Recombinant Human Fibroblast growth factor 23 protein (FGF23) (Active)

What is the molecular structure of recombinant human FGF23?

Recombinant Human FGF23 is a 30-32 kDa member of the FGF family within a subfamily that also includes FGF-19 and FGF-21. The mature secreted protein spans from Tyr25 to Ile251 (after removal of a 24-amino acid signal peptide) and contains a 120 amino acid core FGF domain with a beta-trefoil structure . The protein contains several functional regions including an atypical (very low affinity) heparin binding site (amino acids 134-162), a proteolytic cleavage site (Arg179-Ser180), and multiple O-linked glycosylation sites, with Thr178 being particularly important for preventing cleavage and maintaining FGF23 activity .

How does FGF23 function in phosphate homeostasis?

FGF23 is primarily produced by osteocytes and osteoblasts in bone and functions as an endocrine phosphatonin by regulating phosphate levels in the circulation . It exerts its effects through a ternary complex that includes Klotho (a co-receptor) and an FGF receptor (primarily FGF R4 or the "c" isoforms of FGF R1 or FGF R3) . Its interaction with renal proximal tubular epithelium decreases the renal reabsorption of phosphate by down-regulating phosphate transporters like SLC34A1 and by suppressing vitamin D production . FGF23 also decreases intestinal absorption of phosphate, thereby comprehensively regulating phosphate balance in the body .

What are the primary stimulators of FGF23 production?

FGF23 production is mainly stimulated by:

• High circulating phosphate levels

• 1,25(OH)₂D (active vitamin D)

• Elevated parathyroid hormone (PTH)

• Inflammatory states

Research has also revealed that insulin signaling plays an important inhibitory role in regulating FGF23 production. Specifically, insulin and insulin-like growth factors downregulate FGF23 production in osteocytes by inhibiting the transcription factor FOXO1 through the PI3K/PKB/Akt signaling pathway . Additionally, factors like circulatory volume loading and aldosterone can influence FGF23 levels .

What are the optimal conditions for handling and storing recombinant FGF23?

Recombinant Human FGF23 is typically provided in lyophilized form and requires proper handling to maintain biological activity:

For carrier-free preparations (without BSA), reconstitute in sterile PBS without additional proteins. This is particularly important for applications where the presence of BSA might interfere with experimental outcomes .

How should researchers choose between intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23) assays?

The choice between intact and C-terminal FGF23 assays depends on the specific research question:

What are the key considerations for sample collection and processing for FGF23 measurements?

Sample stability is a critical concern for accurate FGF23 measurement:

• Sample type controversy: Some assay manufacturers recommend EDTA anticoagulated plasma, while others recommend serum

• Stability findings: Research suggests intact FGF23 is significantly more stable in plasma (with either lithium heparin or EDTA) than in serum

• Degradation risk: FGF23 may be degraded by proteases or modified after blood withdrawal, necessitating standardized handling procedures

• Processing time: Minimize the time between collection and processing/freezing

• Storage temperature: For long-term storage, -80°C is recommended

Standardizing these factors across studies is essential for generating reliable and comparable data, particularly in multi-center clinical trials.

How does FGF23 interact with glucose homeostasis?

Research has revealed an important bidirectional relationship between FGF23 and glucose metabolism:

• Effects of glucose loading on FGF23: Glucose loading causes a decrease in plasma FGF23 levels, and intriguingly, changes in FGF23 precede changes in plasma phosphate (p time-lag = .04), indicating phosphate-independent effects

• Associations with glucose parameters: In population-based cohorts, FGF23 is positively associated with plasma glucose (β = .13 [.03-.23]; p = .01), insulin (β = .10 [.03-.17]; p < .001), and proinsulin (β = .06 [0.02-0.10]; p = .01)

• Insulin suppression of FGF23: Insulin and insulin-like growth factors downregulate FGF23 production in osteocytes through PI3K/PKB/Akt signaling and inhibition of FOXO1

• Negative correlation in humans: Baseline insulin concentration in fasted subjects is inversely correlated with baseline FGF23 (r = -0.282, p = 0.005) and HOMA-IR (r = -0.293, p = 0.003)

These findings suggest a complex regulatory relationship that may have important implications for understanding metabolic disorders.

