1. Alport Syndrome – Summary of Current Clinical and Basic Science Research Christoph Licht The Hospital for Sick Children, Toronto, ON Alport Syndrome Family Conference 2012 Minneapolis, MN - 21.-22.7.2012

2. Altered podocyte actin cytoskeleton & cell adhesion Proteolysis of embryonic collagen IV by MMP Nuclear PPAR  downregulation & podocyte apoptosis Albumin GBM with embryonic (  1.  2.  2) collagen IV Atypical GBM laminin isoforms (  1 and  5) Transferrin Podocyte DDR1 & Integrin α2β1 detect abnormal collagen IV in the GBM C3 Biomechanical Strain Pathological proteinuria Intravascular Space Urinary Space Splitting of GBM }

3. Proteinuria Excessive protein in the glomerular filtrate Albumin endocytosed by PTECs via the megalin-cubilin  signaling cascade leading to gene transcription & production of various chemotactic, inflammatory and profibrotic mediators as well as PTEC apoptosis Other plasma proteins such as transferrin, immunoglobulin and complement proteins

4. Tubular Lumen C3 convertase Profibrotic Mediators TGFβ Collagen I & IV Fibronectin CTGF Inflammatory Mediators Cytokines TNFα Chemokines MCP-1 RANTES C5b9 AP Albumin C3 C3a Receptor Transferrin Megalin-Cubulin Endocytosis C3a Ig PTEC Tubulointerstitium Gene Transcription C3a

5. Inhibition Glomerulopathy of Alport Syndrome Persistence of embryonic collagen IV in GBM Subject to proteolysis by MMP & biomechanical strain Biomechanical strain leads to altered GBM/cell adhesion, disrupted actin cytoskeleton & MMP induction Splitting of GBM & proteinuria Disease Initiation 1.RAAS Blockade 2.Aldosterone Inhibitors 3.Aliskiren 4.Calcineurin Inhibitors 5.Endothelin Receptor Blockers 6.HMG CoA Reductase Inhibitors 7.Sulodexide 8.PPAR  agonists 9.Vasopeptidase inhibitors 10.Matrix metalloproteinases 11.DDR1 antagonism PTEC and Tubulointerstitial Pathology Pathological proteinuria Albumin endocytosis via megalin- cubulin Activation of complement cascade on PTEC Signaling, gene transcription of cytokines, profibrotic mediators & chemokines PTEC apoptosis EMT Tubulointerstitial fibrosis Disease Transmission & Progression 1.Bone Morphogenetic Protein-7 2.Chemokine Receptor Antagonists 3.Complement Inhibition 4.Sulodexide 5.Matrix Metalloproteinases 6.TNF alpha blockade and Pentoxifylline 7.Vitamin D 8.DDR1 antagonism PathomechanismTargeted Therapies

6. The role of complement Almost thirty years ago, a urinary protein thought to be unique to AS and termed hereditary nephritis protein (HNP), was purified and discovered to be a split product of complement protein C3

7. Tubular Lumen C3 convertase Profibrotic Mediators TGFβ Collagen I & IV Fibronectin CTGF Inflammatory Mediators Cytokines TNFα Chemokines MCP-1 RANTES C5b9 AP Albumin C3 C3a Receptor Transferrin Megalin-Cubulin Endocytosis C3a Ig PTEC Tubulointerstitium Gene Transcription C3a

8. Complement & the PTEC Complement C3 in the ultrafiltrate increases with proteinuria & localizes to the PTECs PTECs  lack key complement regulatory proteins  complement convertase-like capabilities  can also actually synthesize C3 independently Abbate et al, J Am Soc Nephrol 1999 Ichida et al, Kidney Int 1994 Biancone et al, Kidney Int 1994 Tang et al, J Am Soc Nephrol 1999

9. C6 deficient rat Inherently unable to generate MAC/C5b-9 Completely protected from tubulointerstitial injury  intraluminal formation of the MAC/C5b-9 an essential mediator of tubulointerstitial disease Nangaku et al, J Am Soc Nephrol 2002

