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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