What is the evidence linking FGF23 to diabetes and obesity?

Longitudinal studies have revealed significant associations between FGF23 and metabolic disorders:

• Incident diabetes: Higher baseline FGF23 is independently associated with development of diabetes (199 events [4%]; fully adjusted hazard ratio [HR] 1.66 [95% CI, 1.06-2.60]; p = .03)

• Incident obesity: FGF23 is independently associated with development of obesity (241 events [6%]; fully adjusted HR 1.84 [95% CI, 1.34-2.50]; p < .001)

• BMI-dependent effect: The association between FGF23 and incident diabetes lost significance after additional adjustment for BMI, suggesting the effect may be mediated through adiposity

• Sex differences: The association between FGF23 and obesity appears stronger in men and postmenopausal women than in premenopausal women

• Insulin resistance link: Insulin-resistant individuals have higher FGF23 serum levels, suggesting that intact insulin signaling is required for the suppression of FGF23 secretion by insulin

These findings support the hypothesis that elevated FGF23 may not only be a consequence but also a trigger in the development of metabolic syndrome and type 2 diabetes.

How should experimental designs account for the complex interplay between FGF23, insulin signaling, and phosphate metabolism?

When designing experiments to investigate FGF23 in metabolic disorders, researchers should consider these methodological approaches:

• Time-course analyses: Implement time-lag analyses to determine temporal relationships between changes in FGF23, phosphate, glucose, and insulin

• Comprehensive parameter measurement:

  • Mineral metabolism markers: phosphate, calcium, PTH, vitamin D

  • Glucose metabolism markers: glucose, insulin, proinsulin, HOMA-IR

  • Inflammatory markers

  • Iron status (which affects FGF23 transcription and cleavage)

• Animal models with controlled variables:

  • STZ-induced diabetes models with and without insulin replacement

  • Diet-induced obesity models

  • Models with controlled phosphate status to isolate direct effects of FGF23

• Cell culture systems:

  • Osteocyte cultures to study FGF23 production

  • Kidney cell lines expressing Klotho and FGF receptors to study signaling

• Statistical approaches:

  • Multivariate adjustments for potential confounders

  • Mediation analysis to determine whether effects are direct or mediated through other factors

  • Stratification by relevant parameters (e.g., BMI, sex, menopausal status)

These approaches can help elucidate the complex roles of FGF23 beyond its classical function in phosphate homeostasis.

How can researchers distinguish between intact FGF23 and its cleaved fragments in experimental systems?

Distinguishing between different forms of FGF23 requires specific technical approaches:

• Antibody-based methods:

  • Western blotting using antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Sandwich ELISA systems with capture/detection antibodies specific to intact or cleaved forms

  • Immunoprecipitation followed by mass spectrometry

• Recombinant protein variants:

  • Use of mutant FGF23 resistant to cleavage (e.g., R179Q mutation)

  • Tagged versions that allow tracking of specific fragments

• Analysis of O-glycosylation:

  • O-linked glycosylation at Thr178 blocks the cleavage of FGF23, thereby preventing loss of FGF23 activity

  • Glycosylation analysis can provide insights into the proportion of cleavage-resistant protein

• Mass spectrometry approaches:

  • Liquid chromatography-mass spectrometry (LC-MS/MS) to identify and quantify specific fragments

  • Multiple reaction monitoring (MRM) for targeted quantification of distinct peptides

Understanding the balance between intact and cleaved forms is crucial, as O-linked glycosylation and subsequent cleavage represent important regulatory mechanisms for FGF23 bioactivity.

What are the key considerations when studying FGF23 in chronic kidney disease (CKD) models?

