Amelioration of the premature ageing-like features of Fgf-23 knockout mice by genetically restoring the systemic actions of FGF-23 - PubMed (original) (raw)

Comparative Study

. 2008 Nov;216(3):345-55.

doi: 10.1002/path.2409.

Affiliations

Comparative Study

Amelioration of the premature ageing-like features of Fgf-23 knockout mice by genetically restoring the systemic actions of FGF-23

S DeLuca et al. J Pathol. 2008 Nov.

Abstract

Genetic ablation of fibroblast growth factor 23 from mice (Fgf-23(-/-)) results in a short lifespan with numerous abnormal biochemical and morphological features. Such features include kyphosis, hypogonadism and associated infertility, osteopenia, pulmonary emphysema, severe vascular and soft tissue calcifications, and generalized atrophy of various tissues. To determine whether these widespread anomalies in Fgf-23(-/-) mice can be ameliorated by genetically restoring the systemic actions of FGF-23, we generated Fgf-23(-/-) mice expressing the human FGF-23 transgene in osteoblasts under the control of the 2.3 kb alpha1(I) collagen promoter (Fgf-23(-/-) /hFGF-23-Tg double mutants). This novel mouse model is completely void of all endogenous Fgf-23 activity, but produces human FGF-23 in bone cells that is subsequently released into the circulation. Our results suggest that lack of Fgf-23 activities results in extensive premature ageing-like features and early mortality of Fgf-23(-/-) mice, while restoring the systemic effects of FGF-23 significantly ameliorates these phenotypes, with the resultant effect being improved growth, restored fertility, and significantly prolonged survival of double mutants. With regard to their serum biochemistry, double mutants reversed the severe hyperphosphataemia, hypercalcaemia, and hypervitaminosis D found in Fgf-23(-/-) littermates; rather, double mutants show hypophosphataemia and normal serum 1,25-dihydroxyvitamin D(3) levels similar to pure FGF-23 Tg mice. These changes were associated with reduced renal expression of NaPi2a and 1 alpha-hydroxylase, compared to Fgf-23(-/-) mice. FGF-23 acts to prevent widespread abnormal features by acting systemically to regulate phosphate homeostasis and vitamin D metabolism. This novel mouse model provides us with an in vivo tool to study the systemic effects of FGF-23 in regulating mineral ion metabolism and preventing multiple abnormal phenotypes without the interference of native Fgf-23.

(c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Conflict of interest statement

No conflicts of interest were declared.

Figures

Figure 1

Figure 1

Macroscopic characterization. (A) Macroscopic image of control (CTRL), Fgf -_23_−/− (KO), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM), and _col 1_-_FGF_-_23_-Tg (TG) male littermates at 3 weeks of age. (B) Survival curve of mice with various genotypes until death or 6 months of age (n = 7 for CTRL, ◆; n = 10 for KO, ■; n = 5 for DM, ▲; n = 18 for TG, ●). Body weight curve of male (C) and female (D) control (CTRL, ◆), Fgf -_23_−/− (KO, ■), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM, ▲) and _col 1_-_FGF_-_23_-Tg (TG, ●) mice from 3 to12 weeks of age; n > 5 for each group of mice. ****p < 0.0001, statistically significant difference compared with controls. ####p < 0.0001, statistically significant difference compared with Fgf -_23_−/− mice

Figure 2

Figure 2

Skeletal phenotype. (A) X-ray autoradiographs of the hind limbs from male littermates at 9 weeks of age. Graphic display of total body mineral content (BMC) (B) and hind-limb bone mineral density (BMD) (C) of control (CTRL, n = 8), Fgf -_23_−/− (KO, n = 8), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM, n = 4), and _col 1_-_FGF_-_23_-Tg (TG, n = 4) mice as measured by PIXImus analysis at 9 weeks of age. Each value obtained for BMC was normalized to the body weight of the corresponding animal. **p < 0.01 and ****p < 0.0001, statistically significant difference compared with controls. ####p < 0.0001, statistically significant difference compared with Fgf -_23_−/− mice

