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Regular Articles |
From the Division of Nephrology*
and Department
of Pathology,
University Hospital St. Radboud,
Nijmegen, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-
and ß-dystroglycan, sarcoglycans, and
utrophin/dystrophin, links the cytoskeleton to agrin and
laminin in the basal lamina in muscle and epithelial cells.
Recently, agrin was identified as a major heparan sulfate
proteoglycan in the glomerular basement membrane. In the present
study, we found mRNA expression for agrin,
dystroglycan, and utrophin in kidney cortex, isolated
glomeruli, and cultured podocytes and mesangial cells. In
immunofluorescence, agrin was found in the glomerular basement
membrane. The antibodies against - and ß-dystroglycan and utrophin
revealed a granular podocyte-like staining pattern along the glomerular
capillary wall. With immunoelectron microscopy, agrin was found
in the glomerular basement membrane, dystroglycan was diffusely
found over the entire cell surface of the podocytes, and
utrophin was localized in the cytoplasm of the podocyte foot processes.
In adriamycin nephropathy, a decrease in the glomerular
capillary wall staining for dystroglycan was observed probably
secondary to the extensive fusion of foot processes. Immunoelectron
microscopy showed a different distribution pattern as compared to the
normal kidney, with segmentally enhanced expression of
dystroglycan at the basal side of the extensively fused podocyte foot
processes. In passive Heymann nephritis we observed no changes in the
staining intensity and distribution of the dystrophin-glycoprotein
complex by immunofluorescence and immunoelectron microscopy. From these
data, we conclude that agrin, dystroglycan, and
utrophin are present in the glomerular capillary wall and their
ultrastructural localization supports the concept that these molecules
are involved in linking the podocyte cytoskeleton to the glomerular
basement membrane.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-DG, a heavily glycosylated peripheral membrane protein and the
transmembrane protein ß-DG. In skeletal muscle, -DG is a major
binding protein for agrin as well as for laminins.6-9
-DG remains noncovalently linked to ß-DG, which through its
cytoplasmic tail, binds directly to the C-terminal portion of
dystrophin10,11
whereas the N-terminal domain of
dystrophin binds to the subsarcolemmal actin
cytoskeleton.12,13
Utrophin is an autosomal homologue of
dystrophin and can also bind ß-DG.14
Recent in
vitro studies support the role of the DGC in adhesion. Adhesion of
a rat schwannoma cell line to laminin could be inhibited by antibodies
against -DG in vitro.15
Furthermore,
myotubules of patients with Duchenne muscular dystrophy were unable to
adhere to laminin 2 in vitro.16 Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (GBM).17 Various other components of the DGC were also identified in the kidney.18-20 Therefore, we hypothesized that glomerular visceral epithelial cells or podocytes are linked to the GBM in a similar way as the myocyte to the basal lamina. The podocytes and GBM, together with the fenestrated endothelium form the glomerular capillary wall (GCW), the barrier preventing passage of plasma proteins into the urinary space during glomerular ultrafiltration. During heavy proteinuria in various human and experimental glomerulopathies, the podocytes show dramatic morphological changes like fusion of foot processes and/or detachment from the GBM.21-26 Several observations suggest that podocyte dysfunction and subsequent detachment contributes to the development of proteinuria. In adriamycin nephropathy (ADN), puromycin aminonucleoside nephrosis, and serum sickness nephritis, proteinuria correlates with podocyte detachment,27,28 but not with changes in GBM charge density.29 Passage of albumin through the GCW was localized to regions of saponin-induced detachment of podocytes in the single nephron model.30 In another study it was found that injection of a monoclonal antibody (mAb) directed against a component of the slit diaphragm resulted in an acute massive proteinuria.31,32 Therefore, it is generally assumed that the extent of podocyte detachment is related to the severity of proteinuria. However, no conclusive data are available on the mechanism of podocyte detachment during proteinuria.
The present study focuses on the distribution of agrin, - and
ß-DG, and utrophin in the kidney. Expression and localization was
evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR)
on RNA isolated from renal cortex, isolated glomeruli, and cultured
podocytes and mesangial cells, and by immunofluorescence (IF) and
immunoelectron microscopy (IEM) with monoclonal antibodies against the
N- and C-terminus of agrin, -DG, ß-DG, and utrophin. These studies
show that apart from agrin, - and ß-DG and utrophin are also
present in the GCW and suggest a link between the podocyte and the GBM.
