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Ariela Benigni

1

Dipartimento di Medicina MolecolareIstituto di Ricerche Farmaclogiche Mario Negri,

Laboratori Negri Bergamo

Torino, 23 gennaio 2009

Glomerular hypertension

GBM

Podocytes

2Disease progression

GBM

Endothelial cells

Mechanical strain

Podocyte number0.4

0.6

0.8

1.2

Ang

II(p

g pe

r µg

of c

ell l

ysat

e)

1.0

*

0.5

1.0

1.5

2.0

2.5

AT1R

leve

l(a

djus

ted

for

tubu

lin)

Glomerular hypertension

3

Durvasula et al, Kidney Int, 2004

Ctr0

0.2

(pg

per

µg o

f cel

l lys

ate)

MS

Pore dimension

0

0.5

(adj

uste

d fo

r tu

bulin

)

Ctr MS

Disease progression

Proteinuria

PLC

IP3DAG

AT1R

4Winn et al, Science, 2005 Nitschke et al., Kidney Int, 2000

Ca2+

TRPC6

Cytoskeleton

TRPC6: Transient Receptor Potential cation channel

actin ZO-1 merge

Control

40

0

10

20

30

Alb

umin

flux

(µµ µµg

/hou

r)

5

Ang II

*40

0

10

20

30

Alb

umin

flux

(µµ µµg

/hou

r)

Macconi et al., Am J Pathol, 2006

1 - apoptosis

6

1 - apoptosis

2 - phenotypechanges

Urin

ary

Pro

tein

Exc

retio

n(m

g/24

hrs

)

0

100

200

300

400

500

600

700

UNxControl UNx + Lis

*

**

UNINEPHRECTOMIZED MWF/ZTM RATS - 1

7

Per

cent

age

of g

lom

erul

i af

fect

ed b

y sc

lero

sis

0

20

40

60

80

100

UNxControl UNx + Lis

*

**

* p < 0.05, **p < 0.01 vs control

Remuzzi A. et al., Kidney Int, 1995

UNINEPHRECTOMIZED MWF/ZTM RATS - 2

Sur

viva

l (%

)

ControlUNx + Lis

60

80

100

8 Remuzzi A. et al., Kidney Int, 1995

Time (months after UNx)

Sur

viva

l

UNx

0 3 6 9 1 2 1 50

20

40

45

40

35GF

R(m

l/min

/mon

th)

RamiprilRamipril

∆ GFR = -0.44 ± 0.54

∆∆∆∆ GFR = - 0.10 ± 0.50

9

30

25

(ml/m

in/m

onth

)

∆ GFR = -0.81 ± 1.12 ∆ GFR = -0.14 ± 0.87

RamiprilConventional

CORE FOLLOW-UP

Ruggenenti et al., Lancet, 1998

REGRESSION10 patients with increasing GFR

65

60

55

50

45

(ml/m

in/m

onth

)

P = 0.01

-0.21 + 0.09 +0.49 + 0.19

∆ ∆ ∆ ∆ Proteinuria(pre vs post break point)

0∆∆∆∆GFR(ml/min/month)

10

45

40

35

30

25

20

months

-30 -20 -10 0 10 20 30

GF

R (m

l/min

/mon

th)

Ruggenenti et al., J Am Soc Nephrol, 1999

Break point

%

-40

-20

-60

MWF+LIS 50-60 wMWF 60WMWF 50W

11 Remuzzi A. et al., Kidney Int, 2006

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

50

75

100

MWF 50W

12

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

0

25

10 20 30 40 50 60 70 80 90 1000

Reconstructed Glomeruli

MWF 60W

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

50

75

100

MWF 50W

13

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

0

25

10 20 30 40 50 60 70 80 90 1000

Reconstructed Glomeruli

MWF 60W

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

50

75

100

MWF 50W

14

% o

f Cap

illar

y T

uft V

olum

eA

ffect

ed b

y S

cler

osis

0

25

10 20 30 40 50 60 70 80 90 1000

Reconstructed Glomeruli

MWF + LIS 50-60W

Sclerosis was effectively reabsorbed and aconsistent amount of glomerular tissueregained normal structure

15

This suggests neoformation of glomerularcapillary segments

Adult differentiated

Resident progenitor/stem

INSIGHT INTO ACE-INDUCED RENALREPAIR/ANGIOGENESIS

Renal cells

16

Endothelial progenitor and/or bone marrow-derived stem

Extra renal cells

endo

thel

ial c

ells

(%

)

