Glycated albumin stimulation of PKC-
activity is linked to
increased collagen IV in mesangial cells
Margo P.
Cohen1,
Fuad N.
Ziyadeh2,
Gregory T.
Lautenslager1,
Jonathan A.
Cohen1, and
Clyde W.
Shearman1
1 Institute of Metabolic
Research and Exocell, University City Science Center; and
2 Renal-Electrolyte and
Hypertension Division, Department of Medicine, and Penn Center for the
Molecular Studies of Kidney Diseases, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
Albumin modified by Amadori-glucose adducts
induces coordinate increases in the expression of extracellular matrix
proteins, transforming growth factor (TGF)-
1, and the TGF-
type
II receptor in glomerular mesangial cells. Because activation of
protein kinase C (PKC) accompanies the increased mesangial cell
expression of matrix proteins and TGF-
1 induced by high ambient
glucose, we postulated that glycated albumin (GA) modulates PKC
activity and that PKC participates in mediating the GA-induced
stimulation of matrix production. To test this hypothesis, we examined
the effects of PKC inhibitors on collagen type IV production by mouse or rat mesangial cells incubated with GA, and the influence of GA on
PKC activity in these cells. Increased collagen type IV production
evoked by GA in 5.5 and 25 mM glucose in mouse mesangial cells was
prevented by both general (GF-109203X) and
-specific (LY-379196) PKC
inhibitors. Total PKC activity, measured by phosphorylation of a
PKC-specific substrate, increased with time after exposure of rat
mesangial cells to GA compared with the nonglycated, glucose-free counterpart. GA caused an increase in PKC-
1 membrane-bound fraction and in total PKC activity in media containing physiological (5.5 mM)
glucose concentrations in rat mesangial cells, confirming that the
glucose-modified protein, and not a "hyperglycemic" milieu, was
responsible. The findings indicate that Amadori-modified albumin stimulates mesangial cell PKC activity, and that activation of the
PKC-
isoform is linked to the stimulation of collagen type IV production.
glucose; protein kinase C; amadori; glycation; diabetic nephropathy
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INTRODUCTION |
WE HAVE BEEN INVESTIGATING the pathobiology of diabetic
nephropathy, with particular emphasis on the role of nonenzymatically glycated albumin (GA) and its molecular mediators in the accumulation of extracellular matrix, which is characteristic of this complication of diabetes. GA is formed from a condensation reaction between glucose
and reactive protein amino groups, yielding stable Amadori-glucose adducts in a deoxyfructosyllysine construct (5). This modification confers biological properties to the glycated protein that are not
possessed by its nonglycated counterpart and that can modulate glomerular cell function. Glomerular mesangial and endothelial cells
incubated in concentrations of GA that are found in clinical specimens
manifest increased gene expression of the extracellular matrix proteins
collagen type IV and fibronectin (9, 12, 13, 38). Further, GA induces
increased mesangial cell expression and bioactivation of the fibrogenic
transforming growth factor (TGF)-
1 and its primary signaling
receptor, the TGF-
type II receptor, thus linking activation of the
TGF-
system to the glycated protein-induced stimulation of
extracellular matrix production (41). Additional evidence supporting a
role for GA in the genesis of matrix overproduction in diabetes derives
from experiments in which diabetic
db/db mutant mice were treated with
monoclonal antibodies (A717) that specifically neutralize excess plasma
concentrations of GA. This protocol prevented renal cortical
overpression of mRNAs encoding
1(IV) collagen and fibronectin,
reduced mesangial matrix expansion, and improved changes reflecting
compromised renal function (6, 8, 10). These salutary effects were observed despite marked and persistent hyperglycemia.
Recent studies have implicated the activation of protein kinase C (PKC)
in the abnormalities in glomerular cell function associated with
diabetes (16). PKC is a ubiquitous family of related intracellular serine-threonine kinases that are involved in signal transduction pathways used by cells to respond to various extracellular stimuli, and
in the regulation of diverse cellular functions, such as contractility, proliferation, and hemodynamic control (32). Evidence suggesting a role
for PKC in the pathogenesis of diabetic nephropathy includes the
observations that mesangial cells cultured in high media glucose concentration exhibit increased PKC activity and a cytosol-to-membrane shift of various classical PKC isoforms that is associated with activation (2, 29), and that glomeruli from diabetic rodents exhibit
elevated PKC activity and membrane translocation (3, 16, 26, 30).
