Glomerular laminin isoform transitions: errors in metanephric
culture are corrected by grafting
Patricia L.
St. John1,
Ruixue
Wang1,
Yong
Yin2,
Jeffrey H.
Miner3,
Barry
Robert1, and
Dale R.
Abrahamson1
1 Department of Anatomy and Cell Biology, University of
Kansas Medical Center, Kansas City, Kansas 66160-7400; and
2 Department of Anatomy and Neurobiology and 3 Renal
Division, Department of Internal Medicine, Washington University
School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Glomerular basement membrane (GBM)
assembly and maturation are marked by the replacement of laminin-1
(containing
1-,
1-, and
1-chains) with laminin-11 (consisting
of
5-,
2-, and
1-chains). Similarly, the
1- and
2-chains
of type IV collagen are replaced by collagen
3-,
4-, and
5(IV)-chains. The cellular origins of these molecules and mechanisms
for isoform removal and substitution are unknown. To explore glomerular
laminin isoform transitions in vitro, we assessed metanephric organ
cultures. Standard culture conditions do not support endothelial cell
differentiation, and glomerular structures that form in vitro are
avascular. Nevertheless, extensive podocyte development occurs in these
cultures, including the formation of foot processes and assembly of a
GBM-like matrix. Here, we show that the podocyte-specific markers,
glomerular epithelial protein 1 and nephrin, which are normally
expressed in capillary loop stage glomeruli in vivo, are also expressed
by glomerular figures that form in organ culture. However, the GBM-like
segments that form in vitro do not undergo normal laminin isoform
switching. Instead, both laminin
1- and
5-chains are present, as
is the
1-chain, but not
2. When avascular organ-cultured kidneys
are grafted into anterior eye chambers, however, kidney-derived
angioblasts establish extensive vasculature by 6 days, and glomeruli
are lined by endothelial cells. We evaluated embryonic day
12 (E12) vascular endothelial growth factor receptor
(Flk1)-lacZ kidneys that had first been grown in
organ culture for 6-7 days and then grafted into wild-type mice.
Correct laminin isoform substitution occurred and correlated with the
appearance of endothelial cells expressing Flk1. Our
findings indicate that endothelial cells, and/or factors present in the
circulation, mediate normal GBM laminin isoform transitions in vivo.
endothelial cells; glomerular basement membrane; podocytes
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INTRODUCTION |
DEVELOPMENT OF THE
PERMANENT, metanephric kidney in the mouse begins at ~embryonic
day 10 (E10), when the ureteric bud emerges from
the Wolffian duct and projects dorsolaterally to engage mesenchymal cells of the metanephric blastema. Reciprocal signals from the ureteric
bud and blastema, respectively, induce growth and repeated branching of
the bud and formation of mesenchymal cell aggregates at each branch tip
(13, 27). Subsequently, each mesenchymal aggregate
converts to an epithelial vesicle, which elongates to form a short
tubule that then develops a crevice to resemble a comma-shaped figure.
The glomerulus originates as endothelial cells migrate into this
crevice, or vascular cleft. Together with mesangial progenitors, these
cells give rise to the glomerular capillaries, which mark the capillary
loop stage of glomerular development (reviewed in Ref. 2).
Epithelial cells located immediately beneath the original vascular
cleft differentiate into podocytes, and those above the cleft develop
into tubular segments of the nephron, which join ureteric bud branches
that go on to form the collecting system. The induction of nephric figures in mice continues until ~1 wk after birth and then ceases once the full complement of nephrons has formed (27).
Electron microscopic examination of the vascular cleft has shown that
the early glomerular endothelial cells are resting on a subendothelial
basement membrane that lies parallel to but is distinct from an
overlying basement membrane beneath developing epithelial podocytes
(2). Immunoelectron microscopy has also shown that both
endothelial cells and podocytes are engaged in synthesis of the key
basement membrane proteins: laminin, collagen type IV, and entactin at
this stage (1, 8). As the glomerular capillaries begin to
form, the endothelium flattens and forms fenestrae, a process that is
coincident with development of foot processes by podocytes. The
double basement membranes between the two cell layers fuse to produce
the glomerular basement membrane (GBM), which is shared by the
endothelium on the vascular surface and podocytes on the urinary
surface (reviewed in Ref. 2). Mechanisms accounting for
the union of the subendothelial and subpodocyte basement membrane
layers into the GBM are not known, but the incorporation of
intravenously injected, soluble mouse laminin into newborn rat GBM
indicates that specific binding sites for laminin exist in the immature
GBM (31). Hence, binding interactions occurring between
basement membrane proteins in the separate layers of the dual basement
membrane may promote GBM fusion, but the details of this process remain
unclear (31).
