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

- 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_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

Retinol deficiency aggravates tubular damage (7d)

RBP-KOwt Megalin-KO

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

Retinol deficiency inhibits tubular regeneration (Ki67, 7d)

RBP-KOwt Megalin-KO

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

RBP-KOwt Megalin-KO

Retinol deficiency reduces renal interstitial T-cell infiltration (CD3, 7d)

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

Retinol deficiency reduces renal interstitial FoxP3+T-regs (FoxP3, 7d)

RBP-KOwt Megalin-KO

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

Retinol deficiency reduces renal tubular TGF-beta expression (TGF-1, 7d)

wt Megalin-KO RBP-KO

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

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