Increased activity of PKC in mesangial cells is associated with
increased expression of TGF-
1 (15, 23) and of the extracellular
matrix proteins fibronectin, laminin, and collagen type IV (15, 17, 30,
35, 40). Inhibition of PKC-
isoforms in streptozotocin-diabetic rats
has been reported to ameliorate glomerular hyperfiltration, increased
urine albumin excretion (26), and the stimulation of gene expression
for TGF-
1 and matrix molecules in the kidney (30).
Because the alterations in glomerular cell biology induced by GA
resemble those evoked by high media glucose concentration, we
postulated that GA might also activate PKC isoforms in glomerular mesangial cells. To test this hypothesis, we examined the effects on
PKC activity of Amadori-modified albumin, the principal form in which
GA exists in vivo (5, 13). We report that PKC activity increases when
mesangial cells are incubated in concentrations of Amadori-modified GA
that are found in clinical specimens, even when the glucose
concentration in the culture media is normal (5.5 mM), and that PKC
signaling, particularly through the PKC-
isoform, participates in
the increased collagen type IV production by these cells on exposure to GA.
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MATERIALS AND METHODS |
Cell culture.
Mesangial cells in culture were derived from glomeruli isolated with a
graded sieving technique and plated to explant. Mesangial cells were
selected and subcultured according to previously described criteria
(13), including morphology, ability to grow in medium containing
D-valine instead of
L-valine, and the presence of
cytoplasmic filaments (desmin and vimentin), angiotensin II binding
capacity, and contractile response to angiotensin II. Phenotypically
stable nontransformed rat (RMC) or SV40-transformed murine mesangial cells (MMC) were grown in DMEM containing 10 mM glucose and 10% FCS.
MMC were employed for studies of collagen synthesis because their
parallelism with RMC, with respect to growth and response to elevated
glucose and GA media manipulations, has been documented (13, 38,
41). RMC were employed for studies of PKC activity and
immunoreactivity because preliminary experiments indicated that these
cells yielded reproducible and reliable measurements with the PKC assay
and immunoblotting techniques. To initiate experiments for collagen
synthesis, we seeded 2 × 105
cells into 48-well microtiter plates, allowed them to attach, rested
them for 24 h in serum-free media containing 10 mM glucose, and then
grew them for 48 h in fresh media under the experimental conditions
described below. For assay of PKC activity, 2 × 105 mesangial cells were seeded
into 24-well microtiter plates. After attachment, the cells were grown
for 24 h in DMEM/10 mM glucose/2% FCS. The media were then changed to
1% FCS containing 5.5 or 25 mM glucose, and the cells were grown for
6-48 h under the described experimental conditions.
Experimental culture conditions.
The experimental conditions were introduced on the addition of fresh
media containing 5.5 mM or 25 mM glucose, without or with the described
supplements in the indicated concentrations. Media supplements
consisted of purified glycated or nonglycated albumin (250-600
µg/ml) and the general PKC inhibitor GF-109203X (500 nM) or the
selective PKC-
inhibitor LY-379196 (100 nM) (a gift to F.N. Ziyadeh
from Eli Lilly). LY-379196 was added at a 100-nM concentration, because
-specific inhibitory activity may be lost if this concentration is
exceeded (ED50, ~600 nM for nonspecific PKC inhibition;
ED50, ~30 nM for selective PKC-
inhibition) (28). For
studies of collagen production, media also contained 50 µg each of
L-ascorbic acid and
-aminopropionitrile (43). In the presence of
-aminopropionitrile,
very little collagen type IV synthesized by mesangial cells in culture
is cell associated, and ~80% is recovered in the media (22). The
concentrations of GA used in these studies have been shown to stimulate
glomerular endothelial and mesangial cell expression of collagen type
IV, fibronectin, and TGF-
1 (9, 12, 39, 41), and represent those
found in clinical specimens. In nondiabetic individuals, ~1% of
serum albumin is in the glycated form, which is equivalent to
concentrations of 300-400 µg/ml of GA. The concentration of GA
is increased one-and-a-half- to threefold in diabetic subjects, according to recent glycemic status (5, 7).
Glycated protein preparation.
GA was prepared from human albumin that was purified by chromatography
on Affi-gel Blue and DEAE-Sepharose, and incubated for 5 days at
25°C in buffered saline containing 500 mg/dl (27.8 mM) glucose.
