Alport Syndrome – Summary of Current Clinical and Basic Science Research

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

2. Intravascular Space Biomechanical Strain Altered podocyte actin cytoskeleton & cell adhesion Proteolysis of embryonic collagen IV by MMP Splitting of GBM Nuclear PPAR downregulation & podocyte apoptosis GBM with embryonic (1.2.2) collagen IV } C3 Pathological proteinuria Podocyte DDR1 & Integrinα2β1 detect abnormal collagen IV in the GBM Albumin Atypical GBM laminin isoforms (1 and 5) Transferrin Urinary Space

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 PTECapoptosis • Other plasma proteins such as transferrin, immunoglobulin and complement proteins

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

5. Pathomechanism Targeted Therapies • 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 Inhibition Disease Initiation RAAS Blockade Aldosterone Inhibitors Aliskiren Calcineurin Inhibitors Endothelin Receptor Blockers HMG CoA Reductase Inhibitors Sulodexide PPAR agonists Vasopeptidase inhibitors Matrix metalloproteinases 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 Bone Morphogenetic Protein-7 Chemokine Receptor Antagonists Complement Inhibition Sulodexide Matrix Metalloproteinases TNF alpha blockade and Pentoxifylline Vitamin D DDR1 antagonism

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

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 Nephrol1999

9. C6 deficient rat • Inherently unable to generate MAC/C5b-9 • Completely protected from tubulointerstitialinjury  intraluminal formation of the MAC/C5b-9 an essential mediator of tubulointerstitial disease Nangaku et al, J Am Soc Nephrol2002

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 Immunol2005

11. Crosstalk between the abnormal collagen IV and the podocytevia 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 Biol2010

13. DDR1 • Podocyte detects altered collagen protomers via DDR1receptors • 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 Biol2010

14. Additional role of DDR1 • Facilitates macrophage/leukocyte adhesion and migration • 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 & tubulointerstitialfibrosis of unilateral ureteral obstruction • macrophage receptor CCR2 is downregulated & macrophages have diminished migration ability • TNF- and TGF-1 are reduced in the kidneys Flamantet al, J Am Soc Nephrol 2006 Guerrotet al, Am J Pathol 2011 Hachehoucheet al, Mol Immunol2010

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 & tubulointerstitialfibrosis

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

17. Gross et al, Matrix Biol2010

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 Ninichuket al, Kidney Int 2006 Floegeet 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) Sedrakyanet 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 tubulointerstitialfibrosis • improved ultrastructural glomerulararchitecture • 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 NatlAcadSci USA 2006

24. BM-derived cells contribute to podocyte regeneration in AS mice • 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

25. Irradiation prolongs survival of AS mice Katayama et al, J Am Soc Nephrol2008

26. Type IV collagen expression and restored GBM architecture are shown in Col4A3 knockout mice that received blood transfusions at 8 wk LeBleuet al, J Am Soc Nephrol2009

27. Still a long way off • Cell engraftmentis 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