70 µmol per 24 h; gouty diathesis [defined as the idiopathic formation of pure or mixed uric acid stones, in the presence or absence of gouty arthritis (1)]; renal tubular acidosis [defined as urinary pH > 6, hypokalemia, and positive urinary net charge (11)]; hyperthyroidism (TSH serum concentration < 0.1 mIU/liter); creatinine clearance less than 0.75 ml/sec·m2 or less than 0.85 ml/sec·m2 for women and men, respectively; chronic diarrhea states; and intake of thiazide diuretics, angiotensin-converting enzyme inhibitors, glucocorticoids, or estrogens. None of the stone formers and controls had had rickets during childhood, and none had abnormal height or bone deformity as adults.

We also determined serum FGF23 concentration in six patients affected by X-linked hypophosphatemic rickets [three males and three females; mean age, 27.1 (16.1–38.1) yr; BMI, 26.9 (25.1–28.7) kg/m2] and five patients affected by oncogenic osteomalacia [three males and two females; mean age, 58.9 (51.3–66.5) yr; BMI, 27.1 (25.4–28.8) kg/m2].

Patients and control subjects were born and living in Campania, a region of southern Italy, and gave written informed consent before entering the study, which was conducted according to the Declaration of Helsinki. The study protocol received the approval of the Ethical Committee of the Medical School of University of Naples "Federico II."

Methods

All stone-forming patients and controls were maintained on a fixed diet containing 1000 mg calcium, 100 mEq sodium, 900 mg phosphorus, 300 g carbohydrates, and 70 g proteins (total 2200 calories) daily for 7 d. Diet standardization is expected to lead to a metabolic steady state after 3 d of adaptation so that urinary values of the main relevant parameters reach constant levels (12). On d 8 at 0900 h, a fasting urine specimen was collected 3 h after primary void and its pH determined. Twenty-four-hour urine collections were obtained according to Tiselius et al. (13) and analyzed for their calcium, magnesium, phosphate, sodium, potassium, chloride, citrate, creatinine, oxalate, uric acid, and cystine contents. Fasting venous blood samples were taken for the measurement of serum total calcium, magnesium, phosphate, chloride, sodium, potassium, intact PTH (iPTH), 25 hydroxyvitamin D3 [25(OH)D3], 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], uric acid, creatinine, and FGF23 levels. Serum samples were separated within 1 h of collection and kept frozen at –80 C; urines were stored at –20 C until analysis. Maximal tubular renal phosphate reabsorption normalized for glomerular filtration rate (TmPi/GFR) was assessed with the use of the nomogram described by Walton and Bijvoet (14). Serum FGF23 concentrations were determined by ELISA [human FGF23 (C-term) ELISA kit; Immutopics, Inc., San Clemente, CA]. The intra- and interassay coefficients of variation were 5.0 and 7.3%, respectively. The FGF23 assay used detects both intact and C-terminal fragments of the molecule, and it was the only one available at the time of the study. Recent reports have shown a high degree of concordance between the results obtained by this assay and those obtained with assays using the intact molecule in healthy individuals (15, 16). The determination of other serum and urinary parameters were performed as previously described (17). The stones were analyzed using Fourier-transform infrared spectroscopy and high-resolution x-ray diffraction (17).

Diagnostic criteria

In accordance with Prié et al. (18), the patients with calcium nephrolithiasis and renal phosphate leak were identified based on the occurrence of idiopathic hypophosphatemia [serum phosphate concentration < 2.50 mg/dl (<0.80 mmol/liter)] and reduced TmPi/GFR [<2.2 mg/liter (<0.70 mmol/liter)].

