1. Renal retinol reabsorption is essential in tubular damage and regeneration Jan Hinrich Bräsen, Philine Böckmann, Volker Maus, Melina Nieminen 1, Duska Dragun 1, Friedrich C. Luft 2, Thomas Willnow 2 Institute for Pathology Christian-Albrechts-Universität Kiel, 1 Dept. for Nephrology, Charité, and 2 Max Delbrück Center for Molecular Medicine, Berlin, Germany

2. - 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

3. RAR-RXR RARE RBP-retinol Megalin receptors Lysosomal processing injured PTC „anti -fibrosis“ inflammation / fibrosis? normal expression of receptors regeneration differentiation polarity TGF-ß? disturbed expression of receptors loss of polarity EMT? TGF-ß? RAR-RXR RARE normal PTC

4.  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

5. Megalin RBP Ischemia-reperfusion damage in proximal renal tubuli: loss of polarity and reduced cytoplasmic RBP

6. Retinol deficiency aggravates tubular damage RBP-KO number of tubuli / cross section proximal tubular damage score 7d wt Megalin-KO * ** * * * p<0.01 compared to control and Megalin-KO renal function creatinine (mg/dl) wt Megalin RBP 2d 7d

7. Retinol deficiency aggravates tubular damage (7d) RBP-KO wt Megalin-KO

8. Retinol deficiency inhibits tubular regeneration P < 0.001 P < 0.01 Ki-67 - positive tubular cells / HPF proliferation 2dproliferation 7d control Megalin-KORBP-KO wt Megalin-KORBP-KO proliferation 2d wt Megalin-KO

9. Retinol deficiency inhibits tubular regeneration (Ki67, 7d) RBP-KO wt Megalin-KO

10. Retinol deficiency reduces renal interstitial T-cell infiltration P < 0.01 P < 0.05 P < 0.01 P < 0.05 CD3 - positive cells / HPF CD3 -positive T-cell infiltrates 2d CD3 - positive cells / HPF CD3 -positive T-cell infiltrates 7d wt Megalin-KORBP-KO wt Megalin-KORBP-KO

11. wt Megalin-KO Retinol deficiency reduces renal interstitial T-cell infiltration (CD3, 7d)

12. P < 0.01 P < 0.05 FoxP3 - positive cells / cross section FoxP3 -positive T-reg infiltrates 2d FoxP3 - positive cells / cross section FoxP3 -positive T-reg infiltrates 7d wt Megalin-KORBP-KO wt Megalin-KORBP-KO Retinol deficiency reduces renal interstitial FoxP3-T-regs

13. Retinol deficiency reduces renal interstitial FoxP3+T-regs (FoxP3, 7d) RBP-KO wt Megalin-KO

14. Retinol deficiency reduces renal tubular TGF-beta expression P < 0.05 TGF-beta immunostained area (%) TGF-beta expression 2d TGF-beta immunostained area (%) TGF-beta expression 7d wt Megalin-KORBP-KO wt Megalin-KORBP-KO

15. Retinol deficiency reduces renal tubular TGF-beta expression (TGF-  1, 7d) wt Megalin-KO RBP-KO

16. wt Megalin-KORBP-KO ICAM-1 positive capillaries / HPF ICAM-1 expression 2d wt Megalin-KORBP-KO ICAM-1 positive capillaries / HPF ICAM-1 expression 7d Retinol deficiency and renal peritubular ICAM-1 expression

17. 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

18. - Max Gottesman - Wolfgang Schneider - Bärbel Kuhlmann - Wolfram Klapper - Jan Sperveslage - Monika Stepanowski - Sylvia Iversen - Monika Hauberg - Maike Pacena Acknowledgements