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1ß1 and Transforming Growth Factor-ß1 Play Distinct Roles in Alport Glomerular Pathogenesis and Serve as Dual Targets for Metabolic Therapy
From the Department of Genetics,*
Boys Town National
Research Hospital, Omaha, Nebraska; the Biogen
Corporation,
Cambridge, Massachusetts; the
Nephrology Division,
Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts; and the
Scripps Institute,§
La Jolla, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1ß1, affect Alport glomerular pathogenesis in
distinct ways. In Alport mice that are also null for integrin 1
expression, expansion of the mesangial matrix and podocyte foot
process effacement are attenuated. The novel observation of nonnative
laminin isoforms (laminin-2 and/or laminin-4) accumulating in the
glomerular basement membrane of Alport mice is markedly reduced in the
double knockouts. The second pathway, mediated by
TGF-ß1, was blocked using a soluble fusion protein comprising
the extracellular domain of the TGF-ß1 type II receptor. This
inhibitor prevents focal thickening of the glomerular basement
membrane, but does not prevent effacement of the podocyte foot
processes. If both integrin 1ß1 and TGF-ß1 pathways are
functionally inhibited, glomerular foot process and glomerular
basement membrane morphology are primarily restored and renal function
is markedly improved. These data suggest that integrin 1ß1 and
TGF-ß1 may provide useful targets for a dual therapy aimed at slowing
disease progression in Alport glomerulonephritis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5(IV) gene,3
accounting for 
80% of the
cases. Mutations in the collagen 3(IV) or 4(IV) genes lead to the
recessive forms of the disease.4,5
The absence of any one
of these type IV collagen chains can result in the absence of all three
chains in the glomerular basement membrane (GBM), presumably due to an
obligatory association of the three chains in forming the type IV
collagen superstructure.6,7
Normal distribution of the
three chains is observed in approximately one third of
patients.8
The adult GBM contains a thin
subendothelial network of collagen 1(IV) and 2(IV) chains, and a
thick subepithelial network of collagen 3(IV), 4(IV), and
5(IV) chains.9
These networks are thought to be
physically separate from one another.10,11
In Alport
syndrome the entire width of the GBM is comprised of collagen 1(IV)
and 2(IV) chains, which is the normal collagen composition of the
embryonic GBM.12,13
These changes result in progressive
loss of glomerular function because of alterations in the GBM, podocyte
effacement, and mesangial matrix expansion.
Type IV collagen networks comprised of only 1(IV) and 2(IV)
chains are more susceptible to endoproteolysis than GBM containing all
five type IV collagen chains,13
which is likely because of
the greater number of crosslinks formed in a network of collagen
3(IV), 4(IV), and 5(IV) chains.11
Based on
these observations, it has been proposed that the irregular
ultrastructure of Alport GBM might be attributed to focal
endoproteolysis of the GBM.
Two independently produced gene knockout murine models for Alport syndrome have been described,14,15 as well as one resulting from a random transgene insertion event.16 These models have proven to have progressive renal disease that is remarkably similar to that in humans.
Expansion of the mesangial matrix occurs early in Alport renal
pathogenesis. The most abundant integrin on mesangial cells is the
1ß1 heterodimer.17,18
An 1 integrin knockout has
been produced that shows no renal abnormalities and no phenotype
detrimental to the survival of the animal.19
Considering
the recently described roles for 1ß1 integrin in
collagen-dependent cell proliferation, cell adhesion, mesangial matrix
remodeling, and mesangial cell migration,19-21
we
suspected that integrin 1ß1 might play a specific role in Alport
renal disease progression. To test this notion, we produced a mouse
null at both the collagen 3(IV) gene (Alport mouse) and the 1
integrin gene. These double-knockout mice have delayed onset and slowed
progression of glomerular disease, attenuated expansion of the
mesangial matrix, and markedly improved foot process architecture,
illustrating a major role for 1ß1 integrin in Alport glomerular
disease progression.
