Regulation of renal EPO production

Transcription

1 Actualités Néphrologiques Jean Hamburger Hôpital Necker April 2008 Regulation of renal EPO production Michael Wiesener Dept. Nephrology and Hypertension University Erlangen-Nuremberg Chair Kai-Uwe Eckardt

2 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications

3 Regulation of red cell volume by EPO the prototype of oxygen regulated genes Hb O 2 EPO

4 Hb (g/dl) mean ± SD, n=12 EPO production high sensitivity of oxygen sensing EPO (mu/ml) d Lorentz, et al., Transfusion 1991

5 Stimulation of EPO by oxygen deprivation High amplitude of regulation RPA EPO in rat kidney

6 EPO Production in the kidney Interstitial fibroblasts in the renal cortex produce EPO Regulation of EPO by oxygen availability - mechanism?! Eckardt et al., Kidney Int 1993

7 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications

8 Regulation of EPO by the Hypoxia-inducible Factor, HIF TSS 5 EPO 3 Promotor HIF-1 Enhancer, HRE 5`- RCGTG - 3` NLS-N ODDD Pro Pro NLS-C Asn 803 N 2 HIF-1α COOH bhlh A PAS B TAD-N TAD-C Wang & Semenza, JBC 1995

9 Regulation of EPO by the Hypoxia-inducible Factor, HIF TSS 5 EPO 3 Promotor Enhancer, HRE 5`- RCGTG - 3` Stockmann & Fandrey, CEPP 2006

10 O 2 Regulation of HIF HIF-1α HIF-2α E3-Ubiquitin Ligase Proteasomal Destruction HIF-1α Immunoblot O 2 (%) HIFβ HIF-2α H + Reoxygenation HIF-1 HIF-2 HIF-2α N H HRE HIF-1α 5`- RCGTG - 3`

11 HIF as master regulator of hypoxic adaptation Growth and apoptosis Insulin-like growth-factor-binding-1 Nip3 Endoglin Wilms tumour suppressor α-fetoprotein (neg. regulation) Calcitonin-receptor-like receptor Transcriptional regulation DEC1 and DEC2 ETS1 p35srj Virus related Retrotransponson VL30 Cell migration Chemokine receptor CXCR4 C-Met -1α/-2α HIF? Angiogenic signalling Vascular endothelial growth factor A Endothelial-gland-derived vascular endothelial growth factor Vascular endothelial growth factor receptor-1 (Flt-1) Plasminogen-activator inhibitor-1 Energy metabolism Glucose-transporter-1 and 3 Hexokinase-2 6-Phosphofructo-1-kinase L Glyceraldehyde-3-phosphate dehydrogenase Aldolase A Enolase-1 Phosphoglycerate kinase-1 Lactate dehydroxenase A 6-Phosphofructo-2-kinase Carbonic anhydrase 9 Transport Transferrin Transferrin receptor Ceruloplasmin Multidrug-resistance P-glycoprotein Matrix and barrier functions Procollagen prolyl hydroxylase-α1 Intestinal trefoil factor Ecto-5 -nucleotidase Vasomotor regulation Endothelin-1 Adrenomedullin Tyrosin hydroxylase α1b-adrenergic receptor Inducible nitric-oxide synthase Endothelial nitric-oxide synthase Haem oxygenase Atrial natriuretic peptide Hormonal regulation Erythropoietin Leptin Adapted from Schofield and Ratcliffe, Nat Reviews 2004

12 Gene targeting of HIFα embryonic lethality HIF-1α KO Iyer et al. Genes & Dev 1998 Ryan et al. EMBO J 1998 embryonic lethality by E8-E11 cardiovascular malformations neural tube defects cephalic mesenchyme cell death Different phenotype of HIFα KO animals NON-REDUNDANT SYSTEMS! HIF-2α KO Tian et al. Genes & Dev 1998 embryonic lethality in mid-gestation reduced catecholamine synthesis bradycardia Peng et al. PNAS 2000 embryonic lethality E severe vascular defects yolk sac and embryo proper Scortagagna et al. Blood 2003 Scortagagna et al. Nat. Genetics 2003 incomplete lethality pancytopenia, hepatomegaly, cardiac hypertrophy, retinopathy, sceletal myopathy, biochemical alterations, azoospermia

13 Regulation of red cell volume by EPO the prototype of oxygen regulated genes Hb O 2 Oxygen sensing EPO

14 Physiological oxygenation of the kidney Region or O 2 delivery Blood flow O 2 consumption organ rate O 2 consumption O 2 delivery mll/min/100g % Hepato-portal Kidney Brain Skin Skeletal muscle Heart > abundant oxygenation

15 HIFα activation in kidney Normal oxygenation of the kidney HIF-1α po2 in renal medulla < 10 mmhg Leichtweiss et al., Pflügers. Arch 1969 control / normoxia due to: shunt diffusion between arterial and venous vessels heterogenous oxygen consumption

16 HIFα activation HIF-1α in kidney HIF-1α tubular cells HIF-2α glomerular cells interstitial cells control / normoxia hypoxia

17 EPO Production in the kidney Interstitial fibroblasts in the renal cortex produce EPO HIF-2α EPO Regulation by HIF-2α?

