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Hepcidin, Vitamin D and Anemia of CKD
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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?