1. Can we restore the response to erythropoietin resistance ? Iain C Macdougall BSc, MD, FRCP Consultant Nephrologist and Honorary Senior Lecturer Renal Unit, King’s College Hospital, London, UK

2. 024681012 6 8 10 12 14 90-95% of patients respond well to ESAs Time on therapy (months) Hb (g/dl)

4. 024681012 6 8 10 12 14 Time on therapy (months) Hb (g/dl) Type 1 poor response to ESAs

5. 024681012 6 8 10 12 14 Type 2 poor response to ESAs Hb (g/dl) Time on therapy (months)

6. Poor response to ESAs - causes MajorMinor Iron deficiencyBlood loss Infection / inflammationHyperparathyroidism UnderdialysisAluminium toxicity B 12 / folate deficiency Haemolysis Marrow disorders, e.g. MDS Haemoglobinopathies ACE inhibitors Carnitine deficiency Anti-EPO antibodies

7. Factors influencing response to ESAs AT = Adjuvant therapy

8. Epoetin dose and Hb levels in patients with chronic infection/inflammation (ESAM) 0 25 50 75 100 125 150 175 1 2 3 4 5 6 0 2 4 6 8 10 12 14 1 2 3 4 5 6 Epoetin dose Haemoglobin Mean (IU/kg/week) Mean (g/dl) CRP  50 mg/l on 3 or more months (n=201) CRP<50 mg/l on 4 or more months (n=3,014) MonthMonth Hörl W et al., NDT 2000;15(Suppl 4): 45–45.

9. Inflammatory response and EPO dose Sitter et al NDT 15:1207,2000.

10. 30–50% of CKD patients have serological evidence of an activated inflammatory response (CRP > 8–10 mg/l). Influence of CRP concentration on EPO dose 180 160 140 120 100 80 60 40 20 0 EPO dose [IU/kg/week] Group I: CRP > 10 mg/l Group II: CRP < 10 mg/l P<0.01 138.7 92 Stenvinkel P, NDT 2002;17 Suppl 5:32–37. Nitta K, Acta Haematol 2002;108;168–170. 34% difference in EPO dose

11. What do you do about the patient who is responding poorly to ESAs in the absence of any obvious cause ?

12. The explanation ? Uraemia Chronic inflammation (c.f. anaemia of chronic disease, e.g. RA, malignancy) IL-1 IL-6 TNF-  IFN-  Suppression of erythropoiesis

13. Factors affecting erythropoiesis Erythroid progenitor cell EPO +ve BFU-E CFU-E Erythroblast Red blood cells Bone Marrow Pro-inflammatory cytokines -ve

14. Where do cytokines come from ? T-cell Th 0 Th 1 IFN-  Th 2 IL-4 IL-10 IL-2 CD8 CD8+ T-cell IL-2 TNF-  CD4 Monocyte IL-6 IL-12 TNF- 

15. Aim To examine whether HD patients responding poorly to EPO have altered T cell and monocyte activation states compared with: –Patients responding well to EPO –Normal controls

16. 3 groups of subjects: 1 - HD patients not responding to EPO (Hb 250 IU/kg/wk) 2 - HD patients responding well to EPO (Hb > 11g/dl, EPO dose < 200 IU/kg/wk) 3 - Normal healthy controls

17. Demographic and haematological characteristics of subjects (mean±SD)

18. Hb levels and EPO dose requirements 6005004003002001000 4 6 8 10 12 14 16 18 Hb (g/dl) Good responders Controls Poor responders EPO dose (IU/kg/wk)

23. Intracellular cytokine methodology Whole blood Peripheral blood mononuclear cells Culture for 48h @ 37°C, 5% CO 2 3 colour flow cytometric analysis PMA/ionomycin Brefeldin A Surface stain cells with fluorescent mabs to CD3 & CD8 Permeabilize cells and stain for intracellular cytokines

24.  CD4+ T-cell expression of IFN  stimulated by PMA/ionomycin 0 10 20 30 CD4+ T cells expressing IFN  (%) 0 10 20 30 Normal controls Good responders Poor responders Normal controls Good responders Poor responders P <0.005 ns

25.  CD8+ T-cell expression of IFN  stimulated by PMA/ionomycin 0 25 50 75 100 0 25 50 75 100 CD8+ T cells expressing IFN  (%) Normal controls Good responders Poor responders Normal controls Good responders Poor responders P <0.005 ns

26.  CD4+ T-cell expression of TNF  stimulated by PMA/ionomycin 0 20 40 60 80 CD4+ T cells expressing TNF  (%) 0 20 40 60 80 Normal controls Good responders Poor responders Normal controls Good responders Poor responders P <0.05 P <0.01ns

