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Nephrol Dial Transplant (2014) 29: iv124–iv130
doi: 10.1093/ndt/gfu028
Full Review
Alport syndrome from bench to bedside: the potential of
current treatment beyond RAAS blockade and the
horizon of future therapies
Oliver Gross1,*, Laura Perin2,*and Constantinos Deltas3,*
1
Clinic of Nephrology and Rheumatology, University Medicine Goettingen, Goettingen, Germany,
2
Saban Research Institute, Children’s
Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA and
3
Molecular Medicine Research Center and Laboratory
of Molecular and Medical Genetics, Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
Correspondence and offprint requests to: Oliver Gross; E-mail: gross.oliver@med.uni-goettingen.de
*
All authors contributed equally to this work.
ABSTRACT
The hereditary type IV collagen disease Alport syndrome (AS)
always leads to end-stage renal failure. Yesterday, for the past
90 years, this course was described as ‘inevitable’. Today,
RAAS blockade has changed the ‘inevitable’course to a treata-
ble disease. Tomorrow, researchers hope to erase the ‘always’
from ‘always leads to renal failure’in the textbooks. This
review elucidates therapeutic targets that evolve from research:
(i) kidney embryogenesis and pathogenesis; (ii) phenotype-
genotype correlation and the role of collagen receptors and
podocytes; (iii) the malfunctioning Alport-GBM; (iv) tubu-
lointerstitial fibrosis; (v) the role of proteinuria in pathogenesis
and prognosis; and (vi) secondary events such as infections,
hyperparathyroidism and hypercholesterolaemia. Therefore,
moderate lifestyle, therapy of bacterial infections, Paricalcitol
in adult patients with hyperparathyroidism and HMG-CoA-
reductase inhibitors in adult patients with dyslipoproteinemia
might contribute to a slower progression of AS and less cardio-
vascular events. In the future, upcoming treatments including
stem cells, chaperon therapy, collagen receptor blockade and
anti-microRNA therapy will expand our perspective in pro-
tecting the kidneys of Alport patients from further damage.
This perspective on current and future therapies is naturally
limited by our personal focus in research, but aims to motivate
young scientists and clinicians to find a multimodal cure
for AS.
Keywords: Alport syndrome, chaperon therapy, discoidin
domain receptor 1, kidney fibrosis, microRNA-21
ESSENTIALS FOR FUTURE THERAPIES:
PATHOGENESIS OF ALPORT SYNDROME
AND KIDNEY EMBRYOGENESIS
Alport syndrome (AS) is a hereditary type IV collagen disease,
which always leads to progressive renal fibrosis and end-stage
renal failure [1]. Three different type IV collagen trimers are
deposited in basement membranes: α1/α1/α2, α3/α4/α5 and
α5/α5/α6 (IV) [2]. The mature glomerular basement mem-
brane (GBM) predominantly contains α3/α4/α5 type IV col-
lagen chains. Mutations in the type IV collagen genes
COL4A3/4/5, which encode the α3/α4/α5 chains, cause AS.
These mutations interfere with the correct assembly of the α3/
α4/α5 (IV) collagen network in the GBM and hinder the de-
velopmental switch from the embryonic α1/α1/α2(IV)
network to the mature α3/α4/α5 (IV) network, causing the
persistence of an immature GBM [3,4]. Consequently, a
thickening and splitting of the GBM in AS causes progressive
renal fibrosis leading to end-stage renal failure [5]. Maturation
of the GBM develops in the neonatal age; therefore, a child
with AS is not born with an abnormal GBM (but develops
AS during maturation of the GBM)—leaving a therapeutic
© The Author 2014. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
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window for very early therapy. In fact, the defective Alport-
GBM can be restored in mice [6].
GENOTYPE-PHENOTYPE-CORRELATION
POINTS TO THE ACHILLES’HEEL IN
ALPORT PATHOGENESIS: PODOCYTES AND
THEIR COLLAGEN RECEPTORS
The α3/α4/α5 type IV collagen chains are exclusively pro-
duced by podocytes [7]. The genotype-phenotype correlation
in AS [8] points to a role of the α3/α4/α5 (IV) producing and
sensing cells in AS pathogenesis: the podocytes sense their
GBM via collagen receptors such as α1β1 and α2β1 integrins
and discoidin domain receptor 1 (DDR1). All of these collagen
receptors have been shown to influence Alport pathogenesis
[5,9,10], rendering them as possible therapeutic targets [11].
