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


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


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


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


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


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


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


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


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


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


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

Renal retinol reabsorption is essential in tubular damage and regeneration Jan Hinrich Bräsen,...

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