Renal-Electrolyte and Hypertension Division, Department of Medicine, and Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6144
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ABSTRACT |
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Transforming growth factor- (TGF-
) is important in
the pathogenesis of diabetic nephropathy, but little is known about the regulation of the ligand-binding TGF-
type II signaling receptor (T
IIR). There were significant increases in T
IIR protein and mRNA
levels in kidney cortex after 1-6 wk of streptozotocin-induced diabetes. Mouse mesangial cells cultured in high glucose demonstrated significantly increased T
IIR protein and mRNA levels compared with
normal glucose. This effect was independent of stimulation of TGF-
bioactivity by high glucose. Consistent with transcriptional activation
by high glucose, the half-life (~4 h) of T
IIR mRNA was not
affected by glucose concentration. Moreover, mouse mesangial cells
transiently transfected with reporter constructs containing the first
47- or 274-bp promoter fragments of T
IIR demonstrated significantly
increased reporter activity in high glucose. Cells grown in high
glucose demonstrated increased responsiveness to a relatively small
dose of exogenous TGF-
1 (0.5 ng/ml):
[3H]proline incorporation and
1(IV) collagen mRNA were significantly greater in cells
cultured in high than in normal glucose. Hence, the expression of
T
IIR is increased in the diabetic kidney and in mesangial cells
cultured in high glucose, primarily because of stimulation of gene
transcription. T
IIR upregulation by high ambient glucose may
contribute to the increased sensitivity of mesangial cells to the
profibrogenic action of TGF-
1.
diabetic nephropathy; glomerulosclerosis; type IV collagen; glomerulus; transforming growth factor-
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INTRODUCTION |
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DIABETIC NEPHROPATHY is the single most frequent cause
of end-stage renal disease, and hyperglycemia is a crucial factor in the development of diabetic kidney disease in susceptible individuals. Many in vitro studies using kidney-derived cells have demonstrated that
the cellular abnormalities seen in diabetic kidney disease, such as
cellular hypertrophy, enhanced extracellular matrix production, and
altered production of growth factors, can be reproduced by high ambient
glucose in the culture media (40). High glucose exerts its deleterious
effects by numerous pathways; in particular, the multifunctional
cytokine transforming growth factor- (TGF-
) has been implicated
as a principal mediator of diabetic nephropathy (2, 13, 28). We have
reported that high ambient glucose in proximal tubular cells and
mesangial cells stimulates expression and bioactivity of
TGF-
1, the most ubiquitous isoform in the mammalian
kidney (7, 22, 41). Elevated renal TGF-
1 mRNA and
protein levels have also been found in various animal models and the
human form of diabetic nephropathy (9, 25, 28, 36, 39). There is
compelling evidence that the prosclerotic and hypertrophic effects of
high ambient glucose in glomerular mesangial cells in culture are
mediated by increased production and activation of TGF-
1
(5, 33, 41), since they can be prevented by neutralizing anti-TGF-
antibodies (41).
Virtually all cell types produce one or more isoforms of TGF- and
also express TGF-
receptors. Three major types of TGF-
receptors
have been identified: type I (T
IR), type II (T
IIR), and type III
(T
IIIR). The latter lacks an identifiable cytoplasmic signaling
domain and is thought to act as a reservoir or capacitor of TGF-
on
the cell surface. T
IR and T
IIR are the signaling receptors that
belong to the transmembrane serine/threonine kinase receptor family.
T
IIR is the primary or ligand-binding receptor, because it forms a
high-affinity complex with TGF-
and then binds the type I receptor,
resulting in phosphorylation of T
IR protein by the type II receptor
kinase and subsequent transduction of the signal (14, 35).
Modulation of the expression of TIR and T
IIR is associated with
altered cellular responses in disease states. For instance, reduced
expression of these receptors contributes to the loss of sensitivity to
TGF-
and the increased proliferation of some cancer cells (10, 31),
whereas upregulation of T
IIR is associated with increased
sensitivity to TGF-
-mediated growth inhibition (20). Receptor
upregulation has been reported in animal models of glomerulosclerosis:
T
IR and T
IIR are increased in experimental membranous
nephropathy, and T
IIR and T
IIIR are increased in adriamycin-induced nephropathy (24, 30). In different forms of human
glomerulonephritis, there is increased T
IIR expression in the
sclerotic lesions (37). We previously reported that the early
development of diabetic renal hypertrophy in streptozotocin (STZ)-induced diabetes in mice is likely linked to the coordinate upregulation of TGF-
1 and T
IIR in the kidney (27).
