AGEs induce oxidative stress and activate protein kinase
C-
II in neonatal mesangial cells
Vincenzo
Scivittaro1,
Michael B.
Ganz2, and
Miriam F.
Weiss1
1 Division of Nephrology, Department of
Medicine, Case Western Reserve University and University Hospitals
of Cleveland, Cleveland 44106; and 2 Division of
Nephrology, Department of Medicine, Case Western Reserve University
and the Cleveland Veteran's Administration Medical Center, Cleveland,
Ohio 44106
 |
ABSTRACT |
Increased activation of specific protein kinase C (PKC)
isoforms and increased nonenzymatic glycation of intracellular and extracellular proteins [the accumulation of advanced glycation end products (AGEs)] are major mechanistic pathways implicated in
the pathogenesis of diabetic complications. Blocking
PKC-
II has been shown to decrease albuminuria in animal
models of diabetes. To demonstrate a direct relationship between AGEs
and the induction and translocation of PKC-
II, studies
were carried out in rat neonatal mesangial cells, known to express
PKC-
II in association with rapid proliferation in
post-natal development. Oxidative stress was studied by using the
fluorescent probe dichlorfluorescein diacetate. Translocation of
PKC-
II was demonstrated by using immunofluorescence and
Western blotting of fractionated mesangial cells. Induction of
intracellular oxidative stress, increase in intracellular calcium, and
cytosol to membrane PKC-
II translocation (with no change
in PKC-
) were demonstrated after exposure to AGE-rich proteins.
These data support the hypothesis that AGEs cause mesangial oxidative
stress and alterations in PKC-
II, changes that may
ultimately contribute to phenotypic abnormalities associated with
diabetic nephropathy.
advanced glycation end products; protein kinase C isoforms; intracellular oxidative stress; mesangial cells; neonatal rat mesangial
cells
 |
INTRODUCTION |
THE PATHOLOGICAL HALLMARK of diabetic nephropathy in
the renal glomerulus is the expansion of the mesangial matrix and
thickening of the capillary basement membrane. These critical changes
are believed to lead to loss of renal function. As the mesangium
expands the glomerular capillary lumen is narrowed and glomerular
filtration becomes restricted. A number of growth factors are
associated with activation of mesangial cells and the increased
synthesis of components of the mesangial matrix, with a critical role
played by transforming growth factor-
(TGF-
) particularly in the
late stages of diabetic nephropathy (35). External growth factors, hormones and cytokines work through the transduction of second messenger molecules such as protein kinase C (PKC). PKC activity is
increased in the renal glomeruli of animals with diabetes (19). PKC-
(
I and
II) are the isoforms of PKC most
frequently associated with alterations in mesangial cell phenotypic
behavior such as proliferation and matrix deposition (41). Blocking
PKC-
decreases albuminuria in animal models of diabetes (19).
The degree of hyperglycemia is an important factor associated with the
progression of diabetic nephropathy, as demonstrated in the Diabetes
Control and Complications Trial (38). However, the mechanisms by which
hyperglycemia causes nephropathy are not well defined. Mesangial cells
grown in high-glucose culture medium show enhanced cellular
proliferation (3) as well as direct increases in both the mRNA for, and
the synthesis of, matrix components. The mechanism for direct mesangial
cellular stimulation by glucose is believed to relate to increased
synthesis of diacylglycerol (DAG), a signaling molecule for classic
isoforms of PKC (7).
Hyperglycemia also results in increased nonenzymatic glycation of
intracellular and extracellular proteins, and the eventual formation of
advanced glycation end products (AGEs) on these proteins. A growing
literature associates the accumulation of AGEs on proteins with the
pathogenesis of diabetic nephropathy (29). The glomerular basement
membrane content of the structurally defined AGE pentosidine correlates with nephropathy (4). Immunohistochemical studies demonstrate the accumulation of
N
(carboxymethyl)lysine (CML) and pentosidine in
mesangial matrix and nodular lesions of diabetes (37). Infusion of
AGE-rich proteins into experimental animals causes diabetes-like
complications, including increased vascular permeability, extracellular
matrix deposition, and glomerulosclerosis (30). Mesangial cells are stimulated to increase production of matrix proteins after exposure to
protein with a high AGE content (9). Aminoguanidine, an inhibitor of
AGE formation, blocks mesangial expansion in streptozotocin-treated rats (36). Thus AGES have a direct effect on the mesangial cell, independent of hyperglycemia.