Furthermore, the expression of the DGC was studied in two experimental
models of proteinuric glomerulopathy: ADN in rats, which serves as a
model for the nephrotic syndrome, characterized by extensive fusion of
podocyte foot processes; and passive Heymann nephritis (PHN), a model
for human membranous nephropathy, characterized by IgG depositions
subepithelially in the GCW. Only in ADN were changes seen in the
distribution of DG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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The experimental protocol for the animal studies was approved by the local ethical committee. For induction of ADN and PHN and for the isolation of mesangial cells, we used male Wistar-Unilever rats that were bred at our animal laboratory and weighed ~150 g at the start of the experiments. The animals were given standard food and tap water ad libitum. For primary podocyte culture, female Sprague-Dawley rats (Charles-River, Sulzfeld, Germany) ~120 g were used.
Adriamycin Nephropathy
ADN was induced in four rats as described.33 As controls, four rats were injected with the same volume of saline.
Passive Heymann Nephritis
Antibodies against renal tubular epithelium raised in a sheep were used for the induction of PHN. Serum of four normal sheep was pooled and used as control. The immunoglobulin (Ig) from the antiserum and the normal sheep serum was purified by ammonium sulfate precipitation followed by ion-exchange chromatography using DEAE-Sepharose. The Ig was dialyzed extensively against phosphate-buffered saline (PBS), concentrated to one-fifth of the original volume, passed through a 0.2-µm filter and used for injection. PHN was induced in four rats by three intravenous injections on subsequent days of 40 mg IgG of this purified sheep anti-renal tubular epithelium antiserum. As a control, four rats were injected with the same amount of normal sheep IgG.
Determination of Urine Albumin Concentration
Urinary albumin excretion was measured as described previously.34
Isolation and Culture of Glomerular Cells
Primary Culture of Rat Podocytes
The primary culture of rat podocytes was performed as described by Mundel and co-workers.35 Briefly, glomeruli were isolated from female Sprague-Dawley rats by the differential sieving procedure and cultured for 4 days in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L glutamine, 1 mmol/L pyruvate (all from Gibco, Paisley, Scotland). The outgrowing epithelial cells were trypsinized and passed through a sieve with a pore size of 32 µm to remove the remaining glomerular cores. The epithelial cells were cultured in plastic tissue culture flasks (Greiner, Frickenhausen, Germany) or plastic slide flasks (Nunc, Roskilde, Denmark) coated with collagen I (Seromed, Berlin, Germany) for 3 to 4 weeks without splitting. Medium was changed twice a week.
Isolation and Culture of Rat Mesangial Cells
Isolation and culture of rat mesangial cells was performed as described by Wolthuis and co-workers.36 For IF and RT-PCR mesangial cells between passage 12 and 15 were used.
Reverse Transcriptase-Polymerase Chain Reaction and Analysis of Polymerase Chain Reaction Products
Isolation of RNA
Total RNA was extracted from kidney cortex and glomeruli by RNAzol (Campro Scientific, Veenendaal, The Netherlands) according to the instructions of the manufacturer. RNA extractions from podocytes and mesangial cells were performed using the guanidinium isothiocyanate acid phenol chloroform procedure37 with small modifications. In all samples, RNA was dissolved in DEPC-treated water for 10 minutes at 65°C and stored at -80°C. RNA concentration was determined by measuring the absorbance of a diluted aliquot at 260 nm. Quality of the RNA samples was checked by electrophoresis on a 0.8% agarose gel and staining with ethidium bromide. No degradation was observed.