Figure 3

Figure 3

Serum biochemistry. Comparison of serum parameters for control (CTRL), Fgf -_23_−/− (KO), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM), and _col 1_-_FGF_-_23_-Tg (TG) littermates; n > 3 for each group of mice. (A) Serum phosphate and (B) serum calcium levels for the various groups of mice at 3, 6, and 9–12 weeks of age. (C) Serum 1,25(OH)2D3 and (D) serum PTH levels of the various groups of mice at 6–9 weeks of age. *p < 0.05, **p < 0.01, and ****p < 0.0001, statistically significant difference compared with controls. #p < 0.05, ##p < 0.01, and ####p < 0.0001, statistically significant difference compared with Fgf -_23_−/− mice

Figure 4

Figure 4

Immunofluorescence staining for NaPi2a. (A) Immunohistochemistry of renal NaPi2a protein (green) at 9 weeks of age for control (CTRL), Fgf -_23_−/− (KO), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM), and _col 1_-_FGF_-_23_-Tg (TG) littermates (n = 3 for each group of mice). In contrast to the increased expression of NaPi2a protein observed in Fgf -_23_−/− mice, reduced expression is observed in the double-mutant and pure transgenic mice compared with their littermate controls. Nuclear counterstaining was performed with DAPI (blue); original magnification × 400. Quantification of protein expression was performed by considering both the area (B) and the staining intensity (C) of the fluorescent signal. All data were quantified on images taken under identical conditions. The relative area of staining was determined by calculating the number of positively stained pixels per mm2 of cells. The relative staining intensity was determined by considering the mean luminosity value on background corrected images. *p < 0.05 and **p < 0.01, statistically significant difference compared with controls. ###p < 0.001 and ####p < 0.0001, statistically significant difference compared with Fgf -_23_−/−

Figure 5

Figure 5

Quantitative real-time PCR. RT-PCR for renal (A) 1α(OH)ase and (B) klotho mRNA of control (CTRL), Fgf -_23_−/− (KO), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM), and _col 1_-_FGF_-_23_-Tg (TG) littermates at 9 weeks of age (n = 3 for each genotype). **p < 0.01 and ****p < 0.0001, statistically significant difference compared with controls. ####p < 0.0001, statistically significant difference compared with Fgf -_23_−/−

Figure 6

Figure 6

Histological evaluation. (A) Haematoxylin and eosin staining of various tissues from control (CTRL, n = 9), Fgf -_23_−/− (KO, n = 7), Fgf -_23_−/−/_hFGF_-_23_-Tg double-mutant (DM, n = 7), and _col 1_-_FGF_-_23_-Tg (TG, n = 9) littermates at 9–12 weeks of age. The images shown represent typical findings for each genotype. Please note the difference in magnification between the Fgf -_23_−/− and littermates of other genotypes for the images of the skin. Quantification of changes in (B, C) intestinal, (D) skin, and (E, F) lung morphology. Compared with controls, Fgf -_23_−/− mice exhibit extensive atrophy of the intestinal mucosa with a significant decrease in the intestinal villus height and intestinal villus area. A significant reduction in skin, subcutaneous fat layer, and muscle layer thicknesses is also apparent in the Fgf -_23_−/− mice. Moreover, the lungs of Fgf -_23_−/− animals exhibit typical features of emphysema, including significant enlargement of the mean alveolar air space area and a reduction in the number of alveolar air spaces compared with controls. Such features were consistently absent in all double-mutant and transgenic mice examined, with normalization of the intestine, skin, and lung morphology

Figure 7

Figure 7

Soft tissue calcifications. Von Kossa staining of various tissues from control (CTRL, n = 9), Fgf -_23_−/− (KO, n = 7), Fgf -_23_−/−/_col 1_-_FGF_-_23_-Tg double-mutant (DM, n = 7), and _col 1_-_FGF_-_23_-Tg (TG, n = 9) littermates at 9 weeks of age. Fgf -_23_−/− animals exhibit extensive soft tissue calcifications in multiple tissues; calcifications can be noted within areas depicted by black circles. Kidney calcifications are primarily present in the tubules, while mineral deposition in the heart is mainly localized in the valves. In the lungs, most of the calcifications can be detected in the alveolar septal wall. No soft tissue calcifications were observed in any tissues examined for control, double-mutant, and pure transgenic animals

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