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Renal retinol reabsorption is essential in tubular damage and regenerationJan 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
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- 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
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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
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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
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Megalin RBP
Ischemia-reperfusion damage in proximal renal tubuli:loss of polarity and reduced cytoplasmic RBP
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Retinol deficiency aggravates tubular damage
ATN_0ATN_1ATN_2ATN_3ATN_4
Tubulusschaden 7d
Anz
ahl T
ubul
i / Q
uers
chni
tt
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KORBP-KO
num
ber o
f tub
uli /
cro
ss s
ectio
n
proximal tubular damage score 7d
wt Megalin-KO
* * * * *
* p<0.01 compared to control and Megalin-KO
renal function
crea
tinin
e (m
g/dl
)
wt Megalin RBP wt Megalin RBP
2d 7d
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Retinol deficiency aggravates tubular damage (7d)
RBP-KOwt Megalin-KO
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Retinol deficiency inhibits tubular regeneration
Mean+SEMean-SEMean
Proliferation 2d
Anz
ahl K
i-67-
posi
tiver
Tub
ulus
epith
elie
n/H
PF
0
5
10
15
20
25
30
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SEMean
Proliferation 7d
Anz
ahl K
i-67-
posi
tiver
Tub
ulus
epith
elie
n/H
PF0
5
10
15
20
25
30
Kontrolle Megalin-KO RBP-KO
P < 0.001
P < 0.01
Ki-6
7 - p
ositi
ve tu
bula
r cel
ls /
HPF
Ki-6
7 - p
ositi
ve tu
bula
r cel
ls /
HPF
proliferation 2d proliferation 7d
control Megalin-KO RBP-KO wt Megalin-KO RBP-KO
proliferation 2d
wt Megalin-KO
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Retinol deficiency inhibits tubular regeneration (Ki67, 7d)
RBP-KOwt Megalin-KO
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Retinol deficiency reduces renal interstitial T-cell infiltration
Mean+SEMean-SEMean
CD3-positive T-Zellinfiltration 2d
Anz
ahl C
D3-
posi
tiver
Zel
len/
HPF
0
20
40
60
80
100
120
140
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SEMeanOutliers
CD3-positive T-Zellinfiltration 7d
Anz
ahl C
D3-
posi
tiver
Zel
len/
HPF
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 - p
ositi
ve c
ells
/ H
PF
CD3 -positive T-cell infiltrates 2d
CD
3 - p
ositi
ve c
ells
/ H
PF
CD3 -positive T-cell infiltrates 7d
wt Megalin-KO RBP-KO wt Megalin-KO RBP-KO
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RBP-KOwt Megalin-KO
Retinol deficiency reduces renal interstitial T-cell infiltration (CD3, 7d)
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Mean+SEMean-SEMean
FoxP3-Zellinfiltration 2d
Anz
ahl F
oxP3
-pos
itive
r Zel
len/
Que
rsch
nitt
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SEMean
FoxP3-Zellinfiltration 7d
Anz
ahl F
oxP3
-pos
itive
r Zel
len/
Que
rsch
nitt
0
10
20
30
40
50
Kontrolle Megalin-KO RBP-KO
P < 0.01
P < 0.05P < 0.05
FoxP
3 - p
ositi
ve c
ells
/ cr
oss
sect
ion
FoxP3 -positive T-reg infiltrates 2d
Fo
xP3
- pos
itive
cel
ls /
cros
s se
ctio
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
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Retinol deficiency reduces renal interstitial FoxP3+T-regs (FoxP3, 7d)
RBP-KOwt Megalin-KO
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Mean+SEMean-SEMean
TGF-beta-1-Expression 2d
TGF-
beta
-1-E
xpre
ssio
n [F
läch
e %
/HPF
]
0
2
4
6
8
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SEMean
TGF-beta-1-Expression 7d
TGF-
beta
-1-E
xpre
ssio
n [F
läch
e %
/HPF
]0
2
4
6
8
Kontrolle Megalin-KO RBP-KO
Retinol deficiency reduces renal tubular TGF-beta expression
P < 0.05
P < 0.05
TGF-
beta
imm
unos
tain
ed a
rea
(%)
TGF-beta expression 2d
TG
F-be
ta im
mun
osta
ined
are
a (%
)
TGF-beta expression 7d
wt Megalin-KO RBP-KO wt Megalin-KO RBP-KO
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Retinol deficiency reduces renal tubular TGF-beta expression (TGF-1, 7d)
wt Megalin-KO RBP-KO
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Mean+SEMean-SEMean
ICAM-1-positive Kapillaren 2d
Anz
ahl I
CA
M-1
-pos
itive
r Kap
illar
en/H
PF
0
10
20
30
40
50
60
70
Kontrolle Megalin-KO RBP-KO
Mean+SEMean-SEMean
ICAM-1-positive Kapillaten 7d
Anz
ahl I
CA
M-1
-pos
itive
r Kap
illar
en/H
PF0
10
20
30
40
50
60
70
Kontrolle Megalin-KO RBP-KO wt Megalin-KO RBP-KO
ICA
M-1
pos
itive
cap
illar
ies
/ HPF
ICAM-1 expression 2d
wt Megalin-KO RBP-KO
ICA
M-1
pos
itive
cap
illar
ies
/ HPF
ICAM-1 expression 7d
Retinol deficiency and renal peritubular ICAM-1 expression
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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
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- Max Gottesman- Wolfgang Schneider - Bärbel Kuhlmann- Wolfram Klapper- Jan Sperveslage- Monika Stepanowski- Sylvia Iversen- Monika Hauberg- Maike Pacena
Acknowledgements