Reverse Transcriptase-Polymerase Chain Reaction
Primers and probes listed in Table 1
were designed using the Primer Express 1.0 program (Perkin-Elmer Cetus,
Norwalk, CT) based on the rat agrin and dystroglycan cDNA
sequence38-40
and the homological stretches of mouse and
human utrophin cDNA sequence.41,42
The primer sets for the
agrin N-terminus and C-terminus are exon-spanning (based on the mouse
gene), with introns of 165 and 270 bp, respectively. Primers and probes
were synthesized and purified by the Eurogentec Corporation (Seraing,
Belgium). Synthesis of single-stranded cDNA was performed in a solution
containing 1 µg of total RNA, 50 mmol/L Tris-HCl (pH 8.3), 7.5 mmol/L
KCl, 6 mmol/L MgCl2, 10 mmol/L DTT, 0.2 mmol/L of
dNTPs (Boehringer Mannheim, Mannheim, Germany), 2.5 µmol/L of random
hexamers (Perkin-Elmer Cetus), 1000 U/ml RNase inhibitor (RNasin;
Promega, Madison, WI), 20,000 U/ml Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) in a
total volume of 20 µl. Reactions were overlaid with mineral oil and
were subsequently incubated in a DNA thermal cycler (Eppendorf Master
Cycler 5330, Eppendorf, Hamburg, Germany) at 20°C for 10
minutes, 42°C for 50 minutes, and 95°C for 5 minutes. Amplification
of cDNA was performed by addition of 80 µl of a mixture containing 50
mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.9), various concentrations of
MgCl2 ranging between 0.1 and 2.5 mmol/L (see
Table 1
), 0.2 mmol/L of dNTPs, 150 nmol/L of upstream primer, 150
nmol/L of downstream primer, and 2.5 U/ml Thermoperfect DNA polymerase
(Integro, Zaandam, The Netherlands). Amplification started with an
initial denaturation step at 94°C for 2 minutes, followed by 34
cycles of denaturation at 94°C for 1.5 minutes, annealing at various
temperatures ranging between 54 and 62°C (see Table 1
) for 1.5
minutes, and extension at 72°C for 1.5 minutes. After the last cycle,
the extension phase was prolonged for 10 minutes at 72°C and the
samples were cooled to 15°C. To optimize the PCR reaction, for each
product the MgCl2 concentration and the annealing
temperature were varied and the conditions yielding maximal signal
together with minimal background were chosen for the experiments.
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After amplification, 25 µl of PCR products were analyzed
on a 1.5% agarose gel and stained with ethidium bromide. To check the
specificity of the PCR products, they were blotted to a Hybond
N+ nylon membrane (Amersham, Buckinghamshire,
UK), using the alkali blotting procedure according to the manufacturer.
Probes were 5' end-labeled with 
-32P-dATP using T4
kinase (Boehringer Mannheim). Unincorporated radioactivity was removed
using a Sephadex G-25 spin column. The Southern blots were
prehybridized with 1 mg of herring sperm DNA in 0.5 mol/L
NaH2PO4/Na2HPO4,
7% SDS, 1 mmol/L EDTA buffer (pH 7.2; hybridization solution) for 1
hour at 58°C, and then 50 pmol/L radiolabeled oligomer probe was
added and incubation was continued overnight at 58°C. To remove
unbound probes, blots were washed with 0.5 mol/L
NaH2PO4/Na2HPO4,
1% SDS, 1 mmol/L EDTA (pH 7.2), for 10 minutes at 58°C and with 0.25
mol/L
NaH2PO4/Na2HPO4,
1% SDS, 1 mmol/L EDTA (pH 7.2), for 10 minutes at 58°C and exposed
to X-ray film (Eastman-Kodak, Rochester, NY) for 1 hour at -80°C.
Immunohistology
Immunofluorescence
Indirect IF was performed as described previously34
on 2-µm cryostat sections of rat or human kidney and rat soleus
muscle or human quadriceps muscle. Sections were fixed in acetone at
4°C during 10 minutes, except for incubations with the
anti-dystrophin and anti-sarcoglycan antibodies, which was performed on
nonfixed sections. Details on the used antibodies are given in Table 2
.17,43-48
Primary
antibodies were diluted in PBS containing 1% bovine serum albumin
(BSA) and 0.05% sodium azide (IF-buffer), fluorescein
isothiocyanate-labeled secondary antibodies were diluted in IF-buffer
containing 10% normal rat serum (for IF on rat tissue), all antibodies
were incubated during 45 minutes at room temperature. To evaluate the
deposition of sheep and rat IgG and complement factor C3c in the GCW of
rats with PHN, sections were directly incubated with fluorescein
isothiocyanate-labeled rabbit anti-sheep IgG (Southern, Birmingham, AL)
diluted 1:100 in IF-buffer, goat anti-rat IgG and goat anti-rat C3c
(both from Nordic, Tilburg, The Netherlands) both diluted 1:50 in
IF-buffer. After incubation with antibodies, the sections were washed
with PBS, fixed with 1% paraformaldehyde in PBS for 15 minutes,
washed, and embedded in Vectashield mounting medium H-1000
(Vector Laboratories Inc., Burlingame, CA) and examined with a Zeiss
Axioskop microscope equipped with an epi-illuminator. The staining of
the antibodies in the GCW of the rats (four ADR rats and four controls;
four PHN rats and four controls) were evaluated in 25 glomeruli on a
scale between 0 and 4+ by two independent observers on coded sections
and the mean of the two scores was used for further analysis.