THEROLEOFASPONGYALPORT-GBM
FOR THERAPY
The GBM in most AS patients consists of α1/α2 (IV) chains
only, making this altered GBM more porous [12] (resulting in
acanthocyte formation) and more susceptible to endopro-
teolysis [3]. The GBM in AS patients is thought to be more
vulnerable by increased (or even normal) filtration pressure.
Therefore, thickening and splitting of the GBM in AS in part
is a stress response of the podocytes. Any medication reducing
the mechanical stress on the podocyte, such as RAAS blockade,
reduces the risk of GBM ruptures and focal segmental glomer-
ulosclerosis, which is a common light microscopical glomeru-
lar feature in AS.
THEROLEOFTUBULOINTERSTITIAL
FIBROSIS IN THE COURSE AND
PROGNOSIS OF AS
AS is a glomerular disease; however, tubulointerstitial fibrosis is
the key feature in progressive renal damage leading to end-stage
renal failure. For example, RAAS blockade is not able to hinder
thickening and splitting of the GBM in AS, but markedly delays
tubulointerstitial fibrosis [13]. The glomerular disease and the
podocyte stress response lead to distribution and secretion of
profibrotic chemokines and cytokines in the primary urine that
are re-absorbed by the tubular cells. The re-absorbed profibrotic
chemokines lead to tubular scar tissue formation that finally de-
molishes the kidney. Therefore, until now, the amount of tubu-
lointerstitial fibrosis is the most accurate histological prognostic
factor regarding the evaluation of kidney function.
THEROLEOFPROTEINURIAIN
PATHOGENESIS AND PROGNOSIS OF AS
Proteinuria reflects ongoing glomerular inflammatory damage
in all autoimmune diseases such as lupus nephritis and most
types of glomerulonephritis. Therefore, the amount of
proteinuria serves as an important prognostic factor in order
to evaluate response to therapy. In contrast, the amount of
proteinuria is a poor prognostic marker in AS, because it does
not automatically correlate with inflammation and scar tissue
formation. For example, loss of 5 g protein per day (without
TGFβand CTGF) can have a better prognosis than ‘only’1g
per day with relatively high levels of profibrotic chemokines.
We think that progression from haematuria to microalbumi-
nuria and from microalbuminuria to overt proteinuria rep-
resent very important steps in the course of Alport disease.
However, once the patient has reached the level of ‘overt pro-
teinuria’, increasing amounts of proteinuria do not necessarily
result in a worse prognosis or disease progression (unpub-
lished data from the European Alport registry [13]). In con-
trast, there is increasing evidence that the quality and quantity
of proinflammatory and profibrotic proteins in the urine de-
termines the course of AS [14].
THE UNDERESTIMATED ‘COLLATERAL
DAMAGES’: RECURRENT INFECTIONS,
HYPERPARATHYROIDISM,
CARDIOVASCULAR DISEASE AND
HYPERCHOLESTEROLAEMIA
In mice with AS, bacterial CpG-DNA accelerates glomerulo-
sclerosis by inducing a M1 proinflammatory macrophage
phenotype and podocyte loss [15]. Vice versa, patients with
ongoing renal failure are immuno-compromised and are more
likely to get recurrent bacterial infections. Therefore, these
bacterial infections should be treated rigorously and good
dental health might contribute to a slower progression of AS.
Most patients with progressive renal failure develop secondary
hyperparathyroidism due to their impaired calcium-phosphate-
vitamin D balance. In mice with AS, the vitamin D receptor
activator Paricalcitol (but not Calcitriol) showed a synergistic
nephroprotective effect on top of early ACE inhibition [16].
Paricalcitol is board-approved for therapy of secondary hy-
perthyroidism. Therefore, in adult patients with AS and incipi-
ent hyperparathyroidism, Paricalcitol might be an additional
treatment option to delay renal failure.
Young patients with chronic renal disease have a 1000-fold
higher risk of cardiovascular effects compared with healthy sub-
jects. Proteinuria in a nephrotic range causes hypercholestero-
laemia. In mice with AS, the HMG-CoA-inhibitor cerivastatin
(statin) prolonged the lifespan until renal failure and delayed
uraemia [17]. These effects were associated with decreased
renal fibrosis and a reduction of inflammatory cell infiltration.
Statins are board-approved for therapy of dyslipoproteinemia.
Therefore, in adult patients with AS and incipient hypercholes-
terolaemia, statins might be an additional treatment option to
delay renal failure and prevent cardiovascular events.
Figure 1summarizes the key features of possible therapies
on top of RAAS blockade. All medications in Figure 1are
board-approved for other medical conditions, but all possible
therapies are off-label in AS and are very likely to stay off-label
in the future. Medications in Figure 1do not represent an
expert recommendation for therapy of AS, but summarizes
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medical therapies currently used (or could be used) in adult
patients with AS.