After dialysis to remove free glucose, the glycated species were
separated from nonglycated albumin by affinity chromatography on
phenylboronate agarose (PBA), which binds Amadori adducts and not
advanced glycation end products (AGE). This protocol has been shown to
yield GA containing ~1 mol glucose/mol albumin and in which glycated
moieties are represented as deoxyfructosyllysine residues (9, 39). The
PBA pass through, used as source material for nonglycated albumin in
these experiments, contained <0.05 mol glucose/mol albumin. The
purified glycated and nonglycated albumin preparations migrated on
SDS-PAGE as homogeneous bands of ~66 kDa, and had distinct mobilities
on agarose gel electrophoresis, wherein the glycated protein exhibited
greater electronegativity consequent to glycation of lysine amino groups.
Collagen type IV measurement.
Media were collected at the end of the experimental period and the
cells were harvested for counting in a cell chamber. We measured
collagen type IV by competitive ELISA (13, 39) using collagen type IV
from an Engelbreth-Holm-Swarm (EHS) tumor as standard, rabbit
anti-mouse collagen type IV as primary antibody (both from
Collaborative Research), and horseradish peroxidase-conjugated goat
anti-rabbit IgG (Bio-Rad) for development. The assay is sensitive to 5 ng/well.
PKC-
immunoreactivity.
A modification of the method described by Thomas et. al (36) was used
for analysis of immunoreactive PKC-
in particulate (membrane-associated) and cytosolic fractions prepared from RMC. After
washing the cells three times with ice-cold PBS, we added 600 µl of
ice-cold homogenization buffer [10 mM
Tris · HCl, pH 7.5, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mM EGTA, and 25 µg/ml
leupeptin] to each sample. Cells were lifted with a cell scraper
and transferred, in the buffer, to a homogenizer. The homogenized
preparations were centrifuged at 2,000 rpm for 5 min at 4°C. The
supernatants were separated from undisrupted cellular elements
remaining in the pellet and centrifuged at 100,000 g at 4°C for 1 h. The supernatants
from this centrifugation were retained as the cytosolic fractions, and
the pellet was suspended in the above homogenization buffer, made 1%
in Triton X-100, and shaken for 30 min at 4°C to solubilize
particulate proteins. This material was then centrifuged
at 10,000 rpm for 1 h at 4°C, and the supernatants were retained as
the particulate fractions. Protein was measured with the Bio-Rad
(Hercules, CA) protein assay.
Immunoblotting was performed according to described techniques (1),
with some modifications. Samples were electrophoresed on 8% SDS-PAGE
and transferred to nitrocellulose membranes. Prestained molecular
weight markers were electrophoresed in parallel. Transfers were blocked
in a solution of 5% nonfat milk, 5 mM Tris, pH 7.5, 200 mM NaCl, and
0.1% Triton X-100, and then probed with primary antibody (rabbit
anti-PKC-
1; GIBCO). Incubation with primary antibody was conducted
overnight in 1% milk, 50 mM Tris, pH 8.0, 200 mM NaCl, and 0.1%
Tween. After washing, transfers were developed with horseradish
peroxidase-conjugated anti-rabbit IgG and the enhanced chemiluminescent
detection system (Amersham), followed by exposure to X-Omat film
(Eastman Kodak, Rochester, NY). Equal loading and transfer of samples
was assessed by staining with Ponceau Red. Authentic PKC-
1 peptide
was electrophoresed, transferred, and immunoblotted to serve as the control.
PKC activity.
PKC activity was measured with the PepTag assay system (Promega), in
which the change in charge of a fluorescent-tagged, PKC-specific substrate (PLSRTLSVAAK), which occurs with phosphorylation, is detected
on separation with agarose gel electrophoresis at a neutral pH. At the
end of the incubation period, the cells were washed with Hanks'
balanced salt solution containing 1 mM PMSF and 1 µM leupeptin,
placed in 100 µl of the same buffer, and then freeze thawed for
lysis. Aliquots were taken for the assay, which was performed according
to the manufacturer's instructions. Photographs of the gels were
scanned into a densitometer program for quantitation (Scion Image;
National Institutes of Health). Each assay included a positive control
for PKC activity, wherein PKC supplied by the manufacturer was
simultaneously subjected to the assay and electrophoretic procedure. A
negative control also was run with each assay.
