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

Abstract

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. 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. see commentary on page 668 see commentary on page 668 Translational StatementFerric citrate (FC) has been shown to be an effective source of enterally absorbed iron in chronic kidney disease—a condition with high hepcidin levels in which enterocyte ferroportin function is inhibited, limiting transcellular iron absorption. Here, in murine models with and without impaired kidney function, we demonstrate that enteral FC absorption is dependent predominantly on conventional enterocyte iron transport by ferroportin, with any alternative pathways unable to compensate when ferroportin is inactivated. Therefore, in patients, enteral FC absorption is likely reliant on the regulated transcellular physiologic pathway, lessening concern about possible unregulated paracellular iron absorption. Ferric citrate (FC) has been shown to be an effective source of enterally absorbed iron in chronic kidney disease—a condition with high hepcidin levels in which enterocyte ferroportin function is inhibited, limiting transcellular iron absorption. Here, in murine models with and without impaired kidney function, we demonstrate that enteral FC absorption is dependent predominantly on conventional enterocyte iron transport by ferroportin, with any alternative pathways unable to compensate when ferroportin is inactivated. Therefore, in patients, enteral FC absorption is likely reliant on the regulated transcellular physiologic pathway, lessening concern about possible unregulated paracellular iron absorption. Auryxia (ferric citrate [FC]; Akebia Therapeutics, Inc.) is a novel compound that is approved for clinical use as an enteral phosphate binder in adult patients with chronic kidney disease (CKD) on dialysis. In randomized, placebo-controlled trials conducted in patients with non–dialysis-dependent CKD, FC significantly decreased serum phosphate concentrations.1Block G.A. Fishbane S. Rodriguez M. et al.A 12-week, double-blind, placebo-controlled trial of ferric citrate for the treatment of iron deficiency anemia and reduction of serum phosphate in patients with CKD Stages 3-5.Am J Kidney Dis. 2015; 65: 728-736Google Scholar,2Yokoyama K. Hirakata H. Akiba T. et al.Ferric citrate hydrate for the treatment of hyperphosphatemia in nondialysis-dependent CKD.Clin J Am Soc Nephrol. 2014; 9: 543-552Google Scholar In randomized, active-controlled trials conducted in patients with dialysis-dependent CKD, FC produced similar reductions in serum phosphate concentrations.3Lewis J.B. Sika M. Koury M.J. et al.Ferric citrate controls phosphorus and delivers iron in patients on dialysis.J Am Soc Nephrol. 2015; 26: 493-503Google Scholar,4Yokoyama K. Akiba T. Fukagawa M. et al.A randomized trial of JTT-751 versus sevelamer hydrochloride in patients on hemodialysis.Nephrol Dial Transplant. 2014; 29: 1053-1060Google Scholar Auryxia is also approved in the US for clinical use as an iron-replacement product in patients with non-dialysis CKD and iron-deficiency anemia. In the 4 aforementioned randomized controlled trials, conducted in both non–dialysis-dependent and dialysis-dependent CKD cohorts, FC significantly increased serum transferrin saturation (TSAT).1Block G.A. Fishbane S. Rodriguez M. et al.A 12-week, double-blind, placebo-controlled trial of ferric citrate for the treatment of iron deficiency anemia and reduction of serum phosphate in patients with CKD Stages 3-5.Am J Kidney Dis. 2015; 65: 728-736Google Scholar, 2Yokoyama K. Hirakata H. Akiba T. et al.Ferric citrate hydrate for the treatment of hyperphosphatemia in nondialysis-dependent CKD.Clin J Am Soc Nephrol. 2014; 9: 543-552Google Scholar, 3Lewis J.B. Sika M. Koury M.J. et al.Ferric citrate controls phosphorus and delivers iron in patients on dialysis.J Am Soc Nephrol. 2015; 26: 493-503Google Scholar, 4Yokoyama K. Akiba T. Fukagawa M. et al.A randomized trial of JTT-751 versus sevelamer hydrochloride in patients on hemodialysis.Nephrol Dial Transplant. 2014; 29: 1053-1060Google Scholar Thus, FC can function as both an effective enteral phosphate binder and an enteral iron source. It is remarkable that FC corrects iron deficiency in the setting of CKD, and even end-stage kidney disease, as these are states with high hepcidin levels.5Zaritsky J. Young B. Wang H.J. et al.Hepcidin—a potential novel biomarker for iron status in chronic kidney disease.Clin J Am Soc Nephrol. 