Transforming growth factor (TGF)-ß has been shown to promote accumulation of extracellular matrix in both wound repair and fibrotic diseases, including glomerulonephritis.22 In recent studies, we demonstrated a likely role for TGF-ß1 in Alport glomerular and tubulointerstitial disease.23 Herein, we extend these earlier studies by illustrating that inhibition of TGF-ß1, by injecting a type II TGF-ß soluble receptor as a competitive inhibitor, prevents the irregular thickening of the GBM.
Treating the double knockouts with the TGF-ß1 soluble receptor
provides synergistic benefits, restoring podocyte foot process
architecture, inhibiting matrix deposition in the GBM, and slowing
mesangial matrix expansion. Based on this new evidence, we conclude
that renal pathogenesis in Alport syndrome involves biochemical
pathways modulated by TGF-ß1 and integrin 1ß1, and that the two
pathways affect distinct aspects of glomerular pathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The collagen 3(IV) knockout mice were described
previously.14
These mice, which were originally in the 129
Sv/J background, were successively back-crossed 10 times with 129 Sv
mice. Heterozygotes were then crossed with homozygote integrin 1
knockouts, also described previously,19
which were on a
pure 129 Sv background. A breeding stock of mice heterozygous for the
collagen 3(IV) mutation and homozygous for the integrin 1
mutation was established. No difference in the kinetics of renal
pathogenesis was observed for Alport mice on the 129 Sv/J
versus 129 Sv backgrounds.
Urinary Protein Analysis
Semiquantitative measurements of urinary protein were performed using Albustix reagent strips (Bayer Corporation, Elkhart, IN), and reading the relative amounts from the color chart provided with the kit. Successive readings were performed weekly on fresh urine from the same experimental animals. Microhematuria was assessed using Hemastix reagent strips (Bayer Corporation).
Fractions of the above urine samples were subjected to gel analysis. Samples (the equivalent of 0.5 µl of undiluted urine) were fractionated by electrophoresis through 10% denaturing acrylamide gels. Urine creatinine levels were measured when sufficient urine was collected for meaningful readings (>100 µl). We observed no significant effect of the TGF-ß1 inhibitor on urine creatinine levels, and these levels fluctuated by no more than 20% throughout the time course. The protein in the gels was stained with Coomassie blue and photographed. Bovine serum albumin was used as a molecular weight standard.
Determination of End-Stage Renal Disease
End-stage renal disease was defined as the point in the disease progression where a weight loss of >15% of the body mass was observed. Animals were euthanized at this point and end stage was confirmed by visual examination. Blood urea nitrogen levels at this stage were consistently >200 mg/dl. The kidneys are visibly smaller with a granular surface and are pale in color. When such visual characteristics were present, histological examination invariably confirmed advanced fibrosis.
Transmission Electron Microscopy
Fresh external renal cortex was minced in 4% paraformaldehyde, allowed to fix for 2 hours, and stored at 5°C in phosphate-buffered saline (PBS). The tissue was washed extensively (5 times for 10 minutes each at 4°C) with 0.1 mol/L Sorenson’s buffer (Sorenson’s buffer was made by combining 100 ml of a 200 mmol/L monobasic sodium phosphate with 400 ml of 200 mmol/L dibasic sodium phosphate, with 500 ml of water, and adjusted to pH 7.4), and postfixed in 1% osmium tetroxide in Sorenson’s buffer for 1 hour. The tissue was then dehydrated in graded ethanol (70%, then 80%, then 90%, then 100% for 10 minutes each), and finally in propylene oxide and embedded in Poly/Bed 812 epoxy resin (Polysciences, Inc., Warrington, PA) following the procedures described by the manufacturer. Glomeruli were identified in 1-µm sections stained with toluidine blue, and thin sections were cut at 70-nm thickness using a Reichert Jung Ultracut E ultramicrotome (Cambridge Instrument Co., Vienna, Austria). Sections were mounted onto grids and stained with uranyl acetate and lead citrate. Grid-mounted sections were examined and photographed using a Phillips CM10 electron microscope.
Scanning Electron Microscopy
Small pieces (approximately 2-mm cubes) of kidney cortex were fixed in 3% phosphate-buffered glutaraldehyde, then postfixed in 1% phosphate-buffered osmium tetroxide. Samples were then dehydrated in graded ethanols, and critical point-dried in carbon dioxide. The cubes were then cracked in pieces by stressing them with the edge of a razor blade, and mounted with glue onto stubs with the cracked surface facing upward. The surface was sputter-coated using gold/palladium and visualized with a scanning electron microscope.
