The kidney hepcidin/ferroportin axis controls iron reabsorption and determines the magnitude of kidney and systemic iron overload

Abstract

The hepcidin/ferroportin axis controls systemic iron homeostasis by regulating iron acquisition from the duodenum and reticuloendothelial system, respective sites of iron absorption and recycling. Ferroportin is also abundant in the kidney, where it has been implicated in tubular iron reabsorption. However, it remains unknown whether endogenous hepcidin regulates ferroportin-mediated iron reabsorption under physiological conditions, and whether such regulation is important for kidney and/or systemic iron homeostasis. To address these questions, we generated a novel mouse model with an inducible kidney-tubule specific knock-in of fpnC326Y, which encodes a hepcidin-resistant ferroportin termed FPNC326Y. Under conditions of normal iron availability, female mice harboring this allele had consistently decreased kidney iron but only transiently increased systemic iron indices. Under conditions of excess iron availability, male and female mice harboring this allele had milder kidney iron overload, but greater systemic iron overload relative to controls. Additionally, despite comparable systemic iron overload, kidney iron overload occurred in wild type mice fed an iron-loaded diet but not in hemochromatosis mice harboring a ubiquitous knock-in of fpnC326Y. Thus, our study demonstrates that endogenous hepcidin controls ferroportin-mediated tubular iron reabsorption under physiological conditions. It also shows that such control is important for both kidney and systemic iron homeostasis in the context of iron overload. The hepcidin/ferroportin axis controls systemic iron homeostasis by regulating iron acquisition from the duodenum and reticuloendothelial system, respective sites of iron absorption and recycling. Ferroportin is also abundant in the kidney, where it has been implicated in tubular iron reabsorption. However, it remains unknown whether endogenous hepcidin regulates ferroportin-mediated iron reabsorption under physiological conditions, and whether such regulation is important for kidney and/or systemic iron homeostasis. To address these questions, we generated a novel mouse model with an inducible kidney-tubule specific knock-in of fpnC326Y, which encodes a hepcidin-resistant ferroportin termed FPNC326Y. Under conditions of normal iron availability, female mice harboring this allele had consistently decreased kidney iron but only transiently increased systemic iron indices. Under conditions of excess iron availability, male and female mice harboring this allele had milder kidney iron overload, but greater systemic iron overload relative to controls. Additionally, despite comparable systemic iron overload, kidney iron overload occurred in wild type mice fed an iron-loaded diet but not in hemochromatosis mice harboring a ubiquitous knock-in of fpnC326Y. Thus, our study demonstrates that endogenous hepcidin controls ferroportin-mediated tubular iron reabsorption under physiological conditions. It also shows that such control is important for both kidney and systemic iron homeostasis in the context of iron overload. Translational StatementThe findings of the present study have implications that must be considered in the management of iron disorders and in the development of new iron therapies. First, strategies targeting the hepcidin/ferroportin (FPN) axis for the treatment of iron overload disorders could affect renal iron levels both directly, by inhibiting renal FPN, and indirectly, by inhibiting FPN in the gut and spleen. Second, increased hepcidin levels in chronic kidney disease could impinge on the progression of renal injury by blocking iron export from renal tubules. Third, the action of hepcidin on renal FPN could modify the outcomes of parenteral iron treatment by promoting iron retention in renal tubules. The findings of the present study have implications that must be considered in the management of iron disorders and in the development of new iron therapies. First, strategies targeting the hepcidin/ferroportin (FPN) axis for the treatment of iron overload disorders could affect renal iron levels both directly, by inhibiting renal FPN, and indirectly, by inhibiting FPN in the gut and spleen. Second, increased hepcidin levels in chronic kidney disease could impinge on the progression of renal injury by blocking iron export from renal tubules. Third, the action of hepcidin on renal FPN could modify the outcomes of parenteral iron treatment by promoting iron retention in renal tubules. Ferroportin (FPN) is the only known mammalian iron export protein. It mediates iron release into the circulation from duodenal enterocytes and splenic reticuloendothelial macrophages, the respective sites of iron absorption and recycling.1Donovan A. Lima C.A. Pinkus J.L. et al.The iron exporter ferroportin/slc40a1 is essential for iron homeostasis.Cell Metab. 