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45,49,50
were diluted in
blocking solution and incubated at room temperature for 60 and 45
minutes, respectively. Thereafter, the sections were washed in PBS,
fixed with 1% paraformaldehyde in PBS for 15 minutes, washed, and
mounted in Vectashield mounting medium. To stain vital cells, the cells
were incubated first with primary antibodies on ice, washed in PBS,
fixed in 2% paraformaldehyde and 4% sucrose in PBS, washed, blocked,
incubated with secondary antibodies, washed, fixed with 1%
paraformaldehyde in PBS, washed, and mounted in Vectashield mounting
medium as described above. The whole procedure was performed on ice.
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Normal rats or rats with ADN or PHN were anesthetized with 60
mg/kg pentobarbital. One kidney was removed and snap-frozen in liquid
nitrogen for IF, the other kidney was perfused for 10 minutes via the
retrograde aortic route with PBS, followed by perfusion with a mixture
of 10 mmol/L sodium periodate, 75 mmol/L lysine HCl, and 2%
paraformaldehyde (PLP) for 10 minutes. Small pieces of renal cortex
were immersed in the same fixative for another 3 hours, washed in PBS
for 1 hour, cryoprotected in 2.3 mol/L sucrose in PBS for 45 minutes,
and snap-frozen in liquid nitrogen. Twenty-five-µm cryostat sections
of PLP-fixed renal tissue were incubated with mAbs MI-90, Agr-131,
IIH6, 43-DAG, and DRP-2 (Table 2)
, diluted in PBS containing 1% BSA
for 18 hours at 4°C and washed with PBS. The sections were then
incubated with peroxidase-labeled secondary antibodies diluted in
PBS containing 1% BSA and 10% normal rat serum for 90 minutes. After
washing with PBS, the sections were preincubated with 0.05%
diaminobenzidine medium containing 0.6% Tris for 10 minutes, followed
by the same medium with
H2O2 in a final
concentration of 0.0001%. After three washes in distilled water, the
sections were postfixed in 1% OsO4, pH 7.4, for
30 minutes at room temperature, dehydrated, and embedded in Epon 812.
Ultrathin sections were prepared on a LKB Ultratome and examined in a
Jeol 1200 EX2 electron microscope (Jeol Ltd, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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First, we tested whether mRNA for agrin, DG, and utrophin was
present in kidney cortex. Amplification of cDNA with primers for
C-terminal agrin yielded a PCR product of 101 bp (Table 1
and Figure 1
A, lane 1). Similarly, amplification
with primers for N-terminal agrin and DG yielded products of 101 bp
(Figure 1
, B and D, lanes 1) and with primers for utrophin yielded a
product of 154 bp (Figure 1C
, lane 1). This shows that mRNA for agrin,
DG, and utrophin is present in the kidney cortex. No PCR product was
obtained in samples that were not reverse-transcribed (Figure 1
, A–D,
lanes 2), confirming that the RNA samples were not contaminated with
genomic DNA. Omitting RNA from the samples also yielded no PCR product
(Figure 1
, A–D, lanes 9). To confirm the identity of the PCR products,
the products were transposed to nylon blots that were hybridized with
radiolabeled probes specific for the PCR products. All probes
specifically hybridized with their PCR product (not shown).
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,
A–D, lanes 3). No PCR product was obtained in samples that were not
reverse-transcribed (Figure 1
, A–D, lanes 4).
To find out which cells in the glomerulus are responsible for the
production of agrin, DG, and utrophin, we cultured rat podocytes and
rat mesangial cells. The phenotype of the cells was confirmed by IF
with various markers as shown in Table 4
.
The podocytes showed staining for WT-1, synaptopodin, vimentin, and
smooth muscle actin, and a weak staining for -DG early after
isolation, but this staining disappeared with prolonged culture,
probably as a result of dedifferentiation. The podocytes were negative
for podocalyxin, O-acetylated ganglioside, desmin, Thy 1.1, and
coagulation factor VIII. The mesangial cells showed staining for
desmin, vimentin, Thy 1.1, smooth muscle actin, and -DG, but were
negative for WT-1, synaptopodin, podocalyxin, O-acetylated ganglioside,
and factor VIII. Thus, both cultured cell types had some but not all
features expressed by podocytes and mesangial cells in vivo,
which is consistent with a lower differentiation state of these cells
in vitro.51
Amplification of RNA extracted from
both podocytes and mesangial cells with all four primer sets yielded
PCR products comparable to kidney cortex and glomeruli (Figure 1
, A–D,
lanes 5, podocytes and lanes 7, mesangial cells). RNA samples from
podocytes and mesangial cells that were not reverse-transcribed yielded
weak bands of 266 bp for N-terminal agrin, indicating that these
samples were slightly contaminated with genomic DNA (Figure 1B
, lanes 6
and 8). For C-terminal agrin, DG, and utrophin, however, no PCR product
was obtained in the samples that were not reverse-transcribed (Figure 1
, A, C, and D, lanes 6 and 8). These data show that both podocytes and
mesangial cells contain message for agrin, DG, and utrophin.