Immunofluorescence Analysis
Fresh kidneys were removed and embedded in Tissue Tek OCT aqueous
compound. They were frozen at -150°C and sectioned at 4 µm on a
Microm cryostat. Slides were fixed in cold 100% acetone for 10 minutes
then air-dried overnight. Slides were washed three times in cold 1x
PBS. Primary antibodies were diluted together in 7% nonfat dry milk
(1:200 for entactin, 1:10 for laminin 2), applied to the sections,
and incubated overnight at 4°C in a humidified dish. Slides were then
washed successively for 5, 7, and 10 minutes with cold 1x PBS.
Secondary antibodies were diluted together at 1:00 (Texas red
-rabbit for lam-2 and fluorescein isothiocyanate -rat for
entactin) in the blocking agent and applied for 4 hours at 4°C. After
PBS washes, Vectashield (Vector Laboratories, App Imaging, Santa Clara,
CA) anti-fade mounting media was applied and sections were
coverslipped, sealed, and imaged. Images were collected using a
Cytovision Ultra image analysis system (Applied Imaging, Inc.)
interfaced with an Olympus BH-2 fluorescence microscope.
For the remaining laminin chain-specific antibodies, slides were
brought to room temperature, then postfixed in cold (-20°C) acetone
for 15 minutes, and air-dried overnight at 5°C. The specimens were
rehydrated by three successive washes in PBS for 10 minutes each at
room temperature, then denatured by immersing them in 0.1% sodium
dodecyl sulfate at 37°C for 45 minutes. This step greatly enhanced
immunoreactivity, presumably by exposing the masked laminin epitopes
(B. Patton, personal communication). The specificity of the laminin
chain-specific antibodies have been established in previous
publications.24,25
The anti-laminin 1 (8B3) and ß1
(5A2) antibodies were mouse monoclonals, and a gift from D. Abrahamson
(Department of Anatomy and Cell Biology, University of Kansas Medicine
Center, Kansas City, KS).26
Anti-laminin 2
chain-specific rabbit antibodies were a gift from Dr. Peter Yurchenco
(Robert Wood Johnson Medical School, Piscataway, New
Jersey).27
The anti-laminin 3 chain-specific antibodies
were a gift from Dr. Bob Burgeson (anti laminin-5 rabbit
antiserum No. 4101) and described previously.28,29
The
anti-laminin 4 (C0877) and 5 (8948) rabbit antisera were provided
by Jeff Miner (Washington University School of Medicine Department of
Nephrology, St Louis, MO).24
The anti-laminin ß2 (Gpb1)
chain-specific guinea pig antisera were a gift from Joshua Sanes
(Washington University School of Medicine Department of Anatomy and
Neurobiology, St. Louis, MO).31
Anti-laminin ß3
antibodies were provided by Yoshi Yamada (National Institute of Dental
and Craniofacial Research, Bethesda, MD).31
A rat
monoclonal antibody for the laminin 
1 chain was purchased from
Chemicon (Temecula, CA). All secondary reagents were purchased from
Vector Laboratories.
For fibronectin immunostaining, the antibody was purchased from Sigma
(St. Louis, MO; catalogue no. F3648). The same procedure was used as
used for the laminin 2 chain.
Construction, Purification, and Activity of the Murine TGFßR:Fc
The extracellular domain of the murine TGF-ß type II receptor was amplified from a murine lung cDNA library (Clontech, Palo Alto, CA) by PCR, and engineered to contain a 5' NotI, and a 3' SalI restriction site. The Fc region of murine IgG2a was amplified by PCR from a murine hybridoma, and engineered to contain a 5' SalI restriction site and a 3' NotI restriction site. The receptor and Fc fragments were purified, digested with the appropriate restriction enzymes, and ligated into the expression vector pEAG347, which contains tandem SV40, and adenovirus major late promoters; the SV40 late polyA termination signal; an ampicillin resistance gene; and a pSV2 dihydrofolate reductase-derived selection marker. The resulting construct (pAA002) was transformed into competent JM109, and plasmids were selected for the correct orientation of the receptor-Fc fusion gene. Proper sequence and alignment was confirmed by DNA sequencing.
