Renal retinol reabsorption is essential in tubular damage and regeneration Jan Hinrich Bräsen,...
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Transcript of Renal retinol reabsorption is essential in tubular damage and regeneration Jan Hinrich Bräsen,...
Renal retinol reabsorption is essential in tubular damage and regeneration
Jan Hinrich Bräsen, Philine Böckmann, Volker Maus, Melina Nieminen1, Duska Dragun1, Friedrich C. Luft2, Thomas Willnow2
Institute for PathologyChristian-Albrechts-Universität Kiel, 1Dept. for Nephrology, Charité, and 2Max Delbrück Center for Molecular Medicine, Berlin, Germany
- low molecular retinol binding protein (RBP)–retinol complexes undergo free glomerular filtration and complete reabsoption by receptor-mediated endocytosis via Megalin in proximal renal tubuli (Christensen et al. JASN 1999)
- Vitamin D is finally hydroxylated during endo-lysosomal processing (Nykjaer et al. Cell 1999)
Does proximal tubular retinol deficiency influence renal tubular regeneration following ischemia-reperfusion injury?
Background and Hypothesis
RAR-RXRRARE
RBP-retinol RBP-retinol
Megalin
receptors
Lysosomalprocessing
injured PTC
„anti -fibrosis“ inflammation / fibrosis?
normal expression ofreceptors
regenerationdifferentiationpolarity
TGF-ß?
disturbed expression ofreceptors
loss ofpolarity
EMT?
TGF-ß?
RAR-RXRRARE
normal PTC
mouse model of renal tubular retinoid deficiency: wild type (C57BL\6), RBP-KO, Megalin-KO (n=10 each) 45 minutes ischemia by bilateral renal pedicle clamping sacrifice at day 2 and 7 post reperfusion
- renal function (creatinine level)- tubular damage scoring (debris, brush border, polarity, BM disruption)- immunohistochemistry (Ki67, CD3, FoxP3, TGF-ICAM-1)
Methods
Megalin RBP
Ischemia-reperfusion damage in proximal renal tubuli:loss of polarity and reduced cytoplasmic RBP
Retinol deficiency aggravates tubular damage
ATN_0
ATN_1
ATN_2
ATN_3
ATN_4
Tubulusschaden 7d
An
zah
l Tu
bu
li /
Qu
ers
ch
nit
t
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KORBP-KO
nu
mb
er
of
tub
uli
/ c
ross
se
cti
on
proximal tubular damage score 7d
wt Megalin-KO
* * * * *
* p<0.01 compared to control and Megalin-KO
renal function
crea
tin
ine
(mg
/dl)
wt Megalin RBP wt Megalin RBP
2d 7d
Retinol deficiency aggravates tubular damage (7d)
RBP-KOwt Megalin-KO
Retinol deficiency inhibits tubular regeneration
Mean+SEMean-SE
Mean
Proliferation 2d
An
zah
l K
i-6
7-p
os
itiv
er
Tub
ulu
se
pit
he
lie
n/H
PF
0
5
10
15
20
25
30
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
Proliferation 7d
An
zah
l K
i-6
7-p
os
itiv
er
Tub
ulu
se
pit
he
lie
n/H
PF
0
5
10
15
20
25
30
Kontrolle Megalin-KO RBP-KO
P < 0.001
P < 0.01
Ki-
67
- p
os
itiv
e t
ub
ula
r c
ell
s /
HP
F
Ki-
67
- p
os
itiv
e t
ub
ula
r c
ell
s /
HP
F
proliferation 2d proliferation 7d
control Megalin-KO RBP-KO wt Megalin-KO RBP-KO
proliferation 2d
wt Megalin-KO
Retinol deficiency inhibits tubular regeneration (Ki67, 7d)
RBP-KOwt Megalin-KO
