Suppression of Iron-Regulatory Hepcidin by Vitamin D

Wisit Cheungpasitporn

March 28, 2013

Disclosure

• None

Objective

• To discuss on the role of vitamin D in regulation of hepcidin and anemia of chronic kidney disease.

Background

• Patients with CKD require iron supplementation and erythropoiesis stimulating agents (ESAs) to correct disease-associated anemia.

Background

• ESA hyporesponsiveness ~ 5-10% (in HD pts)1.

• Hepcidin (encoded by the HAMP gene) emerging as a possible culprit.

1 Icardi A, et.al. Nephrol Dial Transplant. 2013 Jul;28(7):1672-9..

• 25 aa peptide hormone.

• Chromosome 19.

• Synthesized by hepatocytes.

• Intrinsic antimicrobial activity.

Hepcidin

Background

• Elevated plasma hepcidin: • Common in CKD or inflammation • causes intracellular sequestration of iron and

increases risk of anemia.

• Patients with hemochromatosis or iron deficiency exhibit decreased hepcidin.

Zaritsky J. Clin J Am Soc Nephrol. 2009 Jun;4(6):1051-6.

Diseases of Hepcidin Dysregulation

Hereditary haemochromatosis

Iron-loading Anaemias

Anaemia of Inflammation

Iron-refractory iron-deficiency anaemia

Hepcidin-secreting tumors

HepcidinIron

Normal homeostasis

Ganz T. J Am Soc Nephol. 2007;18:394-400.Ganz T, Nemeth E. Am J Physiol Gastrointest Liver Physiol. 2006;290:G199-G203.

Background

• Hepcidin suppresses membrane expression of ferroportin, the only known exporter of intracellular iron.

Fpn

Fe

ferritin

Low Hepcidin High Hepcidin

Fe

hepcidin

ferritin

Iron releaseinto plasma

Iron-exporting cells (duodenal enterocytes,

macrophages, hepatocytes)

X

Fpn

Iron uptakeIron uptake

Nemeth E, et al. Science. 2004;306:2090-2093.

Spleen

Liver

Duodenum

Hep

cidin

Hepcidin

Hepcidin

Fpn

Fpn

Fpn

PlasmaFe-Tf

How Hepcidin Regulates Iron

Bone marrowand other sitesof iron usage

Nemeth E, et al. Science. 2004;306:2090-2093.

Icardi A, et.al. Nephrol Dial Transplant. 2013 Jul;28(7):1672-9..

142 HD patients

Kiss Z, et.al. Nephron Clin Pract. 2011;117(4):c373-8.

N = 153 All CKD not on dialysis

Lac PT, et.al. Clin Nephrol. 2010 Jul;74(1):25-32.

at 4 mo

Carvalho C. et al. Clin Nephrol. 2011 Aug;76(2):90-8.

N = 125 CKD not on dialysis

Background

• Vitamin D: a potent inducer of antimicrobial proteins such as cathelicidin antibacterial protein (encoded by the cathelicidin [CAMP] gene). 

• Hepcidin: described as an antimicrobial peptide (encoded by the gene for hepcidin antibacterial protein, HAMP)

Liu PT et. el. Science 311: 1770–1773, 2006

JASN March 2014 vol. 25 no. 3 564-572

Bacchetta J. et. el. J Am Soc Nephrol. 2014 Mar;25(3):564-72. 

HYPOTHESIS

• 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.

Vitamin D metabolite suppression of HAMP

HAMP: Hepcidin

Vitamin D suppresses expression of hepcidin (HAMP) in human monocytes and hepatocytes.

