Division of Nephrology and Hypertension, Research and Education Institute at Harbor-UCLA Medical Center, Torrance, California 90502
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exogenous administration of
recombinant human bone morphogenetic protein (BMP)-7 was recently shown
to ameliorate renal glomerular and interstitial fibrosis in rodents
with experimental renal diseases. We tested the hypothesis that BMP7
functions by antagonizing profibrogenic events that are induced by
transforming growth factor (TGF)- in cultured mesangial cells.
Incubation of murine mesangial cells with TGF-
(50-200 pM)
increased cell-associated collagen type IV and fibronectin, soluble
collagen type IV, thrombospondin, and connective tissue growth factor
(CTGF). Coincubation with recombinant human BMP7 (200 pM) reduced the
increase of these ECM proteins and CTGF. The changes in
collagen type IV and fibronectin proteins occurred without concomitant
changes in collagen type
1IV and fibronectin mRNA
levels, suggesting that TGF-
and BMP7 act primarily by affecting ECM
protein degradation. Indeed, TGF-
decreases the levels and activity
of matrix metalloprotease (MMP)-2, the major metalloprotease that is
secreted by mesangial cells. Moreover, BMP7 inhibits TGF-
-induced
activation of MMP2. Because TGF-
reduces the activity of MMPs
through increasing plasminogen activator inhibitor (PAI)-1, we tested
whether BMP7 interferes with this TGF-
effect. BMP7 reduces, by
about two-thirds, the activation of a PAI-1 promoter/luciferase
reporter in cells stably transfected with this construct. The findings
from these studies indicate that BMP7 reduces TGF-
-induced ECM
protein accumulation in cultured mesangial cells primarily by
maintaining levels and activity of MMP2 partially through prevention of
TGF-
-dependent upregulation of PAI-1.
bone morphogenetic protein 7; transforming growth factor-; kidney fibrosis; matrix metalloprotease-2; plasminogen activator
inhibitor-1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RENAL FIBROSIS,
I.E., ACCUMULATION of ECM proteins in glomeruli and renal
interstitium, is the hallmark of most advanced chronic renal diseases.
Progressive fibrosis is the result of an imbalance between synthesis
and degradation of ECM proteins. Several cytokines contribute to
increased matrix accumulation, but transforming growth factor (TGF)-
has evolved as the single most important profibrogenic mediator in
renal diseases.
Recent studies showed that another member of the TGF- superfamily of
cysteine-knot cytokines, bone morphogenetic protein (BMP)-7, can
prevent or reduce the progression of renal fibrosis in animals with
experimental renal diseases (7, 12, 13, 16, 18, 20).
Morrissey and associates (18) showed that exogenously
administered recombinant human (rh)BMP7 may even resolve, at least
partially, early stages of established glomerular and interstitial
fibrosis in experimental diabetic nephropathy.
BMP7 plays major roles during embryonic development, but in adult
organisms expression of BMP7 and its receptors is retained in only few
tissues, most prominently in the kidney (3, 4, 22, 29).
Its functions in adult kidney are presently unknown but may include
promotion of differentiated epithelial phenotype in tubular cells
(7, 8, 31). Moreover, BMP7 may counteract some of the
profibrogenic actions of TGF-. Consistent with this latter
hypothesis are previous findings indicating that a decrease in renal
BMP7 expression predates the onset of glomerular sclerosis and
interstitial fibrosis in experimental obstructive nephropathy and
diabetic nephropathy (12, 29).