Hyperglycemia and the effects of AGEs probably act through different
pathways. Hyperglycemia induces direct free radical stress through
generation of reactive
-dicarbonyl intermediates and autoxidation.
Thus hydroxyl radicals created during sugar autoxidation degrade
proteins by breaking covalent bonds, or lead to cell membrane lipid peroxidation (26). In contrast AGEs are believed to induce cellular oxidative stress through interaction with specific
cellular receptors (23, 30).
PKC-
II expression is temporally associated with rapid
mesangial cell proliferation during development (1, 33). With further
renal maturation this isoform becomes downregulated in mesangial cells
(33). Conversely, inhibition of PKC-
II expression in
vitro blocks the rapid proliferation of rat glomerular mesangial cells
(1). Because neonatal rat mesangial cells consistently express
PKC-
II, our experiments demonstrate a direct
relationship between AGEs, the induction of oxidative stress, and the
translocation of a PKC isoform with demonstrated pathophysiological
importance in diabetic nephropathy.
 |
METHODS |
Isolation and culture of mesangial cells.
Mesangial cells were obtained from kidneys of neonatal Sprague-Dawley
rats (days 1-5 after birth), as previously described for
adult mesangial cells (33). Pregnant females were purchased from Harlan
(Indianapolis, IN). Kidneys were dissected out from anesthetized rats
under aseptic conditions. After the capsule was removed, the entire
kidney was homogenized in ice-cold Hanks' balanced salt solution
(HBSS; GIBCO, Grand Island, NY) and passed through sieves (Tyler,
Mentor, OH) to isolate glomeruli. Glomeruli were then washed twice with
HBSS and were plated on to 75-cm2 tissue culture flasks
(Falcon, Lincoln Park, NJ) in 15 ml of DMEM (GIBCO) containing 5 µg/ml each of insulin, transferrin, and selenious acid (Collaborative
Research, Bedford, MA), 25 mM glucose, 400 ng/ml penicillin (Sigma
Chemical, St. Louis, MO), 500 ng/ml streptomycin (Sigma Chemical), and
25 mM NaHCO3 with 20% FBS (GIBCO).
For all experiments only primary (unpassaged) neonatal cells were used.
The method of obtaining unpassaged neonatal mesangial cells was as
follows. Glomerular explants from neonatal rats were continued in
flasks (for Western blot experiments) or plated onto 8-well chamber
slides (Nunc, Naperville, IL; for determination of intracellular
calcium, or immunofluorescence), or into 96-well plates (for
determination of oxidative stress). Once the glomerular explants were
attached to the surface (within 24 h), the flasks or wells were washed
thoroughly every other day with HBSS, followed by the addition of fresh
growth medium containing 20% FBS. Neonatal mesangial cells were ready
for immunostaining when they became 60-70% confluent (usually
2-3 wk). As previously described for adult mesangial cells,
neonatal rat mesangial cells in culture were also immunostained for the
Thy 1.1 epitope, a defining characteristic of rat mesangial cells (14),
and vascular smooth muscle myosin antigen (13). Seventy-two hours
before experiments, the growth medium was changed from 20% to 0.5%
FBS to halt cell growth.
Production and characterization of AGE proteins.
AGE proteins were produced using pyrogen-free human serum albumin (HSA)
approved for human clinical use (Albumark, Baxter, McGaw Park, IL).