Statistical analysis

All data were expressed as mean and 95% confidence intervals. Statistical analysis was performed with a SPSS statistical package (version 11.5; SPSS, Inc., Chicago, IL). According to the criteria proposed by Glantz (19), contingency table {chi}2 tests and ANOVA with Bonferroni correction for multiple comparisons were used to test for between-group differences in nonparametric and parametric variables, respectively. Correlations among FGF23 serum levels, serum phosphate levels, and TmPi/GFR were assessed using a Spearman nonparametric test. The relationship between FGF23 serum levels and serum phosphate levels or TmPi/GFR in the different study groups was tested by linear regression analysis (19). All statistical tests were two tailed. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
According to the predefined diagnostic criteria, we found 22 stone formers with renal phosphate leak [20%, 12 males, 10 females; mean age, 38.8 (34.8–42.8) yr, BMI, 25.9 (24.9–26.9) kg/m2] and 88 stone formers without renal phosphate leak [80%, 50 males, 38 females; mean age, 40.8 (36.2–45.4) yr; BMI, 26.6 (25.5–27.7) kg/m2]. No case of hypophosphatemia or renal phosphate leak was observed among control subjects. Mean age, BMI, and gender ratio were not different among the three study groups.

The biochemical characteristics of stone formers with renal phosphate leak, stone formers without renal phosphate leak, and controls are shown in Table 1Go. Stone formers with renal phosphate leak showed significantly higher serum FGF23 levels, compared with both stone formers without renal phosphate leak and controls [83.3 (65.6–101.1) vs. 32.1 (26.8–37.4) and 24.5 (19.8–29.1) reference units (RU)/ml, respectively, P < 0.01]. FGF23 serum levels were not different between stone formers without renal phosphate leak and controls. Nevertheless, the FGF23 serum levels observed in all stone formers and controls were significantly lower and without overlap, compared with those observed in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets [572.3 (235.9–908.7) RU/ml, P < 0.001]. Serum levels of calcium, magnesium, PTH, 25(OH)D3, and 1,25(OH)2D3 were in the normal range and not significantly different among the study groups. No difference in the distribution of types of calculi between stone formers with renal phosphate leak (16 of 22, 72.7% calcium-oxalate stones) or without renal phosphate leak (70 of 88, 79.6% calcium-oxalate stones) was observed.


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  		TABLE 1. Biochemical characteristics of stone-forming patients with or without renal phosphate leak and control subjects

 
Overall, FGF23 serum levels were inversely related to phosphate serum levels (r = –0.784, P = 0.009) and TmPi/GFR (r = –0.791, P = 0.008). These inverse correlations were confirmed within each of the study group: stone formers with renal phosphate leak (r = –0.887 and r = –0.968 for phosphate serum levels and TmPi/GFR, respectively, P = 0.001), stone formers without renal phosphate leak (r = –0.887 and r = –0.968, for phosphate serum levels and TmPi/GFR, respectively, P = 0.001), and controls (r = –0.911 and r = –0.852, for phosphate serum levels and TmPi/GFR, respectively P = 0.001). The between-group comparison of the linear regression equations for both parameters indicated that the slope of the stone-forming group with renal phosphate leak was significantly different, compared with that of stone formers without renal phosphate leak and that of controls (P < 0.01 in all cases) (Fig. 1Go, A and B). No other significant correlations between FGF23 serum levels and the others biochemical parameters examined were observed.


Figure 1
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  		FIG. 1. A, Regression of FGF23 on phosphate serum levels in calcium stone-forming patients with renal phosphate leak ({circ}, y = –235.59X + 629.45); calcium stone-forming patients without renal phosphate leak ({triangleup}, y = 40.729X + 174.38) as well as healthy control subjects ({square}, y = –32.59X + 139.69). Dotted lines indicate phosphate serum levels (2.50 mg/dl). Multiplication factor to convert metric units to SI units is 0.323 for phosphate serum levels. B, Regression of FGF23 on maximal TmPi/GFR in calcium stone-forming patients with renal phosphate leak ({circ}, y = –179.7X + 417.39), in calcium stone-forming patients without renal phosphate leak ({triangleup}, y = –37.751X + 142.19) as well as healthy control subjects ({square}, y = –28.839X + 109.65). Dotted lines indicate TmPi/GFR (2.2 mg/liter). Multiplication factor to convert metric units to SI units is 0.323 for TmPi/GFR. SF, Stone formers.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Epidemiological data indicate that a disorder of phosphate homeostasis with hypophosphatemia and renal phosphate leak affects approximately 20% of calcium stone formers with normal parathyroid function (2, 4). Its has been suggested that the urinary phosphate excretion relatively high might affect the urinary saturation of calcium salts, thereby increasing the probability of stone formation (2, 3, 4). Our study suggests a possible mechanism for this disorder.