The present studies were performed to examine the hypothesis that BMP7
reduces ECM accumulation in mesangial cells and antagonizes (some of
the) profibrogenic events that are induced by TGF-.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse mesangial cells derived from a SV40-expressing mouse were
obtained from ATCC, Manassas, VA (17). Cells were grown to
>90% confluence in DMEM/F-12 (3:1) containing 14 mM HEPES and 5%
FCS. Before each experiment, cells were incubated in serum-free medium
containing 0.1% BSA. In individual experiments, cells were incubated
for 24 to 72 h with rhTGF- (Biosource, Camarillo, CA), 50, 100, or 200 pM, in the presence or absence of 200 pM rhBMP7 (kind gift from
Dr. J. McCartney, Curis, Cambridge, MA). The concentrations of
TGF-
were selected to be within the physiological range that has
been found in serum (9, 11) but less than 1 nM, which usually induces maximal effects in most cell types. In individual experiments, we examined the effects of TGF-
and BMP7 on collagen type IV (col IV), fibronectin (FN), and thrombospondin (TSP), on
connective tissue growth factor (CTGF) and matrix metalloproteases (MMP) and their activity regulator plasminogen activator inhibitor (PAI)-1.
Effects of BMP7 on collagen 1IV and FN mRNA
levels.
Near-confluent mesangial cells in six-well plates were growth arrested
by serum starvation in medium containing 0.1% BSA. Cells were then
incubated with 200 pM rhBMP7, 200 pM rhTGF-
1, or both
for 72 h (n = 6 each). Total RNA was extracted
with RNA-Stat-60 reagent and the procedure recommended by the
manufacturer (Tel Test, Friendswood, TX). mRNA levels encoding FN or
collagen
1IV (col4A1) were measured by quantitative
reverse-transcription polymerase chain reaction. Random-primed first
strand cDNA was synthesized from RNA with Omniscript Reverse
Transcriptase and random primers (Qiagen, Valencia, CA). Aliquots of
cDNA were amplified with Taq polymerase (Qiagen) and the following
species- and gene-specific primers. Col4A1: 5'-TAGGTGTCAGCAATTAGG-3'
(sense) and 5'-TCACTTCAAGCATAGTGGTCCG-3' (antisense); FN:
5'-ATGCACCGATTGTCAACAGA-3' (sense) and 5'-TGCCGCAACTACTGTGATTC-3' (antisense). The optimal number of cycles for amplification in the
linear range was determined in pilot assays. For col4A1, 42 cycles of
94°C for 45 s, 60°C for 45 s, and 72°C for 60 s;
and for FN, 30 cycles of 94°C for 45 s, 58°C for 45 s,
and 72°C for 60 s were found optimal followed by 72°C for 7 min to complete the last cycle. 18S rRNA was used as endogenous
coamplification standard. The optimal ratio of 18S primers to 18S
compatimers (Qiagen) for col4A1 was 1:9 and for FN it was 3:7 to derive
similar 18S yields compared with that of col4A1 and FN, respectively. PCR products were electrophoretically resolved in 2% agarose gels containing ethidium bromide, illuminated with UV light and analyzed by
digital densitometry using Alpha DigiDoc 100 (Alpha Innotech, San
Leandro, CA).
Effects of BMP7 on TGF--induced accumulation of
cell-associated FN and col IV.
Mesangial cells were incubated without or with rhTGF-
1
(200 pM) in the presence or absence of rhBMP7 (200 pM) for 72 h,
n = 4 each. Media were removed and cell layers were
washed three times with ice-cold PBS. Cells and matrix were lysed with
2× reducing Laemmli buffer, scraped off the plates, sonicated with
three short 1-s bursts at 4 W, and heated at 80°C for 10 min.
Proteins were resolved by SDS-PAGE in 5% gels. Resolved proteins were
electrotransfered onto nitrocellulose. Membranes were blocked with 5%
dry milk (DM) in Tris-buffered saline containing 0.05% Tween 20 (TTBS). For visualization of FN, membranes were incubated with anti-FN
monoclonal antibody (1:1,500; BD-Biosciences, Palo Alto, CA) and
subsequently conjugated with horseradish peroxidase (HRP) anti-mouse
IgG. Bands were visualized by enhanced chemiluminescence and captured
on X-ray film. For col IV Western blot, blocked membranes were
incubated with biotin-conjugated goat anti-col IV antibody (1:2,000;
Southern Biotechnology, Birmingham, AL). Bands were
visualized with Neutralite Avidin-HRP (Southern Biotechnology) and
subsequent chemiluminescence.
Effect of BMP7 on TGF--induced soluble col IV and TSP.
