mesangium consists of mesangial cells and a surrounding extracellular matrix, the mesangial matrix. In gbomerular diseases of differing etiologies, mesangial.
Non-Fibrillar Mesangial Norman
Collagenous Cells
D. Rosenblum,
M.D.,1
Morris
Proteins J. Karnovsky,
Synthesized
M.B.B.Ch.,
D.Sc.,
Key Words:
ND. Rosenblum, ments of Anatomy
M.J. Karnovsky, BR. Olsen, Departand Cellular Biology and Pathology,
/agen
Harvard
School,
T
Medical
Children’s
Hospital,
Soc.
(J. Am.
and
Boston,
Nephrol.
Division
of
Nephrology,
MA
1990;
and
Bjorn
Extracellular
cells
and
mesangial
mesangium
accumulation
common
pathways
cell
consists
co/-
of mesangial matrix,
diseases
the
of differing
proliferation
and
thought
to represent
are
Ph.D.
short-chain
extracellular
In gbomerular
mesangial
matrix
M.D.,
mesangium,
a surrounding
matrix.
etiologies,
R. Olsen,
matrix,
he glomerular
1:785-791)
by Rat
mesangial
final
ABSTRACT Recent studies have shown that nonrenal extracellular matrices are composed of collagenous proteins with properties that are different from those of fibrillar collagens and type IV collagen. The structures of these newly described collagens suggest that they may provide connections between specific matrix molecules and, in so doing, partially determine the three-dimensional structure of the matrix. The molecular composition and organization of normal and
capillary loops which, in turn, interferes with normal gbomerular filtration. Given the detrimental effect of mesangial matrix expansion on gbomerular function, it is probable that our understanding of both the diseased mesangial matrix and the mechanisms which determine the progression of gbomerular disease will be enhanced by further defining the com-
diseased
position
mesangial
matrix
are
incompletely
under-
stood. As a means to further understand the structure of the mesangial matrix, we have further defined the collagenous proteins synthesized by rat mesangial cells. By using monospecific antibodies, we have shown that these cells secrete types Ill and IV collagen. Metabolic labeling and electrophoretic analysis of proteins isolated from the culture medium revealed that mesangial cells also synthesize and secrete short-chain collagenous proteins of 80, 75, and 69 kDa which differ in their binding to concanavalin A. Cell-free
translation
of mesangial
mRNA
demon-
not
detected
in the
culture
medium.
These
‘Correspondence
to
Dr. N.D.
300 Longwood
Rosenbium.
Avenue,
Boston,
The
Childrens
MA 02115.
1046-6673/0105-0785S02.00/0 Journal of the American Society of Nephrology copyright © 1990 by the American Society of Nephrology
Journal
of the American
Society
of Nephrology
Hospital.
results
Division
diseases
appears
and
angial The
goal
of
pathogenesis
to
result
in the
of
our
of many
types
which result in renal failure of matrix material in these in
the
three-dimensional
matrix
obliteration
of
structure
normal
and
studies
is
of the
diseased
to
the
mes-
glomerulus.
define
the
types
of
collagenous proteins which are expressed by mesangial cells as a means of further understanding the mesangial matrix The mesangial matrix, as presently understood, is composed of several types of matrix molecules. Studies of intact mesangium by transmission electron microscopy
have
microfibrils and in the of
shown
are present mesanglum
that
hollow
nonbranching
in the normal mesangium (2) of children affected by a va-
gbomerulopathies
unlikely
demonstrate that mesangial cells synthesize several short-chain collagenous proteins which may be similar to short-chain collagens isolated from nonrenal tissues. Further definition of these proteins will lead to a greater understanding of the extracellular matrix elaborated by mesangial cells.
Nephrology.
of gbomerular disease (1). The accumulation
riety
strated the presence of two additional collagenase sensitive proteins, 40 and 35 kDa in size, which were
in the
(3).
to be colbagenous,
These
since
their
microfibrils
are
periodicity
pat-
tern is not typical of collagenous fibrils. Recently, Gibson et at. (4) showed that they contain a 340-kDa glycoprotein Immunofluorescence studies with antibodies directed against known matrix components have shown that the normal mesangium contains .
types
IV and
V collagen,
laminin,
and
fibronectin
(5).
