1. Molecular mechanims of CKD progression following obstructive uropathy Mordi Muorah Clinical Fellow INSERM U845 – Equipe Fabiola Terzi Hopital Necker Enfants- Malades, Paris, FRANCE Paediatric Nephrology Workshop

2. Objectives To discuss – some of the methods available to study CKD in the laboratory – cellular and molecular pathways implicated implicated in the pathophysiology of obstructive nephropathy

3. Introduction CAKUT represent the most common cause of end stage kidney disease in childhood In spite of advances in modern medicine, therapeutic options for these diseases remain few

4. How do we study CKD in the laboratory? In vitro (cell culture) Transgenic mice Mouse models – Unilateral ureteral obstruction – Subtotal nephrectomy model – Streptozotocin induced diabetic nephropathy

5. What are the renal histological effects of obstruction? Strectch induced TGFβ secretion leading to inflammation Apoptosis/ proliferation Interstitial infiltration

6. Eddy et al. Ped Neph 2011 Procollagen III N terminal propeptide is a Potential urinary biomarker for CKD progression Increased collagen deposition

8. Mechanism leading to nephropathy in the UUO model Interstitial inflammation Tubular apoptosis and atrophy Renal fibrosis

9. Cytokines and growth Factors secreted by tubular Epithelial cells attract macrophages ECM Macrophages in turn secrete Growth factors and cytokines

10. Apoptosis Proliferation Fibroblasts either: Infiltrate from the circulation into the Interstitium; Appear by epithelial to mesenchymal transition (controverisial) Or appear by the proliferation of few Resident fibroblasts

11. Cellular infiltration Macrophages F4/80+ Dentritic cells MHC II CD 11b+ Phagocytosis of tissue debris and pathogens Potent source of chemokines and cytokines Can be fibrogenic CD 11c+ Phagocytosis of antigen in the kidney Migration to renal lymph nodes

12. Cellular infiltration and osteopontin Osteopontin is another potential urinary biomarker in the progression of CKD Osteopontin is an adhesion molecule that binds to macrophages via the CD44 receptor; it however has an inhibitory effect on apoptosis Weber Science 1996

13. Cellular infiltration and osteopontin Bascands et al 2005

14. proliferationapoptosis autophagy Koesters AJP, 2010

15. Adapted from Janeways Immunobiology 7 th Ed. Garland Science Apoptosis of T cells activated through cell Surface receptor - FAS FAS is a member of the TNF family CAD – caspase activated DNAse

16. Transforming Growth Factor β1

18. TGFβ Koesters AJP, 2010

19. Epithelial Mesenchymal Transition? Lacz β-galactosidase permanently Stained tubules. Collagen in brown. Post UUO the mesenchyme is not Seen to stain blue suggesting a non- epithelial origin Koesters AJP, 2010

20. TGFβ ovexpressed in Images on the right UUO day 4 Koesters AJP, 2010

21. Bascands et al 2005 Renin Angiotensin system

22. Bascands et al 2005 Renin Angiotensin system

23. Conclusions Complex cellular and molecular pathways which control the processes leading to kidney damage post UUO Potential therapeutic targets or for biomarker validation

24. Thank you