^{Centre de Référence des Maladies Rénales Rares, Institut de Génomique Fonctionnelle à l’Ecole Normale Supérieure de Lyon et Université de Lyon, Lyon, France;}

^{Intrinsic Life Sciences, La Jolla, California; and}

^{Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina}

^{Corresponding author.}

Correspondence: Dr. M. Hewison, Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, 615 Charles E. Young Drive South, Room 410D, Los Angeles, CA 90095-7358. Email: ude.alcu.tendem@nosiwehm

Received 2013 Apr 8; Accepted 2013 Sep 13.

Abstract

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D₂) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

Abstract

Patients with CKD require iron supplementation and erythropoiesis stimulating agents (ESAs) to correct disease-associated anemia.¹ However, ESA hyporesponsiveness is common, with the iron homeostasis factor hepcidin (encoded by the HAMP gene) emerging as a possible culprit.² Hepcidin post-translationally suppresses membrane expression of ferroportin, the only known exporter of intracellular iron.³ Elevated plasma hepcidin, common to patients with CKD⁴ or inflammation,⁵ causes intracellular sequestration of iron and increases risk of anemia. By contrast, patients with hemochromatosis or iron deficiency exhibit decreased hepcidin.⁶

Studies of patients with CKD suggest that vitamin D status (serum concentrations of the prohormone 25-hydroxyvitamin D [25D]) correlates inversely with the prevalence of anemia⁷ and ESA resistance⁸ and directly with blood hemoglobin levels.⁸ In hemodialysis patients with anemia, vitamin D repletion has been shown to correlate with lower ESA requirements.⁹,¹⁰ Vitamin D is known to exert physiologic activities beyond its classic skeletal function, notably as a potent inducer of antimicrobial proteins such as cathelicidin antibacterial protein (encoded by the cathelicidin [CAMP] gene).¹¹,¹² In this respect, it is interesting that hepcidin was initially described as an antimicrobial peptide (encoded by the gene for hepcidin antibacterial protein, HAMP),¹³ with its role in iron homeostasis being a later observation. We therefore hypothesized that vitamin D can act to regulate expression of hepcidin, in a similar fashion to its effects on other antimicrobial proteins. To test this hypothesis, vitamin D–mediated changes in hepcidin and cathelicidin were compared using in vitro and in vivo models.

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Acknowledgments

The authors thank Drs. Thomas Ganz and Ella Nemeth (both UCLA) for kindly providing reagents and technical assistance with this manuscript. They also thank Mrs. Barbara Gales for her help in facilitating the supplementation study.

This work was supported in part by educational grants from the Académie Française/Jean Walter Zellidja, Réunion Pédiatrique de la Région Rhône Alpes, Société Française de Pédiatrie/Evian, Fondation pour la Recherche Médicale, and the Philippe Foundation (to J.B.), as well as grants from the US National Institutes of Health (DK0911672 to M.H.), US Public Health Service (DK 67563 and DK 35423 to I.B.S.), Casey Lee Ball Foundation (to I.B.S.), and National Institutes of Health/National Center for Research Resources/National Center for Advancing Translational Sciences University of California Los Angeles Center for Translational Science Institute (KL2TR000122 to J.J.Z. and UL1 RR-033176 and UL1TR000124 to I.B.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013040355/-/DCSupplemental.