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In indirect IF, anti--sarcoglycan (adhalin) mAb 50-DAG showed
clear staining of myocyte cell membranes of rat soleus muscle, whereas
no staining was seen on rat kidney sections. Anti--dystrophin mAbs
DYS1 and DYS2 also clearly stained myocyte cell membranes of rat muscle
and the vascular myocyte membranes in kidney sections, however, no
staining was observed in the glomeruli and in the tubuli. DYS1, DYS2,
and DYS3 (human-specific) also stained human quadriceps muscle and
human kidney tissue in the same way as described for rat tissue. These
observations are in agreement with previous studies, which have shown
that -sarcoglycan is specifically expressed in muscle52
and dystrophin is only expressed in muscle and neural tissue. These
antibodies were not used in further studies.
On rat kidney cryostat sections, anti-N-terminal agrin mAb MI-90 and
anti-C-terminal agrin mAb Agr-131 stain the GBM in a bright linear way
as described previously.17
mAb MI-90 stains most tubular
basement membranes (TBMs) in a linear fashion, but somewhat less
intense than the GBM, whereas the TBMs are negative or weakly stained
by mAb Agr-131. Bowman’s capsule, basement membranes (BMs) of
endothelial cells in peritubular capillaries, and smooth muscle cells
of arteries and arterioles are negative for both mAbs (Figure 2
, A and B). Anti--DG mAb IIH6 and
anti-ß-DG mAb 43-DAG both show a granular staining along the GCW in a
podocyte-like staining pattern (Figure 2C)
. Some tubular epithelial
cells show a coarse granular staining of IIH6 at the basal side,
whereas most tubular epithelial cells show a granular staining of
43-DAG (anti-ß-DG) at the basal side facing the tubular BMs. However,
the staining of both mAbs in the tubuli is less intense than in the
GCW. The basal sides of parietal epithelial cells near Bowman’s
capsule are negative or weakly stained by both mAbs. The basal cell
surface of smooth muscle cells in arterioles is weakly stained by
43-DAG (not shown). Anti-utrophin mAb DRP-2 shows a granular staining
along the GCW and sometimes of the podocyte cell body. There is a
variable and coarse granular staining of the basal side of the tubular
epithelial cells. The basal sides of parietal epithelial cells near
Bowman’s capsule are negative or weakly stained and the basal cell
surface of smooth muscle cells in arteries and arterioles is stained
brightly (Figure 2D)
. These experiments show that agrin, DG, and
utrophin are present in the GCW. On sections of normal human kidney,
anti-N-terminal agrin mAb MI-90, anti-ß-dystroglycan mAb 43-DAG, and
anti-utrophin mAb DRP-2 showed the same staining pattern as shown
for normal rat kidney (Figure 3)
,
indicating that these components are also important for human
glomerular physiology.
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,A, B, D, and E).
Furthermore, mAb MI-90 stained most TBMs, whereas mAb Agr-131 only
stained TBMs of distal tubuli. Both mAbs slightly stain the basal
in-foldings of the tubular epithelial cells (Figure 4
, C and F). The
regions of the GBM covering the mesangial areas were not stained by mAb
Agr-131 (C-terminal agrin), whereas this part of the GBM was positive
for mAb MI-90 (N-terminal agrin). This suggested that the perimesangial
part of the GBM lacks the C-terminus of agrin. Alternatively, this lack
of staining with mAb Agr-131 could be due to hampered penetration
of the mAb. Therefore, we performed additional IEM using
a rabbit polyclonal antiserum against the recombinant C-terminal 95
amino acids of chick agrin,44
which closely corresponds
with the C-terminus of rat agrin and cross-reacts with rat tissues.