2009; 4: 1051-1056Google Scholar, 6Ashby D.R. Gale D.P. Busbridge M. et al.Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease.Kidney Int. 2009; 75: 976-981Google Scholar, 7Zaritsky J. Young B. Gales B. et al.Reduction of serum hepcidin by hemodialysis in pediatric and adult patients.Clin J Am Soc Nephrol. 2010; 5: 1010-1014Google Scholar, 8Troutt J.S. Butterfield A.M. Konrad R.J. Hepcidin-25 concentrations are markedly increased in patients with chronic kidney disease and are inversely correlated with estimated glomerular filtration rates.J Clin Lab Anal. 2013; 27: 504-510Google Scholar Hepcidin is the master iron-regulatory hormone, binding to and inhibiting ferroportin, the only known cellular iron exporter.9Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Google Scholar In the setting of high hepcidin levels, the activity of ferroportin—located on the basal surface of enterocytes—is decreased, thus inhibiting dietary and medicinal iron absorption from the gastrointestinal lumen (Figure 110Ganz T. Bino A. Salusky I.B. Mechanism of action and clinical attributes of Auryxia ® (ferric citrate).Drugs. 2019; 79: 957-968Google Scholar). The mechanisms by which FC overcomes the enteral iron block induced by high hepcidin and low ferroportin levels are unclear. One hypothesis is that the citrate moiety in FC chelates calcium ions, leading to disruption of enterocyte intercellular tight junctions, allowing for paracellular iron absorption that is independent of the hepcidin-ferroportin pathway. Interestingly, citrate compounds have been noted to facilitate paracellular enteral absorption of aluminum,11Froment D.P. Molitoris B.A. Buddington B. et al.Site and mechanism of enhanced gastrointestinal absorption of aluminum by citrate.Kidney Int. 1989; 36: 978-984Google Scholar,12Gupta A. Ferric citrate hydrate as a phosphate binder and risk of aluminum toxicity.Pharmaceuticals (Basel). 2014; 7: 990-998Google Scholar raising the possibility that iron absorption is enhanced by the same mechanism. In order to assess whether FC-associated enteral iron absorption occurs via a transcellular, ferroportin-dependent route, versus a paracellular, ferroportin-independent route, we evaluated enteral FC absorption in murine models, with or without CKD, with low or absent enterocyte ferroportin activity. Experiments were conducted in accordance with University of California, Los Angeles (UCLA) Division of Laboratory Animal Medicine guidelines, and the study protocols were approved by the UCLA Office of Animal Research Oversight. Mice were housed at UCLA, in standard cages with wood-chip bedding that was changed twice weekly. Animal housing rooms were temperature and humidity controlled, with a 12-hour light cycle. We first assessed the effects of FC in transmembrane serine protease 6 (Tmprss6) knockout mice, a model characterized by high hepcidin levels, low ferroportin activity, and resultant iron-refractory iron-deficiency anemia,13Folgueras A.R. de Lara F.M. Pendás A.M. et al.Membrane-bound serine protease matriptase-2 (Tmprss6) is an essential regulator of iron homeostasis.Blood. 2008; 112: 2539-2545Google Scholar both without and with the addition of adenine diet–induced CKD, which further increases hepcidin levels.14Hanudel M.R. Rappaport M. Gabayan V. et al.Increased serum hepcidin contributes to the anemia of chronic kidney disease in a murine model.Haematologica. 2017; 102: e85-e88Google Scholar To completely inhibit enteral ferroportin activity, we next assessed the effects of FC in a tamoxifen-inducible, enterocyte-specific ferroportin (FPN) knockout model (Villin-Cre-ERT2, Fpnflox/flox). Finally, we assessed the effects of FC in a Villin-Cre-ERT2, Fpnflox/flox model with adenine diet–induced CKD. Tmprss6 knockout mice on a C57BL/6 genetic background were kindly provided by Dr. Jodie Babitt (Massachusetts General Hospital). We first assessed the effects of FC in Tmprss6 knockout mice and wild-type (WT) littermates without CKD. Mice aged 4–7 weeks were fed 50 ppm iron diets without adenine (Envigo, Hackensack, NJ), with or without 0.1% FC (Akebia Therapeutics, Inc.), for 3 weeks, and then were euthanized. We next assessed the effects of FC in Tmprss6 knockout mice and WT littermates with CKD. Mice aged 8–12 weeks were fed 50 ppm iron diets with 0.2% adenine (Envigo) to induce CKD,15Jia T. Olauson H. Lindberg K. et al.A novel model of adenine-induced tubulointerstitial nephropathy in mice.BMC Nephrol. 