Retinol deficiency reduces renal interstitial T-cell infiltration
Mean+SEMean-SE
Mean
CD3-positive T-Zellinfiltration 2d
An
zah
l C
D3
-po
sit
ive
r Z
ell
en
/HP
F
0
20
40
60
80
100
120
140
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
Outliers
CD3-positive T-Zellinfiltration 7d
An
zah
l C
D3
-po
sit
ive
r Z
ell
en
/HP
F
0
20
40
60
80
100
120
140
Kontrolle Megalin-KO RBP-KO
P < 0.01
P < 0.05
P < 0.01
P < 0.05
CD
3 -
po
sit
ive
ce
lls /
HP
F
CD3 -positive T-cell infiltrates 2d
CD
3 -
po
sit
ive
ce
lls /
HP
F
CD3 -positive T-cell infiltrates 7d
wt Megalin-KO RBP-KO wt Megalin-KO RBP-KO
RBP-KOwt Megalin-KO
Retinol deficiency reduces renal interstitial T-cell infiltration (CD3, 7d)
Mean+SEMean-SE
Mean
FoxP3-Zellinfiltration 2d
An
zah
l F
ox
P3
-po
sit
ive
r Z
ell
en
/Qu
ers
ch
nit
t
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
FoxP3-Zellinfiltration 7d
An
zah
l F
ox
P3
-po
sit
ive
r Z
ell
en
/Qu
ers
ch
nit
t
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KO
P < 0.01
P < 0.05P < 0.05
Fo
xP
3 -
po
sit
ive
cel
ls /
cro
ss
se
cti
on
FoxP3 -positive T-reg infiltrates 2d
F
ox
P3
- p
os
itiv
e c
ells
/ c
ros
s s
ec
tio
n
FoxP3 -positive T-reg infiltrates 7d
wt Megalin-KO RBP-KO wt Megalin-KO RBP-KO
Retinol deficiency reduces renal interstitial FoxP3-T-regs
Retinol deficiency reduces renal interstitial FoxP3+T-regs (FoxP3, 7d)
RBP-KOwt Megalin-KO
Mean+SEMean-SE
Mean
TGF-beta-1-Expression 2d
TG
F-b
eta
-1-E
xp
res
sio
n [
Flä
ch
e %
/HP
F]
0
2
4
6
8
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
TGF-beta-1-Expression 7d
TG
F-b
eta
-1-E
xp
res
sio
n [
Flä
ch
e %
/HP
F]
0
2
4
6
8
Kontrolle Megalin-KO RBP-KO
Retinol deficiency reduces renal tubular TGF-beta expression
P < 0.05
P < 0.05
TG
F-b
eta
im
mu
no
sta
ine
d a
rea
(%
)
TGF-beta expression 2d
T
GF
-bet
a i
mm
un
os
tain
ed
are
a (
%)
TGF-beta expression 7d
wt Megalin-KO RBP-KO wt Megalin-KO RBP-KO
Retinol deficiency reduces renal tubular TGF-beta expression (TGF-1, 7d)
wt Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
ICAM-1-positive Kapillaren 2d
An
zah
l IC
AM
-1-p
os
itiv
er
Ka
pil
lare
n/H
PF
0
10
20
30
40
50
60
70
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SE
Mean
ICAM-1-positive Kapillaten 7d
An
zah
l IC
AM
-1-p
os
itiv
er
Ka
pil
lare
n/H
PF
0
10
20
30
40
50
60
70
Kontrolle Megalin-KO RBP-KO wt Megalin-KO RBP-KO
ICA
M-1
po
siti
ve
ca
pil
lari
es
/ H
PF
ICAM-1 expression 2d
wt Megalin-KO RBP-KO
ICA
M-1
po
siti
ve
ca
pil
lari
es
/ H
PF
ICAM-1 expression 7d
Retinol deficiency and renal peritubular ICAM-1 expression
Deficiency of renal tubular retinoid resorption
- inhibits tubular regeneration following ischemia reperfusion
- reduces CD3-positive T-cell infiltration
- reduces FoxP-3-positive T-regs
retinoids may open new treatment options in reperfusion injury and transplantation
Summary and Conclusion
- Max Gottesman- Wolfgang Schneider - Bärbel Kuhlmann- Wolfram Klapper- Jan Sperveslage- Monika Stepanowski- Sylvia Iversen- Monika Hauberg- Maike Pacena
Acknowledgements