Near-confluent mesangial cells were incubated with protein-free medium
containing TGF-
(0, 50, 100, or 200 pM) in the presence or absence
of BMP7 (200 pM), n = 6 each in six-well culture plates for 72 h. Conditioned media were then cleared by centrifugation and concentrated 10-fold with spin concentrators (Amicon, Bedford, MA).
Col IV was examined by Western blotting and quantified by ELISA. For
Western blot analysis, aliquots of concentrated conditioned media were
electrophoresed in 5% SDS-PAGE minigels and transferred to
nitrocellulose. Membranes were blocked and incubated with biotinylated anti-col IV (Southern Biotechnology) and then with
Neutralite-Avidin-HRP (Southern Biotechnology). Bands were visualized
with enhanced chemiluminescence.
Effects of BMP7 on TGF--induced CTGF.
CTGF is thought to mediate some of the fibrogenic actions of TGF-
in
some cell types including mesangial cells (6). Thus we
tested whether coincubation of mesangial cells with TGF-
in the
presence of BMP7 would reduce secreted CTGF levels. Arrested mesangial
cells in six-well plates were incubated with rhTGF-
1 (200 pM), rhBMP7 (200 pM), both, or neither (control) for 48 h. Conditioned media were cleared by centrifugation. Heparin-binding proteins were precipitated as described above. Washed precipitates were
taken up in nonreducing sample buffer and boiled for 3 min. Proteins
were separated in 15% SDS-PAGE gels and electroblotted onto
nitrocellulose. Membranes were blocked and incubated with chicken
anti-CTGF-IgY (0.5 µg/ml; gift from Fibrogen, South San Francisco,
CA) and HRP-anti-chicken IgY. Bands were visualized with
chemiluminescence and captured on X-ray film.
MMP activity in mesangial cell-conditioned media.
To examine whether BMP7 antagonizes the TGF--induced decrease in MMP
activity that contributes to the accumulation of ECM, mesangial cells
were incubated in media containing TGF-
(0-200 pM) or BMP7 (0 or 200 pM) for 24 h. MMP activity was examined by gelatin
zymography in media that had been cleared by centrifugation. Briefly,
10% SDS-PAGE minigels were prepared with 1 mg/ml presolved gelatin as
substrate. Samples in reducing agent-free sample buffer were separated
by electrophoresis and fixed in 2.5% Triton X-100 and then developed
in 50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35, pH
7.5 for 24 h at 37°C. Gels were stained with 0.5% Coomassie
blue R-250 and lightly destained. Additional zymograms were performed
with casein as substrate. However, no protease bands were visualized.
Effect of BMP7 on TGF--dependent PAI-1 promoter activation.
MMPs can be activated by proteolysis of Pro-MMPs by plasmin. This
enzyme, in turn, is derived from plasminogen by a proteolysis step
requiring urokinase-type plasminogen activator (uPA). The activity of
uPA and, hence, MMP2 is negatively regulated by the PAI-1. The PAI-1
promoter contains a TGF-
-regulated response element and TGF-
upregulates PAI-1 transcription (25). We hypothesized that
BMP7 may function by antagonizing TGF-
-dependent activation of MMPs
by reducing PAI-1 promoter activation by TGF-
. This question was
examined in mink lung epithelial cells (MLECs) that had been stably
transfected with a construct containing the PAI-1 promoter and
luciferase reporter (1) (kindly provided by D. Rifkin, NYU). Cells were grown to confluence in DMEM/F-12 containing G418 and
10% FCS. Cells were washed three times with PBS and then incubated for
16 h with serum/G418-free medium containing 0.1% BSA and TGF-
(0, 50, 100, or 200 pM) in the presence or absence of BMP7 (200 pM),
n = 6-8 each. Additional incubations were made
with excess 29 nM BMP7. Cells were washed three times with ice-cold PBS
and lysed in 20 µl/well of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100). Luciferase activity in cell lysates was measured in a luminometer with a commercially available luciferase reagent using the manufacturer's instructions (Promega, Madison, WI).