2013; 14: 116Google Scholar, 16Tamura M. Aizawa R. Hori M. et al.Progressive renal dysfunction and macrophage infiltration in interstitial fibrosis in an adenine-induced tubulointerstitial nephritis mouse model.Histochem Cell Biol. 2009; 131: 483-490Google Scholar, 17Klinkhammer B.M. Djudjaj S. Kunter U. et al.Cellular and molecular mechanisms of kidney injury in 2,8-dihydroxyadenine nephropathy.J Am Soc Nephrol. 2020; 31: 799-816Google Scholar for 6 weeks. For the last 3 weeks of the diets, 0.1% FC (Akebia Therapeutics, Inc.) was added for one group and not added for another. This FC dose in mice corresponds to a typical adult human FC dose; in an adult human, 2 Auryxia tablets taken thrice daily (6000 mg FC) contain a cumulative daily ferric iron dose of 1260 mg, an amount equivalent to approximately one-third of total body iron. In mice, approximately one-third of total body iron is equivalent to ∼0.8 mg of ferric iron daily, or ∼4 mg of FC daily which, given murine daily food consumption of ∼4 g, corresponds to chow containing 0.1% FC. In these experiments, each group contained approximately half males and half females. After sacrifice, we collected whole blood, serum, liver, and duodenal enterocytes. To isolate duodenal enterocytes, the proximal section of the duodenum (∼2 cm) was cut open lengthwise and transferred to ice-cold ethylenediamine tetraacetic acid solution. The solution was vortexed to facilitate cell detachment, and the dispersed cells were collected by filtration. Tamoxifen-inducible, enterocyte-specific ferroportin (FPN) knockout mice were generated by breeding Villin-Cre-ERT2 mice with Fpnflox/flox mice, both of which are on C57BL/6 genetic backgrounds. Villin-Cre-ERT2 mice were kindly provided by Dr. Peter Tontonoz (UCLA). To characterize this model, we assessed Fpnflox/flox mice, with or without Villin-Cre-ERT2, with or without tamoxifen induction body daily for 4 To assess the effects of FC in this model, we Villin-Cre-ERT2, Fpnflox/flox mice aged weeks that were with or tamoxifen body induced daily for 4 Mice were on 50 ppm iron diets and then were weeks of the mice 1% FC added to diets for the 3 weeks to In these experiments, 1% FC was as not to an to FC To demonstrate the efficacy of this we a control experiment in which we assessed whether 3 weeks of 1% FC dietary supplementation can rescue the iron-deficiency anemia in Villin-Cre-ERT2, Fpnflox/flox mice fed a In the enterocyte-specific ferroportin knockout experiments, each group contained both males and females. After sacrifice, we collected whole blood, serum, liver, duodenal enterocytes, and whole duodenum for iron were as For duodenal iron proximal duodenal were in then in from which were were with the for iron and with The of duodenal enterocyte iron was assessed on a by a To the experiment in the setting of CKD, Villin-Cre-ERT2, Fpnflox/flox mice aged 6 weeks were on 0.2% adenine diets (Envigo) to induce CKD. in the adenine diet–induced CKD model are to the effects of dietary V. K. et in adenine-induced chronic kidney disease and in J 2014; Scholar only mice were weeks, mice were as with or weeks, 6 weeks of the adenine mice were to 50 ppm iron diets with or without 1% FC Mice were at weeks, weeks and 3 weeks of treatment with or without FC. blood, for and cell was collected when mice were aged weeks the of weeks of diets with or without and weeks for and iron was collected when mice were aged and weeks. and duodenal were as at were by the were to serum and serum iron were to serum hepcidin as A. E. et al.A mouse model of anemia of with on 2014; Scholar was to serum erythropoietin were in and at of the were and solution and acid in was and the were and concentrations in the were by a then to the of the to iron were in and was to the We using the and specific for mouse We the at for 2 by of at for at for at for and at for was to that of and each was in was using the For protein on Western the was Cell at a of and the was at a of For ferroportin on Western the was at a of and the was at a of For the protein mouse at a of was was using the with Lab and was using data are as and were to group For was to were To assess the efficacy of FC in a model, we evaluated the effects of 3 weeks of a 0.1% FC in Tmprss6 knockout to hepcidin and as et of by Tmprss6 is for of iron homeostasis.Blood. 