 |
RESULTS |
The purpose of these experiments was to determine whether
Amadori-modified GA modulates PKC activity in glomerular mesangial cells, and to probe whether PKC signaling participates in the increased
collagen production by these cells on exposure to GA. Because high
media glucose concentration has been reported to activate PKC and to
augment matrix production in glomerular cells, incubations were
conducted in 5.5 mM glucose concentration to ensure that observed
responses did not accrue from an independent effect of elevated media
glucose. Where indicated, incubations in media containing 25 mM glucose
were performed in parallel for comparative purposes and to document
methodological integrity of the PKC assay by demonstration of high
glucose-stimulated increases. Parallel incubations with nonglycated
albumin, in the same concentrations as the glycated protein, in 5.5 or
25 mM glucose, served as controls. Under the conditions employed, cell
counts were not significantly different in incubations containing
glycated vs. nonglycated protein, as has been previously reported (12).
The concentration of collagen type IV in media from mouse mesangial
cells, incubated with GA in 5.5 mM glucose, was significantly increased
compared with that in the media of cells cultured in 5.5 mM glucose in
the presence of nonglycated albumin (Fig.
1). Relative to cells grown in 5.5 mM
glucose with the nonglycated protein, these increments were dose
related at 190% and 230% of control at the concentrations of GA
studied. High media glucose concentration (25 mM) also stimulated
collagen type IV production. Collagen type IV concentration in media
from cells grown in 25 mM glucose was 170% of that in cells incubated
with 5.5 mM glucose. Collagen type IV concentrations were further
increased when cells were cultured in 25 mM glucose in the presence of
GA. Relative to cells cultured with GA in 5.5 mM glucose, this
increment reached significance at the highest concentration of GA
studied, consistent with an additive effect of high ambient glucose and
GA on matrix protein synthesis (Fig. 1).

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Fig. 1.
Effect of glycated albumin (GA) on collagen type IV production by
mesangial cells. Murine mesangial cells were cultured in 5.5 mM
(left) and 25 mM
(right) glucose in presence of
indicated concentrations of nonglycated (solid bars) vs. glycated
(hatched bars) albumin. Results represent means ± SE of 4 independent cultures; in each, media were collected for immunoassay of
collagen type IV after 48 h of incubation. Results are expressed per
cell number. * P < 0.05 compared with nonglycated albumin at same concentration and under same
glucose condition.
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In the presence of the general PKC inhibitor GF-109203X, concentrations
of collagen type IV in mouse mesangial cells cultured in 5.5 mM glucose
and nonglycated albumin did not differ from those in media from cells
cultured without the inhibitor under the same conditions. However,
GF-109203X prevented the increase in collagen type IV production
induced by 25 mM glucose or by GA under both low and high media glucose
conditions (Fig. 2). Similarly, the
selective PKC-
inhibitor LY-379196 did not affect production of
collagen type IV by cells grown in 5.5 mM glucose and nonglycated
albumin, but prevented the GA-induced increases in collagen type IV
production in cells incubated with either 5.5 mM or 25 mM glucose and
reduced the collagen type IV production in cells incubated with 25 mM
glucose in the presence of nonglycated albumin (Fig. 2).

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Fig. 2.
Effect of protein kinase C (PKC) inhibitors on GA-stimulated collagen
type IV synthesis. Murine mesangial cells were cultured in 5.5 mM
(left) and 25 mM
(right) glucose for 48 h with 0.3 mg/ml of nonglycated (solid bars) or glycated (hatched bars) albumin,
without or with nonselective PKC inhibitor GF-109203X (GFX; 500 nM) or
PKC- -selective inhibitor LY-379196 (LY; 100 nM). Results represent
means ± SE of 4 independent cultures. In each, media were collected
for collagen IV immunoassay after 48 h of incubation. Results are
expressed per cell protein content.
* P < 0.05 compared with
nonglycated albumin under same glucose condition;
# P <0.05 compared with
corresponding albumin and LY-379196;
P < 0.05 compared with
corresponding albumin and GF-109203X.