FUTURE THERAPIES: COLLAGEN IV
PATHOLOGY AND IDEAS FOR NEW
THERAPEUTIC APPROACHES
Collagens are large triple-helical proteins participating in a
variety of processes in connective tissues, including tissue scaf-
folding, cell adhesion and tissue repair, among others. Struc-
turally, there are two major categories, the fibrillar and the
non-fibrillar collagens. Prime examples of the first are the type
I, II, III and V collagens that exert a fundamental gluing role
in extracellular matrix, holding cells together; a prime example
of the latter is type IV collagen, the most abundant component
of all basement membranes (BM), with a crucial role in the
kidney glomerular filtration barrier [18]. A critical character-
istic relating to this review is the participation of fibrillar col-
lagens in higher order structures, through multiple nucleation
events, a process that in case of mutations is susceptible to
strong dominant negative effects. This is exemplified in dis-
eases such as osteogenesis imperfecta where heterozygous
mutations in type I collagen [(α1)
2
,α2)] interfere with triple-
helix formation, affecting 75% of molecules and delaying
protein secretion. These defective helices interfere next with
proper nucleation and fibril formation in bones and other con-
nective tissues [19,20]. One can anticipate that the more de-
fective molecules are secreted, the worse the phenotype is
going to be. Actually, it was shown that the phenotype was
milder if a premature termination of translation prevented
chain association and triple-helix formation, which in turn
prevented secretion of defective molecules.
To the contrary, type IV collagen of BM participates in
network formation which is not the result of molecular nu-
cleation events. Certainly there are interactions with other BM
macromolecules such as laminin, nidogen and proteoglycans
but there is no extensive nucleation and therefore less drastic
dominant-negative effects are expected. For some type IV col-
lagen mutations we hypothesize that should it be possible to
have more molecules secreted in the extracellular matrix and
participate in the meshwork, the phenotype might be milder
compared with no secretion at all. About 50% of AS patients
inherit missense or small in-frame mutations in the X-linked
COL4A5 gene, where mature protomers are either secreted
normally or abnormally, based on immunostaining. Absent or
weak staining is due to poor secretion, most probably because
quality control in the ER recognizes and degrades the mis-
folded chains [21]. This is accomplished through the unfolded
protein response (UPR) pathway which is activated by the ER
stress. UPR activation aims at restoring homeostasis by pro-
moting proper protein folding using chaperones. When this
fails for various reasons, the unfolded or misfolded molecules
are removed by degradation. Prolonged ER stress may lead to
protein translation pause or even to podocyte apoptosis or
death through other mechanisms and foot processes efface-
ment. Mechanisms aimed at enhancing the intracellular cha-
perone machinery or externally administered small chemicals
that mimic chaperones could promote triple-helix formation
and consequently allow hypomorphic mutants to exert their
function, even though not perfectly, once found in the BM.
This has been shown in numerous other occasions, such as
cystic fibrosis and nephrogenic diabetes insipidus [22,23].
A most recent example relating to BM pathology was
the transgenic expression of the mutant rat C321R-LAMB2
gene in Lamb2
-/–
mice, a model that recapitulates Pierson
FIGURE 1: Medical therapies currently used (or could be used) in adult patients with AS and risk factors that might negatively contribute to
progression of disease. Medications do not necessarily represent an expert recommendation for therapy of AS.
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syndrome. This arrangement attenuated the severe proteinur-
ia, while it caused ER stress, noting that mutant protein was
better than no protein. Also, the same authors showed that the
use of chemical chaperone taurodeoxycholic acid in cells facili-
tated protein folding and trafficking and greatly increased
secretion of the mutant LAMB2 [24]. Previously, Ohashi et al.
had shown that mutant podocin due to NPHS2 mutation p.
R138Q could not reach its destination in plasma membrane of
cultured cells due to retention in the ER. Incubation with
chemical chaperones caused folding of the mutant podocin
and redistribution to the plasma membrane [25]. With regard
to collagens, Murray et al. showed recently that treatment with
the chemical chaperone 4-phenyl butyric acid reduced intra-
cellular accumulation of mutant collagen IV in cultured
patient primary cells. This ameliorated the cellular phenotype
of a COL4A2 mutation that caused haemorrhagic stroke [26].