27.  CD8+ T-cell expression of TNF  stimulated by PMA/ionomycin 0 20 40 60 80 CD8+ T cells expressing TNF  (%) 0 20 40 60 80 P <0.001 ns Normal controls Good responders Poor responders Normal controls Good responders Poor responders

28. CD4+ T cell expression of IL-10 stimulated by PMA/ionomycin Normal controls Good responders Poor responders Normal controls Good responders Poor responders CD4+ T cells expressing IL-10 (%) 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 P <0.05 ns

29. CD8+ T cell expression of IL-10 stimulated by PMA/ionomycin 0 1 2 3 4 0 1 2 3 4 Normal controls Good responders Poor responders Normal controls Good responders Poor responders CD8+ T cells expressing IL-10 (%) P <0.001 P <0.05P <0.02

30. Summary of intracellular cytokine staining study TNF  IFN  IL-10 CD4increasedinc asedtrendfor increase CD8trendfor increase trendfor increase inc ased

31. 0510152025 50 Percent survival Time (months) Poor responders Good responders 100 0 * * P < 0.05 poor responders versus good responders Kaplan Meyer survival curve for poor responders versus good responders over 24 months Cooper et al, Nephrol Dial Transplant (2003)

32. Conclusions Poor response to EPO is associated with: –Increased capacity to generate IFN  and TNF  from CD4+ and CD8+ T cells In the absence of any other cause, poor response to ESAs may result from enhanced T cell activation

33. Cytokine-mediated inhibition of erythropoiesis Proposed model of inhibition in poor responders to EPO therapy Erythroid progenitor cell proliferation Uraemia (± other inflammatory conditions) Immune activation CD8+ T cell CD4+ T cell –ve TNF  IL-10 IL-13 IFN  +ve EPO anti-apoptotic pro-apoptotic Cooper et al JASN 2003

34. Blocking cytokine production Increase dialysis prescription Drugs –Pentoxifylline –Thalidomide –Anti-cytokine antibodies –Anti-lymphocyte therapy

35. Pentoxifylline Phosphodiesterase inhibitor Useful in peripheral vascular disease: –Anti-platelet effect –Effect on erythrocyte deformability Anti-TNF  effect Anti-IFN  effect Anti-IL-10 Anti-oxidant effect Anti-apoptotic effect

36. Pentoxifylline trials IBD –Improved disease activity index –Correction of inflammatory markers (Blam et al, 2001) Dilated cardiomyopathy –Improved exercise tolerance –Improved ejection fraction (Skudicky et al, 2001) Septic shock –Improved GFR –Correction of coagulopathy (Jaimes et al, 2001) Membranous nephropathy –Reduction of proteinuria (Duclox et al, 2001)

37. Effect of Pentoxifylline treatment on TNF  production in a patient with poor response to erythropoietin therapy CD3 TNF  60%9% Before treatment After treatment (6 weeks)

38. Effect of Pentoxifylline treatment on IFN  production in a patient with poor response to erythropoietin therapy CD3 IFN  24%6% Before treatment After treatment (6 weeks)

40. Pentoxifylline study Protocol At recruitment blood sample taken for T cell cytokines patients given 400 mg pentoxifylline od Hb monitored monthly 6-8 weeks blood sample taken for T cell cytokines

41. Pentoxifylline study 16 patients were recruited 12 patients completed the study 2 patients were non-compliant 1 patient developed nausea 1 patient developed confusion unrelated to therapy

42. Before treatment After treatment (6–8 weeks) TNF  (%) 0 25 75 p=0.0007 Effect of pentoxifylline treatment on ex vivo cytokine production by CD3+ T cells 50 Before treatment After treatment (6–8 weeks) IFN  (%) p=0.0002 0 10 20 30 40 50

43. Effect of pentoxifylline treatment on Hb level (n=12) Pentoxifylline therapy (months) Hb (g/dl) pentoxifylline -6-5-4-3-201234 0 2 4 6 8 10 12 14 P = 0.0001

44. Conclusions Pro-inflammatory cytokines in renal failure may be an important cause of a poor response to ESAs Pentoxifylline may be a useful adjuvant therapy in patients who respond poorly to ESAs

45. What can we do to restore the response to erythropoietin resistance ? Ensure compliance if self-administering Ensure adequate iron status ? trial of IV iron Exclude / treat other deficiencies e.g. B 12 /folate/thyroxine ? parathyroidectomy if severe hyperparathyroidism Treat any infection/inflammatory cause aggressively ? transplant nephrectomy Increase dialysis prescription ? consider pentoxifylline if “inflammation” present and other causes excluded Pro-inflammatory cytokines in renal failure may be an important cause of resistance to ESAs