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The presence of PKC-
isoforms in rat mesangial cells was confirmed
with immunoblotting experiments. Additionally, because classical
members of the PKC family move to membranes on activation, and because
a cytosol-to-membrane shift of PKC isoenzymes suggests activation of
that isoenzyme, we examined PKC-
1 immunoactivity in the cytosolic
and particulate fractions of mesangial cells to evaluate whether
incubation with GA was associated with cytosol-to-membrane translocation. Immunoblots with anti-PKC-
1 antibodies after
incubation of RMC (Fig. 3) or MMC (data not
shown) confirmed immunoreactivity in both membranes and cytosolic
fractions. Further, Fig. 3 shows that, under normoglycemic conditions,
incubation with GA increased PKC-
1 immunoactivity in the
membrane-bound fraction (with little discernible change in the
cytosolic fraction), consistent with activation and, possibly,
translocation of this isoenzyme.

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Fig. 3.
PKC- 1 immunoactivity in mesangial cells. Rat mesangial cells were
cultured for 48 h in 5.5 mM glucose containing 0.45 mg/ml of either
glycated (GA; lanes 2 and
4) or nonglycated (NA;
lanes 1 and
3) albumin. After harvest, cytosolic
and membrane fractions were prepared and immunoblotting was performed
as described in MATERIALS AND METHODS.
P, PKC- 1 peptide-positive control.
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Stimulation of PKC activity by GA was confirmed by measurement in the
phosphorylation assay. Visual inspection of gels from experiments in
which rat mesangial cells were incubated for 48 h in 5.5 mM glucose
indicated that PKC activity was increased in cells cultured in media
containing GA compared with activity in cells incubated with
nonglycated albumin in the same concentrations (Fig.
4). PKC activity in cells
incubated for 48 h in 25 mM glucose was greater than that in cells
cultured in 5.5 mM glucose, but it was difficult to appreciate visually
whether GA had an additional effect in high glucose medium (Fig. 4).
However, densitometric scanning of the gels confirmed that 48 h
exposure to GA induced a dose-related increase in PKC activity under
both low (5.5 mM) and high (25 mM) glucose conditions (Fig.
5). PKC activity in cells incubated in 5.5 mM glucose with nonglycated albumin was not significantly different
from that in 5.5 mM glucose alone, whereas the relative ratios
(activity compared with 5.5 mM glucose alone, assigned an arbitrary
value of 1.0) with 250 and 500 µg/ml of GA were 1.58 and 1.86, respectively. PKC activity in cells incubated for 48 h with 25 mM
glucose was 169% of that in cells incubated for the same period in 5.5 mM glucose. In the presence of GA, the relative ratios (activity
compared with 25 mM glucose alone, assigned an arbitrary value of 1.0)
with 250 and 500 µg/ml of GA were 1.38 and 1.85, respectively. The
relative ratios when activity was compared with 5.5 mM glucose,
assigned a value of 1.0, were 2.33 and 3.13 for 250 and 500 µg/ml GA,
respectively (Fig. 5).

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Fig. 4.
PKC activity in mesangial cells. Rat mesangial cells were cultured for
48 h in 5.5 mM (lanes C-G) or
25 mM (lanes H-L) glucose
containing glycated vs. nonglycated albumin. In each lane,
nonphosphorylated substrate is at top
and phosphorylated substrate is at
bottom. Negative control
(lane A) shows no phosphorylated
substrate. Lane B is positive control
obtained with PKC supplied by Promega. Lane
G was obtained from cells incubated in 5.5 mM glucose.
Lane H was obtained from cells
incubated in 25 mM glucose. Lanes C
and D, 250 and 500 µg/ml nonglycated
albumin in 5.5 mM glucose, respectively; lanes
I and J, 250 and 500 µg/ml nonglycated albumin in 25 mM glucose, respectively;
lanes E and
F, 250 and 500 µg/ml GA in 5.5 mM
glucose, respectively; lanes K and
L, 250 and 500 µg/ml GA in 25 mM
glucose, respectively.
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Fig. 5.
Effect of GA on mesangial cell PKC activity. Rat mesangial cells were
cultured for 48 h in DMEM containing 5.5 mM
(left) or 25 mM
(right) glucose, 1% FCS, and
indicated concentrations of nonglycated (open bars) or glycated
(hatched bars) albumin. Data were obtained by densitometric scanning of
photographs of gels. Results are expressed as relative ratios compared
with activity in cells incubated with 5.5 mM glucose, assigned an
arbitrary value of 1.0. Results represent means ± SE of 6 independent observations. * P < 0.05 compared with nonglycated albumin at same concentration and
under same glucose condition. #P < 0.05 compared with 0.25 mg/ml GA under same glucose condition.