Metabolic labeling studies of cultured mesangiab cells have demonstrated that these cells synthesize and secrete types I, III, IV, and V collagen (6). In addition, these cells synthesize fibronectin, laminin, and the proteoglycans, chondroitin and heparan sulfate (7). Some of these matrix components are present in increased amounts in diseased gbomeruli. For example, in
the
in the the
diabetic
expression
expanded
rat, of
mesangial
there types
is an III and
matrix
apparent IV collagen
(8).
Mesangial
increase within
cells
785
Mesangial
Cell
Non-fibrillar
Collagens
grown in culture in the presence of a high concentration of glucose express increased amounts of fibronectin mRNA. Studies of human IgA nephropathy have suggested that type III collagen is overproduced in this disorder (9). Taken together, these studies suggest that the altered metabolism of both collagenous and noncollagenous extracellular matrix proteins is important in the pathogenesis of mesanglal matrix expansion seen In gbomerular diseases. Despite the identification of a variety of extracellular matrix molecules in normal and diseased mesangium
and
the
evidence
which
implicates
some
of
these molecules in gbomerular disease, the mechanisms by which these matrix molecules interact to determine normal mesangial structure and function are unknown. Moreover, In the absence of this information, the precise mechanisms by which altered matrix molecule expression determines alterations in mesanglal structure which are, in turn, deleterious to normal gbomerular function are unclear. Recently, several novel classes of collagens with unique domain structures, distinct from previously described fibrillar and basement membrane collagens, have been described in diverse nonrenal tissues. The discovery of these collagens has greatly expanded our view of matrix organization. One such class, the fibrin-assoclated collagens with interrupted triple helices (FACIT) colbagens, consists of collagens with short triple-helical domains interrupted by non-triple-helical domains (for a review, see ref. 1 0). These collagens are associated with fibrils, have large amino-terminal domains, and are thought to Interact with other molecules in the extracellular matrix. Another class, the short-chain coblagens, consists of collagens with triple-helical domains, which are much shorter than those found in the fibrillar collagens. In contrast to the fibrillar collagens, these collagens have intact non-triple-helical
domains
at
their
amino
and
carboxyl
termini.
One member of this class, type VIII collagen, is thought to be the principal component of the hexagonal lattice framework in Descemet’s membrane ( 1 1). Both the FACIT and short-chain collagen families are thought to function as connectors in the extracellular matrix and, in so doing, determine the relationship of matrix components to each other, thereby establishing a stable three-dimensional architecture (10). Just as these molecules are expressed In a variety of extracellular matrices In nonrenal tissues, it Is reasonable to expect that molecules with similar properties are present in the mesanglal matrix, where they may function to connect matrix components in an ordered three-dimensional framework. In the studies described below, we have analyzed the collagenous cells and have tified, collagenous
786
proteins Identified chains
synthesized by several, previously which are similar
mesangial unidenin size to
short-chain collagens described in nonrenal tissues. We have also shown that these colbagenous chains differ from each other in size and in their content of mannose-contalnlng carbohydrate side chains.