pAA002 was transfected in Chinese hamster ovary cells (CHO DUKX-B1) by electroporation. After selection in 200 nmol/L methotrexate, single clones were selected and screened for the expression of mTGFßR:Fc. The clone with the highest titer was picked for expansion to 20 L fermentors, and the expressed protein was purified over protein A-Sepharose (Pharmacia, Piscataway, NJ), under sterile, endotoxin-free conditions. The protein is >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contains <1 U endotoxin per mg protein.
Activity of the mTGFßR:Fc was assessed in the mink lung epithelial cell assay as described.32-34 Briefly, Mv1Lu cells (ATCC CCL-64) were maintained in minimal essential medium supplemented with 100 U/ml penicillin, 100 Tg/ml streptomycin, 10% fetal bovine serum, and 4 mmol/L L-glutamine. To test, serial dilutions of TGFßR:Fc were incubated with 0.1 ng/ml TGF-ß1, 0.5 ng/ml TGF-ß2, and 0.05 ng/ml TGF-ß3 (R&D Systems, Minneapolis, MN) for 1 hour in assay medium (minimal essential medium supplemented with 100 U/ml of penicillin, 100 Tg/ml of streptomycin, and 10% fetal bovine serum) in a 96-well microtiter tissue culture plate. Mv1Lu cells were resuspended in assay medium and added to the assay plate at a concentration of 6,000 cells per well. The cells were incubated at 37°C for 48 hours and pulsed with [3H]thymidine (70 to 86 Ci/mmol; Amersham) for an additional 6 hours. DNA synthesis, which reflects cell proliferation, was determined by measuring incorporation of [3H]thymidine. As reported for similar TGF-ß receptor antagonists,32,33 mTGFßR:Fc blocks TGF-ß1 (IC50 = 1 nmol/L), and TGF-ß3 (IC50 = 1 nmol/L), but not TGF-ß2-mediated inhibition of Mv1Lu cell proliferation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1ß1 Integrin Slows Renal Disease Progression,
Attenuates Expansion of the Mesangial Matrix, and Rescues Podocyte Foot
Process Architecture in Alport Mice
The collagen 3(IV)-deficient mouse developed in this
laboratory14
was crossed with the integrin 1-deficient
mouse19
to produce an animal null at both alleles.
Comparative analysis of renal disease pathogenesis of Alport mice
versus these double-knockout mice revealed a markedly slower
rate of renal disease in the double knockouts. Both the time of onset
and the rate of progression of proteinuria was delayed relative to the
Alport mice (Figure 1A
and Table 1
), suggesting improved function of the
glomerular filter. The data in Table 1
represent successive analysis of
urinary albumin in three sets of experimental animals, and illustrate
that differences in onset and progression of proteinuria in the Alport
mouse versus the double knockout are highly reproducible.
Microhematuria was also assessed, and did not vary significantly among
the experimental groups. The mean age of end-stage renal failure (as
defined in the Methods section), based on analysis of 10
experimental sets (10 controls and 10 Alport, 10 integrin 1 null
mice, and 10 double-knockout animals) was extended from 8.5 ± 0.5
weeks in the Alport mice to 14.5 ± 0.9 weeks in the double
knockouts.
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with D), thus the absence of 1 integrin appears to have provided a
protective effect on GBM damage in the Alport mouse model. The
morphology of the podocyte foot processes was normal in the double
knockout as compared with the Alport mouse both by transmission
electron microscopy (Figure 2
; A, D, and G) and scanning electron
microscopy (Figure 2
; B, E, and H). Effacement of the podocyte foot
processes, as observed in the Alport mouse (Figure 2, D and E)
,
obliterates the slit diaphragms, potentially blocking the glomerular
filter.