These observations suggest the possibility that increased T
IIR
expression in the kidney may enhance the responsiveness to TGF-
and
contribute to extracellular matrix accumulation.
The aim of our study was to examine the expression of TIIR protein
and mRNA levels in the diabetic mouse kidney and to define the role of
high ambient glucose in the regulation of T
IIR in cultured mesangial
cells. To further clarify the biological significance of T
IIR
upregulation, we examined collagen type IV expression and the relative
responsiveness of the cells to exogenous TGF-
1 in
normal- vs. high-glucose media. We report that the expression of
T
IIR is increased in the diabetic kidney and in mesangial cells
cultured in high-glucose media, primarily because of stimulation of
gene transcription, and that T
IIR upregulation by high glucose may
contribute to the increased sensitivity of mesangial cells to the
profibrogenic action of TGF-
1. The data are consistent with the notion that the upregulation of TGF-
1 and
T
IIR by high ambient glucose in kidney cells is correlated with the
increased bioactivity of the TGF-
system in diabetic nephropathy.
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RESEARCH DESIGN AND METHODS |
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Experimental animals.
C57Bl female mice were fed a standard pellet laboratory chow and were
provided with water ad libitum. Diabetes was induced in weight-matched
8-wk-old mice by two consecutive daily injections of STZ (200 mg/kg ip;
Sigma Chemical, St. Louis, MO) dissolved in 10 mmol/l sodium citrate,
pH 5.5; controls were injected with buffer alone. This high dose of STZ
is required to induce diabetes in mice (27), as opposed to rats. Once
glucosuria was detected, 0.3-0.5 U NPH insulin (Eli Lilly,
Indianapolis, IN) was administered daily to prevent ketonuria and
maintain the blood glucose concentration at near the moderately
hyperglycemic level of 25 mmol/l. In an additional group of diabetic
mice, the insulin dose was increased up to 1.0 U daily to maintain the
blood glucose concentration at around the normal level of ~6 mmol/l.
Groups of diabetic and control mice were killed 1-6 wk after the
detection of glucosuria. The kidney cortex was excised, immediately
frozen in liquid nitrogen, and stored at 70°C for subsequent
RNA and protein extraction.
Cell culture.
Murine mesangial cells (MMCs) were isolated and transformed with
non-capsid-forming SV-40 virus to establish a permanent cell line with
a stable, differentiated phenotype, including the typical spindle-like
appearance, positive staining for vimentin and desmin, and contraction
in response to ANG II and expression of AT1 receptor (33,
41). The cells were maintained in DMEM (GIBCO BRL, Gaithersburg, MD)
containing a normal D-glucose concentration of 5.5 mmol/l, 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/l glutamine (42). The cells were incubated in a humidified atmosphere of
5% CO2 at 37°C and passaged every 3-4 days by
trypsinization. Subconfluent cells were made quiescent by reducing the
concentration of FCS to 0.5% for 24 h. The glucose concentration of
experimental conditions was increased to 25 mmol/l by the addition of a
small volume of 1.0 mol/l D-glucose. Cells were cultured
for different time periods in DMEM containing 5.5 or 25 mmol/l glucose.
In some experiments, cells were incubated with a rabbit neutralizing
panselective anti-TGF- antibody (R & D Systems, Minneapolis, MN) or
purified rabbit IgG (Sigma Chemical). Recombinant human
TGF-
1 protein (R & D Systems) was used to examine the
effect of exogenous TGF-
1.
Northern blot analysis.
