Abbiamo farmaci che sappiano riparare il danno ai podociti?

1. Abbiamo farmaci che sappiano
riparare il danno ai podociti?
Ariela Benigni
Dipartimento di Medicina Molecolare
Istituto di Ricerche Farmaclogiche Mario Negri,
Laboratori Negri Bergamo
Torino, 23 gennaio 2009
1

2. Glomerular hypertension
Podocytes
GBM
Endothelial cells
Disease progression
2

3. Glomerular hypertension
Mechanical strain
* 2.5
1.2
(pg per µg of cell lysate)
(adjusted for tubulin)
1.0 2.0
0.8
AT1R level
Ang II
1.5
Podocyte number 0.6
1.0
0.4
0.5
0.2
0
0
Ctr MS Ctr MS
Durvasula et al, Kidney Int, 2004
Pore dimension
Proteinuria
Disease progression
3

4. AT1R
PLC
DAG IP3
TRPC6
Ca2+
Cytoskeleton
TRPC6: Transient Receptor Potential cation channel
Winn et al, Science, 2005 Nitschke et al., Kidney Int, 2000
4

5. actin ZO-1 merge
40
Albumin flux (µg/hour)
30
µ
Control 20
10
0
40
Albumin flux (µg/hour)
*
30
µ
Ang II 20
10
0
Macconi et al., Am J Pathol, 2006
5

6. 1 - apoptosis
2 - phenotype
changes
6

7. UNINEPHRECTOMIZED MWF/ZTM RATS - 1
*
700
Urinary Protein Excretion
600
500
(mg/24 hrs)
400
300
200
100
**
0
Control UNx UNx + Lis
*
100
Percentage of glomeruli
affected by sclerosis
80
60
40
20
**
0
Control UNx UNx + Lis
* p < 0.05, **p < 0.01 vs control
7 Remuzzi A. et al., Kidney Int, 1995

8. UNINEPHRECTOMIZED MWF/ZTM RATS - 2
Control
100
UNx + Lis
80
Survival (%)
60
40
20
UNx
0
0 3 6 9 12 15
Time (months after UNx)
8 Remuzzi A. et al., Kidney Int, 1995

9. Ramipril
Ramipril
45
∆ GFR = -0.44 ± 0.54
40
(ml/min/month)
∆ GFR = - 0.10 ± 0.50
GFR
35
∆ GFR = -0.81 ± 1.12
30
∆ GFR = -0.14 ± 0.87
25
Conventional Ramipril
FOLLOW-UP
CORE
Ruggenenti et al., Lancet, 1998
9

10. REGRESSION
10 patients with increasing GFR
∆ Proteinuria
P = 0.01
65
(pre vs post break point)
∆GFR -0.21 + 0.09 +0.49 + 0.19
60
(ml/min/month)
GFR (ml/min/month)
0
55
50
45
-20
40
%
35
-40
30
25
Break point
20
-60
-30 -20 -10 0 10 20 30
months
Ruggenenti et al., J Am Soc Nephrol, 1999
10

11. MWF 50W MWF 60W MWF+LIS 50-60 w
Remuzzi A. et al., Kidney Int, 2006
11

12. 100
% of Capillary Tuft Volume
Affected by Sclerosis
75
MWF 50W
50
25
0
0 10 20 30 40 50 60 70 80 90 100
Reconstructed Glomeruli
12

13. 100
% of Capillary Tuft Volume
Affected by Sclerosis
75
MWF 50W
MWF 60W
50
25
0
0 10 20 30 40 50 60 70 80 90 100
Reconstructed Glomeruli
13

14. 100
% of Capillary Tuft Volume
Affected by Sclerosis
75
MWF 50W
MWF 60W
50
25
MWF + LIS 50-60W
0
0 10 20 30 40 50 60 70 80 90 100
Reconstructed Glomeruli
14

15. Sclerosis was effectively reabsorbed and a
consistent amount of glomerular tissue
regained normal structure
This suggests neoformation of glomerular
capillary segments
15

16. INSIGHT INTO ACE-INDUCED RENAL
REPAIR/ANGIOGENESIS
Renal cells
Adult differentiated
Resident progenitor/stem
Extra renal cells
Endothelial progenitor and/or bone marrow-
derived stem
16