Effects of TGF- and BMP7 on PAI-1 and MMP2 protein levels.
To examine protein levels of secreted PAI-1 and MMP2 in media,
mesangial cells were incubated with serum/BSA-free medium for 48 h
without (control) or with TGF-
or BMP7, each at 200 pM, or both
(n = 4 each). Media were cleared by centrifugation. For PAI-1 Western blot assay, aliquots of cleared media were concentrated in spin concentrators (Amicon), taken up in 1× reducing sample buffer,
boiled for 5 min, electrophoresed in 10% SDS-PAGE gels, and
transferred to nitrocellulose. Blocked membranes were incubated with
polyclonal anti-PAI-1 antibody (1:800; Santa Cruz Biotechnology) in
TTBS/4% BSA overnight at 4°C, washed, and then incubated with HRP-conjugated second antibody (1:12,000 in TTBS/5% BSA) for 1 h.
Bands were visualized with enhanced chemiluminescence and captured on
X-ray film.
Statistical analysis. Results are expressed as means ± SE. Significance of differences was examined by analysis of variance and subsequent Newman-Keuls multicomparison test. P < 0.05 is considered to reflect significance of difference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BMP7 reduces TGF--induced accumulation of cell-associated FN and
col IV.
At the concentrations tested in the present studies, TGF-
did not
significantly raise steady-state FN or col
1IV mRNA
levels in cultured murine mesangial cells. Both were also unaffected by
BMP7 (Fig. 1). Despite the lack of
significantly increased mRNAs, both FN and col IV proteins accumulated
during incubation with TGF-
. Cell-associated col IV moderately
increased with each of the three TGF-
levels by up to 2.2-fold (Fig.
2). BMP7 did not reduce baseline levels
of col IV but amerliorated the TGF-
-induced increase in
cell-associated col IV by ~25%.
|
|
|
BMP7 reduces TGF--induced soluble col IV and secreted TSP.
Collagens are secreted by cells and assemble into complex matrix
structures that become cell associated. Some of the secreted collagen
in cell cultures is present in soluble forms. As shown previously by
other investigators, TGF-
raises col IV in mesangial cell media. The
soluble col IV that was immunodetected in concentrated, conditioned
media increased more than threefold with the highest TGF-
concentration and increased significantly by ~50%, even with the
lowest TGF-
concentration (Fig. 4).
Coincubation with BMP7 reduced the levels of col IV in media by
~30-40% (Fig. 4). The rise in col IV that is induced by the
lowest concentration of TGF-
(50 pM) is actually quantitatively
inhibited by BMP7. The rise in col IV that is associated with the two
greater levels of TGF-
was significantly reduced but not completely
prevented by BMP7 (Fig. 4). Incubation of cells with BMP7 (200 pM) in
the absence of TGF-
does not significantly reduce secreted or
cell-associated col IV levels.
|
|
BMP7 reduces TGF--induced, secreted CTGF.
CTGF is a secreted mediator of many (but not all) effects of TGF-
in
fibroblasts and also in mesangial cells (6, 15, 19).
Consistent with previous findings from other laboratories, TGF-
increases the levels of CTGF in mesangial cell media in the present
studies about threefold (Fig. 6). The
accumulation of CTGF in conditioned media was significantly, although
not completely, reduced by coincubation with BMP7 at equimolar
concentrations (Fig. 6).
|
BMP7 antagonizes the reduction in MMP2 activity by TGF-.
TGF-
is known to reduce the activity of MMPs in various cell types.
Mesangial cells primarily secrete MMP2, and its activity and perhaps
levels are downregulated by TGF-
(2, 23, 24). To test
whether BMP7 alters the reduced activation that occurs with TGF-
,
mesangial cells were incubated with both proteins and zymography was
performed on conditioned media. Zymograms with gelatin as substrate
showed a single band at
70 kDa, known to represent MMP2 (gelatinase
A) (Fig. 7).