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illustrate a dramatic shift in
laminin 2 (Figure 3, C
versus D) and laminin ß1 (Figure 3, G
versus H) chain expression from the mesangial matrix in
control mice to the GBM in Alport mice. No changes in the glomerular
localization of the 5, ß2, or 1 chains were observed in normal
versus Alport mice. Consistent with earlier
reports,36
the 3, 4, and ß3 chains are not
expressed in the renal glomerulus. By co-localization inference, these
data suggest that either laminin-2 (a heterotrimer comprised of the
2, ß1, and 1 chains) or a combination of laminin-2 and
laminin-4 (comprised of the 2, ß2, and 1 chains), whereas they
are completely absent from the GBM of normal mice, are abundant in the
GBM of Alport mice.
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, the GBM is stained in green, and laminin 2
chain is stained in red to illustrate that the laminin 2 chain,
which normally localizes specifically to the glomerular mesangium
(Figure 2C)
localized heavily in the GBM of Alport mice (Figure 2F)
. In
double-knockout mice, the laminin 2-chain localization was similar
to that in normal mice, however some laminin 2 staining is observed
in the GBM (Figure 2I)
. Similar results were obtained using antisera
specific for the ß1-laminin chain (data not shown). The changes in
laminin composition of the GBM in normal versus Alport mice
may be causally linked to effacement of the podocyte foot processes.
As 1ß1 integrin plays a key role in cell adhesion, spreading, and
migration in other systems,19,37,38
it seemed plausible
that expansion of the mesangial matrix, an early marker of Alport
glomerular pathogenesis, might be attenuated in the double-knockout
mice. In Figure 4
a fibronectin
immunostain was used to illustrate that this is indeed the case. This
abundant mesangial matrix marker reveals extensive expansion of the
mesangial matrix in Alport versus normal mice at 7 weeks of
age (Figure 4, A
versus B). Age matched double-knockout
mice, however, do not exhibit noticeable expansion of the glomerular
mesangium (Figure 4C)
. By 12 weeks of age, however, the mesangial
matrix in double-knockout mice is expanded (data not shown),
illustrating that the absence of 1 integrin attenuates, but does not
inhibit expansion of the mesangial matrix.
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Recently we described induction of TGF-ß1 mRNA as well as
fibronectin, laminin ß1, and collagen 1(IV) mRNAs in the
podocytes of the Alport mouse.23
We further evaluated the
role of TGF-ß1 on renal disease progression in the Alport mouse by
using a newly designed soluble receptor inhibitor. A number of groups,
including ours, have reported the development of soluble TGF-ß type
II receptors as in vitro and in vivo antagonists
of TGF-ß.32,33,39
As reported for these similar TGF-ß
receptor antagonists, mouse TGFßR:Fc blocks TGF-ß1
(IC50 = 1 nmol/L), and TGF-ß3
(IC50 = 1 nmol/L), but not TGF-ß2-mediated
inhibition of mink lung tumor (Mv1Lu) cell proliferation (Figure 5)
. We noted no inflammation or immune
infiltration in normal mice chronically administered effective doses of
this inhibitor (data not shown).
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. Scanning
electron microscopy reveals the podocyte foot processes are effaced in
Alport animals treated with mTGFßR:Fc (Figure 6F)
. Laminin 2 is
present in the GBM of Alport mice treated with mTGFßR:Fc (Figure 6G)
.
Alport mice treated with mTGFßR:Fc die of renal failure at
approximately the same time as untreated Alport mice, presumably
because of effacement of the podocyte foot processes which results in
massive proteinuria (data not shown).
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1ß1 in Alport Mice Restores
Both GBM and Podocyte Architecture
The results shown in Figures 2 and 6
suggest 1ß1 integrin and
TGF-ß1 play distinct roles in Alport glomerular pathogenesis. If so,
blocking TGF-ß1 in Alport mice null for 1ß1 integrin should
prevent both podocyte foot process effacement and GBM thickening. To
test this hypothesis, mTGFßR:Fc was administered to double-knockout
mice starting at 4 weeks of age, and the kidneys were analyzed at 10
weeks of age. The treated animals were compared directly with untreated
double-knockout littermates. Data shown in Table 1
and Figure 1B
illustrates that both the time of onset and progression of proteinuria
are markedly attenuated in double knockouts treated with mTGFßR:Fc
relative to untreated double-knockout mice. Double-knockout animals
treated with mTGFßR:Fc die at 18 to 24 weeks of age. As shown in
Figure 7
, the GBM of the double-knockout
mice at 10 weeks of age contains many areas of focal thickening,
indicating progression of the glomerular pathology relative to
7-week-old double knockouts (compare Figures 7B and 2G
). The podocyte
foot processes have normal morphology, except for areas where the GBM
is focally thickened. In these regions, the foot processes appear
fused. In the double knockouts treated with the soluble receptor, both
basement membrane thickening and effacement of the podocyte foot
processes are virtually absent. Most of the glomeruli in the treated
double knockouts reflect that illustrated in Figure 7C
, where some
irregularities in the glomerular basement membrane are visible.