CKD represents a complex context for FGF23 research that requires special considerations:

• Disease progression monitoring:

  • FGF23 increases in very early stages of CKD, often before changes in other mineral metabolism markers

  • Staging-specific analysis is crucial to understand dynamic changes

• Assay interpretation challenges:

  • C-terminal fragments accumulate in CKD due to reduced renal clearance

  • Ratio of intact to C-terminal FGF23 may provide additional insights

• Confounding factors to control:

  • Phosphate binders and other CKD treatments

  • Vitamin D status and supplementation

  • Secondary hyperparathyroidism

  • Klotho levels (which decrease in CKD)

  • Inflammatory status

• Cardiovascular assessment:

  • Integrate measurements of cardiac structure and function

  • Assess vascular calcification

  • Monitor blood pressure changes

• Bone metabolism markers:

  • Include measurements of bone formation and resorption markers

  • Consider bone histomorphometry in animal models

FGF23 is considered both an early marker for CKD-related bone disease and a potent cardiovascular risk factor in this population .

What are the most promising approaches for targeting FGF23 in experimental therapeutic studies?

Research into FGF23-targeted therapies might consider these approaches:

• Direct FGF23 neutralization:

  • Anti-FGF23 antibodies

  • FGF23 receptor antagonists

  • Soluble Klotho administration

• Modulation of FGF23 production:

  • Targeting FOXO1 signaling pathway

  • Vitamin D receptor modulators

  • Manipulation of O-glycosylation to affect processing

• Downstream signaling intervention:

  • FGF receptor tyrosine kinase inhibitors with appropriate specificity

  • Targeting specific downstream pathways relevant to particular disorders

• Mineral metabolism modulation:

  • Phosphate binders

  • Vitamin D analogs

  • Calcimimetics

• Combined approaches:

  • Targeting both FGF23 and inflammatory pathways

  • Combining FGF23 modulation with insulin sensitization in metabolic disorders

When evaluating these approaches, researchers should monitor comprehensive endpoints related to mineral metabolism, cardiovascular health, and in cases of metabolic disorders, glucose homeostasis and adiposity measures.

How should researchers interpret contradictory findings regarding FGF23 levels in different metabolic studies?

When facing contradictory findings across studies, consider these factors:

• Assay methodology differences:

  • Intact vs. C-terminal FGF23 assays measure different forms

  • Different manufacturers' assays may have variable detection limits and specificities

• Study population heterogeneity:

  • Disease complexity: Patients with complex metabolic and/or cardiorenal diseases have many confounding factors with unpredictable effects on FGF23 secretion

  • Studies in healthy individuals may yield clearer associations than those in patients with multiple comorbidities

• Temporal considerations:

  • Cross-sectional vs. longitudinal designs

  • Time of day for sample collection (potential diurnal variation)

  • Acute vs. chronic effects of conditions on FGF23

• Confounding factors:

  • Renal function status

  • Iron deficiency (stimulates FGF23 transcription but also increases FGF23 cleavage)

  • Inflammatory status

  • Hormonal factors (e.g., aldosterone levels increase FGF23 transcription)

• Statistical approach:

  • Inadequate adjustment for confounders

  • Insufficient power for subgroup analyses

  • Different statistical models yielding different associations

Resolution often requires carefully designed studies that specifically address these potential sources of discrepancy.

What are the bioactivity considerations when using recombinant FGF23 in experimental systems?

When using recombinant FGF23 in experiments, researchers should consider:

• Cofactor requirements:

  • Klotho is essential for canonical FGF23 signaling

  • Heparin can enhance FGF23 activity in some systems

• Concentration considerations:

  • Effective concentration range: 0.1-0.4 µg/mL in the presence of Klotho and heparin

  • Supra-physiological concentrations may activate non-canonical pathways

• Variant selection:

  • Wild-type vs. cleavage-resistant variants (e.g., R179Q mutation)

  • Tagged vs. untagged versions

  • Full-length vs. core domain constructs

• Quality control assessments:

  • Confirm bioactivity through functional assays:

    • Phosphate uptake in kidney cells

    • ERK1/2 phosphorylation

    • EGR1 upregulation in the presence of Klotho

  • Verify protein integrity through analytical methods (SDS-PAGE, MS)

• System-specific considerations:

  • Cell types expressing appropriate receptors and co-receptors

  • Tissue-specific effects may require different experimental readouts

  • Species compatibility (human FGF23 shows 72% amino acid identity to mouse FGF23)

These factors are critical for ensuring that experimental results accurately reflect physiological FGF23 activity.