Glycated human serum albumin (AGE-HSA) was prepared by incubation of
HSA (50 mg/ml) in 0.5M glucose at 37°C for 30 days (25). Control
HSA consisted of HSA (50 mg/ml) incubated without additional glucose at
37°C. HSA with a high specific content of
N
(carboxymethyl)lysine (CML) was prepared
according to the method of Dunn (CML-HSA) (10). Before incubation, the
glucose-HSA solution was filtered sequentially over 0.44- and 0.2-µm
Gelman Acrodisc filters. After incubation the solutions were dialyzed
against deaerated Chelex-treated phosphate buffered saline, aliquoted and stored at
80°C. At the time of storage, all batches were checked for potential contamination by lipopolysaccharide (LPS), and
batches containing >0.25 µU endotoxin/ml in the limulus lysate assay (Sigma Chemical) were discarded. The CML (16) and pentosidine (11) content of the preparations and controls were determined by HPLC
as previously described. The CML content of the CML-HSA was 122,450 pmol/mg protein; the pentosidine content, 2.8 pmol/mg protein. The CML
content of the AGE-rich HSA was 22,512 pmol/mg; the pentosidine
content, 24.9 pmol/mg. The control-HSA contained CML at 495 pmol/mg
protein, and pentosidine at 2.9 pmol/mg
Determination of intracellular calcium concentration
([Ca2+]i)
by using the fura 2 loading procedure.
[Ca2+]i was ascertained by using
the fluorescent calcium indicator fura 2 (Molecular Probes, Eugene, OR)
as described by Grynkiewicz et al. (18). Fura 2 was initially dissolved
in DMSO at 5 mM and diluted to a final concentration of 5 µM in a
saline solution containing 0.1% BSA. Neonatal mesangial cells grown on
glass coverslips were loaded with fura 2 for 30 min. at 37°C and
inserted into a thermostatically controlled cuvette holder in the
Perkin-Elmer LS-5B spectrofluorometer (Norwalk, CT). The contribution
of dye leakage (extracellular fura 2) to the fluorescence signal was eliminated by continuous superfusion of the coverslips with the bathing
solution at a rate of 2.5 ml/min. The dye was then excited at two
different excitation wavelengths, 340 and 380 nm, with a constant
emission of 510 nm. Once a stable baseline had been established, the
AGE protein or control protein was added. The Perkin-Elmer LS-5B is a
single- wavelength instrument that necessitates shuttling between the
two different excitation wavelengths. However, the interval delay
between changing wavelengths is short enough to permit an accurate
determination of the time course of change in cytosolic calcium
concentration. Using the ratio of fluorescence at 340 and 380 nm, the
fractional increase in [Ca2+]i can
be determined, as previously described (13).
Measurement of oxidative stress in neonatal mesangial cells.
AGE-induced cellular oxidative stress was detected in neonatal cells by
using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA;
Molecular Probes) (17, 20, 21) according to methods previously
described for the detection of reactive oxygen metabolites in mesangial
cells (6). DCFH-DA is a membrane-permeant diacetate derivative of
dichlorofluorescein (DCFH). On entering the cells, the diacetate group
is cleaved off enzymatically and DCFH is trapped inside the cell. Both
DCFH-DA and DCFH are nonfluorescent fluorescein analogs that are
oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by reactive oxygen species. DCFH oxidation may be caused by
several reactive intermediates (HO ·,
O
2, NO, and so on). Consequently the
fluorescent signal produced by DCFH is an index of overall oxidative
stress in biological systems.
For each experiment, mesangial cells were washed once with Dulbecco's
phosphate-buffered saline (DPBS) and preloaded with 20 µM DCFH-DA in
DPBS. After 15-min incubation at 37°C, cells were washed with DPBS,
and HSA-control, AGE-HSA, or CML-HSA was added. The development of
fluorescence at different time points was measured in a microplate
fluorometer (Cytofluor II, Perspective Biosystems, Framingham, MA)
interfaced with a computer for data processing. For each experiment,
proteins were also added to empty wells to detect protein
autofluorescence. The autofluorescence signal was subtracted from the
signal generated by the mesangial cells in response to the test
proteins. Results were expressed as relative fluorescence units (RFU)
monitored at 485-nm excitation and at 530-nm emission wavelength.
Because the cells in the plates were not disturbed by the measuring
procedure, the plates could be incubated for additional time periods
and reread. Fluorescence photobleaching under this condition was negligible.
Western blot analysis of PKC isoforms.