As glomeruli mature, a complex series of laminin and collagen type IV
isoform transitions occur in GBM composition (reviewed in Ref.
15). Specifically, laminin-1, consisting of the laminin
1-,
1-, and
1-chains, and laminin-8 (
4-,
1-, and
1-chains) are found in the most immature GBM region of vascular
clefts (9, 18, 28). However, these laminins are replaced
by laminin-11, containing the laminin
5-,
2-, and
1-chains,
and this is apparently the only laminin isoform found in the fully
mature GBM (15). Similarly, the
1- and
2-chains of
type IV collagen are gradually replaced by
3(IV)-,
4(IV)-, and
5(IV)-chains (15, 19). In general, the appearance of
the laminin
5- and
2-chains, and the
3-chain of type IV
collagen, all first occurs in capillary loop stages of glomerular
development, although trace amounts of laminin
5-chain have been
reported in earlier, S-shaped stages (9, 15, 28).
Although very little is known about the regulation of GBM protein
substitutions at either the gene or protein level, these changes in
matrix composition are extremely important functionally. Mice
genetically deficient in laminin
5- or
2-chains display prominent
glomerular phenotypes. Beginning at the capillary loop stage, mutants
for laminin
5 display disorganization of the podocyte cell layer,
absence of an ultrastructurally definitive GBM, and a failure of
glomerular endothelial cells and mesangial cells to form a capillary
tuft (17). In mice lacking the laminin
2-chain, the
laminin
1-chain persists in the GBM, which appears ultrastructurally normal (21). However, the podocyte foot processes are
abnormally broadened, and animals develop massive proteinuria
(21). These findings therefore emphasize pivotal roles for
laminin
5- and
2-chains in GBM structure and function and an
inadequacy of their forerunners, laminin
1,
4, and
1, for
establishing and maintaining the normal glomerular filtration barrier.
In previous work, we used a metanephric organ culture system in which
avascular glomerular epithelial tufts form in vitro. By using
metanephroi dissected from heterozygous Flk1-lacZ transgenic mice, endothelial cells expressing the vascular endothelial growth factor receptor, Flk1, are identified by the expression of
the lacZ transgene. Defined glomerular endothelial cells are
not seen in these cultures, but when cultured metanephroi are
subsequently grafted into anterior eye chambers, graft-derived
angioblasts differentiate into lacZ-positive endothelial
cells and vascularize glomeruli that develop in oculo (23,
24). In the present study, we evaluated the laminin
1-,
5-,
1-, and
2-chains synthesized by the avascular
glomeruli that form in organ culture, as well as in the vascularized
glomeruli that develop after organ-cultured kidneys are grafted. Our
results show that, despite significant podocyte differentiation in
organ culture, incomplete laminin chain transitions occur in
vitro. These errors are corrected, however, when
organ-cultured kidneys are grafted and vascularized glomeruli develop.
Our findings therefore implicate glomerular endothelial cells, and/or
factors present in the circulation, as important mediators for normal
GBM laminin assembly.
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MATERIALS AND METHODS |
Animals.
Mice for these experiments came from our colonies of C57BL/6J and
Flk1-lacZ transgenic (Flk1tm1Jrt)
animals that we obtained originally from the Jackson Laboratory (Bar
Harbor, ME). Flk1-lacZ trangenics were genotyped as before (24).
Antibodies.
Monoclonal rat anti-mouse laminin-1 antibodies (MAb; 5A2 and 8B3) were
prepared and characterized as previously described (3).
Furthermore, MAb 5A2 has been shown to react specifically with the
laminin
1-chain and not
2 (14). Similarly, MAb 8B3 binds specifically to laminin
1-chain and does not recognize
5
(16, 17). To define the MAb epitopes more precisely, we prepared and then screened a newborn mouse kidney
gt11 cDNA
expression library with antibodies by using methods described before
for preparing and screening a newborn rat kidney library with polyconal anti-laminin IgG (30). Positive colonies reacting with MAb
5A2 were then purified, expanded, and sequenced.
Further characterization of the laminin chain specificity of MAb 8B3
was carried out by binding of the antibody to cells transfected with
laminin
1-cDNA. In brief, the quail fibroblast cell line QT-6
(20) was transfected with a full-length mouse laminin
1-cDNA expression vector (a gift from Peter Yurchenco, Robert Wood
Johnson Medical School, Piscataway, NJ) or mock transfected. After
36 h, cells were rinsed in PBS, fixed in 1% paraformaldehyde with 100 mM L-lysine and 10 mM metaperiodate, rinsed again, and
then permeablized with 1% Triton X-100. Cells were then incubated with MAb 8B3, washed, and treated with secondary antibody.
