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*
Department of Pediatrics, University of Minnesota, Minneapolis,
Minnesota
Fred Hutchinson Cancer Research Center, Seattle, Washington.
Correspondence to Dr. Clifford E. Kashtan, University of Minnesota Medical School, Department of Pediatrics, Box 491, 515 Delaware Street, S.E., Minneapolis, MN 55455. Phone: 612-626-2922; Fax: 612-626-2791; E-mail: kasht001{at}tc.umn.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3ß1, which mediates attachment of these cells to extracellular
matrix proteins including type IV collagen. T-SV 40 immortalized human
glomerular epithelial cells were used to study PAN's effects on
3ß1 expression, as well as that of podocalyxin and the slit
diaphragm component ZO-1. Glomerular epithelial cells were seeded into plastic
flasks and allowed to attach and proliferate for 48 h. The cells were then
incubated for another 48 h in media containing 0, 0.5, or 5.0 µg/ml PAN.
PAN exposure resulted in dose-dependent decreases in 3 and ß1
expression, both at the protein level and at the mRNA level. This was
accompanied by a significant decrease in the adhesion of glomerular epithelial
cells to type IV collagen. PAN did not affect ZO-1 protein expression.
Treatment with PAN increased the expression of podocalyxin at the protein and
mRNA levels. Reduced glomerular epithelial cell expression of 3ß1
integrins and impaired adhesion to type IV collagen may contribute to the
glomerular epithelial cell detachment from glomerular basement membrane seen
in the PAN nephrosis model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major morphologic changes in PAN nephrosis are fusion of the foot processes of the glomerular visceral epithelial cells, or podocytes, and focal detachment of these cells from the glomerular basement membrane (GBM) (4,5,6,7). In PAN nephrosis, the onset of proteinuria coincides with and is believed to result fundamentally from the development of focal defects in the podocytic epithelium that expose the outer surface of the GBM to the urinary space. Rat glomerular epithelial cells that were exposed to PAN in vitro were unable to proliferate and had reduced adhesion to plastic (2). However, the pathways by which PAN interferes with glomerular epithelial cell adhesion have not been elucidated.
The integrins are a major family of cell-surface proteins that mediate
binding of cells to the extracellular matrix
(8). Several authors have
demonstrated 3ß1 to be the major integrin present in vivo
on podocytes
(9,10,11,12,13).
We have shown that the 3ß1 integrin is expressed on the surface of
immortalized human glomerular epithelial cells and that specific blocking of
the 3ß1 integrin inhibits adherence of these cells to a type IV
collagen matrix (14). The
experiments described in this article were performed to test the hypothesis
that exposure of glomerular epithelial cells to PAN inhibits expression of the
3ß1 integrin and that this effect is associated with a reduced
capacity of the cells to adhere to type IV collagen. We also examined the
effect of PAN on expression of podocalyxin, a major glomerular sialoprotein,
and ZO-1, a protein present in the slit diaphragm.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and 5 chains of type IV collagen
but not the 6(IV) chain
(16), and they express WT1, a
marker for differentiated visceral epithelial cells. For propagation, 56/10 A1 cells were grown in standard medium that comprised a 1:1 mixture of Dulbecco's modified Eagle's medium:Ham's/F12 with 0.8% fetal calf serum (Hyclone, Logan, UT) supplemented with 5 mM glucose, 10 mM HEPES, 2 mM glutamine, 5 µg/ml insulin, 5 x 10-8 M dexamethasone, 3 x 10-8 M sodium selenate, 5 µg/ml transferrin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B (Life Technologies BRL, Gaithersburg, MD). Cultures were incubated in a 37°C humidified incubator with 5% CO2. For experiments, cells were grown until confluence in 25-cm2 or 75-cm2 flasks (Corning Costar, Cambridge, MA) under standard culture conditions with 5 mM D-glucose.
Cell Number
Cells were seeded at a density of 1 x 104/cm2
in 9.4-cm2 area wells for 2 d. Subsequently, the cells were exposed
to 0, 0.5, 5.0, or 50.0 µg/ml PAN for 24 or 48 h. The cells were
trypsinized and incubated with 0.2% trypan blue for 2 min. The total number of
cells and the number of viable cells were counted on a hemocytometer, three
times for each condition. The experiment was performed three times. These
experiments showed a substantial reduction in the viability of cells exposed
to 50 µg/ml PAN but no differences in cell viability at the other PAN
concentrations, which was approximately 97%.
