24 th European Congress of Pathology, Prague, Czech Republic, 8 - 12 September 2012

1. Podocyte Loss and Glomerulosclerosis in Inducible Mouse Model of Podocin Mutation-Related Nephrotic Syndrome Ivana Simic, Mansoureh Tabatabaeifar, Geraldine Mollet, Barbara Bruehl, Corinne Antignac, Franz Schaefer Division of Pediatric Nephrology, University of Heidelberg, Neuroanatomy, University of Heidelberg, and Department of Human Genetics, Hopital Necker, Paris 24th European Congress of Pathology, Prague, Czech Republic, 8 - 12 September 2012

2. N-term C-term R138Q The NPHS2 Gene and Hereditary Nephrotic Syndrome • Mutations in the NPHS2 gene, encoding podocin, cause autosomal recessive steroid-resistant nephrotic syndrome • R138Q, the most common podocin mutation in Europeans, causes early disease onset and rapid progression to end-stage renal disease

3. N-term C-term R138Q The NPHS2 Gene and Hereditary Nephrotic Syndrome • The R138Q mutation causes retention of podocin in the ER, leading to impaired SD formation and foot process effacement • Knock-in mice carrying the R140Q mutation, the mouse analogue of human R138Q, show developmental arrest of podocytes and lethal renal failure at neonatal age (A. Philippe et al, 2008)

4. Cre Cre Cre Cre Creation of a Conditional Knock-in Mouse Model of R140Q Mutation X Bl6 Bl6 Nphs2R140Q/+ Nphs2lox2/lox2 Cre+/+ Nphs2lox2/R140Q Cre+ Tamoxifen induction Nphs2R140Q/-

5. Proteinuria Develops within 1 Week and Peaks 3-4 Weeks after Induction

6. control R140Q / - Reduced Weight Gain from Week 4

7. Moderate Blood Pressure Increase in Sick Animals

8. Renal Failure, Hypoalbuminemia and Hypercholesterolemia at 12 to 16 Weeks control R140Q / - control R140Q / - control R140Q / -

9. Progressive Decrease of Podocin Level in the Kidney of Sick Animals After Cre Induction Control 1 WK 2 WKS 12 WKS 16 WKS KO 16 WKS kDa Podocin 37 37 GAPDH

10. week 2 week 2 week 2 week 2 control control L FP En L L L L FP FP FP FP En En En En week 16 FP En L Foot Process Effacement upon Podocin Loss control L FP En

11. PAS Staining - Determination of Glomerular Sclerosis Index (GSI) combining the semiquantitative scoring system (el Nahas et al 1991) and Image ProPlus Software to quantify the sclerotic area control PAS staining 100 µM week 4

12. p<0.05 Glomerular Sclerosis Index (GSI) p<0.01 p<0.01 p<0.01 p<0.01

13. control week 4 Sirius Red Staining - Determination of TIF • The percentage of Sirius Red – stained fibrotic tubulointerstitial area was measured using Image-Pro Plus software on 30 randomly sampled fields of kidney (magnification 300x) (Zhang G et al, 2003)

14. p<0.01 Tubulointerstitial Fibrosis

15. Podocyte Counting and Density Analysis Protocol recommended by Animal Models of Diabetic Complications Consortium (AMDCC) (WT1 Ab and immunoperoxidase) control week 4

16. p<0.01 p<0.01 Average Podocyte Number at Different Time-Points after the Induction p<0.01

17. Summary • Postnatal induction of R140Q hemizygosity rapidly leads to massive proteinuria, which is maximal at 4 weeks, hypoalbuminemia, hypercholesterolemia and moderate hypertension • At the later stage, kidneys show global glomerulosclerosis, tubular atrophy and severe interstitial fibrosis • Podocyte loss occurred within the second week after the induction • The inducible NPHS2-R140Q mouse is an ideal model for pharmacological studies

18. Dr. Ivana Simic Dr. Mansoureh Tabatabaeifar Prof. Franz Schaefer Barbara Brühl Hopital Necker, France Dr. Geraldine Mollet Prof. Corinne Antignac