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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 Pathology
Christian-Albrechts-Universität Kiel,
1Dept. for Nephrology, Charité, and 2Max Delbrück Center for Molecular Medicine, Berlin,
Germany
Background and Hypothesis
- 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?
normal PTC
RBP-retinol
injured PTC
RBP-retinol
Megalin
receptors
Lysosomal
processing
disturbed
expression of
receptors
normal
expression of
receptors
RAR-RXR
RAR-RXR
RARE
TGF-ß?
„anti -fibrosis“
regeneration
differentiation
polarity
RARE
loss of
polarity
EMT?
TGF-ß?
inflammation / fibrosis?
Methods




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-b, ICAM-1)
Ischemia-reperfusion damage in proximal renal tubuli:
loss of polarity and reduced cytoplasmic RBP
Megalin
RBP
Retinol deficiency aggravates tubular damage
proximal
tubular damage7d
score 7d
Tubulusschaden
renal function
* * * * *
Anzahl of
Tubuli
cross section
tubuli/ /Querschnitt
number
40
creatinine (mg/dl)
50
wt
30
Megalin
RBP
2d
20
10
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
AT N_0
AT N_1
AT N_2
AT N_3
AT N_4
* p<0.01 compared to control and Megalin-KO
wt
Megalin
7d
RBP
Retinol deficiency aggravates tubular damage (7d)
wt
Megalin-KO
RBP-KO
Retinol deficiency inhibits tubular regeneration
Proliferation 2d
proliferation
2d
Proliferation 7d
7d
proliferation
30
Ki-67 - positive tubular cells / HPF
Anzahl Ki-67-positiver Tubulusepithelien/HPF
Ki-67 - positive tubular cells / HPF
Anzahl Ki-67-positiver Tubulusepithelien/HPF
30
25
20
15
10
5
0
Kontrolle
control
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
P < 0.001
25
P < 0.01
20
15
10
5
Mean+SE
Mean-SE
0
Mean
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
Retinol deficiency inhibits tubular regeneration (Ki67, 7d)
wt
Megalin-KO
RBP-KO
Retinol deficiency reduces renal interstitial T-cell infiltration
CD3CD3-positiv
-positive eT-cell
infiltrates7d
7d
T-Zellinfiltration
CD3
-positive
T-cell infiltrates
CD3-positiv
e T-Zellinfiltration
2d 2d
140
140
P < 0.01
P < 0.01
120
P < 0.05
Anzahl
CD3-positiver
/ HPF
cellsZellen/HPF
CD3
- positive
/ HPF
cellsZellen/HPF
CD3 - positive
Anzahl
CD3-positiver
120
100
80
60
40
20
P < 0.05
100
80
60
40
20
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
Outliers
Retinol deficiency reduces renal interstitial T-cell infiltration (CD3, 7d)
wt
Megalin-KO
RBP-KO
Retinol deficiency reduces renal interstitial FoxP3-T-regs
FoxP3 -positive
T-reg infiltrates
FoxP3-Zellinfiltration
2d 2d
FoxP3 -positive
T-reg infiltrates
FoxP3-Zellinfiltration
7d 7d
50
50
Anzahl
FoxP3-positiver
Zellen/Querschnitt
section
cells / cross
FoxP3
- positive
FoxP3
- positive cells / cross section
Anzahl FoxP3-positiver Zellen/Querschnitt
P < 0.01
P < 0.05
40
30
20
10
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
P < 0.05
40
30
20
10
Mean+SE
Mean-SE
0
Mean
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
Retinol deficiency reduces renal interstitial FoxP3+T-regs (FoxP3, 7d)
wt
Megalin-KO
RBP-KO
Retinol deficiency reduces renal tubular TGF-beta expression
TGF-beta-1-Expression
2d
TGF-beta
expression 2d
TGF-beta-1-Expression
7d
TGF-beta
expression 7d
8
8
immunostained area (%)
TGF-beta
TGF-beta-1-Expression [Fläche %/HPF]
immunostained area (%)
TGF-beta
TGF-beta-1-Expression [Fläche %/HPF]
P < 0.05
6
4
2
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
P < 0.05
6
4
2
Mean+SE
Mean-SE
0
Mean
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
Retinol deficiency reduces renal tubular TGF-beta expression (TGF-b1, 7d)
wt
Megalin-KO
RBP-KO
Retinol deficiency and renal peritubular ICAM-1 expression
ICAM
-1-positiv
e Kapillaten
ICAM-1
expression
7d7d
70
60
60
/ HPF
positive capillaries
ICAM-1ICAM-1-positiver
Anzahl
Kapillaren/HPF
/ HPF
positive capillaries
ICAM-1
Anzahl
ICAM-1-positiver
Kapillaren/HPF
ICAM
-1-positiv
e Kapillaren
ICAM-1
expression
2d2d
70
50
40
30
20
10
0
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
50
40
30
20
10
Mean+SE
Mean-SE
0
Mean
Kontrolle
wt
M egalin-KO
Megalin-KO
RBP-KO
RBP-KO
Mean+SE
Mean-SE
Mean
Summary and Conclusion
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
Acknowledgements
- Max Gottesman
- Wolfgang Schneider
- Bärbel Kuhlmann
- Wolfram Klapper
- Jan Sperveslage
- Monika Stepanowski
- Sylvia Iversen
- Monika Hauberg
- Maike Pacena