On the basis of these results, all subsequent experiments compared the effects of 0, 0.5, and 5.0 µg/ml PAN for 2 d. Cells were seeded in 75-cm2 flasks at a density of 0.01 x 106 cells/cm2. After 48 h, the medium was replaced by medium containing 0, 0.5, or 5.0 µg/ml PAN and incubated for an additional 48 h.
Cell Proliferation
The effect of PAN on proliferation of 56/10 A1 cells was assessed by an
enzyme-linked immunosorbent assay method, using 5-bromo-2'-deoxyuridine
(BrdU), according to the manufacturer's recommendations (Roche Diagnostics,
Indianapolis, IN). Cells were seeded at 0.01 x
106/cm2 in 96-well plates and incubated for 2 d at
37°C. PAN was added to the media at a concentration of 0, 0.5, or 5.0
µg/ml (n = 12 for each PAN concentration), and the cells were
incubated for another 48 h. For labeling, BrdU was added to the cells (final
concentration, 10 µM) for the last 24 h of PAN incubation. At the end of
the incubation period, the labeling medium was removed from the wells. The
cells were fixed and their DNA was denatured by incubation in FixDenat
solution (Roche Diagnostics), 200 µl/well, for 30 min at room temperature.
The denaturing solution was removed, and 100 µl of peroxidase-conjugated
anti-BrdU antibody was added to each well, followed by a 90-min incubation at
room temperature. The antibody was then removed, and the wells were rinsed
three times with 200 µl of solution. The substrate solution (100 µl) was
added to each well, followed by incubation at room temperature for 10 min.
Absorbance at 450 nm was measured in a Microplate Autoreader (Bio-Tek,
Winooski, VT).
Leucine Incorporation
The effect of PAN on protein synthesis was measured by leucine
incorporation, according to the method of Ziyadeh et al.
(17). 56/10 A1 cells were
seeded at a density of 0.01 x 106/cm2 in 24-well
plates. PAN was added at a concentration of 0, 0.5, or 5.0 µg/ml, and the
cells were incubated at 37°C for 48 h. The cells were pulsed with 2
µCi/ml [3H]-leucine (Amersham, Arlington Heights, IL) during the
last 14 h of the incubation period. The medium was removed, and the cells were
washed twice with ice-cold phosphate-buffered saline (PBS). The cells were
precipitated in 5% TCA for 10 min, then washed with ice-cold deionized water.
The acid-precipitated monolayers were solubilized with 0.5 N NaOH-0.1% Triton
X-100 (500 µl/well) and counted for radioactivity. [3H]-leucine
incorporation is expressed per 105 cells. Cell number was
determined in replicate wells cultured as above in 0, 0.5, or 5.0 µg/ml PAN
(n = 8 for each PAN concentration). Cells were trypsinized and
incubated in 0.2% trypan blue for 2 min. Cells were then counted in a
hemocytometer.
Flow Cytometry
Expression of integrin subunits was evaluated by flow cytometry. Cells were
cultured in the presence or absence of PAN for 48 h, and integrin expression
was studied using anti-integrin antibodies. Monoclonal antibodies P3D11, to
the 3 integrin subunit, and P5D2, to the ß1 integrin subunit, have
been described previously
(18). Monoclonal antibody
W6/32 against class I HLA was obtained from ATCC (Rockville, MD).
FITC-conjugated goat anti-mouse F(ab')2 fragment purchased from Tago Biosource
(Camarillo, CA) served as the secondary antibody. Cells were trypsinized,
washed, and resuspended in FACS buffer (HBSS, 2% goat serum, 0.02% sodium
azide). Cells (1.5 x 105) were incubated with the primary
antibody for 60 min at 4°C and then washed twice with FACS buffer. The
cells were then stained with F(ab')2 goat antimouse Ig-FITC (1:200) for 30 min
on ice. After the cells were washed twice with FACS buffer, they were fixed in
3% paraformaldehyde in PBS. Flow cytometric analysis was performed on a FACS
Calibur; 6000 cell events were analyzed using the Cell Quest program for each
experimental condition. Fluorescence was determined on a four-decade log scale
and log F1 was expressed.