2005; 1: 191-200Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar,2Ganz T. Cellular iron: ferroportin is the only way out.Cell Metab. 2005; 1: 155-157Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar FPN-mediated iron release is antagonized by the hormone hepcidin, also known as hepcidin antimicrobial peptide (HAMP). Produced primarily in the liver, hepcidin binds to and induces internalization of FPN, thereby limiting iron release into the circulation and its availability to peripheral tissues.3Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3359) Google Scholar,4Qiao B. Sugianto P. Fung E. et al.Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination.Cell Metab. 2012; 15: 918-924Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar Thus, the HAMP/FPN axis operates at the sites of absorption and recycling to control systemic iron homeostasis. The kidney is the site of iron reabsorption. Both non–transferrin-bound and transferrin-bound iron can cross into the glomerular filtrate.5Wareing M. Ferguson C.J. Green R. et al.In vivo characterization of renal iron transport in the anaesthetized rat.J Physiol. 2000; 524: 581-586Crossref PubMed Scopus (76) Google Scholar The vast majority of this iron is taken up back into the tubular epithelia. Several transporters have been implicated in this reuptake, including multiligand megalin-cubilin receptor complex, transferrin receptor 1, divalent metal transporter 1, zinc transporter ZIP8, and zinc transporter ZIP14.5Wareing M. Ferguson C.J. Green R. et al.In vivo characterization of renal iron transport in the anaesthetized rat.J Physiol. 2000; 524: 581-586Crossref PubMed Scopus (76) Google Scholar, 6Christensen I. Birn H. Storm T. et al.Endocytic receptors in the renal proximal tubule.Physiology. 2012; 27: 223-236Crossref PubMed Scopus (170) Google Scholar, 7Zhang D. Meyron-Holtz E. Rouault T. Renal iron metabolism: transferrin iron delivery and the role of iron regulatory proteins.J Am Soc Nephrol. 2007; 18: 401-406Crossref PubMed Scopus (65) Google Scholar, 8Canonne-Hergaux F. Gruenheid S. Ponka P. Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.Blood. 1999; 93: 4406-4417Crossref PubMed Google Scholar, 9Wang C. Jenkitkasemwong S. Duarte S. et al.ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.J Biol Chem. 2012; 287: 34032-34043Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 10Martines A. Masereeuw R. Tjalsma H. et al.Iron metabolism in the pathogenesis of iron-induced kidney injury.Nat Rev Nephrol. 2013; 9: 385-398Crossref PubMed Scopus (90) Google Scholar Once in the renal epithelia, iron is reabsorbed into the circulation. FPN is abundant in the kidney and has been implicated in iron reabsorption.11Wolff N. Liu W. Fenton R. et al.Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking.J Cell Mol Med. 2011; 15: 209-219Crossref PubMed Scopus (49) Google Scholar, 12Pan S. Qian Z.M. Cui S. et al.Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney.Biochem Biophys Res Commun. 2020; 522: 322-327Crossref PubMed Scopus (3) Google Scholar, 13Moulouel B. Houamel D. Delaby C. et al.Hepcidin regulates intrarenal iron handling at the distal nephron.Kidney Int. 2013; 84: 756-766Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14Wang X. Zheng X. Zhang J. et al.Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.Am J Physiol Renal Physiol. 2018; 315: F1042-F1057Crossref PubMed Scopus (22) Google Scholar However, it remains unknown if renal FPN is also subject to regulation by endogenous HAMP under normal physiological conditions, and if so, whether such regulation is important for systemic and/or renal iron homeostasis. To address these questions, we generated a novel mouse model with an inducible renal tubule–specific knock-in of fpnC326Y, which encodes a HAMP-resistant FPNC326Y protein. In addition, to confirm the previously reported role of FPN in iron reabsorption, we also generated a mouse model with an inducible renal tubule–specific deletion of the fpn gene. Our results demonstrate that endogenous HAMP directly regulates FPN-mediated iron absorption and that this regulation is important for both renal and systemic iron homeostasis, particularly in the setting of excess iron availability. This study is the first to utilize mice harboring renal-specific loss of HAMP responsiveness to formally determine the importance of the renal HAMP/FPN axis. It provides new insights into the role of iron reabsorption in determining the degree of renal and extrarenal iron overload in the setting of hemochromatosis. All animal procedures were compliant with the UK Home Office Animals (Scientific Procedures) Act 1986 and approved by the University of Oxford Medical Sciences Division Ethical Review Committee. The conditional fpnfl and fpnC326Yfl alleles were generated as described previously.15Lakhal-Littleton S. Wolna M. Chung Y.J. et al.An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5e19804Crossref PubMed Scopus (87) Google Scholar,16Lakhal-Littleton S. Wolna M. Carr C.A. et al.Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function.Proc Natl Acad Sci U S A. 2015; 112: 3164-3169Crossref PubMed Scopus (99) Google Scholar Mice harboring the Pax8.CreERT2+ transgene were a gift from Dr. Athena Matakidou, Cancer Research UK Cambridge Institute, University of Cambridge. These mice were generated as described previously.17Espana-Agusti J. Zou X. Wong K. et al.Generation and characterisation of a Pax8-CreERT2 transgenic line and a Slc22a6-CreERT2 knock-in line for inducible and specific genetic manipulation of renal tubular epithelial cells.PLoS One. 2016; 11e0148055Crossref PubMed Scopus (8) Google Scholar All mice were on a C57BL/6 background. Unless otherwise stated, animals were provided with a standard rodent chow diet containing 200 parts per million (ppm) iron. In iron manipulation experiments, mice were given an iron-loaded diet (5000-ppm iron; Teklad TD.140464) or a matched control diet (200-ppm iron; Teklad TD.08713) from weaning for 3 months. Serum iron and ferritin levels were determined using the ABX-Pentra system (Horiba Medical). Hemoglobin values were determined by HemoCue Hb 201 Hemoglobin Microcuvettes. Serum erythroferrone levels were measured by enzyme-linked immunosorbent assay (Intrinsic Lifesciences). Determination of total elemental iron in tissues was performed by inductively coupled plasma mass spectrometry, as described previously.15Lakhal-Littleton S. Wolna M. Chung Y.J. et al.An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5e19804Crossref PubMed Scopus (87) Google Scholar,16Lakhal-Littleton S. Wolna M. Carr C.A. et al.Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function.Proc Natl Acad Sci U S A. 2015; 112: 3164-3169Crossref PubMed Scopus (99) Google Scholar,18Lakhal-Littleton S. Crosby A. Frise M. et al.Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.Proc Natl Acad Sci U S A. 2019; 116: 13122-13130Crossref PubMed Scopus (27) Google Scholar Calibration was achieved using the process of standard additions, where spikes of 0 ng/g, 0.5 ng/g, 1 ng/g, 10 ng/g, 20 ng/g, and 100 ng/g iron were to of a iron standard was and measured to confirm the of the was also and as an standard at a of 1 iron was from mice a of and subject to inductively coupled plasma mass levels in the were measured using a assay and iron values were to was performed in using FPN at and at The of the FPN and hepcidin were using Pax8.CreERT2+ animals and animals as Renal were using at at or at were and were using an were in and using to the were by at for 10 at in the was measured by and to the for were in and at for was was membrane using the system, and were for an in blocking containing were at with FPN at or at were using the were by and the the FPN and the was to were using and in were for 1 with in was and were with and with were using a standard expression was measured using expression assay for and The for the of was first by for to a values of were to the of the values for control to expression levels were as as were performed using the were using of first to the site of FPN expression in the To that we mouse for FPN and 1 of proximal and of of and and of distal and and that FPN is expressed in the with 1 to the proximal tubules. was FPN in the with to the was of FPN with in These results confirm that FPN is abundant in the proximal tubules. To determine whether renal FPN is by we mice harboring a Pax8.CreERT2+ knock-in which expression of the under control of the in proximal and distal and in J. Zou X. Wong K. et al.Generation and characterisation of a Pax8-CreERT2 transgenic line and a Slc22a6-CreERT2 knock-in line for inducible and specific genetic manipulation of renal tubular epithelial cells.PLoS One. 2016; 11e0148055Crossref PubMed Scopus (8) Google Scholar Pax8.CreERT2+ mice with harboring a conditional knock-in allele which encodes a hepcidin-resistant In addition, and to confirm the previously reported role of renal FPN in iron reabsorption, we Pax8.CreERT2+ mice with mice harboring the treatment to the renal FPN levels were increased in female and male mice relative to controls that renal FPN is subject to regulation by endogenous HAMP under normal physiological conditions, and in the of the Pax8.CreERT2+ renal FPN levels were in female and male mice relative to the of the Pax8.CreERT2+ transgene The of this Pax8.CreERT2+ transgene was also by the of the deletion allele in the kidney but not in the or of mice we to determine whether the renal HAMP/FPN axis controls iron reabsorption. To that we measured renal and iron levels at 1 1 3 and that renal iron was in in control at Renal iron in was not to iron also the in iron levels proximal of in we that renal iron was in in control from 1 Renal iron in was not to iron also iron proximal of in Serum iron levels were increased transiently in both male and female mice relative to controls at the iron levels were decreased in relative to control at