METHODS Mesangial Labeling
Cell
Culture
and
Mesangial cells were isolated the method of Harper et at. (1 2). removed from anesthetized 50gue-Dawley rats and were placed balanced salt solution buffered plemented with penicillin (1 00 (100 g/mL), and amphotericin cortices
were
separated
Metabolic from rat gbomerull by BrIefly, kidneys were to 1 00-g female SpraInto sterile Hanks’ with HEPES and supU/mL), streptomycin (0.25 ,g/mL). The
from
the
medullae
and
passed through a series of sieves of decreasing pore size. Gbomerull were washed twice with buffered Hanks-HEPES (GIBCO Laboratories, Grand Island, NY) and were then treated with 0. 1 % collagenase (Sigma Chemical Co. , St. Louis, MO) for 20 mm and then with 0.2% trypsln (GIBCO) for 10 mm at 37#{176}C. After one wash In Hanks-HEPES, the resulting pellet, which contained both whole and dissociated gbomeruli, was plated in Dulbecco’s modified Eagle medium supplemented with 20% fetal calf serum (Hyclone, Logan, UT), penicillin (100 U/mL), and streptomycin (1 00 g/mL). This medium is optimal for the growth of mesanglal cells, which were Identified by their typical morphology. Typical-appearing cells appeared within 3 to 5 days and were passaged twice before the concentration of serum was decreased to 1 0%. All experiments were performed on primary cells passaged not more than five times. For metabolic labeling, [35Sjmethionlne (5 zCi/mL) was added to the culture medium and the cells were incubated for 24 h In the presence of (per mL) 50 g of ascorbate and 64 g of fl-amlnopropionitrlle. At the end of the incubation, N-ethylmaleimide (10 mM, final concentration), p-amlnobenzamidlne ( 1 mM, finab concentration), phenylmethybsubfonyl fluoride (1 mM, final concentration), and 1 76 mg of ammonlum sulfate per mL were added to the medium. After being stirred overnight at 4#{176}C, the medium was centrifuged at 20,000 x g for 20 mm at 4#{176}C to recover precipitated proteins. The precipitate was suspended by being stirred overnight at 4#{176}C in a buffer containing 50 mM Tris-HC1 (pH 7.6), 1 mM EDTA, 0.5% Nonldet P-40, and 6 M urea. After spinning at 12,000 x g for 2 mm at 4#{176}C, the supernatant was used for digestion with cobbagenase and gel electrophoresls.
Enzyme
Digestions
Coblagenase performed
digestion in a buffer
of Precipitated
Proteins
of precipitated proteins was consisting of 0.1 M TrIs-HC1
Volume
I ‘Number
5.
1990
Rosenblum
(pH 7.5) and 1 0 mM CaCl to which 1 8 U of highly purified bacterial collagenase (Advance Blofactures, Lynbrook, NY) was added per 20 tL of reaction mixture. Collagenase digestion was carried out for 1 h at 37#{176}C. Precipitated proteins were digested with 1 00 zg pepsin per mL (Sigma) in 0.5 M acetic acid for 20 h at 4#{176}C. Pepsin was inactivated by the addition of a fivefold molar excess of pepstatmn (Sigma).
with 30% ammonium sulfate and suspended as described above. Proteins bound to the column were eluted by a 1 6-h exposure to 1 M methyl a-D-mannopyranoside (Sigma) in 0.2 M NH4HCO3. The ebuate was
dialyzed
phylized.
were tion
of Precipitated
Precipitated amide slab sodium
Proteins
proteins were gel electrophoresis
dodecyl
sulfate
under
of
reducing
conditions. Molecular size was estimated by comparIson with globular molecular weight standards (DuPont, NEN Research Products, Boston, MA; BloRad Laboratories, Richmond, CA), and type I collagen chains were extracted from rat tail. Proteins separated by gel electrophoresis were transferred to Immobilon membranes (polyvinylidene difluoride membranes; Millipore Corp. , Bedford, MA) at 4#{176}C in a buffer consisting of 1 0 mM 3-cyclohexylamino]-lpropane-sulfonic acid (CAPS)1 0% methanol (pH 11) at 0.3 A for 45 mm. After transfer of proteins, the membranes were stained for 5 mm with 0. 1 % Coomassle
blue
In
50%
methanol
and
were
Isolation
Confluent serum were
analyzed by polyacrylin the presence
(SDS-PAGE)
against Proteins
then
de-
by
0.2 In the
M NH4HCO3
incubating
and
with
and
directly
Cell-Free
mesangial removed
and
flow-through
separated by SDS-PAGE with bacterial collagenase.