-Casein zymography did not visualize any band (not shown). Incubation of cells with TGF-
, 50 and 100 pM, tended to reduce MMP2 activity moderately (P
= not significant), but at 200 pM, TGF-
substantially reduced
MMP2 activity (P < 0.05; Fig. 7). BMP7, 200 pM, almost quantitatively prevented the reduction in MMP2 activity that
was induced by the two lower concentrations of TGF-
and maintained
MMP2 activity at about three-quarters of control even in the presence
of TGF-
, 200 pM (Fig. 7).
|
BMP7 blocks TGF--induced PAI-1 promoter activation.
A major mechanism through which TGF-
downregulates MMP activities
has been demonstrated previously, namely, through increasing PAI-1
levels that blocks upstream steps required for MMP activation. Specifically, PAI-1 reduces the generation of active plasminogen activator (PA) from inactive pro-PA. TGF-
upregulates PAI-1
transcription through smad3 and a smad response element in the PAI-1
promoter. We tested the question of whether BMP7 blocks this action of
TGF-
in MLECs stably expressing a PAI-1 promoter/luciferase reporter construct. As expected, TGF-
substantially increases PAI-1 promoter activity (Fig. 8). Coincubation of the
cells with TGF-
in the presence of BMP7, each at 200 pM, reduced
PAI-1 promoter activation by ~65% compared with incubation with
TGF-
alone (Fig. 8). At excessively high levels, 29 nM, BMP7
downregulates PAI-1 promoter activity by ~50% below control levels
even in the absence of TGF-
(not shown).
|
BMP7 reduces TGF--induced PAI-1 levels in mesangial cell media.
Consistent with the findings in MLECs, incubation of mesangial cells
with TGF-
increases accumulation of PAI-1 protein in media (Fig.
9). This rise in accumulated PAI-1 was
substantially less in media from cells that were coincubated with
TGF-
in the presence of BMP7. These findings indicate that BMP7 also
blocks TGF-
-induced PAI-1 in mesangial cells, presumably also by
antagonizing the increase in PAI-1 transcription.
|
BMP7 blocks TGF--induced decrease in MMP2 levels in mesangial
cells.
Incubation of mesangial cells with TGF-
also substantially reduces
the levels of MMP2 that were recovered from media by precipitation with
heparin-Sepharose (Fig. 9). The reduction in secreted MMP2 was
prevented, in part, by coincubation with BMP7. In the MMP2 Western
blot, only a single band corresponding to ~70 kDa apparent molecular
weight band in the zymograms but not higher molecular weight forms was
found. This may suggest that perhaps only free but not complexed MMP2
was detected.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glomerular sclerosis and renal interstitial fibrosis are features
of most progressive renal diseases and cause renal failure. TGF- has
been recognized as an important profibrogenic agent in the kidney, and
its antagonism such as with administration of neutralizing antibodies
or soluble receptor decoy proteins ameliorates nephron fibrosis in
experimental glomerular and renal diseases (14, 32).
TGF-
contributes to progressive glomerular and interstitial
fibrogenesis by increasing gene expression of some ECM proteins, but a
major action of this cytokine is the reduction of degradation and
thereby increasing half-lives and, hence, progressive extracellular
deposition of matrix proteins (2, 26, 30). To this end, in
mesangial cells, TGF-
reduces MMP2 activation but also its levels as
shown in the present studies and in previous experiments by other
investigators (2). MMP2 contributes to the proteolytic
degradation of several different matrix proteins (including collagens,
elastin, FN, and laminin) and contributes to the proteolytic activation
of several other MMPs (5). Downregulation of MMP2
activity, therefore, contributes to accumulation of several different
ECM proteins. Moreover, maintenance of MMP2 activity by BMP7 may have
important antifibrogenic effects.
BMP7 is another member of the TGF- superfamily of cysteine-knot
growth factors. It plays major roles during renal and eye development
(8). In adults, BMP7 expression is very limited, and
tissues with greatest levels include the kidney (7, 22). Its function in the kidney is largely unknown. During progression of
chronic renal diseases such as diabetic nephropathy and obstructive nephropathy, renal BMP7 levels decrease substantially, which may be
caused, in part, by rising TGF-
(12, 29).