the comparable in of at of iron levels to in control animals at 3 of could not be to of normal renal FPN the in renal FPN levels were at 3 treatment and renal iron and increased iron levels in demonstrate that iron reabsorption in proximal is subject to regulation by HAMP under normal physiological conditions. renal iron and decreased iron levels in confirm findings that renal FPN to iron reabsorption. In addition, under normal physiological conditions, the control of iron reabsorption by the renal HAMP/FPN axis to be important in in at in the C57BL/6 we to determine the of the renal HAMP/FPN axis to systemic iron homeostasis. that had a in ferritin iron and iron at the with control levels comparable to of controls at of these was and controls levels were not to we that have ferritin levels at the and iron at the and and iron at the and with control also had a and in levels at the of these was and controls levels were also not to these demonstrate under normal physiological conditions, the renal HAMP/FPN axis to systemic iron levels but in is not essential for the of normal systemic iron homeostasis. we to determine the role of the renal HAMP/FPN axis in the setting of iron overload. To that animals and controls were with and provided a control chow containing 200 or an iron-loaded containing for 3 months. that of iron-loaded diet increased and iron in and of both However, animals had greater in iron ferritin and iron in the and controls. In had a in renal iron in the degree of iron proximal and in the were also in iron and in In line with greater systemic iron overload, animals also had greater in expression with controls Hemoglobin levels were not by diet or and with erythroferrone levels also and These findings demonstrate under conditions of excess iron availability, control of iron reabsorption by the renal HAMP/FPN axis systemic iron overload renal iron overload. we to the role of the renal HAMP/FPN axis in the context of a genetic of iron overload by in hepcidin or hepcidin C. iron homeostasis genetic of hemochromatosis and 2005; PubMed Scopus Google Scholar To that we mice generated harboring a ubiquitous knock-in of the allele had previously that these mice the of S. Wolna M. Chung Y.J. et al.An essential cell-autonomous role for hepcidin in cardiac iron homeostasis.Elife. 2016; 5e19804Crossref PubMed Scopus (87) Google Scholar,18Lakhal-Littleton S. Crosby A. Frise M. et al.Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.Proc Natl Acad Sci U S A. 2019; 116: 13122-13130Crossref PubMed Scopus (27) Google Scholar the of iron overload in these mice with that in mice fed an iron-loaded diet from weaning for 3 months. Both and mice fed an iron-loaded diet had increased iron in the liver, and with respective controls and Renal iron in was normal at 3 of and only increased by relative to controls at of In animals provided an iron-loaded diet had a in renal iron with on a normal diet in renal and iron overload the were also in iron and in The of extrarenal iron overload was comparable the the of renal overload was in animals provided an iron-loaded diet in mice FPN was increased in the in both mice and mice provided an iron-loaded diet and in of the site of FPN expression in proximal that it to as as to the membrane in In FPN to primarily to the and the in mice provided an iron-loaded diet and The of FPN localization from the of iron-loaded diet was using animals as controls These with the that animals have renal iron controls of an iron-loaded demonstrate that loss of HAMP action on renal FPN the kidney from iron in the setting of hemochromatosis. The important of the present study is that endogenous HAMP controls FPN-mediated iron reabsorption. had reported the regulation of FPN by HAMP in renal and an HAMP and FPN levels in the kidney S. Qian Z.M. Cui S. et al.Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney.Biochem Biophys Res Commun. 2020; 522: 322-327Crossref PubMed Scopus (3) Google B. Houamel D. Delaby C. et al.Hepcidin regulates intrarenal iron handling at the distal nephron.Kidney Int. 2013; 84: 756-766Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar However, this is the first that such regulation operates in vivo under normal physiological conditions, and in a that on renal and systemic iron homeostasis. of this study is the of novel generated to a renal-specific loss of HAMP This the study of the renal HAMP/FPN axis the of systemic iron homeostasis, otherwise in ubiquitous animal The important of the present study is that the renal HAMP/FPN the of both renal and systemic iron overload loss of HAMP responsiveness in renal increased the of liver, and iron overload, the of renal iron overload of an iron-loaded with greater renal iron overload was of an iron-loaded diet to animals which the renal HAMP/FPN axis is in hemochromatosis mice which the renal HAMP/FPN axis is also This the that the kidney is not in with C. iron homeostasis genetic of hemochromatosis and 2005; PubMed Scopus Google Scholar both hemochromatosis and dietary iron overload increased FPN in renal to in of localization to the or were in the rat where iron was to the relative of FPN these in duodenal A. et al.Iron membrane of intestinal iron transporters and ferroportin J Physiol Cell Physiol. PubMed Scopus Google Scholar of these results is that in the setting of dietary iron overload, increased FPN by iron regulatory to FPN in the renal tubular increased HAMP the of FPN that can to the It from this that the as to the localization of FPN in proximal could in renal tubular iron and in hepcidin levels animal in M. Ferguson C.J. Green R. et al.In vivo characterization of renal iron transport in the anaesthetized rat.J Physiol. 