Chain-specific rabbit anti-laminin
5 (18)- and guinea
pig anti-laminin
2-antibodies (25) were characterized
previously. Antibodies against nephrin (12) and glomerular
epithelial protein 1 (GLEPP-1) (32) were gifts from Drs.
Lawrence Holzman and Roger Wiggins, respectively (both at the
University of Michigan, Ann Arbor, MI).
Experimental procedures.
Timed pregnant mice at gestational days 12 and 13 (date of vaginal plug being day 0) and newborn mice were
anesthetized and killed by cervical dislocation. For metanephric organ
cultures, embryos were removed and kidneys were aseptically
microdissected and cultured for 4-7 days (Table 1) as described
before (24). In brief, metanephroi were placed in 12-well
tissue culture dishes (Falcon, Oxnard, CA) containing 1:1 DMEM/Ham's
F-12 medium (GIBCO-BRL Life Technologies, Grand Island, NY)
supplemented with 5% FCS, penicillin G sodium, and streptomycin
sulfate (both at 1,000 U/ml). Cultured metanephroi were incubated at
37°C in humidified air with 5% CO2. In some cases,
wild-type and Flk1-lacZ kidneys that had been maintained in
organ culture for 6 or 7 days were implanted into anterior eye chambers
of C57BL/6J hosts as described before (24). Grafts of
these previously cultured kidneys were then harvested 6 or 7 days later
(Table 1).
For immunofluorescence microscopy, kidney tissues were placed in
optimal cutting temperature compound (OCT; Miles, Elkhart, IN) and
frozen in isopentane chilled in a dry ice-acetone bath. Individual,
serially cut cryostat sections, ~4 µm thick, were picked up on
separate slides, air-dried, fixed in 100% methanol at 0°C for 10 min
(in most cases), and washed in PBS. Sections were then incubated with
50 µg/ml rat anti-mouse laminin MAb 5A2 or 8B3, followed by goat
anti-rat IgG-fluorescein. Other sections were labeled with rabbit
anti-laminin
5-chain, anti-GLEPP-1, or anti-nephrin, followed by
goat anti-rabbit IgG-fluorescein or -rhodamine (ICN Biomedicals, Costa
Mesa, CA). Some sections were double labeled with both MAb 8B3
(anti-laminin
1) and anti-laminin
5-chain antibodies and
appropriate fluorescein- and rhodamine-conjugated secondary
antibodies. For labeling with anti-laminin
2-chain, cryostat
sections were fixed with 4% paraformaldehyde (instead of 100%
methanol) for 10 min at room temperature, blocked with 0.1 M glycine in
PBS, and then treated with 0.1% SDS in PBS for 60 min at 50°C to
expose the epitope. Sections were then incubated with guinea pig
anti-laminin
2-chain, followed by goat anti-guinea pig
IgG-rhodamine.
Serial sections of anterior chamber grafts of organ-cultured
Flk1-lacZ kidneys were processed for immunofluorescence as
described in Experimental procedures and for
-galactosidase histochemistry as detailed before (24).
Organ-cultured and cultured-grafted kidney tissues were also prepared
for transmission electron microscopy as described previously
(4).
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RESULTS AND DISCUSSION |
Localization of the MAb 5A2 epitope to the
NH2-terminal domain VI of the
laminin
1-chain.
Earlier work using Western blotting of porcine pancreatic and human
neutrophil elastase digests of laminin had provided evidence that the
5A2 epitope resided on the E4 fragment of laminin, which corresponds to
the NH2-terminal region of the
-chain short arms (3, 29). Electron microscopy of rotary-shadowed immune
complexes consisting of laminin and MAb 5A2 and rotary-shadowed eluates of laminin digests from MAb 5A2 affinity resins confirmed these results
(3, 29). To define the 5A2 epitope more precisely, we
prepared a newborn mouse kidney
gt11 cDNA expression library, and
library screening with MAb 5A2 identified four immunoreactive clones.
Computer sequence analysis throughout their open reading frames showed
that all four clones corresponded precisely with published sequences
for the laminin
1-chain (26) and encoded overlapping
regions contained specifically within the NH2-terminal domain VI of the
1-chain short arm (Fig.