RNA Analysis
et al
then the
or after
lyoeluate
diges-
Translation
cells grown in 1 0% fetal calf from the tissue culture plates 0.2%
trypsin-EDTA
(GIBCO)
and
were then washed three times with PBS. The cell pellet was frozen in liquid nitrogen and stored at -80#{176}C. PolyA’ mRNA was isolated from these cells by
using
a commercial
kit
(Fast
Track;
Invitrogen,
San Diego, CA). RNA transcripts were assayed by cell-free translation by using a commercial reticulocyte lysate (Amersham Corp. , Arlington Heights, IL). Translation products, labeled with [35Sjmethionine were analyzed by SDS-PAGE.
Collagen Type collagenase
Ill -
+
+
IV -
+
stained in 50% methanol1 0% acetic acid and then in distilled water. Identical unstained membranes were blocked with 5% nonfat dry milk in PBS. (pH 7.4)
with
then then
incubated with with a secondary
line
0.02%
sodium
phosphatase.
azide
primary antibody
Protein
overnight
and
antibody for conjugated
bands
reactive
were
1 h and to alkawith
body were detected by a color reaction with substrates for alkaline phosphatase supplied in a commercial kit (Protobbot Immunoscreening System, Promega Biotec, Madison, WI). A mouse monocbonal antibody against human type III collagen was provided by Yasuteru Muragaki. The specificity of this antibody has been and Immunofluorescence
yclonab antibody vided by Rupert body has been nobbotting (14).
shown
by ELISA, staining
against Timpl.
immunoblotting, (13). A rabbit
by
ELISA
and
92kDa
pob-
type IV collagen was The specificity of this
demonstrated
200 kDa
anti-
proantiimmu-
Figure 1 Immunoidentification of types Ill and IV collagen chains. Proteins isolated from cell culture medium were incubated with (+) and without (-) purified bacterial collagenase and electrophoresed in a 7.5% polyacrylamide .
Concanavalin Culture Medium After
to
was
applied
umn
(conA
Proteins
Journal
labeling
the
cell
and
culture
Affigel;
Blo-Rad)
the
of the American
flow-through
Society
addition
of protease
medium,
to a concanavalin in
of Cell
gel under
metabolic
inhibitors
h.
A Chromatography
the
A (conA) at
a flow
were
of Nephrology
medium
affinity rate
of
col10
precipitated
mL/
reducing
conditions.
Left
panel.
Proteins
sepa-
rated by SDS-PAGE, transferred to Immobilon membrane, and stained with Coomassie blue. Middle panel. Identical unstained blot reacted with a mouse monoclonal antibody against type III collagen. Right panel. Identical unstained blot reacted with a rabbit polyclonal antibody against the 7S domain of mouse type IV collagen.
787
Mesangial
Cell
Non-fibrillar
Collagens
coDagenase
RESULTS Mesangial Cells Fibrillar Collagens Electrophoretic teins
separation
Isolated
revealed graded
from
the
of the culture
a large number when the proteins
purified
collagenase.
Identified
as
merous
precipitated
medium
pro-
(Figure
1)
of bands which were dewere treated with highly
By this
representing
means,
such
collagenous
coblagenous
bands
were
bands
were
proteins. identified
Nubetween
the 92- and 200-kDa molecular mass markers at the expected migratory positions of the pro and mature forms of a chains belonging to the fibrillar collagens and type IV collagen. We used monospecific antibodles to Identify the a chains of types III and IV collagen. Proteins
separated
by
SDS-PAGE
Immobibon membranes specific antibodies. panel), a monocbonal man
type
migrating
were
transferred
200kDa. .ai #{149}cY2
to
92kDa.
and were then reacted with As shown in Figure 1 (middle antibody directed against hu-
III
collagen
in
the
detected
expected
a
collagenous
band
A
poby-
position.