Total RNA (20 µg) was electrophoresed through a 1.2% agarose gel
with 0.67 mol/l formaldehyde, blotted onto Gene-Screen Plus nylon
membranes (NEN Research Products, Boston, MA) by the capillary method,
and subjected to ultraviolet cross-linking (17). Membranes were
prehybridized for 1 h at 65°C in a buffer containing 10% dextran
sulfate, 1% SDS, and 1 mol/l NaCl. Murine TIIR and
1(IV) collagen cDNA probes were synthesized by PCR, with
mouse kidney cDNA as template, and then cloned into the pCRII TA
cloning system (Invitrogen, La Jolla, CA). Nucleotide sequencing of the
probes confirmed their identity. The cDNA inserts were separated from the plasmid in low-melt agarose and labeled with
32P-labeled dCTP (3,000 Ci/mmol; Amersham Pharmacia,
Piscataway, NJ) with use of a DNA labeling kit (Amersham Pharmacia).
The membranes were hybridized with 1 × 106 cpm/ml
probe in hybridization buffer (same as prehybridization buffer) for 16 h at 65°C. The membranes were washed for 10 min twice in 2×
saline-sodium citrate (SSC; 20× SSC = 3 mol/l NaCl, 0.3 mol/l
sodium citrate, pH 7.0) at room temperature and in 2× SSC with
0.1% SDS for 15 min at 65°C; then the membranes were subjected to
two 15-min high-stringency washes in 0.1% SSC-0.1% SDS at 65°C if
necessary. The membranes were autoradiographed with intensifying
screens at
70°C (Kodak, Wilmington, DE). Blots were stripped and rehybridized with a probe encoding mouse ribosomal protein L32 (mrpL32) (16) to account for small loading and transfer variations. Exposed films were scanned with a densitometer (Hoefer Scientific Instruments, San Francisco, CA), and RNA levels relative to
those of mrpL32 were calculated.
Immunoblot analysis.
Kidney tissue and cells were homogenized in a lysis buffer containing
50 mmol/l Tris · HCl (pH 8.0), 150 mmol/l NaCl, 1%
NP-40, 0.1% SDS, 1 mmol/l EDTA, 0.5 mmol/l dithiothreitol, 1 mmol/l
phenylmethylsulfonyl fluoride, and 5 µg/ml each of aprotinin and
leupeptin. Protein concentrations of samples were quantitated by
protein assay kit (Bio-Rad, Hercules, CA). For immunoblots, 30 µg of
samples were subjected to SDS-PAGE (12% acrylamide gel). After
electrophoretic transfer to nitrocellulose membrane and blocking, the
membranes were incubated with rabbit antibody directed against TIIR
protein (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 3 h. A horseradish peroxidase-conjugated anti-rabbit IgG was used
to allow the detection of immunoreactive bands with the enhanced chemiluminescence detection system (Amersham Pharmacia). Equal loading
and transfer of protein samples were assessed by staining with Ponceau
S. Blocking experiments to determine the specificity of the T
IIR
immunoreactive bands were performed using blocking peptide (Santa Cruz
Biotechnology). Densitometric analysis was performed as described above.
Message stability assay.
Quiescent MMCs were cultured for 48 h in DMEM containing 5.5 or 25 mmol/l glucose. The cells were then treated with 5 µg/ml actinomycin
D (Sigma Chemical) to inhibit transcription, and total RNA was
extracted after specified time periods (between 0 and 10 h). Northern
analysis was performed using TIIR and mrpL32 as probes. The ratio of
T
IIR to mrpL32 in normal glucose media at time 0 (i.e.,
before actinomycin D treatment) was assigned a relative value of 100%.
Chloramphenicol acetyltransferase constructs, transfection, and
chloramphenicol acetyltransferase assay.
Human TIIR promoter-reporter chimeric constructs were kindly
provided by Dr. S.-J. Kim (National Cancer Institute, Bethesda, MD).