Exogenous administration of rhBMP7 to rats with experimental
obstructive nephropathy, streptozotocin-induced diabetic nephropathy, or other experimental renal diseases ameliorates the development of
glomerular sclerosis (7, 12, 13, 16, 18, 20). Fibrosis in
these models is, at least in part, TGF- dependent, which gives rise
to the possibility that BMP7 antagonizes profibrogenic actions of
TGF-
in the kidney.
The present experimental studies examine this hypothesis. Mesangial
cells are the pivotal cell type in the glomerulus elaborating the ECM
proteins that accumulate to progressive glomerular fibrosis in many
glomerular diseases, and FN and col IV contribute to glomerular scar
formation. TGF--dependent accumulation of these ECM proteins results
from an imbalance between increased production and/or reduced
metabolism of individual ECM proteins. Although the present studies use
an in vitro model of SV40-transfected murine mesangial cells, previous
observations from other laboratories strongly suggest that the findings
apply to the pathogenesis of glomerular fibrosis in vivo (21,
26).
The present in vitro experiments in cultured mesangial cells show that
the TGF--induced accumulation of FN, col IV, and TSP can be reduced
by coincubation with BMP7, suggesting an antifibrogenic role of the
latter protein. A major although perhaps not exclusive mode of action
of BMP7 is to block the TGF-
-induced downregulation of MMP2, i.e.,
to prevent TGF-
's reduction of ECM protein metabolism. MMP activity
is regulated by activators (uPA) that upregulate plasmin activity,
which, in turn, activates MMPs, and by tissue inhibitors of
metalloproteases that block MMPs. PAI-1 is a principal antagonist to
the activity of uPA, and the upregulation of its promoter activity by
TGF-
is a major mechanism through which TGF-
downregulates
glomerular ECM degradation (26). Increased transcription
of the PAI-1 gene by TGF-
is mediated by direct interaction of
TGF-
-activated smad3/4 complex with a smad response element in the
PAI-1 promoter (25). In the present studies, we tested
whether BMP7 inhibits this particular step in the profibrogenic chain
of actions of TGF-
. Indeed, BMP7 ameliorates the activation of PAI-1
transcription that normally occurs on incubation with TGF-
. This
likely causes or importantly contributes to the maintenance of
near-normal MMP activity despite the presence of TGF-
. This cytokine
has previously been shown to also downregulate MMP2 levels in mesangial
cells (24). Findings in the present studies confirm a
reduction of (free) MMP2 in media from TGF-
-conditioned cells (Fig.
9). Thus TGF-
may reduce both levels and PAI-1-driven activation of
MMP2, and both effects are apparently blocked by BMP7.
In addition to its effects on PAI-1 and MMP2, BMP7 also antagonizes
secretion of TSP and CTGF that is induced by TGF-. The moderate
reduction in CTGF may also mediate some of the antifibrogenic effects
BMP7. This may include TSP, which is, in part, regulated through CTGF
(27). Detailed mechanisms of how BMP7 may regulate CTGF
are not revealed by the present studies.
In summary, the present experimental studies indicate that BMP7
partially blocks TGF--induced CTGF as well as accumulation of col
IV, FN, and TSP in cultured mesangial cells. BMP7 antagonizes TGF-
-induced downregulation of MMP2 and increased transcription and
secretion of PAI-1, suggesting that BMP7 opposes downregulation of
matrix degradation by TGF-
. Thus BMP7 blocks several profibrogenic activities of TGF-
in mesangial cells.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors appreciate the kind gift of MLECs stably overexpressing PAI-1/luciferase from Dr. D. Rifkin, New York University, New York. rhBMP7 was a generous gift from Dr. J. McCartney, Curis, Cambridge, MA.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. Hirschberg, Research and Education Institute at Harbor-UCLA Medical Center, 1124 West Carson St., Torrance, CA 90502 (E-mail: rhirschberg{at}rei.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00382.2002
Received 22 October 2002; accepted in final form 14 January 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, M,
Harpel JG,
Metz CN,
Nunes I,
Loskutoff DJ,
and
Rifkin DB.