17. 30
VV endothelial cells (%)
20
10
*
0
40 W 60 W LIS 40-60 W
WGA = cell membranes
RECA-1= endothelial cells Macconi et al., 2008
17

18. WT1+ cells/glom
160
120
**
80
40
0
40W 60W LIS 40-60W
WT1 = podocyte marker (nuclei)
Macconi et al., Am J Pathol, 2009
18

19. Migration of parietal cells to
capillary tuft through the
vascular pole
19

20. Migration of parietal cells from the Bowman’s capsule to capillary tuft
20

21. PARIETAL CELLS WITH PODOCYTE PHENOTYPE
12
*
Parietal podocytes/PEC (%)
10
8
6
4
2
0
MWF MWF MWF + Lis
40 W 60 W 60 W
These cells were identified by staining for PGP 9.5 (parietal epithelial cell
marker) and WT1 (podocyte marker)
Macconi et al., Am J Pathol, 2009
21

22. ISOLATION AND CHARACTERIZATION OF
MULTIPOTENT PROGENITOR CELLS
CD133+CD24+ FROM THE BOWMAN’S CAPSULE
OF ADULT HUMAN KIDNEYS
Sagrinati et al., J Am Soc Nephrol, 2006
22

23. 23

24. Brenner’s seminal paper
NEJM
Retarding renal Promoting kidney
disease progression repair
1983 2003
24

25. The presence of VEGF is crucial
for normal renal development
Differentiating glomerular
epithelia produce VEGF and may
attract endothelial cells into the
glomeruli
Administration of anti-VEGF
antibody during early kidney
development in mice leads to
formation of abnormal glomerular
structures and diminished
nephrogenesis
Kretzler et al., Kidney Int, 1998
25

26. PODOCYTE ARE THE MAJOR SOURCE OF VEGF
IN THE GLOMERULUS
Kretzler et al., Kidney Int, 1998
26

27. PODOCYTE VEGF BINDS TO COGNATE RECEPTORS
EXPRESSED ON GLOMERULAR ENDOTHELIAL CELLS
How podocytes can signal “up stream” in the glomerular endothelium
A concentration gradient
favors diffusion of VEGF
from the podocyte to
glomerular endothelial
cells
Eremina et al., N Engl J Med, 2008
27

28. 28

29. 29

30. Glomerular endothelial cells differ from most
other endothelial cells in that they are
extremely flattened and densely perforated
by transendothelial pores, the fenestrae,
necessary for the unique permeability
characteristics of the glomerular filtration
barrier
Ballerman et al., Nephrol Physiol, 2007
Mature fenenestrated endothelium is located
adjacent to podocytes expressing VEGF at
high levels Breier et al., Development, 1992
30

31. VEGF induces endothelial
fenestrations by activating the
fusion of intracellular
organelles thought to represent
the precursors of fenestrae
31

32. The New York Times - May 3, 1998
32

33. THE CASE OF THE HUMANIZED ANTI-VEGF
ANTIBODY BEVACIZUMAB
Bevacizumab is effective in the treatment of
patients with many cancers, such as metastatic
colorectal cancer, non-small-cell lung cancer,
and breast cancer
It is also promising for renal cell carcinoma and
prostate cancer
Zhu et al., Am J Kidney Dis, 2007
33

34. PROTEINURIA IS A COMMON SIDE-EFFECT OF
HIGH DOSE BEVACIZUMAB
A randomized, double-blind, phase 2 trial in patients with metastatic
renal-cell carcinoma
Bevacizumab: 10 mg/kg every two weeks
Adverse events Bevacizumab Placebo
(n=39) (n=40)
Patients (n)
15
25
Proteinuria *
1
14
Hypertension
6
13
Malaise
0
5
Hematuria
Proteinuria: > 1+ or > 150 mg/24 hours
*
Yang et al., N Engl J Med, 2003
34