The chloramphenicol acetyltransferase (CAT) constructs employed were
pT
IIR47 and pT
IIR274 containing
47 and
274 bp from
the transcription start site of the human T
IIR gene (1). The
-galactosidase-containing plasmid pCH110 was used to control for
transfection efficiency. The transient transfection method was
essentially as previously described (42). Briefly, 10 µg of test
plasmid and 5 µg of pCH110 were used to transfect MMCs by use of the
calcium phosphate DNA precipitation technique. After 16 h of incubation
in 5% CO2 at 37°C, the medium was removed, cells were
washed twice with PBS, and fresh serum-free DMEM containing 5.5 or 25 mmol/l glucose was added for an additional 48 h. Cells were harvested
in TEN buffer (40 mmol/l Tris · HCl, pH 7.5, 1 mmol/l
EDTA, 150 mmol/l NaCl), lysed with three cycles of freeze-thaw, and
spun down, and the supernatant was assayed for
-galactosidase
activity (26). On the basis of equivalent amounts of
-galactosidase
activity, the cell lysates were added to initiate the CAT enzyme assay
with use of [14C]chloramphenicol (Amersham
Pharmacia), as previously described (7, 42). CAT enzyme activity was
expressed as percentage of acetylated chloramphenicol compared with
total chloramphenicol.
Measurement of [3H]proline incorporation.
Cells (105/well) were plated into 24-well plates (Nunclon),
and the media were changed the next day so that cells could be exposed
for another 72 h to 5.5 or 25 mmol/l glucose.
[3H]proline incorporation was then determined
to test the responsiveness of the cells to exogenous
TGF-1. For this purpose, all media were changed to fresh
DMEM containing 5.5 mmol/l glucose with or without the addition of
TGF-
1 (0.5 ng/ml) for 24 h. For the last 16 h, cells
were pulsed with 1 µCi of [3H]proline
{L-(2,3,4,5)-[3H]proline;
Amersham Pharmacia}. Radiolabeled MMCs were washed twice in
ice-cold PBS and precipitated twice in ice-cold 10% TCA redissolved in
0.5 ml of 0.5 N NaOH with 0.1% Triton X-100. After neutralization with
0.5 N HCl, the incorporated radioactivity was counted in a liquid
scintillation counter. Proline incorporation was corrected for the
protein content of cells and expressed as counts per million per
microgram of cellular protein.
Statistical analysis. Values are means ± SE. ANOVA followed by Scheffé's test was used for multiple comparisons. Two groups were compared by Student's unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Renal TIIR expression in diabetic mouse.
We first examined T
IIR protein and mRNA expression in the kidney of
STZ-induced diabetic mice. The characteristics of the experimental mice
are presented in Table 1. The plasma
glucose levels of diabetic mice were maintained at ~25 mmol/l. The
body weights were significantly smaller in diabetic than in control mice. The kidney-to-body weight ratios were also significantly greater
in diabetic than in control mice. All these changes were normalized
when sufficient insulin was given daily for 3 wk to keep the blood
glucose near the normal level (~6 mmol/l).
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TIIR expression in MMCs cultured in high glucose.
To explore the role of high ambient glucose in T
IIR expression, we
opted to utilize cultured mesangial cells as a study system given the
central role of these cells in the development of diabetic glomerulosclerosis (11, 40). For immunoblot analysis, protein was
isolated from MMCs cultured under normal (5.5 mmol/l)- or high (25 mmol/l)-glucose conditions for 72 h, and the blot was probed with a
polyclonal rabbit anti-T
IIR antibody. Figure
3 demonstrates that T
IIR protein level
was significantly increased in cells cultured in high ambient glucose
compared with normal glucose concentration. In parallel studies,
Northern blot analysis also showed significant increases in T
IIR
mRNA levels in MMCs cultured for 48 or 72 h in high-glucose media (Fig.
4).
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TIIR message stability and ambient glucose
concentration.
To determine whether the increased T
IIR mRNA level is
transcriptional in origin or is secondary to increased
posttranscriptional stability, Northern blot analysis was performed in
the presence of an inhibitor of gene transcription. MMCs were first
grown in normal- or high-glucose media for 48 h until the steady-state T
IIR mRNA level was increased by the high-glucose media, and then
the cells were exposed to actinomycin D (5 µg/ml) for up to 10 h,
which results in a progressive decay in message level over time. Figure
5 shows that the rate of decay of the
T
IIR message was of equivalent magnitude in cells grown in normal- and high-glucose media, and the message half-life was ~4 h in normal-
and high-glucose media. Thus the increase in the steady-state level of
T
IIR mRNA induced by high ambient glucose in mesangial cells is
unlikely to be due to increased mRNA stability, although a small
component of message stabilization cannot be excluded.
