The Tmprss6^-/- Mouse Model of Iron Refractory Iron Deficiency Anemia (IRIDA) Exhibits Disrupted Phosphate Homeostasis, Elevated Circulating FGF23 Levels, and Increased Fgf23 Expression in Bone Marrow

While the effects of chronic iron deficiency on erythropoiesis are well documented, the effects of iron deficiency on metabolism remain poorly understood. Recent studies suggest that iron deficiency anemia may impact systemic phosphate regulation (Farrow et al ., PNAS, 2011; Clinkenbeard et al ., J Bone Miner Res, 2014; David et al ., Kidney Int, 2016; Hanudel et al ., Am J Physiol Renal Physiol, 2016). Here we used Tmprss6^-/- mice, a model of iron refractory iron deficiency anemia (IRIDA) characterized by hepcidin elevation, to explore the relationship between systemic iron deficiency and phosphate homeostasis. By intercrossing Tmprss6^+/- mice, we generated Tmprss6^-/- mice and littermate controls, which were raised on a standard rodent diet (Teklad 2018S containing 200 ppm iron and 0.7% phosphorus) prior to study at 8 weeks of age. Tmprss6^-/- mice displayed a trend toward lower serum phosphate concentration, as well as a significant elevation in the urinary phosphate/creatinine ratio. Because of the role of phosphate in bone mineralization, we also analyzed the tibiae of Tmprss6^-/- mice and found that they showed alterations in bone histology and biomechanical properties.

To determine if fibroblast growth factor 23 (FGF23), a key phosphate-regulating hormone, may contribute to these changes in phosphate balance, we compared plasma FGF23 levels of 8-week-old Tmprss6^-/- mice to littermate controls using enzyme-linked immunosorbant assays (ELISAs). Tmprss6^-/- mice showed significantly higher levels of the biologically-active ("intact") form of FGF23 as well as "total" FGF23 (a measure of both the intact form and the C-terminal cleavage product of FGF23). This FGF23 elevation was also observed in 3-week-old Tmprss6^-/- pups (i.e. prior to weaning). Because osteocytes in bone have been described as a major site of FGF23 production, we measured Fgf23 mRNA levels by qRT-PCR in RNA that was prepared from femur bones of Tmprss6^-/- mice or littermate controls after marrow removal by flushing. We observed a four-fold increase in Fgf23 expression in the femurs of Tmprss6^-/- mice as compared to littermate controls. Because Fgf23 expression has previously been reported in Ter119⁺ erythroid cells from wild type mice (Coe et al ., J Biol Chem, 2014), and because our RNA preparations from femur bones showed evidence of residual bone marrow contamination (as assessed by alpha hemoglobin expression), we also measured Fgf23 mRNA expression in RNA prepared from total bone marrow. Tmprss6^-/- mice exhibited significantly higher bone marrow Fgf23 mRNA levels than littermate controls. Notably, when RNA was prepared from bones that had been more effectively depleted of marrow by mechanical disruption and hypotonic lysis, Tmprss6^-/- mice no longer showed an increase in Fgf23 mRNA levels in the femur RNA as compared to littermate controls. Collectively, these results suggest that increased Fgf23 production by one or more bone marrow cell populations contributes to elevated circulating FGF23 in Tmprss6^-/- mice.

FGF23 has been proposed as the basis for a bone-kidney axis that coordinates bone health with renal handling of phosphate. Our findings suggest that altered phosphate homeostasis in Tmprss6^-/- mice may result from increased Fgf23 expression in the bone marrow and a corresponding elevation in circulating FGF23. These results may have relevance to the skeletal health of patients with IRIDA due to TMPRSS6 mutation and those with other forms of iron-restricted anemia resulting from hepcidin elevation. Additionally, as Tmprss6^-/- mice are known to exhibit growth retardation, our results raise the possibility that disrupted phosphate homeostasis may contribute to the commonly observed association of childhood iron deficiency with growth impairment. Future studies will characterize the source of Fgf23 production in the bone marrow of Tmprss6^-/- mice as well as the mechanism by which iron deficiency elevates Fgf23 expression.