It is tempting to hypothesize that once found in its correct des-
tination the mutant protein will function properly or near
properly, depending on the exact nature of the mutation. Hy-
pomorphic and milder mutations that interfere with molecule
folding may be selected against during a very strict quality
control early on in the ER; however, it is reasonable to hypoth-
esize that once folded with the accessory contribution of exter-
nal chaperones, these molecules may reach their destination
and function sub-optimally but adequately and prevent severe
disease progression.
Recently we created a new AS knockin mouse model, carry-
ing missense mutation COL4A3-G1332E that demonstrated
UPR activation in glomeruli. Hopefully, this will serve as a tool
for testing a variety of available chemical chaperones and
other alternative therapeutic approaches for alleviating or
halting disease progression [27].
FUTURE THERAPIES: STEM CELL-BASED
THERAPIES FOR AS
Stem cells and renal progenitors might offer a possible novel
treatment of AS. Being able to deliver to the affected glomeruli
a cell that could potentially become a mature podocyte, and
produce new functional GBM, could be considered the ‘Holy
Grail’in the treatment of AS. Even if different groups [28–30]
have claimed podocyte differentiation from stem cells in
Alport mice, these publications still need validation and do
not convincingly demonstrate that podocyte differentiation and
consequent restoration of the GBM really occurs. In fact, the
concept that direct differentiation of stem cells into organ-
specific mature cell types occurs and can rescue the progression
of disease has almost been abandoned; stem cell integration
and differentiation is a very rare event that cannot sustain the
complete regeneration of kidney [31] or other organs.
In particular, our group has demonstrated [32] that differ-
entiation of injected stem cells (amniotic fluid stem cells) into
podocytes does not occur in vivo in an animal model of AS,
despite significant protection of glomerular structure and
function.The main mechanism of action of stem cells appears
to be via paracrine activity ( possibly affecting the TGFβaxis)
leading to attenuation of fibrosis and chronic inflammation
[32–34], in addition to stimulating ingress of ‘healing’type II
macrophages (M2) [34]. In addition, stem cells seem to induce
blockade of the angiotensin II pathway that prevents further
damage to the glomeruli and favours preservation of podocyte
number. Thus, based on studies published so far, cell-based
therapies have the potential to slow but not prevent renal injury
in AS. Despite these encouraging results and increases in the
lifespan of treated mice, the restoration of a functional GBM is
still the elusive but requisite goal to actually cure AS [6].
In continuing efforts to find a cell source that produces the
proper GBM, we and others have been working to obtain ‘new
podocytes’including in the form of nephron progenitors that
can be induced to become podocytes. Several studies con-
ducted on the differentiation of embryonic stem cells (ESC)
and adult stem cells into intermediate mesoderm or cap me-
senchyme (embryological tissues from which the kidney
originates) have demonstrated the possibility of inducing com-
mitted cell differentiation in renal lineages [6,35–38]. Other
groups [39] have demonstrated the possibility of expanding
nephron progenitors using induced pluripotent stem cell (IPS)
technology, while Song et al. [40] have a newly developed IPS
line of cells that ‘resemble’in vivo podocytes. However, gener-
ation of differentiated renal structures from IPS or ESC has
very low efficiency and is probably insufficient for strategies to
directly translate these cells into potential therapeutic agents
[41]. We have recently reported the isolation of a subpopu-
lation within the human amniotic fluid that possess character-
istics of nephron progenitors and that can be differentiated
into podocytes expressing the collagen IV trimer [42].
With the challenges of fibrosis, chronic inflammation and
podocytes incapable of laying down the proper type IV ‘col-
lagen network’, a cell-based approach would ideally be able to
act simultaneously on all these aspects in order to offer a
robust treatment for AS. In this regard, different cell-based
strategies might be combined together. Stem cells, being more
immune-privileged than adult renal cells, can be systemically
infused and act in reducing fibrosis and inflammation, while
direct replenishment of new non-defective podocytes derived
from nephron progenitors might be the solution to replace the
failing membrane (Figure 2). Nevertheless, one of the biggest
challenges will be getting the cells across the GBM. An impor-
tant feature of the novel podocyte progenitor cells mentioned
above is their potential use in the development of small mol-
ecule-based therapies [6,43] aimed at salvaging disturbed type
IV collagen production. Thus, although we are still far from
being able to harness novel cell sources like stem or progenitor
cells to support new therapeutic approaches, several promising
avenues seem possible.