35. RISK OF PROTEINURIA IN PATIENTS WITH CANCER GIVEN
BEVACIZUMAB IS DOSE-DEPENDENT
A meta-analysis of 5 trials in 1,850 patients (1966-2006 year)
Relative risk
Hurwitz et al., 2004
Johnson et al., 2003
Kabbinavar et al., 2003
Kabbinavar et al., 2005
Yang et al., 2003
Combined P < 0.001
P = 0.003
Bevacizumab
Control
Bevacizumab
Control
high dose
low dose
100
1
100
1
Zhu et al., Am J Kidney Dis, 2007
35

36. PROTEINURIA AFTER BEVACIZUMAB THERAPY
Urinary albumin/creatinine
6
4
ratio
2
0
Post
Pre
Bevacizumab (9 months)
Eremina et al., N Engl J Med, 2008
36

37. VEGF Inhibition and Renal
Thrombotic Microangiopathy
The glomerular microvasculature is particularly susceptible
to injury to thrombotic microangiopathy, but the mechanisms
by which this occurs are unclear
We report the cases of six patients who were treated with
bevacizumab, a humanized monoclonal antibody against
vascular endothelial growth factor, in whom proteinuria and
glomerular disease characteristic of thrombotic
microangiopathy developed
Eremina et al., N Engl J Med, 2008
37

38. Disease Clinical and biochemical Kidney biopsy
parameters
- Normal renal function at Thrombotic
Patient 1 Hepatocellular
baseline microangiopathy
(59 years) carcinoma
- Urinary P/C from 0.5 to 3.4
- New onset hypertension
- Low platelet count
- Normal renal function at Thrombotic
Patient 2 Recurrent
baseline microangiopathy
(74 years) hepatocellular
- Urinary P/C from 0.4 to 2.7
carcinoma
Patient 3 Bronchoalveolar - Normal renal function at Thrombotic
baseline microangiopathy
(56 years) carcinoma
- Minimal proteinuria (0.6 g/24h)
- Hypertension worsened
- Anemia
Eremina et al., N Engl J Med, 2008
38

39. Disease Clinical and biochemical Kidney biopsy
parameters
- Diabetic nephropathy at Thrombotic
Patient 4 Small-cell lung
baseline microangiopathy
(62 years) carcinoma
- Acute renal failure
(s. creat from 1,4 to 5,7 mg/dl)
- Proteinuria 3+
- Normal renal function at Thrombotic
Patient 5 Metastatic
baseline microangiopathy
(61 years) pancretic cancer
- Proteinuria up to 4.6 g/24h
- Low platelet count
Thrombotic
- Proteinuria from 0.2 to 0.8
Patient 6 Metastatic
microangiopathy
mg/24 h
(59 years) ovarian cancer
- Normal platelet count
Eremina et al., N Engl J Med, 2008
39

40. To show that local reduction of VEGF within the
kidney is sufficient to trigger the pathogenesis of
thrombotic microangiopathy, we used conditional
targeting to delete VEGF from renal podocytes in
adult mice
This resulted in pronounced proteinuria and
thrombotic glomerular injury
These observations provide evidence that
glomerular injury in patients who are treated with
bevacizumab is probably due to direct targeting of
VEGF by antiangiogenic therapy
Eremina et al., N Engl J Med, 2008
40

41. VEGF inhibition/blockade 120
ET-1 (pg/106 cells)
Ab-VEGF
60
Loss of glomerular Endothelial
C
endothelial fenestrae ET-1 0
Collino et al., Am J Physiol
Renal Physiol, 2008
Nephrin
Microvascular Protein
expression
injury traffic
on podocyte
Protein overload Podocyte
on podocytes ET-1
Altered
Ct)
2
glomerular
ET-1 gene (2-
permselectivity
Albumin
1
Thrombotic
C
0
microangiopathy Proteinuria
Morigi et al., Am J Pathol, 2005
41

42. 10 YEARS OF MARKET APPROVALS OF
CANCER DRUGS BY EMEA
1995-2004
- 14 cancer drugs
- 27 indications
Survival benefit 0-3.7 months
Apolone et al., Br J Cancer, 2005
42

43. February 15, 2006
A Cancer Drug Show Promise, at a Price
That Many Can’t Pay
By ALEX BERENSON
43

44. 44

45. These slides are belonging to
Ariela Benigni, Ph.D.
Mario Negri Institute for Pharmacological
Research, Bergamo, Italy.
Using these slides is only authorized by
mentioning the source
45