An assay for transforming growth factor- using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
Anal Biochem
216:
276-284,
1994[ISI][Medline].
2.
Baricos, WH,
Cortez SL,
Deboisblanc M,
and
Xin S.
Transforming growth factor- is a potent inhibitor of extracellular matrix degradation by cultured human mesangial cells.
J Am Soc Nephrol
10:
790-795,
1999
3.
Boström, K,
Tsao D,
Shen S,
Wang Y,
and
Demer LL.
Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells.
J Biol Chem
276:
14044-14052,
2001
4.
Bosukonda, D,
Shih MS,
Sampath KT,
and
Vukicevic S.
Characterization of receptors for osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7) in rat kidneys.
Kidney Int
58:
1902-1911,
2000[ISI][Medline].
5.
Briknarova, K,
Grishaev A,
Banyai L,
Tordai H,
Patthy L,
and
Llinas M.
The second type II module from human matrix metalloproteinase 2: structure, function and dynamics.
Structure Fold Des
7:
1235-1245,
1999[ISI][Medline].
6.
Chen, Y,
Blom IE,
Sa S,
Goldschmeding R,
Abraham DJ,
and
Leask A.
CTGF expression in mesangial cells: involvement of SMADs, MAP kinase, and PKC.
Kidney Int
62:
1149-1159,
2002[ISI][Medline].
7.
Dube, P,
Frazier K,
Charette M,
and
Paredes A.
Osteogenic protein-1 treatment induces tubular regeneration in the acute phase of the rat remnant kidney model (Abstract).
J Am Soc Nephrol
8:
614,
1997.
8.
Dudley, AT,
Lyons KM,
and
Robertson EJ.
A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye.
Genes Dev
9:
2795-2807,
1995[Abstract].
9.
Hegele, A,
Varga Z,
von Knobloch R,
Heidenreich A,
Kropf J,
and
Hofmann R.
TGF-1 in patients with renal cell carcinoma.
Urol Res
30:
126-129,
2002[ISI][Medline].
10.
Hirschberg, R.
Bioactivity of glomerular ultrafiltrate during heavy proteinuria may contribute to renal tubulo-interstitial lesions: evidence for a role for insulin-like growth factor I.
J Clin Invest
98:
116-124,
1996
11.
Houi, K,
Kobayashi T,
Kato S,
Mochio S,
and
Inoue K.
Increased plasma TGF-1 in patients with amyotrophic lateral sclerosis.
Acta Neurol Scand
106:
299-301,
2002[ISI][Medline].
12.
Hruska, KA,
Guo G,
Wozniak M,
Martin D,
Miller S,
Liapis H,
Loveday K,
Klahr S,
Sampath TK,
and
Morrissey J.
Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction.
Am J Physiol Renal Physiol
279:
F130-F143,
2000
13.
Ikeda, Y,
Jung Y,
Kim H,
Oda T,
Lopez-Guisa J,
Wing D,
Cai X,
McCartney J,
and
Eddy A.
Effects of BMP-7 on interstitial fibrosis in rats with overload proteinuria (Abstract).
J Am Soc Nephrol
11:
530A,
2000.
14.
Isaka, Y,
Akagi Y,
Ando Y,
Tsujie M,
Sudo T,
Ohno N,
Border WA,
Noble NA,
Kaneda Y,
Hori M,
and
Imai E.
Gene therapy by transforming growth factor- receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis.
Kidney Int
55:
465-475,
1999[ISI][Medline].
15.
Kothapalli, D,
Frazier KS,
Welply A,
Segarini PR,
and
Grotendorst GR.
Transforming growth factor induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway.
Cell Growth Differ
8:
61-68,
1997[Abstract].
16.
Lund, RJ,
Davies MR,
and
Hruska KA.
Bone morphogenetic protein-7: an anti-fibrotic morphogenetic protein with therapeutic importance in renal disease.
Curr Opin Nephrol Hypertens
11:
31-36,
2002[ISI][Medline].
17.