Direct Cell Adhesion Assay
Assays were performed on 96-well microtiter plates. Type IV collagen was
isolated and purified according to previous methods
(19). Type IV collagen was
coated in serial dilutions starting from 25 mg/ml and dried overnight at
29°C, and then the plates were blocked with 200 µl of 2% bovine serum
albumin (BSA) for 2 h. Cells were treated with PAN for 2 d and labeled
overnight with 0.5 mCi 35S methionine. The cells were trypsinized
and were washed twice with and resuspended in binding buffer (Dulbecco's
modified Eagle's medium containing 2 mg/ml BSA at pH 7.4). A total of 7500
cells were added to each well in 100 µl of binding buffer. The plates were
incubated at 37°C in a humidified incubator for 75 min. The wells were
then washed two times with PBS to remove unbound cells, and 100 µl of lysis
buffer (0.5 N NaOH, 1% sodium dodecyl sulfate in distilled water) was added to
each well, followed by incubation at 60°C for 30 min. The lysates were
transferred to scintillation vials and counted. Total input cpm was 100 µl
of cells (7500 cells) added to scintillation vials. The cpm obtained from the
wells of the microtiter plate were expressed as a percentage of total input
cpm to give percentage of adhesion. The nonspecific adhesion of cells to BSA
was subtracted from the percentage of adhesion to type IV collagen. Adhesion
assays were performed in quadruplicate for each PAN concentration, and the
experiment was repeated three times.
Western Blotting
The expression of 3 integrin, ß1 integrin, podocalyxin, and
ZO-1 proteins was measured by quantitative Western blotting. Cells were
cultured as described. After treatment with PAN for 48 h, cells were lysed at
4°C with 700 µl of lysis buffer (50 mM Tris-HCl [pH 7.2] containing 150
mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, 10
µg/ml leupeptin, 1% Triton X-100, 2 µg/ml aprotinin, and 2 mM sodium
orthovanadate). The lysates were clarified by centrifugation at 12,000 x
g for 15 min. Protein estimation was performed by the DC Protein
Assay Kit (Bio-Rad, Hercules, CA). For assessment of ZO-1, 40 µg of cell
lysate was run on a 7.5% polyacrylamide gel. For assessment of podocalyxin, 30
µg of the cell lysate was run on a 5 to 15% gradient gel. For assessment of
3 and ß1 integrin subunits, 8 µg of cell lysate was run on a 5
to 15% acrylamide gel. All samples were nonreduced. After electrophoresis,
proteins were electrophoretically transferred to Immobilon-P membranes
(Millipore, Bedford, MA) for 2.5 h at 0.3 A. The membranes were blocked
overnight with PBS containing 3% skim milk. Membranes were then incubated with
one of the following antibodies, in blocking buffer for 2 h at room
temperature: rabbit anti-human ZO-1 (Zymed, San Francisco, CA; 1:2000);
monoclonal antibody 3D3 against podocalyxin, kindly provided by Dr. David
Kershaw (20); rabbit
anti-human 3 integrin (Chemicon, Temecula, CA; 1:2000 dilution);
monoclonal anti-human ß1 integrin (Chemicon; 1:2000 dilution.). This was
followed by incubation with peroxidase-conjugated goat anti-rabbit IgG or goat
anti-mouse IgG (Amersham). After membranes were washed with TBS containing
0.05% Tween 20, the blots were developed using enhanced chemiluminescence
according to the manufacturer's protocol (Amersham). Equality of loading was
ensured by testing for tubulin, using a monoclonal antibody to ß-tubulin
(Sigma Immunochemicals, St. Louis, MO). Films were scanned using a Bio-Rad
Model GS-700 Imaging densitometer, and densitometric analysis was performed
using Molecular Analyst Version 2.1.