The main finding was that stone-forming patients with renal phosphate leak had significantly increased serum levels of FGF23, compared with stone formers without renal phosphate leak and healthy controls, although these levels remain lower than those achieved in patients with phosphate-wasting disorders such as oncogenic osteomalacia and X-linked hypophosphatemic rickets, two clinical entities in which the pathogenetic role of FGF23 is well described (5, 10, 16). In addition, mutations in the FGF23 gene are also responsible for autosomal dominant hypophosphatemic rickets (5). In these phosphate-wasting disorders, the anomaly appears to reside in a FGF23 cleavage alteration (as in autosomal dominant and X-linked hypophosphatemic rickets) or a FGF23 overproduction (as in oncogenic osteomalacia) (5, 16).

Experimental models indicate that FGF23 regulates vitamin D metabolism and tubular phosphate reabsorption by modulating the activity of type IIa sodium-phosphate cotransporter independently from PTH (6, 20). FGF23 is readily detectable in the bloodstream of healthy subjects, confirming that this growth factor plays a role in phosphate and vitamin D homeostasis and also in physiological conditions (7, 16). Indeed, its role in the regulation of phosphate metabolism is supported by the strong associations observed in our study between serum FGF23 concentrations and phosphate serum levels as well as the rate of phosphate renal tubular reabsorption. Our study protocol, characterized by standardization of the diet for 7 d in patients and controls, rules out the possible influence of dietary factors, including phosphate, on the results we have observed.

The cause for the higher circulating FGF23 in stone-forming patients with renal phosphate leak is unknown. Either a subtle alteration in FGF23 metabolic degradation or an altered regulation of its biosynthesis might cause its abnormal accumulation in these individuals. Although it is unlikely that stone-forming patients with renal phosphate leak have a mutation in FGF23 gene similar to those described in autosomal dominant hypophosphatemic rickets (16), the possibility of genetic variation affecting the rate of synthesis or the clearance of this molecule cannot be excluded. As previously reported in patients with phosphate-wasting disorders, inappropriately normal concentrations of osteotropic hormones were found in our stone-forming patients with the renal phosphate leak (10, 16).

In conclusion, our findings support a role of FGF23 in the pathogenesis of calcium nephrolithiasis and suggest that determination of FGF23 could be useful for a more accurate characterization of recurrent calcium nephrolithiasis. Further studies are necessary to determine the FGF23 serum concentrations that are predictive of increased risk of nephrolithiasis in subjects with renal phosphate leak and the influence of other factors, in particular diet composition and calciotropic hormones, on the serum levels of this molecule.


   Acknowledgments
 
We are indebted to Pasquale and Italia Cornelia Rendina for their statistical advice.


   Footnotes
 
First Published Online December 13, 2005

Abbreviations: BMI, Body mass index; FGF23, fibroblast growth factor 23; iPTH, intact PTH; 1,25(OH)2D3, 1,25 dihydroxyvitamin D3; 25(OH)D3, 25 hydroxyvitamin D3; RU, reference units; TmPi/GFR, tubular renal phosphate reabsorption normalized for glomerular filtration rate.

This work was supported in part by grants from the Italian Ministry of University Research (MUIR), PRIN no. 2002062925-006 and FIRB no. RBNE017hc-005.

None of the authors have a conflict of interest.

Received July 19, 2005.

Accepted December 1, 2005.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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