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TIIR promoter activity in mesangial cells.
To confirm that the increase in the steady-state level of T
IIR mRNA
by high ambient glucose is likely due to increased gene transcription
rate, we performed a reporter assay using chimeric T
IIR promoter/CAT
reporter constructs. MMCs were transiently transfected with the human
promoter constructs pT
IIR47 and pT
IIR274. To account for
transfection efficiency, the cells were also transfected with the
-galactosidase-containing plasmid pCH110. On the next day the media
were changed to 5.5 or 25 mmol/l glucose, and the cells were harvested
48 h later. As shown in Fig. 6, there was a
significant stimulation in T
IIR promoter activity as reflected by
CAT activity when the cells were transfected with pT
IIR47 or
pT
IIR274 constructs and then grown for 48 h in high-glucose media.
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Effect of addition of anti-TGF- antibody or
exogenous TGF-
1 on T
IIR
mRNA level.
High glucose concentration is known to increase TGF-
1
level and bioactivity in mesangial cells (33), and previous studies have been conflicting as to whether TGF-
1 can modulate
the expression of its receptors; exogenous TGF-
1
modestly inhibited the expression of T
IIR mRNA in rat mesangial
cells (3), but there was no change in T
IIR in the
TGF-
1 transgenic mouse (19). To assess the contribution
of endogenous TGF-
1 production in mesangial cells under
high-glucose conditions, we examined the effect of a neutralizing
panselective anti-TGF-
antibody on T
IIR expression. Cells were
incubated with control IgG or anti-TGF-
antibody and exposed for 72 h to normal- or high-glucose media. As shown in Fig.
7A, the increased T
IIR
expression in high glucose was largely unaffected by the addition of
anti-TGF-
antibody. This suggests that high glucose is capable of
increasing the mRNA level of T
IIR independent of the influence of
endogenous stimulation of TGF-
bioactivity. However, addition of
exogenous TGF-
1 inhibited the expression of T
IIR,
especially at a high dose of 2 ng/ml, in cells grown in basal media
containing a normal glucose concentration (Fig. 7B), and in
accord with previous results in rat mesangial cells (3).
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Sensitivity to exogenous TGF-1 protein.
We next examined the possibility that increased expression of T
IIR
is related to increased sensitivity of mesangial cells to exogenous
TGF-
1 protein. To test for enhanced TGF-
1
action, [3H]proline incorporation and type IV
collagen gene expression were measured in MMCs that were grown in 5.5 or 25 mmol/l glucose and then stimulated with a submaximal dose of
TGF-
1 (0.5 ng/ml). Proline incorporation (Fig.
8A) was significantly increased in cells grown in high glucose, as would be expected (41). Addition of 0.5 ng/ml TGF-
1 stimulated proline incorporation into MMCs grown in normal- or high-glucose media, but the extent of this increase
was significantly greater in high-glucose media (average increase of
51% in 25 mmol/l glucose vs. 24% in 5.5 mmol/l glucose; Fig.
8A). Similar results were obtained for type IV collagen
expression. Figure 8B shows that
1(IV) collagen
mRNA was increased to a greater extent by treatment with
TGF-
1 if the cells were grown in high rather than normal
glucose concentration.
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DISCUSSION |
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Our study is the first to demonstrate a significant increase in TIIR
protein and message levels in the kidney of STZ-induced diabetes
mellitus in the mouse and in glomerular mesangial cells cultured under
high-glucose conditions. The upregulation of T
IIR in mesangial cells
is transcriptional and is not due to increased mRNA stability. In
addition, the functional counterpart of the upregulated T
IIR is an
enhanced sensitivity to the stimulation by TGF-
1, since
the incorporation of [3H]proline and the
expression of
1(IV) collagen mRNA are significantly greater in mesangial cells cultured in high than in normal glucose concentration.