PKC-
and -
II isoform expression in serum-starved
mesangial cells were detected by using polyclonal antibodies against
PKC-
and -
II purchased from GIBCO as previously
described (15). Antibody specificity was established by competing with
excess peptide. Neonatal mesangial cells were lysed, and the cytosolic and membrane fractions were isolated. In brief, mesangial cells were
washed twice and incubated with 200 µl of a balanced saline solution
containing 0.8 meq/dl Ca2+. The cells were scraped,
transferred to prechilled Eppendorf tubes, and centrifuged at 5,000 rpm
for 5 min. at 4°C. Cell pellets were resuspended in ice-cold
homogenization buffer (60 mM Tris, 0.25 M sucrose, 10 mM EGTA, 2 mM
EDTA, 10 mM
-mercaptoethanol) containing protease inhibitors, 200 µM phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin. The cell
suspensions were sonicated, and the degree of cell lysis was assessed
by light microscopy. Lysate and membrane fractions were separated by
high-speed centrifugation. The supernatants were used as cytosol
fractions while the pellets were resuspended in ice-cold homogenization
buffer containing 1% Triton X-100 and protease inhibitors and
centrifuged at 100,000 g for 1 h at 4°C. The pellets were
discarded, and supernatants contained crude (i.e., nuclear, plasma, and
mitochondrial membranes) membrane fractions. Rat brain cell PKC
or recombinant PKC-
or -
II was used as positive
controls in appropriate experiments. Membrane and cytosol fractions
from each time period were separated by electrophoresis on 12%
SDS-polyacrylamide slab gels and the separated peptides were
transferred by electrophoresis to nitrocellulose sheets by using Semi
Dry Trans Blot Cell (Bio-Rad, Hercules, CA). Nonspecific binding sites
were blocked by incubating nitrocellulose sheets with 5% nonfat dry
milk in PBS for 1 h at room temperature. Thereafter, the nitrocellulose
strips were incubated with polyclonal isoform-specific antibodies in
appropriate dilutions in PBS (1:1,000) overnight at 4°C. The sheets
were washed three times for 10 min with PBS and incubated with
secondary antibodies (anti-rabbit IgG-horseradish peroxidase; diluted
1:1,000 in PBS) for 1 h at room temperature. The sheets were washed,
and the activity of enzyme peroxidase was detected by the
chemiluminescent reaction of the substrate luminol by using the
enhanced chemiluminescence technique (Amersham, Arlington Heights, IL).
Rat brain and C6 glioma cell PKC were used as a positive confirmation
of the presence of PKC-
or -
II in the neonatal
mesangial cells (12, 15).
Immunofluorescent detection of PKC-
II
translocation.
Rat neonatal mesangial cells were stimulated for 30-180 min in the
presence of two concentrations of control and AGE-proteins at 37°C,
washed with PBS, and then fixed with 37% formaldehyde for 30 min.
After washing and blocking with PBS-1% BSA, rabbit polyclonal
anti-PKC- or rabbit polyclonal anti- PKC-
II (both from
Santa Cruz Biotech, Santa Cruz, CA) were added for 2 h, followed by a
specific secondary antibody conjugated to FITC. Cells were observed by
using the microscope and the fluorescent images captured with an
Optronic Video-digital Image System (Optronics, Goleta, CA) connected
to an IBM computer.
 |
RESULTS |
Intracellular calcium
[Ca2+]i
is increased in neonatal mesangial cells exposed to AGEs.
Stable baseline calcium concentration was achieved in the neonatal
mesangial cells. Then, AGE-HSA, CML-HSA, or control proteins were added
to the cells at protein concentrations ranging from 1 nM to 1 µM. A
representative experiment demonstrates the effects of adding the
AGE-HSA, CML-HSA, and control proteins (arrow) at a concentration of 1 µM (Fig. 1A). A highly
significant increase in intracellular calcium content developed over
the next 1-2 min in response to the glycated proteins, but not the
HSA-control protein. To define whether the increase in intracellular
calcium was due to release from intracellular stores or a
channel-mediated effect, we preincubated cells with 1,2-bis (2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; Sigma Chemical). BAPTA acts inside the cell to chelate calcium on release from smooth endoplasmic reticulum. As shown in a
representative experiment (Fig. 1B), preincubation with BAPTA blocked the increase in intracellular calcium in response to CML-HSA. The effect of AGEs was not altered by adding nifedipine, a calcium channel blocker. The peak calcium response to CML-HSA reached 160 ± 8.3 nM in the presence of 1 mM nifedipine, compared with 163 ± 12.1 nM in its absence.

