Alport Syndrome –Summary of Current Clinical and

Basic Science Research

Christoph LichtThe Hospital for Sick Children, Toronto, ONAlport Syndrome Family Conference 2012

Minneapolis, MN - 21.-22.7.2012

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

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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

Tubular Lumen

C3

C3 convertase

Profibrotic Mediators•TGFβ•Collagen I & IV•Fibronectin•CTGF

Inflammatory Mediators•Cytokines•TNFα

Chemokines•MCP-1•RANTES

C5b9

AP

Albumin

C3

C3a Receptor

TransferrinMegalin-CubulinEndocytosis

C3a

Ig

PTEC Tubulointerstitium

GeneTranscription

C3a

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 Blockade2. Aldosterone Inhibitors3. Aliskiren4. Calcineurin Inhibitors 5. Endothelin Receptor Blockers6. HMG CoA Reductase Inhibitors7. Sulodexide8. PPAR agonists9. Vasopeptidase inhibitors10.Matrix metalloproteinases11. 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 Antagonists3. Complement Inhibition4. Sulodexide5. Matrix Metalloproteinases6. TNF alpha blockade and

Pentoxifylline7. Vitamin D8. DDR1 antagonism

Pathomechanism Targeted Therapies

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

Tubular Lumen

C3

C3 convertase

Profibrotic Mediators•TGFβ•Collagen I & IV•Fibronectin•CTGF

Inflammatory Mediators•Cytokines•TNFα

Chemokines•MCP-1•RANTES

C5b9

AP

Albumin

C3

C3a Receptor

TransferrinMegalin-CubulinEndocytosis

C3a

Ig

PTEC Tubulointerstitium

GeneTranscription

C3a

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 1999Ichida et al, Kidney Int 1994

Biancone et al, Kidney Int 1994Tang et al, J Am Soc Nephrol 1999

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

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 2008Saran et al, Kidney Int 2003

He et al, J Immunol 2005

Crosstalk between the abnormal collagen IV and the podocyte via the Discoidin Domain Receptor 1 (& 21 Integrin)

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

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

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 2006Guerrot et al, Am J Pathol 2011

Hachehouche et al, Mol Immunol 2010

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

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

Gross et al, Matrix Biol 2010

Stem cells

Pleniceanu et al, Stem Cells 2010

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 2006Floege et al, Nephrol Dial Transplant 2006

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

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

Sugimoto et al, Proc Natl Acad Sci USA 2006

• 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

Irradiation prolongs survival of AS mice

Katayama et al, J Am Soc Nephrol 2008

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

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