10. Preclinical studies of complement inhibition C3 null mouse protected from adriamycin induced glomerulopathy, tubulointerstitial injury & renal impairment Rat membranous nephropathy model - eliminated proteinuria & preserved slit diaphragm by preventing nephrin and podocin loss PAN nephrosis rat model - complement inhibitor delivered direct to PTEC inhibited MAC/C5b-9 formation & prevented tubulointerstitial injury Sheerin et al, FASEB J 2008 Saran et al, Kidney Int 2003 He et al, J Immunol 2005

11. Crosstalk between the abnormal collagen IV and the podocyte via the Discoidin Domain Receptor 1 (&  2  1 Integrin)

12. DDR1 Discoidin Domain Receptor 1 is a tyrosine kinase transmembrane receptor that has collagen I - V as its ligand  regulates ECM remodeling & controls adhesion & proliferation of renal cells In COL4A3 -/- mice DDR1 expressed on the podocyte foot processes allowing interaction between the podocyte and GBM Vogel et al, Mol Cell 1997 Gross et al, Matrix Biol 2010

13. DDR1 Podocyte detects altered collagen protomers via DDR1 receptors  upregulation of various cytokines and growth factors (probably in an attempt to repair it)  ultimately lead to disease progression mediated by inflammatory cell infiltration and fibrosis Gross et al, Matrix Biol 2010

14. Additional role of DDR1 Facilitates macrophage/leukocyte adhesion and migration o DDR1 antagonism limits T-cell migration through a collagen meshwork Upregulation of proinflammatory cytokines & chemokines by macrophages DDR1 null mice  Protected against hypertensive renal injury & tubulointerstitial fibrosis of unilateral ureteral obstruction  macrophage receptor CCR2 is downregulated & macrophages have diminished migration ability  TNF-  and TGF-  1 are reduced in the kidneys Flamant et al, J Am Soc Nephrol 2006 Guerrot et al, Am J Pathol 2011 Hachehouche et al, Mol Immunol 2010

15. DDR1 inhibition may be important on a number of levels with respect to AS by maintaining GBM and slit diaphragm integrity, decreasing mesangial cell proliferation/adhesion & decreasing periglomerular & tubulointerstitial fibrosis

16. Wild Type MiceDDR +/+ & COL4A3 -/-DDR-/- & COL4A3 -/-

17. Gross et al, Matrix Biol 2010

18. Stem cells Pleniceanu et al, Stem Cells 2010

20. Stem cell therapy The potential to offer a curative treatment? Bone marrow derived murine mesenchymal stromal cells (MSC) injected into COL4A3 null mice have improved interstitial fibrosis However MSC have not been shown to differentiate into renal cells Ninichuk et al, Kidney Int 2006 Floege et al, Nephrol Dial Transplant 2006

21. Amniotic fluid stem cells Amniotic fluid stem cells injected into AS mice have a similar beneficial effect on disease progression by  preserving podocyte numbers  attenuating macrophage invasion  decreasing fibrosis Through a reduction in various mediators (TNF- , Il-6, RANTES and CCL2) Sedrakyan et al, J Am Soc Nephrol 2012

22. Bone marrow therapy 4 key studies examined the role of allogenic BMT from WT mice into the mouse model of AS  reduction in proteinuria  improved renal function  less tubulointerstitial fibrosis  improved ultrastructural glomerular architecture BM cells incorporate into the glomeruli & differentiate into podocytes capable of expressing & producing normal  3(IV) and  5(IV) collagen protomers  some normal collagen hexamer formation within the GBM

23. Sugimoto et al, Proc Natl Acad Sci USA 2006

24. BM-derived cells detected in all recipients by the presence of a Y chromosome in cell nuclei Fluorescence microscopy revealed occasional Y-positive cells with the characteristic morphology and location of podocytes in glomeruli of Col4A3 – /– mice given+ /+ BM BM-derived cells contribute to podocyte regeneration in AS mice

25. Irradiation prolongs survival of AS mice Katayama et al, J Am Soc Nephrol 2008

26. Type IV collagen expression and restored GBM architecture are shown in Col4A3 knockout mice that received blood transfusions at 8 wk LeBleu et al, J Am Soc Nephrol 2009

27. Still a long way off Cell engraftment is relatively small at about 10-13%, & much greater levels would be necessary to exert a cure Negating the need for radiation as a preconditioning tool as in the study of Le Bleu et al, is of relevance to the future potential of BMT for humans with AS