rabbit
clonal mouse bands marker
antibody directed against the 75 domain of type IV collagen detected two cobbagenous just above the 200-kDa molecular mass (Figure 1 , right panel). These are the expected positions of the a 1 (IV) and a2(IV) collagen chains. In the absence of a monospecific antibody for type I collagen, we digested precipitated proteins with pepsin
and
PAGE.
separated
We
extracted
with
from
is consistent
(6)
that
collagen
the
identified
comigrated
with
cultured
peptic
two
the
rat
fragments
prominant
a 1 (I) and
tails the
(data
a2(I)
not
collagen
shown).
observations
mesangiab
by
bands This
of Harabson
cells
synthesize
Synthesize Proteins
and
Secrete
SDSchains
finding et at. type I
Short-
We metabolically labeled proteins synthesized by mesangiab cells with I35Simethionmne and analyzed the proteins which were precipitated from the cell culture medium by SDS-PAGE (Figure 2). A large number of collagenous chains were identified at or above the positions of the al and a2 chains of type I collagen.
In
addition,
a prominent
collagenous
band
of approximately 80 kDa was identified. Less intense collagenous bands of approximately 75 and 69 kDa were also noted. This 69-kDa collagenous chain migrates In the same position as does a 1(X) collagen from
hypertrophic
Mesangial Collagens Containing
(11).
Cells Secrete Short-Chain with and without MannoseCarbohydrate Side Chains
[35Sjmethionine-labeled from cell culture
788
chondrocytes
medium
69kDa.
which
chains.
Mesangial Cells Chain Collagenous
+
Synthesize and Secrete and Type IV Collagen
mesangial cell proteins were passed through
a
Figure 2. SDS-PAGE of metabolically labeled mesangial cell medium-derived proteins. Proteins isolated from cell culture medium were incubated with (-F) and without (-) purified bacterial collagenase and electrophoresed in a 7.5% polyacrylamide
gel
positions of the left. The lagen chains the position arrow marks
globular molecular mass markers are noted on migratory positions of the al(l) and a2(l) colare noted on the right. The upper arrow marks of the 80-kDa collagenous chain. The lower the position of the 69-kDa collagenous chain.
under
reducing
conditions.
The
migratory
conA affinity column, and the bound and unbound proteins were then analyzed by SDS-PAGE (Figure 3). The 80-kDa coblagenous chain, seen In the starting material was not bound to the column, indicating that it does not contain carbohydrate side chains with exposed mannose side chains. In contrast, the 75-kDa collagenous chain did bind to conA. This chain was more strongly detected in the bound fraction, since the proteins in this fraction were more highly concentrated than those in the starting material. Although this is difficult to discern from the figure, the 69-kDa colbagenous chain was detected only in the bound fraction.
Volume
I ‘Number
5’
1990
Rosenblum
collagnase
F.T.
+
-
ELUATE ±
collagen
+
-
ase
et al
#{247}
92kDa.
92kDa. 69kDa.
69kDa. 46kDa. Figure 3. conA chromatography of metabolically labeled mesangial cell medium-derived proteins. Proteins isolated from the cell culture medium, the unbound fraction, and the bound fraction were incubated with (+) and without (-) purified bacterial collagenase and were electrophoresed in a 7.5% polyacrylamide gel under reducing conditions. The migratory position of the 92- and the 69-kDa globular molecular mass markers are noted on the left. Left panel. Proteins present in the starting material. Middle panel (flow through). Unbound proteins. Right panel (eluate). Proteins bound to the column. The arrow on the left marks the position of the 80-kDa collagenous protein which did not bind
to conA.
the 75-kDa
The arrow
collagenous
on the
protein
right
marks
which
the
did bind
position
of
to conA.
Mesangial mRNA Directs the Translation of Collagenous Chains Approximately 35 and kDa in Size
40
We used cell-free translation of mesanglab cell mRNA to detect colbagenous proteins not necessarily detected by the analysis of proteins In the cell culture medium.