1). Clone 1a is 304 bp in length and is
entirely included in clones 1b and 09. Clones 1b and 5 are 650 and 632 bp in length, respectively, and each contains 60-80 bp of the
laminin
1-5' untranslated region, the 5' signal peptide, as
well as the NH2 terminus of domain VI. Clone 09 is 487 bp
in length and stretches from nucleotide +89 to +576 of laminin
1-chain domain VI. The alignment of the four clones is shown in Fig.
1. Together, they encode 162 of the 248 amino acids that constitute
domain VI, which contains a mixture of short
-helices,
-sheets,
and random coils that form the NH2-terminal globular
structure of the short arm of the laminin
1-chain (26).

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Fig. 1.
Diagram of laminin molecule showing location of region encoded by
cDNA clones identified by anti-laminin monoclonal antibodies (MAb) 5A2.
Clones 1b, 05, 09, and 1a all map to the NH2-terminal
domain VI of the laminin 1-chain. The amino acid sequence in the
overlap region among all 4 clones (dashed line) for the laminin
1-chain (accession number M15525) is shown and compared with the
corresponding region of the laminin 2-chain (accession number
NM-008483), which is unreactive with MAb 5A2 (14). Areas
marked by bars show sites of sequence divergence between these 2 chains. One of these possibly represents the specific 1-chain
epitope recognized by MAb 5A2.
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When the 102-amino acid consensus-overlapping sequence of laminin
1-chain recognized by MAb 5A2 was compared with the sequence in the
corresponding region of laminin
2 (10), a striking
level of homology was observed (Fig. 1). Areas of sequence divergence were clustered within just three sites, each consisting of five to
seven amino acids. Because MAb 5A2 does not crossreact with the laminin
2-chain (14), we speculate that one of these sites on
the laminin
1-chain represents the core epitope for this MAb. At
present, we are carrying out additional studies to confirm this
possibility. Nevertheless, these results are entirely consistent with
our earlier immunomap of the MAb 5A2 epitope on laminin and show that
this MAb recognizes the NH2-terminal domain VI on the laminin
1-chain, specifically.
Confirmation of the chain specificity of MAb
8B3.
Screening of the newborn mouse kidney
gt11 expression library with
MAb 8B3 was unsuccessful and failed to produce any immunoreactive clones. To provide additional evidence that this MAb binds specifically to the laminin
1-chain, we therefore undertook a strategy similar to
what was originally used to demonstrate that MAb 5A2 is specific for
laminin
1-chain (14). In this case, quail fibroblasts
were transfected with a full-length mouse laminin
1-cDNA expression vector. Cells transfected with the laminin
1-cDNA reacted intensely with MAb 8B3, whereas mock-transfected cells did not (Fig.
2). Together with evidence presented
earlier (16, 17), this confirms that MAb 8B3 binds
specifically to the laminin
1-chain.

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Fig. 2.
Quail fibroblasts mock transfected (a) or
transfected with mouse laminin 1-chain cDNA (b). When
cells transfected with anti-laminin 1-chain cDNA are incubated with
MAb 8B3, intense immunofluorescence is seen (b). No
fluorescence occurs in mock-transfected control cells incubated with
MAb 8B3 (a).
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GBM laminin isoform transitions in vivo.
There are striking transitions in laminin distribution within the GBM
during normal glomerular development in vivo (5, 15)
(Figs. 3 and 4). Specifically, labeling
for the laminin
1-chain is abundant in early GBMs within vascular
clefts of comma- and S-shaped figures, but it completely disappears
from GBMs of capillary loop-stage glomeruli (Fig. 3a). In
contrast, double immunolabeling of the same sections showed that the
5-chain, which is absent in vascular clefts, becomes abruptly
prominent in early capillary loops (Fig. 3b). On the other
hand, the laminin
1-chain is present in both vascular clefts and
peripheral GBM of early capillary loop stage glomeruli (Fig.
4a). Laminin
2-chain is not
expressed in vascular clefts, however, and becomes progressively detectable in GBMs of capillary loop-stage glomeruli (Fig.
4b). In later, maturing-stage glomeruli, laminin
1-chain
disappears from the GBM but remains in mesangial matrices, whereas
laminin
2-chain persists in GBMs of maturing glomeruli, but not
mesangial matrices (5, 15).

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Fig. 3.
Double immunofluorescence image of cryostat section of
newborn mouse kidney labeled with anti-laminin 1-chain
(a) and anti-laminin 5-chain (b) primary
antibodies, incubated with appropriate secondary antibodies, and
examined with fluorescein and rhodamine filters, respectively.
Anti-laminin 1-chain labels vascular cleft (a, arrows)
but is completely unreactive with glomerular basement membrane (GBM) of
capillary loop stage glomerulus (a, *). In contrast,
anti-laminin 5-chain reacts intensely with capillary loop GBM
(b, arrowheads) but does not detect vascular cleft GBM of
early nephric figure (b, *).