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Fig. 1.
A: representative experiment demonstrating intracellular
calcium transient induced on addition of 1 µm of advanced glycation
end product (AGE)-human serum albumin (HSA) or
N (carboxymethyl)lysine (CML)-HSA at time
3 min (arrow). Control-HSA 1 µm does not induce a significant
increase in intracellular calcium. B: representative experiment
showing effect of preincubation of mesangial cells with 1,2-bis (2 aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA). Addition of 1 µm CML-HSA at time 3 min (arrow)
to BAPTA-treated cells blocks increase in intracellular calcium in
response to CML-HSA.
|
|
The dose-response relationship between peak intracellular calcium and
the addition of AGE-proteins or control is demonstrated in Table
1. Addition of both CML-HSA and AGE-HSA
resulted in significant increases in intracellular calcium content
compared with the control HSA. The effect of CML-HSA was slightly, but not significantly, greater than the effect of AGE-HSA, reflecting the
greater content of the chemically identified AGE structure, CML. A
threshold effect for the increase in intracellular calcium is noted
between 1 nM and 1 µM of protein for both AGE-HSA and CML-HSA.
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Table 1.
Dose-dependent peak intracellular calcium (nM) or oxidative stress
signal (RFU) in neonatal rat mesangial cells
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Intracellular oxidative stress is increased in neonatal mesangial
cells exposed to AGEs.
The threshold for the dose of AGE-proteins stimulating increased
intracellular oxidative stress was comparable to the dose that
increased intracellular calcium (Table 1). The effect reached a plateau
at 10 µM and demonstrated a decrease in peak intracellular oxidative
stress above 20 µM, reflecting quenching of the fluorescence signal
by increasing concentrations of protein. The time course of stimulation
of increased intracellular oxidative stress by AGE-proteins was
considerably slower than the increase in intracellular calcium. A
maximum increase was seen at 60 min (Fig.
2). At 60 min, the fluorescence signal
produced by neonatal rat mesangial cells with no added protein was 237 ± 30 RFU. In the presence of 100 µM phorbol myristate acetate
(PMA), a maximal signal of 4,032 ± 226 RFU was produced. In contrast
to the rapid increase in intracellular calcium, the time course of
intracellular oxidative stress was comparable to the translocation
response of PKC-
II (see below).

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Fig. 2.
Time course of intracellular oxidative stress in neonatal mesangial
cells exposed to 5 µm AGE-HSA, CML-HSA, or control-HSA.Values are
means ± SD of 4 experiments.
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|
Translocation of PKC-
II in response to
AGEs.
Translocation of PKC-
II was examined across a range of
protein concentrations. Each value represents the mean ± SD
of 14 experiments (12 control experiments), expressed as densitometric readings (Table 2). Highly significant
differences in cytosol to membrane translocation of
PKC-
II were found in response to both CML-HSA and
AGE-HSA at protein concentrations above 1 µM. Similar to the results
for increased intracellular calcium, at least 1 µM of CML-HSA or
AGE-HSA was required to stimulate translocation of
PKC-
II. Translocation of PKC-
II between
cellular and membrane fractions was examined at 0, 10, 30, 60, and 120 min after the serum-starved neonatal mesangial cells were incubated
with either 1 µM CML-HSA, AGE-HSA, or control protein. Each value
represents the mean ± SD of 12 experiments (4 control experiments),
expressed as densitometric readings (Table
3). A greater translocation of
PKC-
II was seen in response to CML-HSA than to AGE-HSA.
In contrast to PKC-
II, no change in translocation of
PKC-
could be detected, at maximal translocation time for any
concentration of the CML-HSA (8 experiments). These results did not
differ from HSA-Control protein (13 experiments; Table
4).
A representative Western blot of the 80-kDa PKC-
II
signal in cytosolic and membrane fractions of neonatal rat mesangial
cells is shown in Fig. 3. In this figure,
the density of the membrane fraction signal for PKC-
II
can be seen to increase at 60 min and remain significantly elevated at
120 min in response to 1 µM CML-HSA.