2000; 524: 581-586Crossref PubMed Scopus (76) Google N. Liu W. Fenton R. et al.Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking.J Cell Mol Med. 2011; 15: 209-219Crossref PubMed Scopus (49) Google Scholar, 12Pan S. Qian Z.M. Cui S. et al.Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney.Biochem Biophys Res Commun. 2020; 522: 322-327Crossref PubMed Scopus (3) Google Scholar, 13Moulouel B. Houamel D. Delaby C. et al.Hepcidin regulates intrarenal iron handling at the distal nephron.Kidney Int. 2013; 84: 756-766Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14Wang X. Zheng X. Zhang J. et al.Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.Am J Physiol Renal Physiol. 2018; 315: F1042-F1057Crossref PubMed Scopus (22) Google T. of the kidney in iron renal expression of and in J Physiol Renal Physiol. PubMed Scopus Google Scholar this could a degree of reported previously in to FPN including the in the present X. Zheng X. Zhang J. et al.Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.Am J Physiol Renal Physiol. 2018; 315: F1042-F1057Crossref PubMed Scopus (22) Google Scholar The of the localization of FPN in renal remains and we could not in iron in iron-loaded mice is that FPN to the in renal of iron-loaded were by in iron-loaded and in the rat The study FPN to be in iron retention as of the acute N. I. N. et is a in rat a with 2012; PubMed Scopus (22) Google F. Jenkitkasemwong S. S. increases ferroportin and levels in PubMed Scopus Google Scholar of the subcellular localization of FPN in iron and in to our of the role of FPN in the The regulation and of renal HAMP also not that expression of the in the kidney was increased in animals of iron-loaded diet but in hemochromatosis mice and in harboring renal tubule–specific loss of FPN or renal tubule–specific loss of HAMP responsiveness and These results not the that renal expression is by renal iron In of the of renal a study in a model of that it is in renal that renal HAMP using an with of distal and and the FPN expression not with in mice harboring renal-specific loss of HAMP responsiveness and These results not with an role for renal HAMP in the regulation of renal renal HAMP be in regulation of renal In the it be to study the functions of renal and to the relative of renal and to the control of iron reabsorption. from the present study is under conditions of normal iron availability, the renal HAMP/FPN axis is not essential for of normal systemic iron homeostasis and is important for regulating renal iron at in female mice of the C57BL/6 the in renal iron levels from loss of FPN or of HAMP responsiveness in the renal were in systemic iron were in The of the systemic the is of dietary iron absorption by we that in mice harboring renal-specific loss of HAMP expression was increased the in iron levels at the and increased at expression was in mice harboring renal-specific loss of FPN the in iron levels at the and at and In addition, increased expression in the first setting was by decreased gut FPN decreased expression in the setting was by gut FPN and These results that the action of HAMP on dietary iron absorption is an in normal systemic iron homeostasis in the of renal iron reabsorption. under conditions of normal iron availability, the renal HAMP/FPN axis to be important in female mice that in male at in the C57BL/6 This could not be to and in the of the Pax8.CreERT2+ in the levels and of transporters the has been reported previously in this mouse J. et of renal transporters and Am Soc Nephrol. PubMed Scopus Google Scholar with we expression of FPN in the of in the of male study using a by a to fpn in the also reported an in renal iron and in iron and iron However, that study was in a mouse and not the to or X. Zheng X. Zhang J. et al.Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.Am J Physiol Renal Physiol. 2018; 315: F1042-F1057Crossref PubMed Scopus (22) Google Scholar the findings of the present study new of the role of the renal HAMP/FPN axis in renal and systemic iron homeostasis. This new has important implications for the management of hemochromatosis and iron All the was the of a was by a Research UK to and and on the with

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