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Fig. 4.
Separate cryostat sections of newborn mouse kidney labeled with
anti-laminin 1-chain (a) and anti-laminin 2-chain
(b). a: Anti-laminin 1-chain recognizes
vascular cleft (arrows) and capillary loop-stage GBMs (arrowheads).
b: Anti-laminin 2-chain reacts only with capillary
loop-stage glomeruli (arrowheads), and GBMs of earlier nephric figures
(arrows) are negative. C, capsule.
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These results therefore show that the transition of laminin
-chains
takes place earlier and occurs far faster than that for the laminin
-chains. Whether the abruptly expressed laminin
5-chain associates with the
1-chain synthesized at that time (together with
the
1-chain to form laminin-10), with the
2-chain (to form laminin-11), and/or with other laminin chains is not yet clear (11, 15, 18). Importantly, the fate of the original
laminin
1-chain, the process for its rapid and selective removal
from the nascent GBM, and the molecular control of the upregulation of
the laminin
5-chain are also unknown.
Laminin synthesis in metanephric organ cultures.
When E12 mouse kidneys were placed in organ culture,
organotypic tubulogenesis and glomerular epithelial tuft formation
occurred under the conditions used here (Fig.
5). Standard organ culture conditions do
not support endothelial differentiation, however, and the glomerular
structures that developed in vitro appeared avascular (Fig. 5), which
has been observed repeatedly (6, 7, 22, 23, 24). When
sections of organ cultures were labeled with anti-GLEPP-1 and
anti-nephrin antibodies, the glomerular epithelial tufts were positive
for both of these podocyte-specific differentiation markers (Fig.
6) that are normally first expressed at
early capillary loop stages (12, 32). Additionally, when cultures were examined by electron microscopy, a substantial amount of
podocyte development was observed, replete with the formation of foot
processes (Fig. 7) spanned by slit
diaphragms (Fig. 8). Moreover, basement
membrane material containing defined layers resembling typical laminae
densae and laminae rarae were found beneath the developing foot
processes (Fig. 8). These basement membrane structures were not
organized into linear loops as they are in normal glomeruli, however,
but instead occurred as segments or cords of material between podocytes
(Figs. 7 and 8). These ultrastructural features therefore appear
identical to those observed earlier when intact kidney rudiments or
dissected metanephric blastema separated from ureteric buds are
maintained in transfilter cultures with appropriate inducer tissues
(6, 7, 22). Despite the extensive development and
differentiation of podocytes in metanephric organ culture, sections of
cultured Flk1-LacZ metanephroi showed that there was no
corresponding development of Flk1-positive endothelial cells
within the glomerular epithelial tufts. The absence of defined
endothelial cells, mesangial cells, and mesangial matrices was
confirmed by electron microscopy (Figs. 7 and 8).

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Fig. 5.
Sections of embryonic day
12 (E12) kidney before (a) and after
(b) 6 days in culture. Note that several "glomerular"
epithelial tufts develop in organ culture (b, arrows) but no
defined vascular structures are seen.
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Fig. 6.
Cryostat sections of E12 kidneys grown in
culture for 6 days and labeled with anti-glomerular epithelial protein
1 (GLEPP-1) (a) and anti-nephrin antibodies (b).
Both podocyte-specific markers of differentiation are expressed by the
"glomerular" epithelial tufts in vitro.
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Fig. 7.
Electron micrograph of organ-cultured kidney showing glomerular
epithelial tuft. A number of visceral epithelial cells resembling
podocytes (Po) are seen, as are segments or cords of basement
membrane-like material (arrows). Note cells corresponding to
parietal epithelium of Bowman's capsule (BC). Magnification:
×5,700.
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Fig. 8.
Higher power electron micrograph from organ-cultured kidney showing
presence of foot process-like interdigitations. Basement membrane
material resembling the GBM is found beneath the foot processes (*).
Structures corresponding to slit diaphragms can be seen between many of
the foot processes (arrows). Magnification: ×38,000.
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Earlier reports have shown that podocytes in metanephric organ culture
express collagen type IV and laminin (22). To examine whether the GBM laminin chain isoform substitutions that normally take
place in vivo might also occur in vitro, cryostat sections of
E12/E13 kidneys that had been maintained in organ
culture for 4-7 days were labeled with chain-specific anti-laminin
antibodies. In sections that were doubly labeled for laminin
1- and
5-chains, glomeruli from organ-cultured kidneys often contained both
chains, sometimes in the same basement membrane segments (Fig.