20

30

17

VV

endo

thel

ial c

ells

0

10*

40 W 60 W LIS 40-60 W

WGA = cell membranesRECA-1= endothelial cells Macconi et al., 2008

80

120

160

**

WT1+ cells/glom

18 Macconi et al., Am J Pathol, 2009

0

40

80

40W 60W LIS 40-60W

WT1 = podocyte marker (nuclei )

19

Migration of parietal cells tocapillary tuft through thevascular pole

Migration of parietal cells from the Bowman’s capsule to capillary tuft

20

PARIETAL CELLS WITH PODOCYTE PHENOTYPE

12

10

8P

arie

tal p

odoc

ytes

/PE

C (

%)

6

4

*

21

MWF 40 W

0MWF 60 W

MWF + Lis 60 W

Par

ieta

l pod

ocyt

es/P

EC

These cells were identified by staining for PGP 9.5 (parietal epithelial cellmarker) and WT1 (podocyte marker)

4

2

Macconi et al., Am J Pathol, 2009

ISOLATION AND CHARACTERIZATION OFMULTIPOTENT PROGENITOR CELLSCD133+CD24+ FROM THE BOWMAN’S CAPSULEOF ADULT HUMAN KIDNEYS

22Sagrinati et al., J Am Soc Nephrol, 2006

23

Brenner’s seminal paperNEJM

Retarding renal Promoting kidney

24

1983

Retarding renal disease progression

2003

Promoting kidney repair

The presence of VEGF is crucialfor normal renal development

Differentiating glomerularepithelia produce VEGF and mayattract endothelial cells into theglomeruli

25 Kretzler et al., Kidney Int, 1998

Administration of anti-VEGFantibody during early kidneydevelopment in mice leads toformation of abnormal glomerularstructures and diminishednephrogenesis

PODOCYTE ARE THE MAJOR SOURCE OF VEGFIN THE GLOMERULUS

26 Kretzler et al., Kidney Int, 1998

PODOCYTE VEGF BINDS TO COGNATE RECEPTORSEXPRESSED ON GLOMERULAR ENDOTHELIAL CELLS

How podocytes can signal “up stream” in the glomerular endothelium

A concentration gradientfavors diffusion of VEGFfrom the podocyte toglomerular endothelial

27

Eremina et al., N Engl J Med, 2008

glomerular endothelialcells

28

29

Glomerular endothelial cells differ from mostother endothelial cells in that they areextremely flattened and densely perforatedby transendothelial pores, the fenestrae,necessary for the unique permeabilitycharacteristics of the glomerular filtrationbarrier

30

barrier

Mature fenenestrated endothelium is locatedadjacent to podocytes expressing VEGF athigh levels

Ballerman et al., Nephrol Physiol, 2007

Breier et al., Development, 1992

VEGF induces endothelialfenestrations by activating thefusion of intracellular

31

fusion of intracellularorganelles thought to representthe precursors of fenestrae

32

The New York Times - May 3, 1998

THE CASE OF THE HUMANIZED ANTI-VEGFANTIBODY BEVACIZUMAB

Bevacizumab is effective in the treatment ofpatients with many cancers, such as metastaticcolorectal cancer, non-small-cell lung cancer,

33

Zhu et al., Am J Kidney Dis, 2007

colorectal cancer, non-small-cell lung cancer,and breast cancer

It is also promising for renal cell carcinoma andprostate cancer

PROTEINURIA IS A COMMON SIDE-EFFECT OFHIGH DOSE BEVACIZUMAB

A randomized, double-blind, phase 2 trial in patients with metastaticrenal-cell carcinoma

Bevacizumab: 10 mg/kg every two weeks

Adverse events Bevacizumab(n=39)

Placebo(n=40)

34 Yang et al., N Engl J Med, 2003

(n=39) (n=40)

Proteinuria *HypertensionMalaiseHematuria

25 14135

15 160

Patients (n)

Proteinuria: > 1+ or > 150 mg/24 hours*

RISK OF PROTEINURIA IN PATIENTS WITH CANCER GIVENBEVACIZUMAB IS DOSE-DEPENDENT

A meta-analysis of 5 trials in 1,850 patients (1966-2006 year)