18 Effect of specific knock down of HIFα isoforms by RNA interference DP sirna HIF-1α HIF-2α 97 kda 97 kda Immunoblot Luc 1α 2α Luc 1α 2α DP sirna - - HIF-1α HIF-2α VEGF LDH-A RNase Protection Assay HeLa luc1α 2α GLUT-1 U6sn

19 Effect of specific knock down of HIFα isoforms by RNA interference DP sirna HIF-1α HIF-2α 97 kda 97 kda Immunoblot Luc 1α 2α Luc 1α 2α RNase Protection Assay Hep3B DP sirna - luc 1α 2α HIF-1α HIF-2α EPO U6sn EPO is a HIF-2α target! (in vitro)

20 Is Erythropoiesis a function of HIF-2? Gene deletion studies Gruber et al., PNAS 2007 Rankin et al., JCI 2007 (hepatic EPO production by HIF-2)

21 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications

22 von Hippel Lindau (VHL) Tumour Suppressor Gene familial Tumour-Syndrome Inactivation in the Germline > VHL Syndrome Identification of the VHL Gene by Linkage Analysis of RCC; Latif et al., Science 1993 Development of tumours by second hit on further allele Manifestation of typical Tumours: Retinal Angioma Haemangioblastoma Phaeochromocytoma Renal Cell Carcinoma 22

23 VHL-associated tumours hypervascular phenotype Clear Cell RCC Retinal Angioma Haemangioblastoma Phaeochromocytoma 23

24 HIF is targeted for degradation by the von Hippel Lindau (VHL) Tumor Suppressor gene product Immunoblot +VHL N H N H +VHL N H N H RCC4 HIF-1 α HIF-2 α Clear Cell RCC Retinal Angioma Haemangioblastoma Phaeochromocytoma 24

25 Regulation of HIFα by the von Hippel Lindau tumour suppressor gene O 2 HIF-1α HIF-2α E3-Ubiquitin Ligase Proteasomal Destruction VHL inactivation pvhl HIFβ HIF-1 HIF-2 E3-Ubiquitin Ligase HRE 5`- RCGTG - 3`

26 Mechanism of oxygen sensing HIF Hydroxylases oxygen, iron and 2-oxoglutarate dependent enzymes Hydroxylation NLS-N ODDD Pro Pro NLS-C Asn 803 N 2 HIF-1α COOH bhlh A PAS B TAD-N TAD-C PHD 1 PHD 2* PHD 3* FIH-1 * Induced by hypoxia HIF-1α HIF-2α Epstein et al., Cell 2001 Bruick et al., Science 2001 Mahon et al., Genes Dev 2001, Lando et al., Science 2002

27 Regulation of HIFα by hydroxylation O 2 PHD/FIH HIF-1α HIF-2α OH -OH E3-Ubiquitin Ligase HIFβ P300/CBP HIF-1 HIF-2 HRE 5`-RCGTG -3`

28 Regulation of HIFα by hydroxylation HIF-1α O 2 PHD/FIH -OH Proteasomal Destruction HIF-2α OH E3-Ubiquitin Ligase HIFβ P300/CBP 1. Protein stability 2. Transcriptional activity HIF-1 HIF-2 HIF-2 EPO Is there evidence in human studies? HRE 5`-RCGTG -3`

29 Is Erythropoiesis a function of HIF-2? Erythrocytosis in humans PHD VHL HIF-2α EPO Percy et al. PNAS 2006 (PHD2) Ang et al. Nat Genet Randi et al. Haematologica 2005 Pastore et al. Blood 2003 Pastore et al. Am J Hum Genet Bento et al. Haematologica 2005 Percy et al. New England J Medicine 2008 Percy et al. Blood 2008

30 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications

31 HIF modulation for clinical exploitation? Ischemia < > Tumours / erythropoiesis O 2 Coronary artery stenosis + HIF-1α - HIF-2α

32 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications ischemic protection

33 HIF Prolyl Hydroxylase Inhibitors Tools for HIF activation in vivo? Ascorbate Regeneration of Fe 2+ 2-oxoglutarate succinate OH CO 2 2-oxoglutarate analogues Fibrogen Inc., San Diego