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Fig. 3.
Representative Western blot analysis of rat neonatal mesangial cells
stained for protein kinase C (PKC)- II in response to
incubation with 1 µm CML-HSA. Arrow, 80-kDa protein band; c,
cytosolic fraction; m, membrane fraction. Each lane was loaded with 5 µg of total protein. Density of membrane fraction signal for
PKC- II increased at 30 and 60 min, with a comcomitant
reduction in density of cytosol signal at 30 and 60 min.
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|
A representative experiment using immunofluorescence to demonstrate the
translocation of the signal for PKC-
II from cytosol to
membrane is shown in Fig. 4. These
photomicrographs were obtained after 60 min of exposure to AGE-proteins
at 10 µM. In four experiments, luminosity measurements comparing the
cytosol with membrane fractions were performed. The membrane luminosity
increased from 38.1 ± 4.1 to 55.8 ± 10.8 arbitrary units
in response to CML-HSA, and to 46.2 ± 8.8 in response to AGE-HSA. A
lesser degree of translocation was also observed by 30 min (to 45.3 ± 9.9 units in response to CML-HSA), and at a protein concentration of 5 µM (data not shown). Immunofluorescence staining for PKC-
showed
no cytosol-to-membrane translocation of signal (not shown).

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Fig. 4.
Representative immunofluoresence photomicrographs demonstrating
PKC- II in a predominantly cytosolic or
membrane-localized pattern in response to 10 µm of protein at 60 min.
A: unstimulated control at time 0. B:
control-HSA. C: CML-HSA. D: AGE-HSA.
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 |
DISCUSSION |
These data demonstrate a dose-response relationship between proteins
with a high content of AGEs, the induction of intracellular oxidative
stress, and translocation of the specific isoform PKC-
II in neonatal mesangial cells. AGEs also cause an immediate increase in
mesangial cell intracellular calcium, independent of induction of a
calcium channel, as demonstrated by blockade with BAPTA but not with
nifedipine. These changes and intracellular oxidative stress are
unrelated to alterations in the glucose concentration of the growth
media, which was kept constant in all control and experimental
conditions. Paradoxically, long-term exposure to AGEs causes a decrease
in [Ca+]i flux, when flux is
stimulated by angiotensin II (27). Further studies are needed to
investigate the relationship between initial and late effects on
[Ca+]i flux induced by AGEs. AGEs
upregulate an array of genes expressed in diabetic glomerular disease
(40). In addition, one of the known receptors for AGE-modified proteins
has significant sequence homology with an 87-kDa protein substrate for
PKC (24). However, the data presented in this paper are the first to
illuminate a direct relationship between AGEs and this important
subcellular signaling pathway, without implicating the effect of
hyperglycemia on DAG.
Most workers are unable to demonstrate translocation of PKC isoforms in
adult rat mesangial cells exposed to AGE-proteins(27). Defining
subcellular processes responsible for changes in mesangial cell
phenotypic behavior in disease is difficult because mesangial cells
from diseased animals are not readily subcultured. As a result a number
of laboratories have compared immature cells to diseased cells.
Expression of specific phenotypic behavior is suppressed after
completion of embryological and early post-natal development, but
reappears in disease states. For example an increase in the PKC
isoforms
I,
II , and
are found in
immature hematopoietic cells. Expression of these isotypes may, in
part, be responsible for the uncontrolled growth seen in leukemic cells
(8). These phenotypes are also responsible for the rapid growth of
fetal epithelial cells and have been reported in association with
changes in differentiation and cellular growth in other tissues as well (31). Cultured adult rat mesangial cells, whether primary adult mesangial cells or passaged cells, can be demonstrated to possess the
-,
I-, and
-isoforms of PKC, but not
PKC-
II. A distinctive temporal pattern of PKC isoform
translocation in mesangial cells is suggestive of the idea that each of
the PKC isoforms subserves a specific biological role. The
-isoform
may be responsible for early membrane events such as Na-H exchange and
channel activity (32). The
I-,
II -, and
-isoforms of PKC are associated with cellular events such as
mitogenesis, matrix production and, differentiation (15). The data in
this report demonstrate that exposure to AGE-HSA or CML-HSA induces
translocation in a PKC isoform, which is more likely to effect
alterations in cell phenotype than those that occur in response to
changes in Na-H exchange or channel activity.