In vitro effect of vitamin D

CAMP: Cathelicidin vitamin D catabolic enzyme CYP24A1

Vitamin D increased expression of Cathelicidin and CYP24A1 in monocytes but not hepatocytes

In vitro effect of vitamin D

In vivo effect of vitamin D

PBMCm Wild type C57BL/6

No change in Hamp expression following 6 hr treatment with 25D or 1,25 D

No change in mouse Hamp expression following 24 hr treatment with increased doses of vit D

Mouse Monocyte cell line J774

In vivo effect of vitamin D 12-wk C57BL/6 mice on vitamin D-deficient diet * 6 wk

4 ppm iron diet for 1 week

Treated with 25D,1,25 D, saline IP injection

HAMP mRNA expression

Vitamin D receptor-mediated transcriptional repression of HAMP

In human cells, the suppression of HAMP expression by 1,25D or 25D appears to be due to direct inhibition of HAMP transcription

- ChIP assay using human PBMC monocytes- 1,25D decreased 0.5-fold for HAMP promoters in VDR enrichment - 1,25D enhanced CAMP and CYP24A1 promoters in VDR and RNA pol II enrichment

Luciferase promoter-reporter construct transfected into VDR-expressing MC3T3 cells

-24% decrease in transcription with 1,25D-In absence of 1α hydroxylase activity in MC3T3, 25 D had no effect

HAMPTarget geneactivity

Vitamin D-induced suppression of HAMP is associated with changes in ferroportin and ferritin expression

The effect of vitamin D on ferroportin expression in Human monocytes and hepatocytes

25D and 1,25D had no effect on level of ferroportin mRNA

25 and 1,25D increased ferroportin protein expression

Western blot

Effect of vitamin D on ferroportin - Post-transcriptional action

25D and 1,25D decreased ferritin mRNA level 25D and 1,25D decreased

ferritin protein expression

Immunohistochemistry

Effect of vitamin D supplementation on circulating Hepcidin in healthy volunteers

- Single arm PK study- 7 healthy volunteers (4 Men, median age 42 years)- Before and after a single dose of vitamin D2 100,000 IU

Increase in 25D serum levels No change in 1,25D serum levels

Circulating hepcidin levels decreased by 34% at 24 hr and 33% at 72 hr

Result

• Baseline and +72 hr after vitamin D supplement• Increase in FGF23 (62.57 vs 74.71 RU/ml, p=0.03)• Increase in phosphate (3.27 vs 3.61mg/dl, p<0.001)• Decrease in PTH (58.86 vs 51.29 pg/ml, p=0.06)• No significant Ca (9.19 vs 9.07 mg/dl, p=0.36)

Discussion

• Vitamin D is a potent regulator of the iron-regulatory protein hepcidin in both monocytes and hepatocytes

• Direct transcriptional suppression of the HAMP gene proximal promoter by 1,25D bound to its nuclear receptor,VDR

• Suppression of HAMP by 25D or 1,25D was not observed in murine models

Discussion

• In all three cell types studied, regulation of HAMP was observed after treatment with either active 1,25D or inactive 25D

• suggesting an intracrine mode of action• PBMC monocytes, THP1 cells, and HepG2 cells express

mRNA for the enzyme that catalyzes conversion of 25D to 1,25D, 1a-hydroxylase/ CYP27B1, as well as the VDR

• Intracrine responses to vitamin D appear to be exquisitely sensitive to the availability of substrate 25D

• hepcidin-ferroportin homeostasis system may be influenced by serum vitamin D

Vitamin D and Hepcidin-ferroportin iron-regulatory axis

Discussion

• Elevated serum concentrations of 25D (but not 1,25D) after a single oral dose of vitamin D2 produced a 34% decrease in serum hepcidin concentrations that persisted for 72 hours

• the regulation of serum hepcidin after vitamin D supplementation in vivo was at least as sensitive as more established markers of serum 25D status such as PTH, suggesting that hepcidin may be a useful marker of vitamin D function for future studies.

Discussion

• Low vitamin D status may be a contributing factor to the anemia of chronic disease.

• It is proposed that by acting to suppress expression of hepcidin in hepatocytes and monocytes, simple vitamin D supplementation may provide a cost-effective and safe adjuvant therapy for managing the anemia associated with this disease.

Questions & Discussion

Clinical implication – Future studies?