FUTURE THERAPIES: COLLAGEN IV
RECEPTOR BLOCKADE IN AS
In general, the GBM structure is maintained by an equilibrium
of synthesis and degradation. In Alport pathogenesis, in-
creased synthesis of defective α3/4/5 type IV collagen, imma-
ture α1/α1/α2 type IV collagen and other basement
membrane components in the GBM results in excessive
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accumulation of matrix proteins [5]. Ablating the function of
collagen receptors such as Discoidin domain receptor 1
(DDR1), integrin α1β1orα2β1 might hinder or slow down
podocytes’matrix accumulation in AS [9,10]. For example,
loss of DDR1 reduced proinflammatory, profibrotic cells via
signalling of TGFβ, CTGF, NFκB and IL-6 and decreased
deposition of extracellular matrix. Immunogold staining and
in situ hybridization identified podocytes as key players in
DDR1-mediated fibrosis and inflammation [10].
This supports our hypothesis that podocyte–matrix inter-
action via collagen receptors plays an important part in pro-
gression of fibrosis. We postulate that specific receptor
blockers such as DDR1 blockers might become a new thera-
peutic option in patients with AS in the near future.
FUTURE THERAPIES: ANTI-MICRORNA
THERAPY IN AS
MicroRNA-21 (miR-21) has been shown to play a distinct role
in the progression of kidney scarring in different animal
models and humans [44]. Mice treated with anti-miR-21 oli-
gonucleotides suffered far less interstitial fibrosis in response
to kidney injury. Analysis of gene expression profiles identified
groups of genes involved in metabolic pathways, including the
lipid metabolism pathway regulated by peroxisome prolifera-
tor-activated receptor-α(Pparα), a direct miR-21 target [44].
Further, miR-21 Sponge inhibited TGFβ-stimulated phos-
phorylation of Akt kinase, resulting in attenuation of phos-
phorylation of different Akt substrates that regulate mesangial
cell hypertrophy [45]. Additionally, inhibition of miR-21
reduced TGFβ-stimulated fibronectin and collagen expression
[45]. TGFβis a very well-known key player in Alport patho-
genesis and progressive kidney fibrosis in AS [13].
These studies demonstrated that miR-21 contributes to fi-
brogenesis and is a promising candidate target for antifibrotic
therapy in AS. We postulate that anti-miR-21 compounds
might become a new therapeutic option in patients with AS in
the near future.
Additional potential future targets of nephroprotective
therapy in AS such as the complement system, chemokine re-
ceptor blockade, TNFα-blockade and inhibition of matrix-
metallo-proteinases are summarized in a recent review [11].
PERSPECTIVE CURRENT AND FUTURE
THERAPY
Yesterday, in the textbooks the course of AS was described as
‘inevitable’for the past 90 years after the first description by
Alport [46].
Today, RAAS blockade has changed the ‘inevitable’course
of AS to a treatable disease [47,48] and led to treatment rec-
ommendations [49,50]. Our review summarized additional
therapies that are currently used in humans and that might
influence the course of AS (Figure 1). There is preliminary
scientific evidence for effectiveness in animal models.
However, the long-term effects of these therapies still need to
be evaluated in humans with AS. These therapies might well
be additive to RAAS blockade and might further delay renal
failure by years. Some medications are already board-approved
for other indications; therefore, international Alport registries
hopefully will generate the evidence for patients with AS.
Tomorrow, our review draws a bright horizon of several
promising new therapies, all with different targets additive to
existing therapies. This revives hope that AS has not only
become a treatable disease, but end-stage renal failure can be
prevented in most patients by multimodal therapy.
FIGURE 2: Schematic representation of possible mechanisms of injury repair in AS by stem cells and progenitors.
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ACKNOWLEDGEMENTS
This article summarizes the presentations on future therapies
of Alport syndrome held at the 2014 International workshop
on Alport Syndrome, 3–5 January 2014, Said Business School,
Oxford, UK. The research activities of CD are supported by a
grant co-funded by the European Regional Development Fund
and the Republic of Cyprus through the Research Promotion
Foundation (Strategic Infrastructure Project NEW INFRA-
STRUCTURE/STRATEGIC/ 0308/24). L.P. was supported by
a grant of the Alport Syndrome Foundation and the Baxter
foundation. The presented research of O.G. is supported by
the Association pour l`Information et la Recherche sur les ma-
ladies rénale Génétiques (AIRG) France, the KfH-Foundation
Preventive Medicine, AbbVie GmbH & Co. KG Germany, the
German Kidney Foundation, the Deutsche Forschungsge-
meinschaft GR 1852/4–1 and 4–2, the European Renal Associ-
ation ERA/EDTA, the Kidney Foundation of Canada and the
Alport Syndrome Foundation.
CONFLICT OF INTEREST STATEMENT
None declared.
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Received for publication: 26.12.2013; Accepted in revised form: 17.1.2014
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