Translation
of mesangiab
RNA
directed
the
translation of collagenous bands of 80 and 69 kDa as expected from our analysis of metabolically labeled proteins (Figure 4). In addition, two prominent collagenase-sensitive bands of approximately 35 and 40 kDa were noted. These chains were not detected by examination of culture medium proteins and may represent proteins which are membrane bound or which are found only in the cell-associated matrix layer.
DISCUSSION We have short
35
shown
that
collagenous
kDa-none
Journal
rat
mesangial
polypeptides
of
of the American
which
Society
cells
of 80,
have
been
of Nephrology
synthesize 40, and previously de-
75,
3OkDa.
69,
Figure 4. SDS-polyacrylamide gel electrophoresis of cellfree translation products synthesized with poly(A) RNA isolated from cultured rat mesangial cells. Cell-free translation products were incubated with (+) and without (-) purified bacterial collagenase and electrophoresed in a 7.5% polyacrylamide
gel
under
reducing
conditions.
The
migratory
positions of globular molecular mass markers are noted on the left. The arrows mark the position of the 40- and 35-kDa collagenous proteins. Collagenous cell-free translation products of 80 and 69 kDa are also present.
scribed as mesangial of these proteins other published type IV collagens difference in the Is due
to the
different
cell products. The identification in our studies stands In contrast to studies in which only fibriblar and were Identified. We believe that the collagens identified in these studies techniques
used
to isolate
these
proteins. Previous studies have used the classical biochemicab techniques of graded sodium chloride fractionation and column chromatography of pepsin-digested proteins (6). These methods were developed to isolate fibrillar collagens and type IV collagen but are not
789
Mesangial
Cell Non-fibrillar
Collagens
suitable for the isolation of collagens with domain structures that are different. For example, types IX and XII collagen consist of short triple-helical domains separated by non-triple-helical domains. Pepsin
digestion
of these
fragments ods
which
(1 0).
The
lost
isolation
concentrations those typically It is therefore rupted
proteins
are
of
short
others
results
in small
routine
isolation
type
VIII
protein meth-
collagen
requires
of sodium chloride far in excess of used to isolate fibrilbar collagens (15). likely that collagens with unintertriple-helical
domains
domains interrupted were present in the by
by
but
were
or
lost
during
collagen
domains studied
80
kDa
(globular
molecular
mass
standards),
product
of the
al(VIII)
gene,
since
both
its
size
and the failure to bind to conA are predicted by the primary structure of al(VIII) collagen. Cornea! endothelial cell a 1 (VIII) collagen is thought to be the subunit which is organized into a hexagonal lattice framework in Descemet’s membrane in the cornea ( 1 1 ). It is possible that the same protein synthesized by mesangial cells is deposited in the mesangial matrix, where It may be organized to form highly
stable
structures.
Smith
and
have shown that undifferentiated carcinoma cells produce a collagen al(VIII)
collagen.
regulated shown
The
by retinoic to direct cell
ment.
In the
presence
entiate
into
parietal
type
VIII-like
collagen
synthesis
of
acid, a factor differentiation of retinoic
endoderm, decreases,
Baldwin
(16)
F9 embroyonal the same size this
acid,
the
F9 the
is
has been developcells
synthesis and
as
collagen
which during
differ-
of the synthesis
of type IV collagen increases. In a similar fashion, It Is possible that when normally quiescent mesangial cells begin to proliferate, they undergo a phenotypic change during which the production of type VIII collagen Increases. This, in turn, could affect the structure and stability of the mesangial matrix. The amino acid sequences obtained from cyanogen bromide peptides of cornea! endothelial cell type VIII colbagen Indicate that It is a heterodimer, since not
790
sequences
cDNA
(1 1).
is
the
same
size
hypertrophic type
are It is
contained
therefore
within
possible
X
as
type
X
chondrocytes
the
that
collagen
is
a
marker
collagen,
a
the
product
(1 7).