Also this antiserum exclusively stained the peripheral GBM loops and
not the perimesangial GBM parts (Figure 5)
. This similar staining pattern
revealed by a mouse IgG2b mAb and a polyclonal rabbit antiserum toward
the C-terminus of agrin suggests that the perimesangial GBM expresses a
truncated variant of agrin, lacking the C-terminus. Both MI-90 and
Agr-131 showed minor staining of mesangial cells, predominantly in the
regions where a mesangial cell adheres to another mesangial or
endothelial cell (Figure 4
, A and D). Anti--DG mAb IIH6 stained the
cell membrane of the podocyte cell bodies as well as the foot processes
with high intensity, however, the regions where the foot processes face
the GBM were stained less intense than the rest of the podocyte cell
membrane (Figure 4
, G and H). mAb IIH6 also stains the basolateral cell
membranes of the epithelial cells of tubuli, including the basal
membrane in-foldings (Figure 4I)
. Anti-ß-DG mAb 43-DAG showed a
similar but weaker staining pattern than mAb IIH6 (not shown), which
also corresponds to the staining observed in IF. Anti-utrophin mAb
DRP-2 stained the cytoplasm of some podocyte foot processes, where the
strongest intensity was observed in the regions near the GBM (Figure 4
,
J and K). At the basal side of epithelial cells of distal tubuli some
staining for utrophin was also observed (Figure 4L)
. The staining
patterns of these mAbs obtained by IEM are in agreement with the
staining observed in IF.
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- and ß-DG are podocyte cell membrane components, and
that utrophin is localized in the cytoplasm of the podocyte foot
processes. Agrin, Dystroglycan, and Utrophin Staining in Adriamycin Nephropathy and Passive Heymann Nephritis
To evaluate the correlation between the development of albuminuria and the expression of agrin, DG, and utrophin, we analyzed glomerular staining of these components in rats with ADN and PHN.
Adriamycin Nephropathy
The injection of 5 mg of adriamycin per kg of bodyweight induced a
heavy albuminuria of 344 ± 106 mg per 24 hours similarly as
previously documented.34
The rats injected with saline had
an albumin excretion of less than 1 mg per 24 hours throughout the
experiment. In ADN, ~20% of the glomeruli showed a slight segmental
decrease in staining of mAb MI-90 against the N-terminus of agrin and
of mAb Agr-131 against the C-terminus of agrin. More notable, however,
was the decreased staining of mAb IIH6 against -DG in ~70% of the
glomeruli. This decreased staining was due to a segmentally or
generally reduced staining of the capillary loops from 3+ to 2+ (Figure 6A)
. In ~50% of the glomeruli the staining with mAb 43-DAG against
ß-DG was decreased in a pattern similar to mAb IIH6 (not shown). The
staining of mAb DRP-2 against utrophin was slightly decreased in
~20% of the glomeruli.
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-DG, we
also performed IEM with this mAb. There were striking morphological
changes in ADN because podocytes showed vacuolization and extensive
fusion of foot processes, however, no detachment of podocyte foot
processes from the GBM was observed (Figure 6B)
. Furthermore, the
expression of -DG on the basal side of the podocytes was locally
more pronounced than in the normal rat kidney, whereas the staining of
the podocyte cell membrane was unaltered (Figure 6
, B and C). There was
also a more pronounced expression of -DG at the basal side of
tubular epithelial cells and the membrane in-foldings (Figure 6D)
. Passive Heymann Nephritis
Injection of rats with 40 mg of anti-renal tubular epithelium IgG
on 3 subsequent days, induced in a mild albuminuria starting at week 2,
that increased steadily, and resulted after 18 weeks in an albumin
excretion of 115 ± 76 mg per 24 hours. The rats injected with the
same volume of normal sheep IgG had an albumin excretion of less than 1
mg per 24 hours throughout the experiment. In rats with PHN, the GCW
was stained in a bright, coarse granular pattern for sheep IgG (Figure 7A)
and was weakly positive for rat IgG
and rat C3c (not shown). In PHN we did not observe alterations in the
staining of mAb MI-90 against the N-terminus of agrin, mAb Agr-131
against the C-terminus of agrin, mAb IIH6 against -DG (Figure 7B)
,
mAb 43-DAG against ß-DG, and mAb DRP-2 against utrophin, despite the
presence of subepithelial immune deposits as shown by a bright course
granular staining for sheep IgG (Figure 7A)
. IEM was performed with
IIH6 (-DG). No obvious changes were observed. Podocytes displayed a
rather normal phenotype and barely any effacement of the foot processes
was seen. Occasionally, large immune deposits were visible
subepithelially. IIH6 stained the cell membranes of the podocytes very
prominently, the basal side of the foot processes were rather weak,
identical to the staining in the control kidney (Figure 7
,
C and D).
