IRON HOMEOSTASIS REGULATION AND ROLE IN HEALTH AND DISEASE

Elizabeta Nemeth, PhD Professor, David Geffen School of Medicine at UCLA Director, UCLA Center for Iron Disorders

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DISCLOSURES

• Co-founder and consultant for: • Intrinsic LifeSciences (hepcidin diagnostics) • Silarus Therapeutics (erythroferrone-targeted therapeutics)

• Consultant for:

• Akebia Therapeutics • Ionis Pharmaceuticals • Vifor • Protagonist Therapeutics

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Why do we need iron?

ATP

Function of iron-containing proteins: • Oxygen transport & storage (hemoglobin, myoglobin) • ATP synthesis (mitochondrial respiratory complexes) • DNA synthesis (ribonucleotide reductase) • DNA repair (XPD, FANCJ, nucleotide excision repair) • miRNA processing (Pasha/Dgsr8) • Amino acid synthesis and degradation (amino acid oxidases) • Lipid metabolism (fatty acid desaturases) • Nitric oxide production (NO synthase) • Protection from oxidative damage (peroxidases, catalase) • Innate immunity (NADPH oxidase and myeloperoxidase) • Hormone, neurotransmitter synthesis (thyroid, dopamine) • Oxygen sensing (prolyl hydroxylase)

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Toxicity of iron

• Excess free iron catalyzes generation of harmful oxygen radicals

Fenton reaction: (1) Fe2+ + H2O2 +H+ → Fe3+ + HO• + H2O (2) Fe3+ + H2O2 → Fe2+ + HOO• + H+

• Damage to the vital cell structures (DNA, RNA, proteins, cell membrane)

Hydroxyl radical

Superoxide radical

heart liver

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Iron disorders

Iron deficiency may lead to • Anemia • Cardiovascular strain • In fetuses and children:

• Developmental defects • Growth retardation • Neurological defects

• Impaired muscle function, exercise tolerance, work performance

• Altered immune function

Iron overload may lead to • Liver cirrhosis, liver cancer • Cardiomyopathy • Endocrine disorders including

diabetes, testicular failure • Arthritis, and bone and joint pain

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Systemic iron homeostasis

Spleen

Bone marrow

RBC

Liver (1000 mg)

Duodenum

Plasma Fe-Tf

Iron loss (1-2 mg/d)

2400 mg 3-4 mg

Total body iron: ~4 grams

(not regulated)

Storage

Utilization

Recycling

Absorption

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Ferroportin - the iron exporter • The only cellular iron exporter known, supplies iron to plasma • The receptor for hepcidin

Spleen

Bone marrow

RBC

Liver (1000 mg)

Duodenum

Plasma Fe-Tf 2400 mg

Storage

Utilization

Recycling

Absorption

Fpn

Fpn

Fpn Fpn

hepcidin

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Blood

Food

Fpn

Fe

ferritin

Low hepcidin High hepcidin

hepcidin

ferritin

Fpn

Dietary iron uptake

Regulation of intestinal iron absorption

Duodenal enterocytes

Dietary iron uptake

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Fpn

Fe

ferritin

Low hepcidin High hepcidin

hepcidin

ferritin

Iron release into plasma

Fpn

Erythrocyte uptake

Hepcidin causes iron retention in macrophages Erythrocyte

uptake Macrophages

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Hepcidin regulation

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

hepcidin

Iron signals

Inflammation

Liver Fe

Erythropoietic signal

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hours relative to iron ingestion-24 -12 0 12 24

seru

m ir

on (m

cg/d

L)

100

120

140

160

180

200

220

240

seru

m h

epci

din

(ng/

ml)

0

10

20

30

40

50

60

Hepcidin response to oral iron in a healthy volunteer

Fe 65mg

Ganz et al, Blood 2008

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Hepcidin production is proportional to iron stores in healthy humans

Ganz et al, Blood 2008

Women Men

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Space of Disse

Hepcidin regulation by iron

hepc hepatocyte

Smad pathway

Smads

BMP2/6

liver sinusoidal endothelium

HJV BMPR

BMP2/6

TfR1

Fe-Tf

TfR2 HFE

Fe-Tf

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CTRL 4h 9h 12h 15h 24h

Live

r H

amp

mR

NA

exp

ress

ion

0.062

0.125

0.25

0.5

1

2

4

phlebotomy EPO

***

****** ******

*

* p

Erythroferrone (ERFE): a hormone produced by erythroid precursors in response to EPO (Kautz et al. Nat Genet 2014, Kautz et al. Blood 2015, Ganz et al. Blood 2017)

Spleen

Bone marrow

Liver

Duodenum

Plasma Fe-Tf Fpn

Fpn

Fpn

hepcidin

EPO Erythroferrone

Hepcidin regulation by erythropoietic activity

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Space of Disse

Hepcidin regulation by ERFE

hepc

hepatocyte Smad pathway Smads

BMP2/6

liver sinusoidal endothelium

ERFE BMPR

BMP2/6

Arezes et al. Blood 2018, Wang et al. Blood 2019

ERFE traps BMP2,5,6,7 KDIG

O

Hepcidin plays a role in host defense: prevents the appearance of NTBI in plasma during infection (protective against some gram-negative and fungal infections)

Hepcidin regulation by inflammation

Ganz and Nemeth. Hematology 2011

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Iron deficiency

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

hepcidin Fe stores

All cells are iron-deficient

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Iron-restricted anemias

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

hepcidin

Inflammation

Oral iron therapy less effective

hepcidin

Maldistribution of iron

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Primary iron overload (hereditary hemochromatosis)