RNA Isolation
Total RNA was isolated by a single-step method using RNA STAT-60 (TEL-TEST
"B," Inc., Friendswood, TX) according to the manufacturer's
instructions. The concentration and the purity of each sample were determined
spectrophotometrically. The integrity of the RNA and the accuracy of the
quantitation were checked by 1% agarose gel electrophoresis.
cDNA Cloning and Antisense RNA Probes
The human podocalyxin-like protein (PCLP) cDNA clone was obtained from a
human kidney 5'-STRETCH PLUS cDNA library (Clontech, Palo Alto, CA) by
PCR using specific primers (5'-CAGATGCCAGCCAGCTCTACGGC-3';
5'-AGTGAGATCAATTTCTCATCCG-3'). Primer sets were designed using the
Oligo and Amplify software programs (NBI Biotechnology, Plymouth, MN), on the
basis of published sequence data
(20). The human integrin
3 and ß1 cDNA clones were produced by PCR amplification from
previously described plasmids
(21,22).
A Genbank search did not reveal significant homologies of these cDNA with
other known genes.
The PCR-amplified cDNA fragments of PCLP (293 bp, nucleotides 1004 to
1296), 3 integrin (236 bp, nucleotides 2761 to 2996), and ß1
integrin (240 bp, nucleotides 481 to 720) were cloned into the Srf I site of
pCR-Script-Amp SK+ (Stratagene, La Jolla, CA). The orientation of
each cDNA clone within the vector was confirmed by automated fluorescence
sequencing using dye-labeled terminators, in an Applied Biosystems DNA
Sequencer Stretch. Appropriate restriction enzymes were used to linearize the
cDNA constructs. The human glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)
gene (316 bp or 154 bp) was purchased from Ambion (Austin, TX) and served as
an internal control.
RNase Protection Assay
In vitro transcription was performed in the presence of
a[32P]UTP (800 Ci/mmol; Amersham) using T3 or T7 RNA polymerase
(MAXIscript, Ambion). Total RNA was hybridized with 1 x 106
cpm of each labeled antisense probe with high specific activity (1.3 x
109 cpm/µg) overnight at 42°C. Subsequent RNase A and T1
digestions were performed using the RPAII Kit (Ambion). Assays for PCLP mRNA
used 40 µg of total RNA, whereas 20 µg of total RNA was used for
3 and ß1 mRNA. Each reaction included the GAPDH probe, labeled to
a 200-fold lower specific activity, as the internal control to which all
quantitation data were normalized. The linearity of quantitative accuracy was
confirmed by pilot experiments. The protected riboprobe fragments were
separated on denaturing 6% polyacrylamide gels. Gels were exposed to
phosphorimage screens, and the quantity of each specific mRNA was expressed as
the ratio of the densities of the specific mRNA and GAPDH protected
fragments.
Statistical Analyses
Mean values were derived from experiments performed in triplicate or
quadruplicate, as described above. The two-tailed paired t test was
used to compare the effects of different PAN concentrations (0, 0.5, and 5.0
µg/ml) on the results of quantitative Western blotting for ZO-1, flow
cytometry, and leucine incorporation. In these experiments, each PAN
concentration was tested on a single microtiter plate, and each experiment was
repeated on three different days. Because two statistical comparisons were
performed for each experiment (0 versus 0.5 and 0 versus
5.0), a P value of <0.025 was considered significant. The
two-factor ANOVA with replication was used to evaluate the results of
quantitative Western blotting for 3 and ß1 integrins and
podocalyxin, ribonuclease protection assays, cell number determinations, and
cell proliferation assays. In these experiments, multiple replicates for each
PAN concentration were carried out on a single microtiter plate. When the
overall effect of concentration was significant (P < 0.05), each
concentration, 0.5 and 5.0, was compared separately to the 0 concentration and
a P value of <0.025 was considered significant. Cell adhesion was
analyzed with a three-factor ANOVA with Tukey's test for multiple comparisons,
where concentration of PAN and type IV collagen were the fixed effects and the
number of times the experiment was repeated was the replicated factor. When
the overall effect of concentration was significant (P < 0.05),
each concentration, 0.5 and 5.0, was compared separately to the 0
concentration and a P value of <0.025 was considered significant.
The statistical analysis was carried out using Microsoft Excel and the
statistical package SAS (version 6.12, SAS Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incorporation of BrdU was measured as an indicator of DNA synthetic activity. At 48 h, the mean ± SD for optical density at 450 nm for 0, 0.5, and 5.0 µg/ml PAN was 0.46 ± 0.08, 0.45 ± 0.07, and 0.42 ± 0.07, respectively. These values were not significantly different (P = 0.07).