Approximately 20% of the glomeruli, however, are morphologically
indistinguishable from those of control animals (Figure 7D)
. By
comparison, out of >50 glomeruli examined by transmission electron
microscopy, none of the glomeruli in 3-week-old Alport mice are
morphologically normal. Laminin 2 in the untreated 10-week-old
double knockouts shows considerable staining in the GBM (Figure 7E)
,
however the staining is focally deposited rather than distributed
throughout the full length of the GBM, as observed in the 7-week-old
Alport mice (compare Figure 7E
with 2F). This is consistent with the
focal thickening observed on transmission electron microscopy
micrographs (Figure 7B)
. In 10-week-old double knockouts treated with
mTGFßR:Fc, laminin 2 staining is restricted to the glomerular
mesangium (Figure 7F)
, identical to that observed in normal animals
(Figure 2C)
. Thus, blocking 1ß1 integrin and TGF-ß1 is
synergistic in slowing the rate of both onset and progression of
glomerular pathology in the Alport mouse.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Herein we describe that at least two pathways play distinct
contributory roles in the glomerular pathology of Alport syndrome. The
first involves integrin 1ß1, which is the most prevalent integrin
found on the surface of mesangial cells.17,18
Our data
illustrates this integrin is involved in the mechanisms of both
podocyte foot process effacement and mesangial matrix expansion. Its
effect on podocyte effacement is likely mediated via GBM deposition of
laminin isoforms not native to the GBM. The second pathway involves
TGF-ß1, which we show is driving focal thickening of the GBM, a
hallmark for diagnosis of Alport GBM pathogenesis. When both TGF-ß1
and 1ß1 integrin are removed from Alport mice, synergistic
improvement in renal morphology and function are observed, indicating
that these two pathways work in distinct ways to drive renal disease
progression in Alport syndrome.
Integrin 1ß1 and Alport Renal Disease
A major indicator of Alport glomerular pathogenesis is expansion
of the mesangial matrix.2
How or whether changes in the
glomerular mesangium have anything to do with observed GBM damage in
Alport syndrome, however, is not clear. Integrin 1ß1 has been
shown to be important for gel contraction of mesangial cells in
vitro.21
It is well documented that mesangial cells
depend on ß1 integrins for adhesion and migration,40
and
1ß1 integrin has been directly implicated in both adhesion and
spreading of chondrocytes.38
Thus, the absence of 1ß1
in the double knockouts might affect the migration of cytoplasmic
processes that interface the mesangium with the glomerular capillary
loops.41,42
These contact points provide direct access for
mesangial enzymes and proteins to be deposited into the GBM. Focal
degradation by matrix metalloproteinase derived from the glomerular
mesangium with concomitant deposition of mesangial matrix proteins
might explain why focal thickening and splitting are observed in Alport
GBM disease. Indeed, the proteins that accumulate in the GBM as a
function of Alport renal disease pathogenesis include many matrix
proteins normally synthesized by mesangial cells,14,23
and
mesangial cells are known to produce both matrix metalloproteinase
(MMP)-2, MMP-9 and the tissue inhibitors of metalloproteinases-I, -II,
and -III.43,44
The mRNAs encoding both the integrin 1
and ß1 subunits are induced by TGF-ß1,45
as are
mesangial expression of laminins, fibronectin, and collagen 1(IV)
and 2(IV). An alternative explanation for the appearance of
typically mesangial proteins in the GBM is gene activation in the
podocytes. Activation in podocytes of the genes encoding collagen
1(IV), fibronectin, and the laminin ß1chain, all typically
mesangial matrix proteins, has been demonstrated in Alport
mice.23
Thus, an integrin 1ß1 regulated paracrine
factor may exist that can activate these genes in podocytes.