In the pre- and postnatal period of kidney development, proliferation
with subsequent functional maturation of intrinsic glomerular mesangial
cells continues within the existing framework. The differential expression of PKC-
II parallels the period of active
proliferative and/or matrix-secreting behavior of mesangial cells
closely in the maturing glomerulus (33). Although all neonatal and
adult mesangial cells from glomerular explants of Sprague-Dawley rat kidneys express PKC-
I, neonatal mesangial cells express
PKC-
II only during postnatal days 1-5. Thus
expression and activation of PKC-
II may play a critical
role in modulating cellular phenotype.
The mechanism(s) by which AGE-proteins induce abnormal cellular
responses remains poorly understood because AGEs are a heterogeneous group of structures, and cells have many AGE "receptors." The group of receptors identified to date is diverse and includes the
receptor for AGE (RAGE), lactoferrin, galectin-3/p60/p90 (24), and the
macrophage scavenger receptor (2). AGE-induced activation has been
demonstrated in a variety of cell types. RAGE expression on vascular
endothelium is increased in diabetes, where it leads to increased
expression of VCAM-1 (34) and is regarded as a hallmark of
atherogenesis. Both antibodies against RAGE and soluble RAGE, a
truncated form of the receptor, inhibit AGE-albumin induced VCAM-1
expression. Incubation of mesangial cells with AGE-proteins induces
mRNA and protein for transforming growth factor-
(TGF-
), insulin
like growth factor-I (IGF-I), and extracellular matrix components
(fibronectin, laminin, collagen IV). An antibody against the p60/OST
AGE receptor can block these effects (30).
AGEs binding to RAGE induce cellular oxidative stress, as demonstrated
in endothelial cells (39) and monocytes (28). Intracellular oxidative
stress leads to the activation of the transcription of NF-
B and
upregulation of various NF-
B-controlled genes, an effect that can be
blocked by transfecting the antisense to RAGE (5). In smooth muscle
cells from rat pulmonary artery, AGE-albumin activated p21ras and
enhanced MAP kinase activity, effects that were enhanced by depleting
intracellular glutathione, and blocked by anti-RAGE antibody (23). The
specific receptor mediating the translocation of PKC-
II
by AGEs remains undefined. The temporal relationship between DCFH
fluorescence and PKC-
II translocation is suggestive of a
cell-signaling process involving intracellular oxidative stress (23).
Independent of AGEs, the link between oxidant stress and activation of
PKC can be broken by antioxidants such as
-tocopherol (22). More
research is needed to define the relative importance of hyperglycemia
with its activation of DAG-PKC pathways, and AGE-mediated activation of
PKC-
II in the development of diabetic nephropathy.
In summary, translocation of PKC-
II can be demonstrated
in neonatal rat mesangial cells after exposure to AGE-rich proteins. The time course parallels the increase in intracellular oxidative stress induced by these proteins. These effects are not mediated by
hyperglycemia but represent a direct response to AGE proteins. In the
present study, the demonstration of a specific AGE effect may not have
been possible without the use of actively proliferating cells that
express PKC-
II. These data lead to the hypothesis that
the dedifferentiation of mature mesangial cells may be necessary before
AGEs can trigger intracellular calcium flux, intracellular oxidative
stress, and the translocation of PKC-
II in diabetic nephropathy.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by the Kidney Foundation of
Northeast Ohio (M. F. Weiss), by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-45619, by a Merit Review Grant
from the VA Medical Center (M. B. Ganz), and by an American Heart
Association Established Investigator Award (M. B. Ganz).
 |
FOOTNOTES |
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: M. F. Weiss,
Div. of Nephrology, Dept. of Medicine, Univ. Hospitals of Cleveland,
11100 Euclid Ave., Cleveland, Ohio 44124 (E-mail:
maf3{at}po.cwru.edu).
Received 14 August 1999; accepted in final form 10 December 1999.
 |
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