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Time-course studies in rat mesangial cells revealed that media
containing either 25 mM glucose concentration or GA in 5.5 mM glucose
concentration elicited steady increases in PKC activity over the 48-h
period studied (Fig. 6). Relative ratios of
PKC activity (compared with 5.5 mM glucose alone) in cells incubated for 6, 18, and 48 h in 5.5 mM glucose with 500 µg/ml of GA were 1.22, 1.51, and 1.86, respectively. The relative ratios of PKC activity in
cells incubated for these periods in 25 mM glucose (compared with 5.5 mM glucose alone) were 1.13, 1.37, and 1.69, respectively. The steady
increases in activity under high glucose conditions were consistent
with a requirement for cellular de novo production of diacylglycerol in
the mediation of glucose-induced effects on PKC activity. The slopes of
the time-course lines of PKC activity under GA/5.5 mM glucose vs. 25 mM
glucose (without GA) were almost identical
(m = 0.013 and 0.014, respectively), in keeping with the foregoing results indicating that the increases in
PKC activity stimulated by high glucose alone or by GA (in low glucose
media) were comparable. Although the slope of the time-course line of
PKC activity under conditions of GA/25 mM glucose was lower
(m = 0.009), values at each time
interval were higher than with 25 mM glucose alone (Fig. 6), consistent
with an additive effect, as illustrated in the experiments depicted in
Fig. 5.

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Fig. 6.
Time course of glucose and GA effects on mesangial cell PKC activity.
Rat mesangial cells were cultured for 6, 18, and 48 h in DMEM
containing 1% FCS and 0.50 mg/ml GA in 5.5 mM glucose ( ) or 25 mM
glucose ( ) or DMEM containing 1% FCS and 25 mM glucose ( ). Data
were obtained by densitometric scanning of photographs of gels. Values
represent relative ratios compared with activity in cells incubated
with 5.5 mM glucose, assigned an arbitrary value of 1.0. Results
represent means ± SE of 4 observations. Slope of top line ( ) is
less steep than slopes for other 2 lines.
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DISCUSSION |
The results of these experiments demonstrate that GA stimulates PKC
activity in renal glomerular mesangial cells, and indicate that
activation of selective PKC isoforms participates in the GA-induced
stimulation of extracellular matrix protein production by these cells.
This interpretation is based on the observations that PKC activity
increases in rat mesangial cells incubated with glycated, but not
nonglycated, albumin; that the glycated protein increases
membrane-associated PKC-
1 immunoreactivity; and that inhibitors of
PKC prevent the increases in collagen type IV production that occur on
exposure of mouse mesangial cells in culture to GA. In this regard,
both the general PKC inhibitor GF-109203X and the selective PKC-
inhibitor LY-379196 were effective in 5.5 mM glucose concentration,
suggesting that PKC-
isoforms predominate in mediating the signal
from a GA stimulus for matrix overproduction. It should be noted that
LY-379196 does not differentiate between the two splice variants of
PKC-
enzyme, though our studies in mesangial cells indicate that GA
can selectively activate PKC-
1, one of the splice variants.
TGF-
1 is a key cytokine for matrix protein production (23, 34, 41,
42). GA has been previously shown to induce coordinate increases in
mRNAs encoding TGF-
1, the TGF-
type II receptor, and the
extracellular matrix proteins fibronectin and collagen type IV (9, 13,
39, 41). Given that PKC activation is believed to promote gene
expression of TGF-
1 and extracellular matrix proteins, the findings
of the current study suggest that GA-induced activation of PKC may be
linked to these responses. This chain of events resembles that induced
by high media glucose concentration, which increases mesangial cell PKC
activity in association with increased gene expression of TGF-
1 and
extracellular matrix proteins (2, 15, 17, 23, 29, 30, 34, 40). However,
in the present experiments, increased PKC activity and collagen type IV
production were observed in a low physiological glucose concentration
(5.5 mM), and cannot be ascribed to an effect of high glucose
concentration in the culture media.
The activation of PKC by high glucose is believed to derive from
conversion of glycolytic intermediates into increased de novo
production of diacylgycerol, a major endogenous activator of some PKC
species (2, 14, 20). PKC activation triggers a series of
phosphorylation events, including mitogen-activated protein kinases
(19, 21, 23, 27) and increased expression of
c-fos and
c-jun protooncogenes (31), which form
a heterodimer, AP-1, that activates the TGF-
1 gene promoter (4, 23).