34 HIF Prolyl Hydroxylase Inhibitors Tools for HIF activation in vivo? HIF-1α HIF-2α Supra-physiological activation of HIFα subunits by PHD-inhibitor FG4487 Testing in acute ischemic renal failure, preconditioning with PHD-I

35 HIF Prolyl Hydroxylase Inhibitors (FG4487) Protection by preconditioning in ischemic renal failure 5 n = 10 4,5 S-creatinine [mg/dl] 4 3,5 3 2,5 2 1,5 1 # # * # * Veh PHD-I Sham 0,5 0 0h 24h 72h # - p < 0.05 vs. Sham * - p < 0.05 vs. Veh

36 HIF Prolyl Hydroxylase Inhibitors (FG4487) Attenuation of tissue damage by ischemic renal failure * Sham Veh PHD-I blinded analysis * - p < 0.05 vs. Veh

37 Rodent models of renal transplantation long term course The allogenic Fisher-to-Lewis Model of KTx in cooperation with B. Yards, U. Göttmann; Nephrology Mannheim

38 Regulation of renal EPO production Erythropoietin (EPO), prototype of oxygen regulated genes Identification, structure and regulation of HIF Mechanism of oxygen sensing Clinical Implications ischemic protection anemia therapy (?) PHDs are drugable targets are they involved in erythropoiesis?

39 Does PHD function affect Erythropoiesis? Gene deletion studies Knockout of all 3 PHDs PHD2 lethal PHD No apparent phenotype PHD2 KO PHD2 KO Takeda et al., MCB 2006

40 Does PHD function affect Erythropoiesis? Gene deletion studies PHD double KO moderate polycythemia PHD 2, inducible KO severely polycythemic Takeda et al., Blood 2008

41 Anemia of renal disease - inadequate capability to induce EPO chronic anemia renal anemia Eckardt et al., 1991

42 Phase 1 study endogenous EPO induction by PHD-I in dialysis patients Single dose study of oral prolyl hydroxylase inhibitor. repo in dialysis patients paused one week prior to dosing A B1 B predose 24h A healthy controls B1 nephric dialysis patients B2 anephric dialysis patients A B1 B2 Induction of endogenous EPO in dialysis patients is feasible Is it safe?! 42

43 Summary HIF is the master regulator of hypoxic adaptation Regulation and function of HIF underlies multiple elements of fine tuning; separate α-subunits, distinct cellular expression, target gene specificity, specific hydroxylases Erythropoiesis is a function of HIF-2α; PHD2 primarily involved Broad spectrum of biological relevance; May lead to multiple approaches of clinical use Anemia therapy?

44 Putative targets for modulating erythropoiesis Tumour Suppressor Gene Tumour growth tumorigenesis PHD2 VHL HIF-2α EPO HIF-2α Syed et al.; Nature 1998

45 Summary HIF is the master regulator of hypoxic adaptation Regulation and function of HIF underlies multiple elements of fine tuning; separate α-subunits, distinct cellular expression, target gene specificity, specific hydroxylases Erythropoiesis is a function of HIF-2α; PHD2 primarily involved Broad spectrum of biological relevance; May lead to multiple approaches of clinical use Anemia therapy? More careful definition of biological role; HIFα specific function First application in acute interventions, such as organ ischemia, transplantation?

46 Thank you for your attention! Nikolaus-Fiebiger-Center for Molecular Medicine: Susanne Gastiger Thomas Hackenbeck Katrin Jozefowski Karl Knaup Philipp Lechler Ruth Schietke Regina Schmitt Xiaoqing Wu Benito Yard / Uwe Göttmann Dept. Nephrology Mannheim Peter Ratcliffe University of Oxford, U.K. Patrick Maxwell Imperial College London, U.K. Eamonn Maher University of Birmingham, U.K. Kai-Uwe Eckardt, Chair Dept. Nephrology Supported by: IZKF Erlangen SFB 423 EU 6th Framework Nephrolog. Laboratories Loschgestrasse: Wanja Bernhardt Björn Buchholz Hans Fees Jonathan Jantsch Bernd Klanke Andrea Kosel Brigitte Rogge Tetsuhiro Tanaka Melanie Volke Christina Warnecke Alexander Weidemann Carsten Willam

47 HIFα is activated in renal polycystic disease Involvement of HIF in cyst formation / growth?! Rat PKD model ADPKD HIF-1α IHC Experimental study with HIF-inhibitor (Topetecan) is on the way Possibly in combination with mtor inhibitor?! 47

48 Deletion of VHL in podocytes > crescentic GN Mei Ding,., S. Quaggin. Nature Medicine 2006