Footnotes

References

1. Kalantar-Zadeh K, Streja E, Miller JE, Nissenson AR: Intravenous iron versus erythropoiesis-stimulating agents: Friends or foes in treating chronic kidney disease anemia?Adv Chronic Kidney Dis16: 143–151, 2009 [[PubMed]
2. Nakanishi T, Hasuike Y, Otaki Y, Kida A, Nonoguchi H, Kuragano T: Hepcidin: another culprit for complications in patients with chronic kidney disease? Nephrol Dial Transplant 26: 3092–3100, 2011 [[PubMed]
3. Ganz T: Hepcidin and iron regulation, 10 years later.Blood117: 4425–4433, 2011
4. Young B, Zaritsky J: Hepcidin for clinicians.Clin J Am Soc Nephrol4: 1384–1387, 2009 [[PubMed]
5. Ganz T: Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation.Blood102: 783–788, 2003 [[PubMed]
6. Ramos E, Kautz L, Rodriguez R, Hansen M, Gabayan V, Ginzburg Y, Roth MP, Nemeth E, Ganz T: Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice.Hepatology53: 1333–1341, 2011
7. Lac PT, Choi K, Liu IA, Meguerditchian S, Rasgon SA, Sim JJ: The effects of changing vitamin D levels on anemia in chronic kidney disease patients: A retrospective cohort review.Clin Nephrol74: 25–32, 2010 [[PubMed]
8. Kiss Z, Ambrus C, Almasi C, Berta K, Deak G, Horonyi P, Kiss I, Lakatos P, Marton A, Molnar MZ, Nemeth Z, Szabo A, Mucsi I: Serum 25(OH)-cholecalciferol concentration is associated with hemoglobin level and erythropoietin resistance in patients on maintenance hemodialysis.Nephron Clin Pract117: c373–c378, 2011 [[PubMed]
9. Kumar VA, Kujubu DA, Sim JJ, Rasgon SA, Yang PS: Vitamin D supplementation and recombinant human erythropoietin utilization in vitamin D-deficient hemodialysis patients.J Nephrol24: 98–105, 2011 [[PubMed]
10. Rianthavorn P, Boonyapapong P: Ergocalciferol decreases erythropoietin resistance in children with chronic kidney disease stage 5.Pediatr Nephrol28: 1261–1266, 2013 [[PubMed]
11. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zügel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL: Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.Science311: 1770–1773, 2006 [[PubMed]
12. Adams JS, Ren S, Liu PT, Chun RF, Lagishetty V, Gombart AF, Borregaard N, Modlin RL, Hewison M: Vitamin D-directed rheostatic regulation of monocyte antibacterial responses.J Immunol182: 4289–4295, 2009
13. Hewison M: Antibacterial effects of vitamin D.Nat Rev Endocrinol7: 337–345, 2011 [[PubMed]
14. Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, Tavera-Mendoza L, Lin R, Hanrahan JW, Mader S, White JH: Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.J Immunol173: 2909–2912, 2004 [[PubMed]
15. Gombart AF, Borregaard N, Koeffler HP: Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3. FASEB J 19: 1067–1077, 2005 [[PubMed]
16. Gombart AF, Saito T, Koeffler HP: Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediated innate immune response in humans and primates.BMC Genomics10: 321, 2009
17. Gal-Tanamy M, Bachmetov L, Ravid A, Koren R, Erman A, Tur-Kaspa R, Zemel R: Vitamin D: An innate antiviral agent suppressing hepatitis C virus in human hepatocytes.Hepatology54: 1570–1579, 2011 [[PubMed]
18. Hewison M: Vitamin D and the intracrinology of innate immunity.Mol Cell Endocrinol321: 103–111, 2010
19. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J: Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science306: 2090–2093, 2004 [[PubMed]
20. Wang L, Cherayil BJ: Ironing out the wrinkles in host defense: Interactions between iron homeostasis and innate immunity.J Innate Immun1: 455–464, 2009
21. Drakesmith H, Prentice AM: Hepcidin and the iron-infection axis.Science338: 768–772, 2012 [[PubMed]
22. Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T: Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood101: 2461–2463, 2003 [[PubMed]
23. Nairz M, Theurl I, Ludwiczek S, Theurl M, Mair SM, Fritsche G, Weiss G: The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium.Cell Microbiol9: 2126–2140, 2007 [[PubMed]
24. Schaible UE, Collins HL, Priem F, Kaufmann SH: Correction of the iron overload defect in beta-2-microglobulin knockout mice by lactoferrin abolishes their increased susceptibility to tuberculosis.J Exp Med196: 1507–1513, 2002
25. Sow FB, Alvarez GR, Gross RP, Satoskar AR, Schlesinger LS, Zwilling BS, Lafuse WP: Role of STAT1, NF-kappaB, and C/EBPbeta in the macrophage transcriptional regulation of hepcidin by mycobacterial infection and IFN-gamma.J Leukoc Biol86: 1247–1258, 2009 [[PubMed]
26. Sow FB, Florence WC, Satoskar AR, Schlesinger LS, Zwilling BS, Lafuse WP: Expression and localization of hepcidin in macrophages: A role in host defense against tuberculosis.J Leukoc Biol82: 934–945, 2007 [[PubMed]