9). There were segments within the same
glomeruli that contained laminin
5-chain alone, however (Fig. 9).
The basement membranes that formed within the glomerular epithelial
tufts in vitro were not arranged in defined, linear loops as they are
in vivo but instead were assembled into broad segments or cords (Figs.
7-9). Also in contrast to what occurs by the capillary loop stage
of glomerular development in vivo, most glomeruli that formed in organ
culture retained abundant amounts of laminin
1-chain-positive
basement membrane material (Fig. 9). Although both laminin
1- and
5-chain expression were observed within the same glomeruli, laminin
1-chain was expressed almost exclusively; little or no
immunolabeling for laminin
2-chain was seen in glomeruli that formed
in organ cultures (Fig. 9). Results from examination of sections taken from eight separate metanephric organ cultures are shown in Table 2, in
which there was usually coexpression of laminin
1- and
5-chains
and almost a complete absence of the laminin
2-chain. Taken
together, these results indicate that, although considerable podocyte
morphogenesis took place in organ cultures, including the expression of
GLEPP-1 and nephrin, normal laminin isoform switching did not occur in
this environment.

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Fig. 9.
Cryostat sections of E12 kidneys grown
for 6 days in culture. Sections were doubly immunolabeled for laminin
1- and 5-chains, or serial sections were labeled for 1- and
2-chains, as indicated. Epithelial tufts that form in organ culture
express both 1- and 5-chains, sometimes in the same basement
membrane segments (arrows, top left and right).
The laminin 5-chain is seen in other segments exclusively
(arrowheads, top right). In contrast, only
laminin 1-chain is synthesized by glomerular epithlelial tufts
in organ culture; laminin 2-chain is not detected in the same
tufts.
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Laminin synthesis in organ-cultured and then grafted metanephroi.
To assess the laminin isoform switching patterns that might occur in
anterior chamber grafts, we dissected E12 kidneys and implanted these into anterior eye chambers for 6 and 7 days (Table 1). In all cases, the normal laminin
substitution sequences occurred: laminin
1-chain was replaced by
laminin
5-chain, and the joint expression of laminin
1- and
2-chains were seen in capillary loop GBMs. When E12 and
E13 kidneys that had first been grown in organ culture for 6 or 7 days were grafted into anterior chambers and examined for
glomerular laminin expression, results from double-labeled sections
also showed that, in most glomeruli, laminin
1-chain was almost
entirely replaced by laminin
5 (Fig.
10, a and b, and Table 2). Additionally, capillary
loop-stage glomeruli within grafts also usually contained both laminin
1- and
2-chains (Fig. 10, c and d, Table
2), as is normally the case at this stage of development. In other
words, laminin isoform switching, which was arrested in organ cultures,
appeared to proceed normally after organ-cultured kidneys were grafted
into anterior chambers.

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Fig. 10.
Immunofluorescence of E12 kidneys that were
first grown in organ culture for 6 days and then grafted into anterior
eye chambers. Grafts were harvested 6 days later. a and
b: Double immunofluorescence image from the same section
labeled with anti-laminin 1- and 5-chain antibodies, as
indicated. After grafting, laminin 1 disappears from capillary
loop-stage glomeruli, whereas laminin 5-chain is abundant, as occurs
normally (cf. Fig. 3). c and d: Serial sections
treated separately with anti-laminin 1- and 2-chain antibodies,
as indicated. Note that the same 4 capillary loop-stage glomeruli (*)
express both laminin 1- and 2-chains in normal patterns (cf.
Fig. 4).
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A role for endothelial cells in GBM laminin isoform
substitution?
Work we carried out previously with Flk1-lacZ kidneys has
shown that metanephric mesenchymal cells expressing Flk1,
which we operationally defined as angioblasts, no longer express
Flk1 in organ culture (24). This may explain,
in part, why kidney microvessels and glomerular endothelial cells fail
to develop under typical culture conditions. On the other hand, when
avascular, organ-cultured kidneys are then grafted into anterior eye
chambers, Flk1 expression by endothelial progenitors is
restored, and glomerular and microvascular endothelial cells develop
organotypically within the grafts (24). To evaluate a
possible role for glomerular endothelial cells in mediating laminin
isoform switching, kidneys from E12 Flk1-lacZ
mice were cultured for 7 days and then grafted into anterior eye
chambers of wild-type host mice. Grafts were harvested after 6 or 7 days of growth in oculo (Table 1) and frozen. Because conditions for
-galactosidase histochemistry were incompatible with the
immunofluorescence procedures required for this particular set of
antibodies, separate, serial sections were processed for
-galactosidase and immunofluorescence.