In contrast to ADN, no up-regulation of
DG was observed between the GBM and the podocyte foot processes.
Staining of -DG in the tubular areas of PHN kidneys did not differ
from control renal tissue (data not shown). We conclude that in PHN the
distribution of -DG is not altered.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Agrin has been identified in the BM in many tissues,53
including the developing kidney.54
Furthermore, agrin is a
major heparan sulfate proteoglycan in the adult GBM.17,55
The anionic heparan sulfate side chains of agrin play an important role
in the maintenance of the charge-dependent permeability of the
GBM.56-58
In the present study, we observed a difference
in ultrastructural localization between the N- and C-terminus of agrin.
The N-terminus of agrin was found in the entire GBM whereas with two
different antibodies the C-terminus was not found in the regions of the
GBM covering mesangial cells. In a previous study, we also observed a
difference in tissue distribution with these antibodies. Most TBMs
lacked staining for the C-terminus of agrin, whereas the N-terminus of
agrin was present in most TBMs.17
The difference in GBM
staining pattern for N- and C-terminal agrin in the present study could
be due to a reduced accessibility of the perimesangial GBM regions for
the antibodies. This is, however, not very likely because the mAb
against N-terminal agrin clearly stained both peripheral and
perimesangial GBM parts. Alternatively, we suggest the presence of a
truncated agrin isoform, missing the C-terminus, in the perimesangial
GBM. Because the C-terminus of agrin contains the binding site for
-DG,6-9
the agrin-DGC link between the GBM and the
podocyte cytoskeleton may only be important in peripheral GCW regions
that are involved in ultrafiltration.
DG has been shown to be present adjacent to BMs in many tissues
including the kidney.18,19
The staining patterns that we
observed for - and ß-DG correspond to the findings of Durbeej and
co-workers,19
who have shown that an antiserum recognizing
DG stains the GCW and the basal side of tubular epithelial and
glomerular parietal epithelial cells near Bowman’s capsule in human
and mouse kidney. The importance of DG in development is underlined by
the observation that DG-deficient mice, which die early in development,
fail to form a functional Reichert’s membrane, suggesting that DG is
involved in organizing BMs.59
Furthermore, antibodies
directed against -DG, that block the binding of -DG to agrin and
laminin,7
perturb kidney development in
vitro.18
Several investigators reported
co-localization of agrin and ß-DG in the developing kidney, lung, and
brain60
and of ß-DG and dystrophin in human, rat, and
mouse myofibers, near or at the plasma membrane.61-63
Several studies have already shown that utrophin is present near BMs in
many tissues,64-67
including the kidney.20
In the present study we found staining for utrophin in some but not all
of the podocyte foot processes. That not all foot processes were
positive could be due to low affinity of the mAb (DRP-2), because
staining in IF was weak as well (see Figure 2D
). The observation in IEM
that staining for utrophin was strongest in the regions near the GBM
confirms the hypothesis that via utrophin the DGC links the
cytoskeleton to the GBM.
By RT-PCR, we could show that cultured podocytes express mRNA for
agrin, DG, and utrophin. mRNA for these components was, however, also
present in the mesangial cells. In IEM however, no staining was
observed in the mesangium for DG and utrophin and only a weak staining
for agrin. By in situ hybridization on adult mouse kidney,
Durbeej et al19
suggested that DG mRNA expression was
predominantly localized in epithelial cells. With this technique
however, it cannot be excluded that mesangial cells also express mRNA
for DG. As known, PCR does not provide information about protein
expression. With IEM we found in the mesangium some staining for the
N-terminus of agrin and also a weaker staining for the C-terminus of
agrin. This staining was seen in regions where mesangial cells adhere
to other mesangial or endothelial cells. Because the mAbs against
ß-DG and utrophin showed a rather weak staining both in IF and IEM,
the possible expression of these proteins by mesangial cells could have
been missed. The staining intensity on the podocyte cell membrane for
-DG was much higher, but no staining was observed with this mAb in
mesangial areas. Furthermore, cultured mesangial cells have a lower
differentiation state than in vivo (Table 4)
and this
dedifferentiation may result in an altered gene expression. Therefore,
it is possible that mesangial cells are to some extent able to
synthesize agrin, DG, and utrophin. On the other hand, we found
cultured podocytes to be weakly positive or negative for DG. Based on
the data obtained by IEM however, it can be concluded that within the
glomerulus the podocyte is the major source for DG and utrophin. With
respect to DG, we conclude that glomerular cell culture systems are not
reliable models for the in vivo situation.
