Enteral ferric citrate absorption is dependent on the iron transport protein ferroportin.

Ferric citrate is approved as an iron replacement product in patients with non-dialysis chronic kidney disease and iron deficiency anemia. Ferric citrate-delivered iron is enterally absorbed, but the specific mechanisms involved have not been evaluated, including the possibilities of conventional, transcellular ferroportin-mediated absorption and/or citrate-mediated paracellular absorption. Here, we first demonstrate the efficacy of ferric citrate in high hepcidin models, including Tmprss6 knockout mice (characterized by iron-refractory iron deficiency anemia) with and without adenine diet-induced chronic kidney disease. Next, to assess whether or not enteral ferric citrate absorption is dependent on ferroportin, we evaluated the effects of ferric citrate in a tamoxifen-inducible, enterocyte-specific ferroportin knockout murine model (Villin-Cre-ERT2, Fpnflox/flox). In this model, ferroportin deletion was efficient, as tamoxifen injection induced a 4000-fold decrease in duodenum ferroportin mRNA expression, with undetectable ferroportin protein on Western blot of duodenal enterocytes, resulting in a severe iron deficiency anemia phenotype. In ferroportin-deficient mice, three weeks of 1% ferric citrate dietary supplementation, a dose that prevented iron deficiency in control mice, did not improve iron status or rescue the iron deficiency anemia phenotype. We repeated the conditional ferroportin knockout experiment in the setting of uremia, using an adenine nephropathy model, where three weeks of 1% ferric citrate dietary supplementation again failed to improve iron status or rescue the iron deficiency anemia phenotype. Thus, our data suggest that enteral ferric citrate absorption is dependent on conventional enterocyte iron transport by ferroportin and that, in these models, significant paracellular absorption does not occur.

Effects of erythropoietin on fibroblast growth factor 23 in mice and humans.

BACKGROUND: Erythropoietin (EPO) has been reported as a novel determinant of fibroblast growth factor 23 (FGF23) production; however, it is unknown whether FGF23 is stimulated by chronic exposure to EPO or by EPO administration in nonpolycystic chronic kidney disease (CKD) models. METHODS: We analyzed the effects of chronic EPO on FGF23 in murine models with chronically high EPO levels and normal kidney function. We studied the effects of exogenous EPO on FGF23 in wild-type mice, with and without CKD, injected with EPO. Also, in four independent human CKD cohorts, we evaluated associations between FGF23 and serum EPO levels or exogenous EPO dose. RESULTS: Mice with high endogenous EPO have elevated circulating total FGF23, increased disproportionately to intact FGF23, suggesting coupling of increased FGF23 production with increased proteolytic cleavage. Similarly, in wild-type mice with and without CKD, a single exogenous EPO dose acutely increases circulating total FGF23 out of proportion to intact FGF23. In these murine models, the bone marrow is shown to be a novel source of EPO-stimulated FGF23 production. In humans, serum EPO levels and recombinant human EPO dose are positively and independently associated with total FGF23 levels across the spectrum of CKD and after kidney transplantation. In our largest cohort of 680 renal transplant recipients, serum EPO levels are associated with total FGF23, but not intact FGF23, consistent with the effects of EPO on FGF23 production and metabolism observed in our murine models. CONCLUSION: EPO affects FGF23 production and metabolism, which may have important implications for CKD patients.

Effects of dietary iron intake and chronic kidney disease on fibroblast growth factor 23 metabolism in wild-type and hepcidin knockout mice.

In the setting of normal kidney function, iron deficiency is associated with increased FGF23 production and cleavage, altering circulating FGF23 levels. Our objective was to determine how chronic kidney disease (CKD) and dietary iron intake affect FGF23 production and metabolism in wild-type (WT) and hepcidin knockout (HKO) mice. For 8 wk, the mice were fed diets that contained adenine (to induce CKD) or no adenine (control group), with either low-iron (4 ppm) or standard-iron (335 ppm) concentrations. The low-iron diet induced iron deficiency anemia in both the WT and HKO mice. Among the WT mice, in both the control and CKD groups, a low-iron compared with a standard-iron diet increased bone Fgf23 mRNA expression, C-terminal FGF23 (cFGF23) levels, and FGF23 cleavage as manifested by a lower percentage intact FGF23 (iFGF23). Independent of iron status, CKD was associated with inhibition of FGF23 cleavage. Similar results were observed in the HKO control and CKD groups. Dietary iron content was more influential on FGF23 parameters than the presence or absence of hepcidin. In the CKD mice (WT and HKO, total n = 42), independent of the effects of serum phosphate, iron deficiency was associated with increased FGF23 production but also greater cleavage, whereas worse kidney function was associated with increased FGF23 production but decreased cleavage. Therefore, in both the WT and HKO mouse models, dietary iron content and CKD affected FGF23 production and metabolism.

Signature of an aggregation-prone conformation of tau.

The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended ß-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended ß-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.