2010; Scholar to (WT) the Tmprss6 knockout mice serum hepcidin concentrations and to have duodenum ferroportin protein levels (Figure and with decreased enteral iron the Tmprss6 knockout mice decreased and serum iron concentrations (Figure and and a anemia (Figure with a in serum erythropoietin concentrations (Figure In this model, FC increased duodenum ferroportin serum and with a decrease in serum (Figure To assess the efficacy of FC in even hepcidin models, we evaluated the effects of 3 weeks of a 0.1% FC in WT and Tmprss6 knockout mice with adenine diet–induced CKD. to mice of the same both the WT and Tmprss6 knockout mice with nephropathy increased serum increased serum increased with iron and a anemia with increases in serum (Figure In these CKD models, FC increased cells and (Figure and as was in the Tmprss6 knockout mice, FC increased duodenum ferroportin protein in the CKD Tmprss6 knockout mice (Figure with the mechanism of binding protein of ferroportin et of of to that iron Scholar, B. et control a block to iron 2013; Scholar, K. The in from mouse 2014; 5: Scholar To whether enteral FC absorption is dependent on ferroportin absorbed via a transcellular, ferroportin-dependent versus a paracellular, ferroportin-independent we assessed the effects of FC in a tamoxifen-inducible, enterocyte-specific ferroportin knockout murine model (Villin-Cre-ERT2, Fpnflox/flox). We first that this model duodenum ferroportin levels, resulting in decreased iron absorption and anemia. We mice with or without Villin-Cre-ERT2 with tamoxifen or weeks control mice without Villin-Cre-ERT2 or that were not tamoxifen in duodenum ferroportin (Figure and and iron-deficiency anemia (Figure mice with Villin-Cre-ERT2 that tamoxifen an decrease in duodenum mRNA (Figure undetectable duodenum protein (Figure and increased duodenal enterocyte iron (Figure with decreased iron from the decreased iron (Figure and anemia (Figure and Next, in a control we that 3 weeks of a 1% FC can iron-deficiency anemia when enterocyte ferroportin is In this WT mice were on weeks of half of the mice diets with 1% FC. Mice were 3 weeks The 1% FC increased iron increased serum iron concentrations and prevented anemia that this iron can WT We then whether 1% at iron-deficiency anemia (Figure rescue the iron-deficiency anemia resulting from enterocyte deletion (Figure ferroportin deletion in a decrease in duodenum ferroportin mRNA (Figure and protein (Figure iron-deficiency anemia the mice, 3 weeks of 1% FC did not iron (Figure or serum iron (Figure as did in WT mice (Figure mice with or without FC similar cell and and FC did not as did in WT mice (Figure data suggest that enteral FC absorption is predominantly dependent on ferroportin, and that in this model, significant paracellular absorption does not occur. that or enterocyte intercellular tight B. R. S. et function in chronic kidney (Basel). Scholar the potential for paracellular we repeated the experiment in the setting of CKD. in we a 0.2% adenine to induce et deletion hyperphosphatemia with Am Soc Nephrol. Scholar then the mice to diets with or without 1% as to possible adenine and high dietary iron in the mice, tamoxifen markedly decreased duodenum ferroportin mRNA (Figure and protein (Figure resulting in decreased iron (Figure After 6 weeks of the adenine serum concentrations were and did not to be with versus without FC concentrations decreased to similar in mice with versus without FC (Figure In the CKD mice, FC treatment in decreased duodenum ferroportin but increased duodenum ferroportin and serum iron (Figure and in the induced CKD mice, FC treatment significant on or serum iron (Figure and the induced mice the mice and FC did not cell or in the or induced mice induced and the of anemia in this CKD FC significantly iron status only in the CKD mice, that enteral FC absorption is predominantly dependent on ferroportin in this CKD of a 1% ferric citrate (FC) in the enterocyte-specific ferroportin knockout model, with adenine diet–induced chronic kidney disease. Villin-Cre-ERT2, Fpnflox/flox mice with adenine nephropathy were with tamoxifen or control at weeks of then diets with versus without 1% FC from to weeks of the duodenum duodenum ferroportin assessed via Western blot in serum from to weeks of in serum iron from to weeks of in cell from to weeks of and in from to weeks of is for are as and were to for FC versus treatment or induced for tamoxifen induction versus induction or mice In clinical FC has been shown to iron deficiency in patients with CKD and end-stage kidney disease, which are with levels.5Zaritsky J. Young B. Wang H.J. et al.Hepcidin—a potential novel biomarker for iron status in chronic kidney disease.Clin J Am Soc Nephrol. 