Non-Fibrillar Collagenous Proteins Synthesized by Rat Mesangial Cells

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.

of the American

Society

of Nephrology

cells.

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