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
Intravascular Space
Proteolysis of
embryonic
collagen IV by
MMP
Biomechanical
Strain
Altered podocyte
actin
cytoskeleton &
cell adhesion
Splitting of
GBM
Nuclear PPAR
downregulation
& podocyte
apoptosis
GBM with embryonic
(1.2.2) collagen IV
Atypical GBM
laminin
isoforms (1 and 5)
Podocyte DDR1 & Integrin
α2β1 detect abnormal
collagen IV in the GBM
Urinary Space
C3
Albumin
Transferrin
}
Pathological
proteinuria
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
PTEC
Tubulointerstitium
Albumin
Transferrin
Megalin-Cubulin
Endocytosis
Ig
C3a
C3
Profibrotic Mediators
•TGFβ
•Collagen I & IV
•Fibronectin
•CTGF
Inflammatory Mediators
C3
C3
convertase
AP
Gene
Transcription
•Cytokines
•TNFα
Chemokines
C5b9
C3a
C3a Receptor
•MCP-1
•RANTES
Pathomechanism
Targeted Therapies
Glomerulopathy of Alport Syndrome
Disease Initiation
•
1. RAAS Blockade
2. Aldosterone Inhibitors
3. Aliskiren
4. Calcineurin Inhibitors
Inhibition
5.
Endothelin Receptor Blockers
6. HMG CoA Reductase Inhibitors
7. Sulodexide
8. PPAR agonists
9. Vasopeptidase inhibitors
10. Matrix metalloproteinases
11. DDR1 antagonism
•
•
•
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
PTEC and Tubulointerstitial Pathology
Disease Transmission & Progression
•
•
1.
2.
3.
4.
5.
6.
•
•
•
•
•
Pathological proteinuria
Albumin endocytosis via megalincubulin
Activation of complement cascade
on PTEC
Signaling, gene transcription of
cytokines, profibrotic mediators &
chemokines
PTEC apoptosis
EMT
Tubulointerstitial fibrosis
Bone Morphogenetic Protein-7
Chemokine Receptor Antagonists
Complement Inhibition
Sulodexide
Matrix Metalloproteinases
TNF alpha blockade and
Pentoxifylline
7. Vitamin D
8. DDR1 antagonism
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
PTEC
Tubulointerstitium
Albumin
Transferrin
Megalin-Cubulin
Endocytosis
Ig
C3a
C3
Profibrotic Mediators
•TGFβ
•Collagen I & IV
•Fibronectin
•CTGF
Inflammatory Mediators
C3
C3
convertase
AP
Gene
Transcription
•Cytokines
•TNFα
Chemokines
C5b9
C3a
C3a Receptor
•MCP-1
•RANTES
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
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 2008
Saran 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 (& 21 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 2006
Guerrot 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 2006
Floege 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
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
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