https://www.haaselab.org

Volker H. Haase M.D.Krick-Brooks Chair in Nephrology

Professor of MedicineVanderbilt University

Hypoxia-inducible factors in erythropoiesis, iron homeostasis and beyond

@HIFpathway

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Outline: HIF in erythropoiesis

u overview

u EPO synthesis in kidney and liver

u iron metabolism

u renal anemia

u HIF beyond erythropoiesis

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Physiologic Responses to Hypoxia:This what happens when you go to high altitude

Andrew M. Luks J Appl Physiol 2015;118:509-519

©2015 by American Physiological Society

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EPO and Hgb responses at high altitude

Faura et al., Blood, 1969

Abbrecht and Littel, J. Appl. Physiol., 1972

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HIF controls the Hypoxic Induction of EPO

Haase, VH

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HIF regulates multiple processes:biological target complexity

HIF

Vasculogenesis/AngiogenesisHeme oxygenase-2NOS-2, PAI - IVEGF, VEGF-R (FLT-1)

Iron metabolism /ErythropoiesisCeruloplasminEPO (HIF-2 > HIF-1)TransferrinTransferrin R

Metabolism: Glycolysis (HIF-1)Fat metabolism (HIF-2)Adenylate kinase-3Carbonic anhydrase-9Glut-1 and -3Glycolytic enzymes(Hexokinase, LDH,PGK, etc.)Leptin

Proliferation/Cell survivalCyclin G2, EPO, Heme oxygenase 1IGF-2, IGFBP1, -2, -3NOS-2, NIP-3, p21TGF-b3, VEGF, WT1Indirect: c-Myc

HYPOXIAvia

ECM production/Cell migrationCxCR4, c-met, CTGF, PAI-1,Procollagen prolyl hydroxylase-a1

Epithelial Barrier FunctionITF, MDR-1, CD73

Transcription factorsEts-1, DEC-1/Stra13

Haase, VH

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Regulation of HIF-α stability: molecular complexity

Angiotensin II

Non-hypoxic stimuli:Interleukin1, TNF-a, Growth factors

HIF: hypoxia-inducible factor Haase, VH, AJP Renal 2006

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• Wang, G.L. and Semenza, G.L. (1995). Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270, 1230-1237.

• Wang, G.L., Jiang, B.-H., Rue, E.A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA. 92, 5510-5514.

• Forsythe, J.A., Jiang, B.-H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., and Semenza, G.L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604-4613.

• Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (1993). Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc. Natl. Acad. Sci. USA. 90, 2423-2427.

• Maxwell, P.H. et al. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 399, 271-275.

• Jaakkola, P. et al. (2001). Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 292, 468-472

• Epstein, A.C.R. et al.. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 107, 43-54.

• Iliopoulos, O., Levy, A.P., Jiang, C., Kaelin, W.G., Jr., and Goldberg, M.A. (1996). Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc. Natl. Acad. Sci. USA. 93, 10595-10599.

• Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.-Y., Huang, L.E., Chau, V., Pavletich, N., and Kaelin, W.G., Jr. (2000). Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423-427.

• Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S., and Kaelin, W.G., Jr. (2001). HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 292, 464-468

Discovery of HIF-oxygen sensing

Gregg L. SemenzaJohns Hopkins

Sir Peter J. RatcliffeOxford

William G. Kaelin, Jr.Harvard

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2OG-dependent dioxygenases:pharmacologic target complexity

Loenarz and Schofield, Nature Chemical Biology, 2008PHD1, PHD2, PHD3FIH

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The HIF-EPO axis

in kidney and liver

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GATA-2/3 (5’-TGATAA-3’)

HIF-2 dependent regulation of EPO

EPO: erythropoietin; HIF: hypoxia-inducible factor.

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EPO-producing cells in human kidneys

Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

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Hct 15

cortex

IM

IS

OS

Hct 26Hct 45

HIF-2 regulates the size of the REPC pool

Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

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Distinct interstitial cell populations regulate renal EPO production: cellular complexity

EPO: erythropoietin; PHD: prolyl-4-hydroxylase domain; REPC: renal EPO-producing cell.Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

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The liver as EPO source:role of individual PHDs

EPO: erythropoietin; PHD: prolyl-4-hydroxylase domain.Tojo Y, et al. Mol Cell Biol. 2015;35(15):2658-2672.