The three panels shown in Fig.
11 reflect the
distribution of lacZ-positive glomeruli (indicating the
presence of endothelial cells expressing Flk1) in serial
section 7, and laminin
1- and
5-chains, respectively,
in the doubly immunolabeled serial section 10. Several
glomerular structures containing endothelial cells (Fig.
11c) correspond precisely to those in which there are marked absences of laminin
1-chain (Fig. 11a) but the presence
of laminin
5-chain (Fig. 11b). Similarly, when serial
sections were processed for laminin
1- and
2-chain immunolabeling
and lacZ histochemistry, glomeruli containing
Flk1-positive endothelial cells coincided exactly with those
in which laminin
1- and
2-chains were both expressed (Fig.
12).

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Fig. 11.
Serial sections of cultured and grafted
Flk1-lacZ kidney. a: Serial section
10 (s10) immunolabeled for laminin 1-chain and viewed
with the fluorescein channel. b: s10 immunolabeled for
laminin 5-chain and viewed with rhodamine. Note that between
a and b, GBMs within 5 glomeruli (arrows) do not
express laminin 1-chain but do express laminin 5-chain,
reflecting the normal pattern of laminin transition in capillary
loop-stage glomeruli. Tubular basement membranes that selectively
express the laminin 1-chain (double arrows in a) and
laminin 5-chain (double arrows in b) can also be
discriminated. art, arteriole. c: Serial section
7 (s7), developed for lacZ histochemistry, which
reveals sites of Flk1 transcription and thereby identifies
endothelial cells. Four glomeruli are found in the same location of the
section as those seen above (arrows), and each contains endothelial
cells. A fifth glomerulus (arrowhead) is also seen, but there are no
corresponding stuctures seen in the later sections in the series
(a and b) that were immunolableled. Similarly, a
glomerulus that is immunolabeled in a and b
(right of center) cannot be resolved by -galactosidase
staining in c. Also, note in c a cross section
through an arteriole. This serves as an additional reference point for
the arteriolar tissue observed in b, coursing through the
bottom portion of the serial section.
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Fig. 12.
Serial sections of a cultured-grafted Flk1-lacZ kidney.
a: Section 15 (s15) immunolabeled for laminin
1-chain. b: Section 14 (s14) immunolabeled for
laminin 2-chain. c: Section 13 (s13),
processed for lacZ histochemistry. A glomerulus containing
endothelial cells also expresses both laminin 1- and 2-chains in
capillary loop GBM.
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Not all glomeruli viewed in the serial sections that were processed for
immunofluorescence were also detected in sections developed for
Flk1-lacZ, however, and a few lacZ-positive
glomeruli were absent in the serial sections processed for
immunofluorescence (cf Fig. 11). We attribute these disparities to the
unavoidable differences that exist from section to section. Had we been
successful in processing the same sections for Flk1-lacZ and
for double-label immunofluorescence, we might have been able to
distinguish between nonvascularized and vascularized glomeruli and
could have determined definitively whether correct laminin isoform
switching occurred only in glomeruli containing endothelial
cells. On the other hand, almost all of the glomeruli that
develop in anterior chamber grafts of cultured kidneys are
vascularized, and avascular glomeruli are rare (23, 24).
To evaluate glomeruli that developed in organ-cultured-grafted kidneys
in more detail, grafts were examined by electron microscopy. As shown
in Fig. 13, perfused glomerular
capillary loops containing erythrocytes were lined by a distinct
endothelial cell layer. The overall glomerular architecture in these
cultured-grafted kidneys was therefore strikingly similar to capillary
loop-stage glomeruli that develop normally and very different from the
avascular glomerular structures that form in organ cultures (Figs. 7
and 8 ). Of interest, a few tubular basement membranes within the grafts were intensely positive for the laminin
1-chain, and others were selectively positive for the laminin
5-chain (Fig. 11). These probably represent proximal and distal tubular segments, respectively (15, 18), and are further indicators of the level of
nephron differentiation that is achieved in this ectopic, anterior
chamber grafting site.

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Fig. 13.
Electron micrograph of glomerular capillary wall from an
E12 kidney that was grown in organ culture for 6 days and
then grafted into anterior eye chamber. The graft was fixed for
microscopy 6 days after implantation. An endothelial cell layer
(En) lines the capillary, and is closely apposed by overlying podocytes
(Po). The capillary is perfused by erythrocytes (E). Magnification:
×9,700.