Effects of PAN on [3H]-Leucine Incorporation
The mean ± SD of cpm/105 cells for 0, 0.5, and 5.0
µg/ml PAN were 2.72 x 104 ± 2900, 2.7 x
104 ± 3200, and 1.64 x 104 ± 500,
respectively. The difference in [3H]-leucine incorporation by cells
that were exposed to 0 or 0.5 µg/ml PAN was not statistically significant.
However, there was a statistically significant decrease in
[3H]-leucine incorporation by cells that were exposed to 5.0
µg/ml PAN, compared with cells that were exposed to 0 or 0.5 µg/ml PAN
(P = 0.02 for either comparison).
Effects of PAN on Protein and mRNA Expression of 3 and ß1
Integrin Subunits
After culturing in the presence or absence of PAN, equal numbers of cells
(1.5 x 105) were analyzed for class I HLA, 3 and
ß1 integrin subunit expression by FACS analysis, and quantitative Western
blotting. By FACS analysis, there was no change in the expression of 3
and ß1 integrin subunits upon exposure of cells to 0.5 µg/ml PAN for
48 h (P = 0.06 and 0.2, respectively, versus 0 µg/ml
PAN). However, treatment with 5.0 µg/ml PAN for 48 h resulted in
significant reductions in the cell-surface expression of 3 and ß1
integrins (P = 0.005 and 0.01, respectively). Neither concentration
of PAN was associated with altered cell-surface expression of class I HLA (0
versus 0.5 µg/ml PAN, P = 0.4; 0 versus 5.0
µg/ml PAN, P = 0.36; Figure
1).
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Treatment with 5.0 µg/ml PAN for 48 h also resulted in decreased overall
cellular expression of 3 and ß1 integrin subunits as measured by
quantitative Western blotting (0 versus 5.0 µg/ml PAN; P
= 2.9 x 10-5 and P = 0.00016, respectively).
However, there was no significant decrease in expression of 3 and
ß1 integrin subunits on treatment with 0.5 µg/ml PAN (P =
0.04 and 0.146, respectively). Two bands were identified by the anti-ß1
integrin antibody. The smaller band is pre-ß1 integrin, which undergoes
N-glycosylation to form mature ß1 integrin, the larger band
(23)
(Figure 2). Both bands were
scanned and quantified for statistical analysis.
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Figure 3 shows the mRNA
expression of 3 and ß1 integrin subunits estimated by the
ribonuclease protection assay. Treatment with 0.5 µg/ml PAN resulted in
decreases in 3 and ß1 integrin subunit mRNA expression of
approximately 20%. These reductions were statistically significant (P
= 1.135 x 10-9 and P = 0.00026, respectively).
Treatment with 5.0 µg/ml PAN decreased both 3 and ß1 integrin
subunit mRNA expression by approximately 50%. These decreases were
statistically significant (P = 1.424 x 10-5 and
P = 2.05 x 10-10, respectively).
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Effects of PAN on Adhesion of 56/10 A1 Cells to Type IV Collagen
Because the 3ß1 integrin mediates the binding of 56/10 A1 cells
to type IV collagen (14), we
investigated the effect of PAN treatment on adhesion of these cells to type IV
collagen. There was no significant change in the adhesion of 56/10 A1 cells to
type IV collagen after treatment with 0.5 µg/ml PAN. However, at all
coating concentrations of type IV collagen, adhesion was significantly reduced
after treatment with 5.0 µg/ml PAN (P < 0.001;
Figure 4).
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Expression of Podocalyxin Protein and mRNA
Figure 5 shows podocalyxin
expression, as estimated by Western blotting, after treatment with PAN. There
was no significant change in the expression of podocalyxin after treatment
with 0.5 µg/ml PAN (P = 0.087). The expression of podocalyxin
after treatment with 5 µg/ml PAN was increased, and this difference was
statistically significant (P = 0.002).
Figure 6 shows podocalyxin mRNA
expression estimated by ribonuclease protection assay after treatment with
PAN. Treatment with 0.5 or 5.0 µg/ml PAN was associated with increased
podocalyxin mRNA expression, and these differences were statistically
significant (0 versus 0.5 µg/ml PAN, P = 0.002; 0
versus 5.0 µg/ml PAN, P = 7.5 x
10-6).
