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Abbiamo farmaci che sappiano riparare il danno ai podociti?
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