2009; 4: 1051-1056Google Scholar, 6Ashby D.R. Gale D.P. Busbridge M. et al.Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease.Kidney Int. 2009; 75: 976-981Google Scholar, 7Zaritsky J. Young B. Gales B. et al.Reduction of serum hepcidin by hemodialysis in pediatric and adult patients.Clin J Am Soc Nephrol. 2010; 5: 1010-1014Google Scholar, 8Troutt J.S. Butterfield A.M. Konrad R.J. Hepcidin-25 concentrations are markedly increased in patients with chronic kidney disease and are inversely correlated with estimated glomerular filtration rates.J Clin Lab Anal. 2013; 27: 504-510Google Scholar In the setting of high hepcidin enteral ferroportin activity is decreased, a block to enteral iron E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Google Scholar The mechanisms by which FC this enteral block are One hypothesis is that the citrate moiety intercellular tight junctions, paracellular, ferroportin-independent enteral iron absorption. We this hypothesis by the of FC in and without which the transcellular iron is or completely In our first model, Tmprss6 knockout mice without CKD, in which hepcidin levels are FC iron status and increased in our WT and Tmprss6 knockout models with CKD, in which hepcidin levels are markedly FC increased cell and data demonstrate the efficacy of FC in states with high hepcidin levels and are with has been in FC clinical these models not suggest the mechanisms are by which FC overcomes the hepcidin In these models, in which ferroportin FC be absorbed via a paracellular pathway, the or be absorbed via a transcellular pathway, To the of FC on enteral ferroportin, we an enterocyte-specific ferroportin knockout murine This model is effective at completely enterocyte ferroportin resulting in enteral iron absorption and severe iron-deficiency a that can be by iron A. et iron is essential for iron Scholar In this model, any of the ferroportin-dependent be In our model, in the of enterocyte ferroportin, an FC dose that been effective in WT mice did not improve iron status or rescue the iron-deficiency anemia phenotype. increases in cell and were not that enteral FC absorption is predominantly dependent on ferroportin, and paracellular absorption. In the setting of CKD, B. R. S. et function in chronic kidney (Basel). Scholar intercellular tight and of paracellular FC absorption. in our CKD model, FC significantly iron status in the of enterocyte ferroportin, but not in its data suggest that, even in the setting of CKD, enteral FC absorption is predominantly by ferroportin transport and not possible paracellular In the CKD mice, FC treatment increased duodenum ferroportin duodenum ferroportin mRNA expression, with an mechanism by which iron of ferroportin et of of to that iron Scholar, B. et control a block to iron 2013; Scholar, K. The in from mouse 2014; 5: Scholar thus cellular iron absorption and to the We 2 CKD serum and concentrations were similar the WT CKD control group from the first experiment and the CKD group from the in the first CKD WT mice with FC a significant in but in the CKD the mice with FC did not have a significant in The FC did not significantly in the mice, iron is possible the of CKD the of mice, and the repeated in this study is that we mouse models to is not the for the of significant paracellular absorption of FC is In in our high hepcidin models, we effects of FC on iron and effective enteral iron absorption high hepcidin To assess whether FC the hepcidin block via potential paracellular we evaluated an enterocyte-specific ferroportin knockout model, in the or of impaired kidney In these models, FC did not rescue the iron-deficiency anemia that enteral FC absorption is predominantly dependent on ferroportin, and that paracellular pathways likely not of physiologic FC absorption. and from Akebia the This study was by an Akebia to This was at the of Kidney with The and of ferric citrate is for the treatment of hyperphosphatemia in patients with chronic kidney disease and is approved as an iron replacement product for patients with iron-deficiency anemia. In this of Kidney and of genetic models with and without chronic kidney injury to demonstrate that the absorption of iron by ferric citrate is dependent on ferroportin and does not paracellular iron

Topics & Concepts