Hurwitz et al., 2004Johnson et al., 2003Kabbinavar et al., 2003

Relative risk

35Zhu et al., Am J Kidney Dis, 2007

Kabbinavar et al., 2003Kabbinavar et al., 2005Yang et al., 2003

CombinedControl Bevacizumab

low dose

P = 0.003 P < 0.001

1 100 1 100

Control Bevacizumabhigh dose

PROTEINURIA AFTER BEVACIZUMAB THERAPY

2

4

6

Urin

ary

albu

min

/cre

atin

ine

ratio

36Eremina et al., N Engl J Med, 2008

0

2

Urin

ary

albu

min

/cre

atin

ine

Pre Post

Bevacizumab (9 months)

VEGF Inhibition and Renal Thrombotic Microangiopathy

The glomerular microvasculature is particularly susceptibleto injury to thrombotic microangiopathy, but the mechanismsby which this occurs are unclear

37

Eremina et al., N Engl J Med, 2008

by which this occurs are unclear

We report the cases of six patients who were treated withbevacizumab, a humanized monoclonal antibody againstvascular endothelial growth factor, in whom proteinuria andglomerular disease characteristic of thromboticmicroangiopathy developed

Disease Clinical and biochemical parameters

Kidney biopsy

Patient 1(59 years)

Patient 2

Hepatocellular carcinoma

Recurrent

- Normal renal function at baseline

- Urinary P/C from 0.5 to 3.4- New onset hypertension- Low platelet count

- Normal renal function at

Thrombotic microangiopathy

Thrombotic

38Eremina et al., N Engl J Med, 2008

Patient 2(74 years)

Patient 3(56 years)

Recurrent hepatocellular carcinoma

Bronchoalveolar carcinoma

- Normal renal function at baseline

- Urinary P/C from 0.4 to 2.7

- Normal renal function at baseline

- Minimal proteinuria (0.6 g/24h)- Hypertension worsened- Anemia

Thrombotic microangiopathy

Thrombotic microangiopathy

Disease Clinical and biochemical parameters

Kidney biopsy

Patient 4(62 years)

Patient 5

Small-cell lung carcinoma

Metastatic

- Diabetic nephropathy at baseline

- Acute renal failure(s. creat from 1,4 to 5,7 mg/dl)

- Proteinuria 3+

- Normal renal function at

Thrombotic microangiopathy

Thrombotic

39

Eremina et al., N Engl J Med, 2008

Patient 5(61 years)

Patient 6(59 years)

Metastatic pancretic cancer

Metastatic ovarian cancer

- Normal renal function at baseline

- Proteinuria up to 4.6 g/24h- Low platelet count

- Proteinuria from 0.2 to 0.8 mg/24 h

- Normal platelet count

Thrombotic microangiopathy

Thrombotic microangiopathy

To show that local reduction of VEGF within thekidney is sufficient to trigger the pathogenesis ofthrombotic microangiopathy, we used conditionaltargeting to delete VEGF from renal podocytes inadult mice

This resulted in pronounced proteinuria and

40Eremina et al., N Engl J Med, 2008

This resulted in pronounced proteinuria andthrombotic glomerular injury

These observations provide evidence thatglomerular injury in patients who are treated withbevacizumab is probably due to direct targeting ofVEGF by antiangiogenic therapy

VEGF inhibition/blockade

Loss of glomerularendothelial fenestrae

Endothelial ET-1

Nephrin expression on podocyte

Proteintraffic

Microvascularinjury

0

60

120

ET-

1(p

g/10

6ce

lls)

Collino et al., Am J Physiol Renal Physiol, 2008

C Ab-

VE

GF

41

on podocyte

Altered glomerular permselectivity

Proteinuria

Protein overloadon podocytes

Podocyte ET-1

Thromboticmicroangiopathy

ET-

1 ge

ne(2

-��

Ct )

0

1

2

C Alb

umin

Morigi et al., Am J Pathol, 2005

10 YEARS OF MARKET APPROVALS OFCANCER DRUGS BY EMEA1995-2004

- 14 cancer drugs- 27 indications

42 Apolone et al., Br J Cancer, 2005

Survival benefit 0-3.7 months

A Cancer Drug Show Promise, at a Price That Many Can’t Pay

February 15, 2006

43

By ALEX BERENSON

44

These slides are belonging to Ariela Benigni, Ph.D.

Mario Negri Institute for Pharmacological Research, Bergamo, Italy.

45

Using these slides is only authorized bymentioning the source