The single most important factor in the development of diabetic kidney
disease is hyperglycemia (18). In general, the effects of high glucose
have been attributed to metabolic alterations related to several
humoral and biochemical processes. Because TGF- has been widely
recognized to be a major promoter of extracellular matrix production
(40), we have postulated that this autacoid may mediate the stimulatory
effects of high glucose on the synthesis of extracellular matrix in
mesangial cells (28). In MMCs, we previously demonstrated that the
addition of neutralizing anti-TGF-
antibody markedly attenuates the
prosclerotic and hypertrophic effects of high ambient glucose (41). In
STZ-induced diabetic kidney disease in mice, we also demonstrated that
inhibition of TGF-
activity with neutralizing anti-TGF-
antibody
leads to significant attenuation of kidney hypertrophy and increased
mRNA levels encoding
1(IV) collagen and fibronectin
(27). Ample evidence has been gathered to support the notion that
increased renal expression of TGF-
1 plays an important
role in experimental models of diabetic kidney disease as well as in
patients with established diabetic nephropathy (9, 18, 25, 29, 36, 39).
However, increased TGF-
bioactivity may be a result of the
upregulation of the ligand itself and/or of its signaling receptors.
Unlike the situation with TGF-
1, little is known about the regulation in the diabetic state of the signaling receptors for
TGF-
1.
In our study we found a sustained increase in the protein and mRNA
levels of TIIR in the kidney after 1-6 wk of diabetes in the
STZ mouse model. The cellular localization of this effect in the kidney
cannot be inferred, but it may include the tubular compartment. Our
mesangial cell culture studies are in accord with a very recent report
that demonstrated that mechanical stretch, especially in conjunction
with high-glucose media, can amplify the expression of TGF-
receptors in rat mesangial cells (21). Previous studies in
LLC-PK1 tubular epithelial cells (6) and Madin-Darby canine
kidney epithelial cells (38) demonstrated increased TGF-
receptor
protein binding in high glucose. In preliminary studies using mouse
proximal tubular epithelial cells, we found a ~50% increase in
T
IIR mRNA when the cells were cultured in high-glucose media for 48 or 72 h (data not shown). However, whether the diabetic state can
further upregulate T
IIR mRNA level in tubular epithelia in vivo may
depend on ambient factors in addition to hyperglycemia, such as ANG II
(34).
Upregulation of TGF- receptors in other experimental animal models
of glomerulosclerosis has been reported. In passive Heymann nephritis
in rats, a model of membranous nephropathy, levels of T
IR and
T
IIR protein and mRNA were increased in glomeruli, and these changes
correlated with fibrotic lesions (25). Tamaki et al. (30) also reported
an increase in T
IIR and T
IIIR in adriamycin-induced nephropathy
with parallel changes in TGF-
1 and fibronectin in the
renal cortex. In addition to these observations, increased levels of
T
IR, T
IIR, and T
IIIR have been reported in the sclerotic
lesions of human glomerulonephritis such as IgA nephropathy and lupus
nephritis (37). These results and our observations suggest that the
increase in receptors for TGF-
1 as well as the
upregulation of TGF-
1 itself might play an important role in various diseases causing renal fibrosis.
We investigated the basis for the stimulation of TIIR expression by
high ambient glucose. By applying actinomycin D, we showed that this
stimulation is not due to an increase in message stability. In fact,
the half-life (~4 h) of T
IIR mRNA in mesangial cells was not
affected by the glucose concentration in culture. In addition, use of
CAT/T
IIR promoter constructs in a reporter assay provided further evidence for transcriptional activation of T
IIR by high ambient glucose.
Although little is known about the mechanisms regulating TIIR, it is
likely that AP-1 and/or Sp-1 binding sites in the T
IIR promoter are
important for stimulation of gene transcription by high glucose. The
CAT constructs that we used contain an Sp-1 binding site in pT
IIR47
and an AP-1 (or cAMP-response element) site in addition to Sp-1 sites
in pT
IIR274 (1, 8). It has been reported that high glucose
stimulates AP-1 binding and expression of c-fos and
c-jun, components of the AP-1 complex, in cultured mesangial
cells (11, 32). Sp-1 has also been shown to mediate expression of the
acetyl-CoA carboxylase by high glucose (4). Longer promoter segments as
well as additional studies are required to establish a link between
increased activity of cis-acting glucose-response elements and
transcriptional activation of the T
IIR promoter by high glucose.