Several investigators have suggested a role for the DGC in linking the
cytoskeleton to the ECM, not only for muscle cells but also for
epithelial cells.1,19
The results from the present study
underline the possibility that GBM agrin is linked via - and ß-DG
to utrophin (Figure 8)
. This hypothesis, however, is weakened by the
observation that although both - and ß-DG are present at the basal
side of the podocytes, the strongest expression is seen outside the
contact sites with the GBM at the lateral and apical regions of the
podocyte membrane. If -DG on podocytes serves as a receptor for
agrin (and laminin), it is unclear why it is more abundantly present on
the luminal than on the basal side of the cells. Furthermore, it is
unlikely that - and ß-DG are complexed to utrophin at the luminal
sides, because we only observed utrophin expression in the foot
processes of the podocytes. The weaker staining for DG at the basal
side of the foot processes could also be the result of a differential
accessibility of the basal and lateral sides of the podocyte cell
membrane to the antibodies. At present, it is unclear what the function
could be of -DG on the luminal membrane of the podocyte cell body. A
similar membrane distribution on the podocyte is found for podocalyxin,
glomerular epithelial protein 1 (GLEPP1), and podoplanin which all are
more heavily expressed on the luminal podocyte membrane than on the
basal side.68-73
Also the functions of these proteins are
not clear yet. The anionic sialoglycoprotein podocalyxin may contribute
to the negative charge of the GCW.74
GLEPP1 is a
transmembrane receptor-like protein-tyrosine phosphatase, which may
have a function in podocyte structure by regulating tyrosine
phosphorylation of podocyte proteins.71,72
Because -DG
is very heavily glycosylated,75
it may have a function
similar to that of podocalyxin and contribute to the negative charge of
the GCW. However, it may also be a receptor for a presently unknown
substance in Bowman’s space. Agrin or laminin fragments that are
released in situations of GBM damage could appear in the urinary space
and bind to -DG on the podocyte cell surface. This interaction could
lead via intracellular signaling to fusion and/or detachment of foot
processes.
|
- and ß-DG. In IEM, an alteration was observed
in the distribution pattern of -DG, with locally a more pronounced
expression at the basal side of the fused podocyte foot processes. The
decrease in staining intensity in IF can be explained by the fusion of
the foot processes in ADN, which results in a decreased cell membrane
surface near the GCW and therefore in a decreased density of
membrane-associated proteins. The significance of the agrin-DG-utrophin
complex for the development of proteinuria is not clear. Although it
has been reported that podocytes detach from the GBM in
ADN,24,29
we did not observe this in the present study. It
is possible that in later stages of ADN podocytes detach, and it may be
interesting to study the relation between the sites of detachment and
-DG expression. In PHN the staining intensity for agrin, DG, and
utrophin did not change. In contrast to ADN, IEM of PHN barely showed
foot process effacement. Besides that, there is no up-regulation of DG
on the basal sides of the podocytes. These data suggest that in this
model of immune-complex-mediated proteinuria, DGC expression and
distribution is not affected. This suggests that alterations in the DGC
complex are not uniformly associated with proteinuria. Variations in DG
expression might be related to differences in the induction of podocyte
injury (toxic in ADN versus immune-complex mediated in PHN).
However, alterations in the expression of agrin, DG, and utrophin may
also be related to the degree of proteinuria, because rats with ADN had
a heavier proteinuria than rats with PHN.
Apart from the DGC which is presently recognized as a possible link
between muscle or epithelial cells and the ECM,1,19
another well-characterized adhesion receptor system for epithelial
cells are the integrin matrix receptors. In the GBM 3ß1 integrin
is predominantly found.76
Because it has been shown that
integrins interact with agrin and sarcoglycans, the DGC may also act
synergistically to integrins to link podocytes to the
GBM.77,78
Taken together, the present study shows that agrin, DG, and utrophin are present in the GCW. Further studies are needed to elucidate their function in normal podocyte physiology and their role in increased glomerular permeability for proteins. They may have a role in maintaining the intricate relation between podocytes and the GBM. It remains to be determined whether alterations in the DGC complex occur in human glomerular diseases and whether specific alterations are related to certain glomerulopathies.
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Supported by the Dutch Kidney Foundation (Grants 93.1318, 95.1530, and 95.1513) and the Dutch Diabetes Foundation (Grant DFN 940–10-009).
Jacob van den Born’s present address is Department of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands.
Accepted for publication January 21, 2000.
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