The mechanism by which GA promotes PKC activation is speculative, but
may relate to an interaction of the Amadori-modified protein with
cell-associated ligand receptor systems (11). Knowing whether GA acts
through diacylglycerol will require further study. In this regard, it
is interesting to note that our studies have shown that GA induces
sustained activation of PKC, which contrasts with the transient
response effectuated by hormonal stimuli or by synthetic diglycerides,
and is consistent with the persistent PKC membrane translocation that
has been observed in tissues from diabetic animals (1, 25, 26, 37).
The characterization and cellular localization of PKC isoforms, and
changes induced by high glucose or associated with diabetes, have not
been completely delineated. Constitutive expression of several
classical PKC isoforms in glomerular mesangial and epithelial cells has
been reported, with some inconsistency regarding the presence of
PKC-
1 and -
2 in mesangial cells (3, 18, 23, 24, 29, 30).
Membrane-associated PKC-
2 has been reported to be increased (26) and
decreased (3) in glomeruli from rats with acute streptozotocin
diabetes, whereas membrane-associated PKC-
1 has been reported to be
increased after 12 wk of streptozotocin diabetes (30). PKC-
has been
more consistently identified in mesangial cells (18, 23, 29), and
membrane associated PKC-
has been found to be increased in glomeruli
of streptozotocin-diabetic rodents after 2, 4, and 12 wk of diabetes
(3, 30). Our findings that GA increases PKC activity, and that PKC-
activation participates in GA-induced stimulation of matrix synthesis
(as evidenced by prevention of this stimulation with both general and
PKC-
-specific inhibitors) resemble observations with mesangial cells
incubated with high media glucose concentration and in glomeruli from
diabetic rats. However, it should be noted that, in the context of the above, the relative importance of the different PKC isoforms in normal
and pathological states may differ in different glomerular cell types.
The findings reported herein are the first to demonstrate that albumin
modified by Amadori glucose-adducts modulates PKC isoenzyme activity,
and does so in a "normoglycemic" milieu and in concentrations of
GA that are typically found in serum specimens of diabetic patients. It
is likely that the results obtained in mouse and rat mesangial cells
can be extrapolated to human mesangial cells, but direct experimental
confirmation is required. These observations afford new insight into
the PKC activation that has been found in tissues from diabetic
animals, and into pathophysiological mechanisms contributory to
extracellular matrix accumulation in diabetic renal disease. Either
elevated glucose or GA can activate mesangial cell PKC, and either can
induce TGF-
1 gene expression, which stimulates extracellular matrix
protein production. Hyperglycemia is the driving force for increased
albumin glycation, but the effects of GA on PKC activation and TGF-
1
expression do not require a hyperglycemic milieu to be operative. Thus
the in vivo influence of GA, which has a circulating half-life of ~2
wk, on PKC isoenzymes can continue after restoration of normoglycemia
and can act independently of hyperglycemia. We have shown that therapy
directed against increased GA beneficially influences nephropathology
in a rodent model of genetic diabetes, despite marked and persistent
hyperglycemia (6, 8, 10). We postulate that the salutary effects of this intervention strategy may derive, at least in part, from a
reduction of PKC activity concomitant with a lowering of circulating GA
concentrations. This approach affords the potential advantage of
avoiding possible untoward effects of systemic PKC inhibition (33).
In summary, we have shown that albumin modified by Amadori
glucose-adducts stimulates PKC activity and collagen type IV production in rodent mesangial cells in culture. These effects are observed in
physiological glucose concentration. The findings link PKC signaling,
the PKC-
isoform in particular, to the increased extracellular matrix production induced by elevated concentrations of GA in diabetes mellitus.
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ACKNOWLEDGEMENTS |
We thank Dr. D. K. Ways for the gift of LY-379196 (Lilly Research
Laboratories). The technical assistance of Ryan Grinkewitz is
gratefully acknowledged.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-54608, DK-54143, DK-49455, DK-45191, DK-44513, and EY-11825.
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,
Renal-Electrolyte & Hypertension Div., Univ. of Pennsylvania, 700 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144 (E-mail: ziyadeh{at}mail.med.upenn.edu).
Received 24 November 1998; accepted in final form 26 January 1999.
 |
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