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

2. 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 N-term C-term R138Q

3. 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) The NPHS2 Gene and Hereditary Nephrotic Syndrome N-term C-term R138Q

4. Creation of a Conditional Knock-in Mouse Model of R140Q Mutation Cre Nphs2 R140Q/+ Nphs2 lox2/lox2 Cre +/+ Nphs2 lox2/R140Q Cre + Nphs2 R140Q/- X Tamoxifen induction Bl6

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

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

7. Moderate Blood Pressure Increase in Sick Animals

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

9. Progressive Decrease of Podocin Level in the Kidney of Sick Animals 37 GAPDH 37 kDa 1 WK2 WKS12 WKS16 WKSKO16 WKS After Cre InductionControl Podocin

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

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 100 µM PAS staining week 4

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

13. 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) control week 4

14. Tubulointerstitial Fibrosis p<0.01

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

16. 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