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Effects of PAN on Expression of ZO-1 Protein
Figure 7 shows ZO-1
expression as estimated by Western blotting after treatment with PAN. Several
bands were identified by the antibody to ZO-1, including a 225-kD band
representing the intact protein and three smaller bands believed to be
degradation products. Only the 225-kD band was quantified for statistical
analysis. There was no change in the expression of ZO-1 with either
concentration of PAN on treatment with either 0.5 or 5.0 µg/ml PAN (0
versus 0.5 µg/ml PAN, P = 0.65; 0 versus 5.0
µg/ml PAN, P = 0.69).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3ß1 to be the major integrin
present in vivo on podocytes
(9,10,11,12,13).
This integrin is present in high density on the whole plasma membrane of
podocyte foot processes, particularly at the interface between the foot
processes and the GBM. In contrast, only sparse positivity is found on the
membranes of the podocyte cell bodies
(24). 3ß1 is also
the major integrin expressed in vitro by both primary and
T-SV40-immortalized human glomerular epithelial cells and mediates the binding
of these cells to type IV collagen and GBM in vitro
(14). Focal detachment of
visceral epithelial cells from the GBM is a prominent morphologic feature of
PAN-induced nephrosis in rats
(4,5,6,7).
We hypothesized that the mechanism of PAN-induced detachment involves
inhibition of the expression of 3ß1 integrin by visceral
epithelial cells.
We found that PAN suppressed the expression of 3ß1 integrin
mRNA and protein by transformed human glomerular epithelial cells and that
this suppression was associated with a reduced capacity to adhere to type IV
collagen. Taken together, the Western blotting and FACS results indicate that
PAN resulted in decreased cellular content of 3ß1 integrin, rather
than simply causing redistribution of 3ß1 integrin from the cell
surface to the cytoplasm.
Although PAN at a concentration of 5.0 µg/ml caused a reduction in
protein synthesis as measured by leucine incorporation, there are several
indications that the observed decrease in 3ß1 integrin expression
was not attributable solely to a generalized decrease in protein synthesis.
First, both 0.5 and 5.0 µg/ml PAN suppressed expression of 3 and
ß1 integrin mRNA. Second, the expression of immunoreactive podocalyxin
was actually increased in the presence of PAN, in association with increased
podocalyxin mRNA, which would not be expected in the face of a generalized
inhibition of protein synthesis. The absence of a change in class I HLA
indicates that PAN's effects on 3ß1 integrin were not the result
of a nonspecific effect on the expression of cell-surface proteins.
These arguments aside, the experiments demonstrate clearly that PAN
suppresses 3ß1 integrin expression by cultured glomerular
epithelial cells, although the precise mechanism of this effect remains to be
elucidated. This effect is associated with a decreased ability of these cells
to adhere to type IV collagen. It is possible that the expression of other
proteins necessary for cell adhesion to type IV collagen is reduced by PAN,
contributing to the adhesion defect.
We found that the 5.0 µg/ml concentration of PAN caused a significant reduction in cell numbers, without affecting cell viability or DNA synthesis. We suspect that the reduced cell numbers at this concentration resulted from detachment of viable cells and that the remaining cells experienced no change in proliferative activity.
Studies of PAN's effect on glomerular integrin expression in vivo
have yielded mixed results. Smoyer et al.
(25) examined glomerular
expression of 3 and ß1 integrins in rats with PAN-induced
nephrosis by Western blotting of solubilized glomerular proteins. These
authors found no statistically significant changes in the expression of either
protein, except for increased 3 integrin expression 10 d after PAN
injection. Conversely, Kojima et al.
(26), using confocal
microscopy, found decreased glomerular 3 integrin expression 4 d after
PAN injection, preceding the development of proteinuria. Both the decrease in
glomerular 3 integrin expression and proteinuria were prevented by
treatment with superoxide dismutase. The absence of a reduction in glomerular
3 integrin expression in the study of Smoyer et al. may
reflect that glomerular mesangial cells and endothelial cells also express
3 and ß1 integrin subunits
(11,12,13,27)
so that a specific reduction in visceral epithelial cell expression of these
proteins may have been obscured.