Absence of 1ß1 integrin results in an inhibition of laminin 2
and ß1 chain deposition in the GBM. This report is the first to
illustrate GBM deposition of these laminin chains in GBM pathogenesis.
The importance of this observation lies with its likely association
with effacement of the podocyte foot processes. Known laminin
heterotrimers that contain the 2 chain include laminin-2
(2ß11) and laminin-4 (2ß21).46
Normal
mature GBM contains only laminin-11 (5ß21).24
Recent studies used a recombinant soluble 3ß1 integrin receptor to
illustrate that the integrin heterodimer binds specifically to laminin
heterotrimers that contain the 5 chain, but not those that contain
the 1 or 2 chains.47
It is widely believed that
adhesion of the podocyte foot processes to the GBM requires 3ß1
integrin.35
Immunogold localization studies place 3ß1
integrin at the interface between the podocytes and the basement
membrane.48
An 3 integrin knockout has been produced,
and dies at birth from acute renal failure, with complete effacement of
the podocyte foot processes.49
Integrin 1ß1 and
2ß1 bind to the amino terminus of the laminin 1 chain and the
laminin 2 chain,50,51
creating precedent for laminin
chains as specific ligands for integrins. Thus, deposition of the
laminin heterotrimers containing the 2 chain might mask binding of
3ß1 integrin to its preferred ligand, laminin-11. Such masking
might lead to progressive loss of focal contact adhesion, resulting in
foot process effacement. Because laminin 2 is found only in the
mesangial matrix of normal mice and humans, how it gets into the GBM in
Alport syndrome is an important unanswered question. Clearly 1ß1
integrin plays an key role in this process.
Role of TGF-ß1 in Alport Glomerular Pathogenesis
TGF-ß1 is strongly implicated as a molecular driver in the rat model for acute mesangial proliferative glomerulonephritis.52 The induced disease can be suppressed by injecting the animals with neutralizing antibodies against TGF-ß1 or antisense oligonucleotides complimentary to the TGF-ß1 mRNA.53,54 More recently, this work has been extended to include gene therapy by transduction with an expression vector expressing a soluble TGF-ß type II receptor/Fc fusion peptide, much like the soluble receptor used in our studies.39 TGF-ß1 has been implicated in a number of glomerular diseases,54,55 including Alport syndrome.23
In this report we show that by inhibiting TGF-ß1 we can inhibit
thickening of the GBM. We do not, however, inhibit effacement of the
podocyte foot processes. Laminin 2 is present in the GBM of
7-week-old Alport mice treated with mTGFßR:Fc, suggesting that the
events resulting in deposition of laminin 2 containing isoforms in
the GBM are not mediated by TGF-ß1. The animals die at about the same
time as untreated animals, however why they die is not clear. They do
develop acute massive proteinuria, so the cause of death may be
hypoalbuminemia and hypovolemia. This is very different from the
results obtained with experimental glomerulonephritis, where inhibition
of TGF-ß1 was sufficient to restore normal glomerular
function.22,53
Thus, although TGF-ß1 might be induced in
a variety of glomerular disease, it is likely that its contributory
role may be unique for each.
Combined, the observations reported herein support a potential
therapeutic paradigm for Alport renal disease by treatment with
inhibitors for both integrin 1ß1 and TGF-ß1.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grants R01 DK55000 (to D. C.) from the National Institute of Diabetes and Digestive and Kidney Diseases, P01 DC01813 (to D. C.) from the National Institute on Deafness and Other Communication Disorders, and by R01 DK51711 (to R. K.); the 1998 Carl Gottschalk award (to R. K.); the 1998 NKF Murray Award (to R. K.); and funds from Beth Israel Deaconess Medical Center.
Accepted for publication July 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 (COLCOL4A3) gene in autosomal recessive Alport syndrome. Hum Mol Genet 1994, 3:1269-12733(IV) and 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat Genet 1994, 8:77-82[Medline]
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