27. Paradkar PN, De Domenico I, Durchfort N, Zohn I, Kaplan J, Ward DM: Iron depletion limits intracellular bacterial growth in macrophages.Blood112: 866–874, 2008
28. Armitage AE, Eddowes LA, Gileadi U, Cole S, Spottiswoode N, Selvakumar TA, Ho LP, Townsend AR, Drakesmith H: Hepcidin regulation by innate immune and infectious stimuli.Blood118: 4129–4139, 2011 [[PubMed]
29. Adams JS, Hewison M: Unexpected actions of vitamin D: New perspectives on the regulation of innate and adaptive immunity.Nat Clin Pract Endocrinol Metab4: 80–90, 2008
30. Yuk JM, Shin DM, Lee HM, Yang CS, Jin HS, Kim KK, Lee ZW, Lee SH, Kim JM, Jo EK: Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.Cell Host Microbe6: 231–243, 2009 [[PubMed]
31. Shin DM, Yuk JM, Lee HM, Lee SH, Son JW, Harding CV, Kim JM, Modlin RL, Jo EK: Mycobacterial lipoprotein activates autophagy via TLR2/1/CD14 and a functional vitamin D receptor signalling.Cell Microbiol12: 1648–1665, 2010
32. Ganz T, Nemeth E: Iron sequestration and anemia of inflammation.Semin Hematol46: 387–393, 2009
33. Andrews NC: Anemia of inflammation: The cytokine-hepcidin link.J Clin Invest113: 1251–1253, 2004
34. Sun CC, Vaja V, Babitt JL, Lin HY: Targeting the hepcidin-ferroportin axis to develop new treatment strategies for anemia of chronic disease and anemia of inflammation.Am J Hematol87: 392–400, 2012
35. Babitt JL, Lin HY: Molecular mechanisms of hepcidin regulation: implications for the anemia of CKD. Am J Kidney Dis 55: 726–741, 2010
36. Sasu BJ, Cooke KS, Arvedson TL, Plewa C, Ellison AR, Sheng J, Winters A, Juan T, Li H, Begley CG, Molineux G: Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.Blood115: 3616–3624, 2010 [[PubMed]
37. Schwoebel F, van Eijk LT, Zboralski D, Sell S, Buchner K, Maasch C, Purschke WG, Humphrey M, Zöllner S, Eulberg D, Morich F, Pickkers P, Klussmann S: The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys.Blood121: 2311–2315, 2013
38. Theurl I, Schroll A, Sonnweber T, Nairz M, Theurl M, Willenbacher W, Eller K, Wolf D, Seifert M, Sun CC, Babitt JL, Hong CC, Menhall T, Gearing P, Lin HY, Weiss G: Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats.Blood118: 4977–4984, 2011
39. Song SN, Tomosugi N, Kawabata H, Ishikawa T, Nishikawa T, Yoshizaki K: Down-regulation of hepcidin resulting from long-term treatment with an anti-IL-6 receptor antibody (tocilizumab) improves anemia of inflammation in multicentric Castleman disease.Blood116: 3627–3634, 2010 [[PubMed]
40. Bachman E, Feng R, Travison T, Li M, Olbina G, Ostland V, Ulloor J, Zhang A, Basaria S, Ganz T, Westerman M, Bhasin S: Testosterone suppresses hepcidin in men: A potential mechanism for testosterone-induced erythrocytosis.J Clin Endocrinol Metab95: 4743–4747, 2010
41. Pilz S, Iodice S, Zittermann A, Grant WB, Gandini S: Vitamin D status and mortality risk in CKD: a meta-analysis of prospective studies. Am J Kidney Dis 58: 374–382, 2011 [[PubMed]
42. Chun RF, Lauridsen AL, Suon L, Zella LA, Pike JW, Modlin RL, Martineau AR, Wilkinson RJ, Adams J, Hewison M: Vitamin D-binding protein directs monocyte responses to 25-hydroxy- and 1,25-dihydroxyvitamin D.J Clin Endocrinol Metab95: 3368–3376, 2010
43. Lagishetty V, Misharin AV, Liu NQ, Lisse TS, Chun RF, Ouyang Y, McLachlan SM, Adams JS, Hewison M: Vitamin D deficiency in mice impairs colonic antibacterial activity and predisposes to colitis.Endocrinology151: 2423–2432, 2010
44. Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL: Determination of vitamin D status by radioimmunoassay with an ^{I-labeled tracer.}Clin Chem39: 529–533, 1993 [[PubMed]
45. Hollis BW, Kamerud JQ, Kurkowski A, Beaulieu J, Napoli JL: Quantification of circulating 1,25-dihydroxyvitamin D by radioimmunoassay with ^{I-labeled tracer.}Clin Chem42: 586–592, 1996 [[PubMed]
46. Bacchetta J, Sea JL, Chun RF, Lisse TS, Wesseling-Perry K, Gales B, Adams JS, Salusky IB, Hewison M: Fibroblast growth factor 23 inhibits extrarenal synthesis of 1,25-dihydroxyvitamin D in human monocytes. J Bone Miner Res 28: 46–55, 2013
47. Lisse TS, Liu T, Irmler M, Beckers J, Chen H, Adams JS, Hewison M: Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling.FASEB J25: 937–947, 2011
48. Maes K, Nemeth E, Roodman GD, Huston A, Esteve F, Freytes C, Callander N, Katodritou E, Tussing-Humphreys L, Rivera S, Vanderkerken K, Lichtenstein A, Ganz T: In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2.Blood116: 3635–3644, 2010