The glomerular capillary wall is negatively charged as a result of the presence of sulfated proteoglycans in the GBM and sialoproteins on glomerular endothelial and epithelial cells. Podocalyxin, first identified in rats as a 140-kD glycoprotein, is concentrated along the apical and lateral plasma membrane of the podocyte, carries a high net negative charge, and is both sialylated and sulfated (28,29,30). The human homologue of podocalyxin has a molecular size of 165 to 170 kD and is referred to as PCLP (20). The nephrosis produced by PAN injection is accompanied by alterations in the polyanionic composition of the glomerulus, as demonstrated by histochemical staining with cationic dyes and by estimation of the glomerular content of sialic acid (7,31,32,33,34). Kerjaschki et al. (30) found a decrease in the sialic acid composition of podocalyxin in rats with PAN-induced nephrosis but no differences in the density of podocalyxin expression on podocytes between control and nephrotic rats. These observations suggest that although sialylation of podocalyxin is affected, podocalyxin synthesis is not impaired in podocytes exposed to PAN in vivo.
We observed increased expression of podocalyxin by 56/10 A1 cells after PAN treatment, at both the mRNA and protein levels. It is difficult to compare our results with those of Kerjaschki et al., given the differences in model systems and time frames (e.g., Kerjaschki's rats received PAN injections daily for 9 to 11 d before they were killed and their kidneys were isolated). The results of both studies are consistent with the notion that PAN does not suppress podocalyxin synthesis directly.
The glomerular slit diaphragm, located between adjacent podocyte foot processes, is believed to be a modified adherens junction and expresses ZO-1, a protein present in tight junctions and adherens junctions (28,35,36). Collapse of the slit diaphragm is another characteristic ultrastructural feature of PAN-induced nephrosis. Our studies showed no change in the amount of ZO-1 as estimated by immunoblotting. Kurihara et al. (36) did not observe differences in the quantity of ZO-1 proteins between control and PAN-nephrotic glomeruli. This result is similar to our finding that ZO-1 expression by 56/10 A1 cells was not altered by exposure to PAN.
The popularity of PAN-induced nephrosis as a model system derives from its
morphologic similarities to minimal change nephrotic syndrome and focal
segmental glomerulosclerosis, the major entities associated with idiopathic
nephrotic syndrome. The causes of these disorders remain mysterious, but
various lines of evidence suggest that circulating factors provoke changes in
visceral epithelial cell function, resulting in breakdown of the glomerular
barrier to protein. The findings of this study suggest that suppression of
3ß1 integrin expression on cultured visceral epithelial cells and
inhibition of adhesion of these cells to type IV collagen may serve to
identify the presence of such factors in the circulation of patients with
idiopathic nephrotic syndrome.
|
Acknowledgments |
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This work was supported by a grant from NIH (AI10704) to A.M.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 C. G. Economou, P. V. Kitsiou, A. K. Tzinia, E. Panagopoulou, E. Marinos, D. B. Kershaw, D. Kerjaschki, and E. C. Tsilibary Enhanced podocalyxin expression alters the structure of podocyte basal surface J. Cell Sci., July 1, 2004; 117(15): 3281 - 3294. [Abstract] [Full Text] [PDF]
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 A. Suzuki, T. Ito, E. Imai, M. Yamato, H. Iwatani, H. Kawachi, and M. Hori Retinoids Regulate the Repairing Process of the Podocytes in Puromycin Aminonucleoside-induced Nephrotic Rats J. Am. Soc. Nephrol., April 1, 2003; 14(4): 981 - 991. [Abstract] [Full Text] [PDF]
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 H. Pavenstadt, W. Kriz, and M. Kretzler Cell Biology of the Glomerular Podocyte Physiol Rev, January 1, 2003; 83(1): 253 - 307. [Abstract] [Full Text] [PDF]
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 B. Doornaert, V. Leblond, S. Galiacy, G. Gras, E. Planus, V. Laurent, D. Isabey, and C. Lafuma Negative impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair Am J Physiol Lung Cell Mol Physiol, January 1, 2003; 284(1): L119 - L132. [Abstract] [Full Text] [PDF]
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