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

BMP6 TfR2

HJV HFE X

Hepcidin deficiency NTBI = non-transferrin-bound iron

Iron loading primarily in parenchyma (hepatocytes, endocrine cells, cardiomyocytes)

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Secondary iron overload (e.g. transfusions)

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

hepcidin

Iron signals

Liver Fe 1ml pRBC =

1mg Fe

Iron loading primarily in macrophages

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Iron (dys)homeostasis in CKD KDIG

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Serum hepcidin is high in CKD

Zaritsky J et al. Clin J Am Soc Nephrol 2009;4:1051–1056; Zaritsky J et al. Clin J Am Soc Nephrol 2010;5:1010–1014

10

100

1000

Paediatric controls

Adult controls

PCKD2-4 ACKD2-4 PCKD5D

Seru

m h

epci

din

(ng/

mL)

N=20 N=24 N=48 N=32

Median 25.3 Median 72.9

Median 127.3

Median 269.9

Median 652.4

N=26

10

100

1000

Paediatric controls

Adult controls

Paediatric HD

Adult HD

N=20 N=24 N=30 N=33

Median 25.3 Median 72.9

Median 240.5 Median 690.2

Hep

cidi

n (n

g/m

L)

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Hepcidin is cleared by HD but recovers rapidly

Zaritsky et al. CJASN 2010 June; 5: 1010–1014.

Polyflux Revaclear dialyzer (Gambro) with a dialysate flow (Qd) of 800 ml/min for an average of 3.2 ± 0.2 and 3.0 ± 0.4 hours in pediatric and adult patients, respectively (NS).

Kuragano et al. Am J Nephrol 2010;31:534–540

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879302/

Hepcidin contributes to the development of CKD anemia • Adenine-induced mouse model of CKD • Iron-normalized Hepcidin KO compared to WT mice

Mark Hanudel, Dept of Pediatrics, UCLA

weeks of adenine diet0 4 8

Hem

oglo

bin

(g/d

L)

6

8

10

12

14

16

WT

Hamp-/-

p

Anemia in CKD

Spleen

Bone marrow

RBC

Liver

Duodenum

Plasma Fe-Tf

Fpn

Fpn

Fpn

hepcidin

Inflammation

hepcidin

Inflammation KDIG

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Pathogenesis of anemia of CKD

• Inflammation and uremic toxins mediate: • Iron restriction due to increased hepcidin • Suppression of erythropoiesis • Shortened erythrocyte lifespan

• True iron deficiency from blood loss and decreased iron absorption from chronic inflammation (EPO resistance)

• Relative EPO deficiency

EPO resistance

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Inflammation (C-reactive protein) predicts EPO resistance

• Groups defined by initial CRP • EPO resistance: Hb < 10 g/dl

x during 12 months despite: • ≥9,000 U/week epoetin-α or

rHuEPO-β • ≥60 μg/week darbepoetin-α

Relative risk (RR) of EPO resistance depends on CRP

Kimachi M et al. Nephron 2015;131:123-130

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Estimates of blood loss in CKD Author Year Normal CKD-nondialysis Hemodialysis Method

Gastrointestinal Rosenblatt et al. 1982 0.8 ml blood/d =

100 mg Fe/y 3.2 ml blood/d = 400 mg Fe/y

6.3 ml blood/d = 800 mg Fe/y

51-chromium

Wizemann et al. 1983 5 ml blood/d = 600 mg Fe/y

51-chromium

Hemodialysis Tsukamoto et al. 2016

500 mg Fe/y Tubing washout, balance

Others (older) 1.2-2.8 g Fe/y Tubing washout, balance

0.5 -3.5 g Fe/year is needed to replace iron losses in most hemodialysis patients

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Artunz and Risler. Nephrol Dial Transplant. 2007

Relative erythropoietin deficiency in CKD

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Conclusions • Hepcidin-ferroportin axis controls systemic iron homeostasis and

is involved in the pathogenesis of multiple iron disorders • CKD iron dyshomeostasis is characterized by both iron restriction

and iron deficiency • Iron restriction is caused by elevated hepcidin:

– malabsorption of iron – sequestration of iron in macrophages

• Iron deficiency is largely a result of HD-related blood loss • Iron deficiency and iron restriction contribute to erythropoietin

resistance • Erythropoietin and ESAs dose-dependently suppress hepcidin • IV iron reduces erythropoietin resistance

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Iron homeostasis regulation and role in health and diseaseDisclosuresWhy do we need iron?Slide Number 4Iron disordersSystemic iron homeostasisFerroportin - the iron exporterRegulation of intestinal iron absorptionHepcidin causes iron retention in macrophagesHepcidin regulationSlide Number 11Slide Number 12Hepcidin regulation by ironSlide Number 14Slide Number 15Hepcidin regulation by ERFESlide Number 17Iron deficiencyIron-restricted anemiasPrimary iron overload (hereditary hemochromatosis) Secondary iron overload (e.g. transfusions) Iron (dys)homeostasis in CKDSerum hepcidin is high in CKDSlide Number 24Hepcidin contributes to the development of CKD anemiaAnemia in CKDSlide Number 27Inflammation (C-reactive protein) predicts EPO resistanceEstimates of blood loss in CKDRelative erythropoietin deficiency in CKDConclusions