MacKay, K,
Striker LJ,
Elliot S,
Pinkert CA,
Brinster RL,
and
Striker GE.
Glomerular epithelial, mesangial, and endothelial cell lines from transgenic mice.
Kidney Int
33:
677-684,
1988[ISI][Medline].
18.
Morrissey, J,
Hruska K,
Guo G,
Wang S,
Chen Q,
and
Klahr S.
Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function.
J Am Soc Nephrol
13, Suppl1:
S14-S21,
2002
19.
Murphy, M,
Godson C,
Cannon S,
Kato S,
Mackenzie HS,
Martin F,
and
Brady HR.
Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.
J Biol Chem
274:
5830-5834,
1999
20.
Nadim, M,
Rennke H,
Taal M,
Brenner B,
Mackenzie H,
and
Sayegh M.
Bone morphogenetic protein-7 (BMP-7) reduces chronic progressive renal injury in 5/6 nephrectomized rats (Abstract).
J Am Soc Nephrol
11:
625A,
2000.
21.
Schafer, L,
Lorenz T,
Daemmrich J,
Heidland A,
and
Schaefer RM.
Role of proteinases in renal hypertrophy and matrix accumulation.
Nephrol Dial Transplant
10:
801-807,
1995[Abstract].
22.
Simon, M,
Maresh JG,
Harris SE,
Hernandez JD,
Arar M,
Olson MS,
and
Abboud HE.
Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.
Am J Physiol Renal Physiol
276:
F382-F389,
1999
23.
Singh, R,
Alavi N,
Singh AK,
and
Leehey DJ.
Role of angiotensin II in glucose-induced inhibition of mesangial matrix degradation.
Diabetes
48:
2066-2073,
1999[Abstract].
24.
Singh, R,
Song RH,
Alavi N,
Pegoraro AA,
Singh AK,
and
Leehey DJ.
High glucose decreases matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor-1.
Exp Nephrol
9:
249-257,
2001[ISI][Medline].
25.
Song, CZ,
Siok TE,
and
Gelehrter TD.
Smad4/DPC4 and Smad3 mediate transforming growth factor- (TGF-
) signaling through direct binding to a novel TGF-
-responsive element in the human plasminogen activator inhibitor-1 promoter.
J Biol Chem
273:
29287-29290,
1998
26.
Tomooka, S,
Border WA,
Marshall BC,
and
Noble NA.
Glomerular matrix accumulation is linked to inhibition of the plasmin protease system.
Kidney Int
42:
1462-1469,
1992[ISI][Medline].
27.
Wang, S,
DeNichilo M,
Brubaker C,
and
Hirschberg R.
Connective tissue growth factor in tubulo-interstitial injury of diabetic nephropathy.
Kidney Int
60:
96-105,
2001[ISI][Medline].
28.
Wang, S,
and
Hirschberg R.
Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis.
Am J Physiol Renal Physiol
278:
F554-F560,
2000
29.
Wang, S,
LaPage J,
and
Hirschberg R.
Loss of tubular bone morphogenetic protein-7 (BMP7) in diabetic nephropathy.
J Am Soc Nephrol
12:
2392-2399,
2001
30.
Wilson, HM,
Reid FJ,
Brown PA,
Power DA,
Haites NE,
and
Booth NA.
Effect of transforming growth factor-1 on plasminogen activators and plasminogen activator inhibitor-1 in renal glomerular cells.
Exp Nephrol
1:
343-350,
1993[ISI][Medline].
31.
Zeisberg, M,
Strutz F,
Mueller G,
and
Kalluri R.
Osteogenic protein-1 (OP-1), an inducer of tubulogenesis in kidney development, mediates anti-fibrogenic effects in vitro (Abstract).
Kidney Blood Press Res
23:
227,
2000.
32.
Ziyadeh, FN,
Hoffman BB,
Han DC,
Iglesias-De La Cruz MC,
Hong SW,
Isono M,
Chen S,
McGowan TA,
and
Sharma K.
Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor- antibody in db/db diabetic mice.
Proc Natl Acad Sci USA
97:
8015-8020,
2000