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

0 2 4 6 8 10 12

6

8

10

12

14

90-95% of patients respond well to ESAs

Time on therapy (months)

Hb

(g

/dl)

0 2 4 6 8 10 12

6

8

10

12

14

Time on therapy (months)

Hb

(g

/dl)

Type 1 poor response to ESAs

0 2 4 6 8 10 12

6

8

10

12

14

Type 2 poor response to ESAsH

b (

g/d

l)

Time on therapy (months)

Poor response to ESAs - causes

Major Minor

Iron deficiency Blood loss

Infection / inflammation Hyperparathyroidism

Underdialysis Aluminium toxicity

B12 / folate deficiency

Haemolysis

Marrow disorders, e.g. MDS

Haemoglobinopathies

ACE inhibitors

Carnitine deficiency

Anti-EPO antibodies

Dialysis adequacy

Infection, Inflammation

Iron supplementation

Blood losses

AT

Dialysis adequacy

Infection, Inflammation

Iron supplementation

Blood loss

AT

Factors influencing response to ESAs

AT = Adjuvant therapy

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 doseEpoetin dose HaemoglobinHaemoglobin

Mea

n (

IU/k

g/w

eek)

Mea

n (

IU/k

g/w

eek)

Mea

n (

g/d

l)M

ean

(g

/dl)

CRPCRP50 mg/l on 3 or more months (n=201)50 mg/l on 3 or more months (n=201)

CRP<50 mg/l on 4 or more months (n=3,014)CRP<50 mg/l on 4 or more months (n=3,014)

MonthMonth MonthMonth

Hörl W et al., NDT 2000;15(Suppl 4): 45–45.

Inflammatory response and EPO doseInflammatory response and EPO doseSitter et al NDT 15:1207,2000.Sitter et al NDT 15:1207,2000.

30–50% of CKD patients have serological evidence ofan 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

EP

O d

os

e [

IU/k

g/w

eek]

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

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

The explanation ?

Uraemia Chronic inflammation (c.f. anaemia of chronic disease,

e.g. RA, malignancy)

IL-1

IL-6

TNF-

IFN-

Suppression of erythropoiesis

Factors affecting erythropoiesis

Erythroidprogenitor

cell

EPO +ve

BFU-E

CFU-E

Erythroblast

Red blood cells

Bone Marrow

Pro-inflammatory cytokines

-ve

Where do cytokines come from ?

T-cell

Th0

Th1IFN-

Th2IL-4

IL-10IL-2

CD8

CD8+T-cell

IL-2

TNF-

CD4

MonocyteIL-6

IL-12

TNF-

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

3 groups of subjects:

1 - HD patients not responding to EPO (Hb < 10g/dl, EPO dose > 250 IU/kg/wk)

2 - HD patients responding well to EPO (Hb > 11g/dl, EPO dose < 200 IU/kg/wk)

3 - Normal healthy controls

Demographic and haematological characteristics of subjects (mean±SD)

Normalcontrols(n=17)

Goodresponders

(n=17)

Poorresponders

(n=15)

Age (yrs) 40±15 58±18# 56±18†

Hb (g/dl) 14.1±1.1 11.8±1.3## 8.9±1.7††**

CRP (mg/l) 3.8±1.1 8.7±8.0# 18.6±20.7††*

EPO dose(U/kg/week)

N/A 71±30 296±147**

EPOdose/Hbratio

N/A 6.3±2.6 33.4±17.8**

Hb levels and EPO dose requirements

60050040030020010004

6

8

10

12

14

16

18

Hb

(g

/dl)

Hb

(g

/dl)

Good responders

ControlsControls

Poor respondersPoor responders

EPO dose (IU/kg/wk)EPO dose (IU/kg/wk)

Intracellular cytokine methodology

Whole blood

Peripheral blood mononuclear cells

Culture for 48h @ 37°C, 5% CO2

3 colour flow cytometric analysis

PMA/ionomycinBrefeldin A

Surface stain cells with fluorescent mabs to CD3 & CD8

Permeabilize cells and stain for intracellular cytokines

CD4+ T-cell expression of IFN stimulated by PMA/ionomycin

0

10

20

30

CD

4+ T

cel

ls e

xpre

ssin

g IF

N

(%)

0

10

20

30

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

P <0.005

P <0.005ns

CD8+ T-cell expression of IFN stimulated by PMA/ionomycin

0

25

50

75

100

0

25

50

75

100

CD

8+ T

cel

ls e

xpre

ssin

g IF

N

(%)

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

P <0.005

nsns

CD4+ T-cell expression of TNF stimulated by PMA/ionomycin

0

20

40

60

80

CD

4+ T

cel

ls e

xpre

ssin

g T

NF

(%

)

0

20

40

60

80

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

P <0.05

P <0.01ns

CD8+ T-cell expression of TNF stimulated by PMA/ionomycin

0

20

40

60

80

CD

8+ T

cel

ls e

xpre

ssin

g T

NF

(%

)

0

20

40

60

80

P <0.001

nsns

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

CD4+ T cell expression of IL-10 stimulated by PMA/ionomycin

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

CD

4+ T

cel

ls e

xpre

ssin

g IL

-10

(%

)

0

1

2

3

0.0

0.5

1.0

1.5

2.0

2.5

3.0

P <0.05

nsns

CD8+ T cell expression of IL-10 stimulated by PMA/ionomycin

0

1

2

3

4

0

1

2

3

4

Normalcontrols

Goodresponders

Poorresponders

Normalcontrols

Goodresponders

Poorresponders

CD

8+ T

cel

ls e

xpre

ssin

g IL

-10

(%

) P <0.001

P <0.05P <0.02

Summary of intracellular cytokine staining study

TNF IFN IL-10

CD4 increased increased trend forincrease

CD8 trend forincrease

trend forincrease

increased

0 5 10 15 20 25

50

Pe

rce

nt

surv

ival

Time (months)

Poor responders

Good responders100

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)

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

Cytokine-mediated inhibition of erythropoiesis

Proposed model of inhibition in poor responders to EPO therapy

Erythroidprogenitor

cellproliferation

Uraemia (± other inflammatory conditions)

Immune activation

CD8+T cell

CD4+T cell

–ve

–ve

–ve

–ve

TNF

IL-10

IL-13

IFN

+veEPOanti-apoptotic

pro-apoptotic

Cooper et al JASN 2003

Blocking cytokine production

Increase dialysis prescription

Drugs

– Pentoxifylline

– Thalidomide

– Anti-cytokine antibodies

– Anti-lymphocyte therapy

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

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)

Effect of Pentoxifylline treatment on TNF production in a patient with poor response to erythropoietin therapy

CD

3

TNFTNF

60% 9%

Before treatment After treatment(6 weeks)

Effect of Pentoxifylline treatment on IFN production in a patient with poor response to erythropoietin therapy

CD

3

IFNIFN

24% 6%

Before treatment After treatment(6 weeks)

Pentoxifylline studyProtocol

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

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

Before treatment

After treatment(6–8 weeks)

TNF (%)

0

25

75p=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

Effect of pentoxifylline treatment on Hb level (n=12)

Pentoxifylline therapy (months)

Hb(g/dl)

pentoxifylline

-6 -5 -4 -3 -2 -1 0 1 2 3 40

2

4

6

8

10

12

14

P = 0.0001

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

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. B12/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