The
synthesis
for
the
differentiated
hypertrophlc
chondrocytes,
It
understanding of the regulation provide important information entiated properties of mesangial conditions.
of of
Finally,
we
have
identified
Is
likely
that
an
of its synthesis will regarding the differcells under varying two
collagenase
sensi-
tive proteins of 40 and 35 kDa, respectively, which are synthesized by RNA-directed cell-free translation but which are not detected in the cell culture medium. While it is possible that these proteins are deposited only
likely thus
which is in agreement with the protein data of Benya and Padilla ( 1 5). In addition, the a 1 (VIII) cDNA sequence does not contain sites for N- or 0-linked oligosaccharides. It is therefore probable that the mesangial cell 80-kDa collagen represents the translatlon
these
mesangiab cell 75-kDa collagen chain is the translation product of the a2(VIII) gene. Purification of this protein and cloning of the a2(VIII) cDNA will be required to demonstrate this. As noted above, the mesangial cell 69-kDa collagen
by
purification
procedures. Given these considerations, we have analyzed nondigested proteins isolated directly from the culture medium. The identity of the short-chain collagen chains which we have identified is unclear at this point. However, these chains may be similar to other collagenous chains of similar size, Identified in other experimental systems. The primary structure of rabbit cornea! endothelial cell a 1(VIII) collagen has recently been published (1 1). The primary sequence predicts a translation product of about
of
al(VIII)
state of these cebbs, and its synthesis is developmentally regulated (1 8). If the mesangial cebl 69-kDa collagen is, indeed, the same protein as that produced
triple-helical
by non-triple-helical crude protein fractions
a!!
into
the
in view far,
can
cell-associated
of the be
fact
found
matrix,
that In
other the
this
seems
co!bagens
culture
un-
studied,
medium
even
when they are predominately deposited into the matrix layer (6). It is more likeby that these two collagenous proteins are not secreted by these cebls and are membrane-associated proteins. Kodama et at. (19) have recently described two cDNA clones which correspond to the macrophage type I scavenger receptor. Two forms of this receptor have been identified; they differ in that only one of them contains a cysteinerich carboxyl-terminal domain. Both forms contain a 72-amino-acid triple-helical domain which renders them sensitive to degradation with bacterial collagenase. The cDNAs for these two forms predict translation products of 45 and 34 kDa, respectively. The mesangial cell-free translation products are very similar in size to these two receptor proteins. It is therefore possible that the mesangial cell 40- and 35-kDa collagenous chains represent forms which are simibar
to these
scavenger
receptors.
The identity of the mesangial short-chain collagens and their relationship to collagens with similar characteristics will be demonstrated only after purification of these proteins and further analysis, including cDNA cloning. This will provide the basis for the study of these collagens in vivo. The discovery of the short-chain collagens in nonrenal
tissues
has
added
a new
dimension
to our
un-
derstanding of matrix structure, as well as to the role of collagenous proteins In cell function. We expect that further delineation of the properties of the mesangial cell short-chain collagens which we have described will expand our knowledge of mesangial cell function and the mesangial matrix.
Volume
I ‘NumberS’
1990
Rosenblum
ACKNOWLEDGMENTS We thank Ruth Schillig for helpful advice and support concerning mesangial cell culture. This work was supported by the Medical Research Council of Canada and the Wolbach Fund (to ND. Rosenblum) and the National Institutes of Health (grants AM13132 and HL17747 (to
(to
BR.
M.J.
Karnovsky)
and
grants
AR36819
and
1 0.
AR36820
11
Olsen).
REFERENCES Klahr
5, Schrelner
of renal
Munde!
2.
disease.
G, Ichikawa N Engl
P, Elger
M, Sakal
are a major component the gbomerulus of the 1988:254:183-187.
3.
Yoshkama ing DM:
5.
9.
T, KrIz
progression 1 2.
W: Microfibrils
the mesangial kidney. Cell
N, Cameron AH, White Microfibrils in gbomerulopathies
Houser MT, MW, Michael
matrix Tissue
in Res
Scheinman JI, AF: Preservation
Haralson
RL:
MA,
Jacobson
HR.
1 4.
Hoover
1 5.
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