Our observations demonstrating that anti-TGF- antibody failed to
significantly modify T
IIR mRNA levels suggest that the stimulatory
effects of high glucose on receptor expression are independent of the
associated stimulation of endogenous TGF-
1 bioactivity.
This finding is also supported by our previous report showing that
treatment with anti-TGF-
antibody in diabetic mice failed to
significantly inhibit the renal T
IIR expression (27). Moreover,
Mozes et al. (19) recently reported no changes in T
IIR in the
TGF-
1 transgenic mouse (19). However, these findings do
not rule out an inhibitory effect of high concentrations of TGF-
1 on T
IIR expression. We showed in the present
study that sufficiently high concentrations of TGF-
1, up
to 2 ng/ml, can dampen the level of T
IIR mRNA in cells grown in
normal-glucose media.
The number of TIIR may be regulated by a variety of hormones and
growth factors. In rat prostate epithelial cells, T
IIR number is
negatively regulated by androgens (12). In IK-90, a human endometrial
cancer cell line, epidermal growth factor increases
TGF-
1 binding sites (23). Modulation of the receptor is
associated with changes in the sensitivity to TGF-
1
protein. For instance, reduced expression of T
IR and/or T
IIR in
some cancer cell lines contributes to the abnormal cell growth (10, 31). On the other hand, stimulation of collagen synthesis by TGF-
1 is enhanced in vascular smooth muscle cells from
atherosclerotic lesions that exhibit a high T
IR-to-T
IIR ratio
(15). Furthermore, regenerating liver cells, which express more TGF-
receptors than resting cells, exhibit high sensitivity to growth
inhibition by TGF-
1 (20). From these observations and
several in vivo studies demonstrating increased expression of TGF-
receptors in experimental renal disease (24, 30, 37), we hypothesize
that the coordinate upregulation of TGF-
receptors, as well as of
TGF-
1 itself (7, 33), may play an important role in
progressive renal fibrosis.
In this study we examined whether high ambient glucose, in addition to
its known upregulating effect on TGF-1 protein and message (33, 41), would also increase the sensitivity of mesangial cells to exogenous TGF-
1 treatment in conjunction with
the increased T
IIR. Indeed, we found that cells grown in high
glucose exhibit greater responsiveness than cells grown in normal
glucose, as assessed by [3H]proline
incorporation and type IV collagen expression. These observations
suggest that receptor upregulation by high glucose in mesangial cells
leads to increased responsiveness to TGF-
1 and, in turn,
promotes the fibrotic response. Thus the increased production of
TGF-
1 in the diabetic state and the greater abundance of
T
IIR in renal cells exposed to high ambient glucose provide an
explanation for the heightened bioactivity of the TGF-
system and
the resultant tissue injury in diabetic nephropathy.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Jia Guo for technical assistance.
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FOOTNOTES |
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* M. Isono and A. Mogyorósi contributed equally to this work.
This study was supported in part by Grants DK-44513, DK-45191, and DK-54608 and Training Grant DK-07006 from the National Institute of Diabetes and Digestive and Kidney Diseases. M. Isono and A. Mogyorósi are supported by fellowships from the Juvenile Diabetes Foundation International. D. C. Han is a visiting scholar at the University of Pennsylvania and is supported by the Korean Research Foundation and the Hyonam Kidney Laboratory at Soon Chun Hyang University Hospital (Seoul, Korea). B. B. Hoffman is supported by an Individual National Research Service Award from the National Institutes of Health.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. N. Ziyadeh, 700 Clinical Research Bldg., Renal-Electrolyte and Hypertension Div., University of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104-6144 (E-mail: ziyadeh{at}mail.med.upenn.edu).
Received 17 May 1999; accepted in final form 7 December 1999.
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