Cont

rol

P13-

L-KO

P23-

L-KO

P123

-L-K

O

P12-

L-KO

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HIF and

iron metabolism

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HIF in iron metabolism

Koury MJ, Haase VH. Nat Rev Nephrol. 2015;11(7): 394-410.

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HIF-2 iron feedback via IRP

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u the hypoxic induction of EPO in the kidney and liver is HIF-2-dependent

u inactivation of PHD2 alone is sufficient to stimulate the production of renal EPO

u there are at least two distinct populations of EPO-producing cells in the kidney that differ in their regulation of HIF-2 activity and EPO production

u inactivation of at least 2 PHD enzymes in the liver is required to stimulate erythropoiesis

u HIF coordinates erythropoiesis with iron metabolismu intracellular iron regulates HIF oxygen sensing

Key Points

EPO: erythropoietin; HIF: hypoxia-inducible factor; PHD: prolyl-4-hydroxylase domain.

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HIF-PHD Inhibitors for renal anemia therapy: overview of compounds and mechanism

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Pathogenesis of Renal Anemia

EPO: erythropoietin.Koury MJ, Haase VH. Nat Rev Nephrol. 2015;11(7):394-410.

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HIF-PHDs (PHD1, PHD2, PHD3)

R +

R = HIF-α

HIF-PHD as pharmacologic target

HAASE, VH

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HIF-PHIs chemical structures

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Sanghani and Haase, ACKD, 2019

Pharmacologic profiles of HIF-PHIs

Others: Enarodustat (JTZ-951, Japan Tabacco), Desidustat (Zyann1, Cadila Healthcare/Zydus Cadila),TP0463518 (Taisho Pharmaceutical)

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HIF-PHIs: mechanisms in renal anemia

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Renal EPO-producing cells in CKD

CKD: chronic kidney disease; EPO: erythropoietin; REPC: renal EPO-producing cell.Koury MJ, Haase VH. Nat Rev Nephrol. 2015;11(7):394-410.

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HIF-Prolyl Hydroxylase Inhibition:potential adverse effects

q VEGF ?q Metabolic effects (glucose, cholesterol, fat

metabolism, uric acid, FGF 23) ?q Pulmonary artery pressure ?q Systemic arterial blood pressure ?q Effects on kidney disease progression ?q Liver toxicity ?q Pro-oncogenic potential ?

HAASE, V.H..

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HIF activation:potential applications in renal injury

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Preclinical studies in AKI – evidence for renoprotection

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Potential mechanisms of HIF-dependent

“Ischemic preconditioning”

Kapitsinou and Haase., AJP Renal, 2015

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Key PointsØ there is strong preclinical evidence for HIF-induced

protection from acute ischemic injury warranting further investigation in patients

Ø HIF protects from renal ischemia-reperfusion injury and transition to CKD

Ø are currently used dosing regimen sufficient to afford cytoprotection (Dapro trial in PVD failed) ?

Ø the effects of HIF activation on chronic kidney injury are controversial. Preclinical studies indicate strong cell type and context dependence

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HIF oxygen sensingcardiovascular disease

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Cardiovascular risk in CKD: is HIF-PHI therapy beneficial ?

Gansevoort RT et al., Lancet, 2013

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Altitude reduces all-cause mortality in incident dialysis

Winkelmayer et al., JAMA, 2009

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HIF activation in CVD – importance of timing and duration

Minamashima et al., Blood 2008

Tanaka and Eckardt, Seminars in Nephrology, 2018

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Key points

Ø HIF activation has the potential to reduce cardiovascular morbidity and mortality in CKD patients

Ø HIF-PHIs may have anti-inflammatory effects

Ø several cardiovascular safety concerns have not been addressed yet in long-term studies

Ø additional clinical studies are needed

Ø acute effects may be protective – timing is critical

Ø chronic HIF activation results in organ dysfunction

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