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In summary, routine organ culture conditions support the
differentiation of avascular glomerular tufts containing
well-differentiated podocytes. In our analysis, these cells expressed
the podocyte-specific differentiation markers GLEPP-1 and nephrin and
assembled epithelial slit diaphragms and lengths of GBM-like material.
Although podocytes that develop in organ culture displayed many
features of their normal counterparts, the basement membrane material
they assembled was compositionally distinct from normal GBM.
Specifically, substitution of laminin
1-chain by
5-chain was
incomplete, and the laminin
2-chain was not installed. Once
avascular, organ-cultured kidneys were grafted into anterior eye
chambers, however, normal laminin isoform-switching patterns were
largely restored: the laminin
1-chain disappeared from capillary
loop-stage glomeruli, and GBMs contained both the
1- and
2-chains. These results correlated with the direct identification of
graft-derived glomerular endothelial cells within the same glomeruli in
which appropriate laminin isoform substitution occurred. Taking all the
data together, we conclude that correct GBM laminin isoform switching
occurred only when glomeruli became vascularized.
Between the organ culture and anterior chamber environments, there are
many potentially important differences affecting kidney development,
and we recognize that neither environment replicates normal
metanephrogenesis in situ. Nevertheless, three of the most prominent
differences are the absence in organ cultures of 1) vascular
endothelial cells, 2) blood-borne growth factors/chemokines and other signaling molecules, and 3) biophysical effects
contributed by hemodynamic pressures and glomerular ultrafiltration.
Although all three of these parameters are present within grafts of
organ-cultured kidneys, we do not know which one figures most
importantly in mediating basement membrane assembly. Clearly, the
grafts become well perfused, but the renovascular dynamics in the
anterior eye chamber grafts are likely to be far different than in
kidneys that develop in the normal, abdominal location. Specifically, given that vessels that deliver blood into the grafts originate chiefly
from the iris, the glomerular filtration pressures in the grafts are a
small fraction of what they are in normal kidneys. Although glomeruli
in grafts are forming a modest ultrafiltrate, there is, of course, no
elimination of the filtrate by normal mechanisms through the ureter.
Hence there are very different physiological conditions that exist
between glomeruli that form in oculo from those that develop normally;
yet, in both cases, GBM laminin isoform transitions occur correctly. As
a consequence, physiological, hemodynamic parameters are probably not
major determinants mediating GBM laminin synthesis. This leaves
circulating growth factors and/or endothelial cells themselves as the
most likely candidates, and we believe that they both are probably crucial.
Identification of specific growth factors and other substances that
selectively direct endothelial cell development would be very important
to achieve. As seen earlier (23, 24), and observed here
again as well, the glomerular endothelial cells that differentiate in
the grafts are in fact derived from the engrafted kidney. Under
conditions used here, the vascular endothelial growth factor receptor
Flk1 is expressed by only a few metanephric cells in organ
culture, and these do not establish a recognizable microvasculature in
vitro (23, 24). Once avascular, organ-cultured kidneys are
grafted, however, there is marked upregulation of Flk1
within the graft, and some of these cells go on to assemble the
glomerular endothelium. The molecular signals for this Flk1 upregulation on grafting are not known, and multiple signaling pathways
are undoubtedly involved. In addition, we suspect that at least some of
the signals are derived from the circulation, but this may be a very
difficult question to resolve further at present.
How might glomerular endothelial cells affect GBM laminin isoform
substitution? First, endothelial cells may be a key biosynthetic source
for certain laminin isoforms. Earlier work carried out before the
advent of isoform- and chain-specific anti-basement membrane protein
antibodies have clearly shown that endothelial cells of immature
glomeruli, in conjunction with podocytes, secrete laminin-1, type IV
collagen, and entactin (1, 8). Whether glomerular
endothelial cells synthesize the separate laminin-1 and laminin-11
chains examined here, and whether they are the sole source for these
chains, now needs to be determined. Second, endothelial cells (possibly
along with circulation-derived factors) may provide signals that
stimulate podocytes to alter their laminin synthetic programs, which
then lead to the appearance of new GBM laminin isoforms. As mentioned
earlier, what becomes of the laminin isoforms laid down during initial
stages of glomerular development is not known, but endothelial cells
might play a role in GBM turnover as well. Perhaps additional
experiments with the organ culture-grafting system used here, which
stops and then restarts kidney endothelial cell development, may
provide new insights into mechanisms governing glomerular endothelial
cell differentiation and function. We are presently using this and
other technologies with the hope of understanding more about how the
endothelial and podocyte cell layers each help establish and maintain
the glomerular capillary wall.
 |
ACKNOWLEDGEMENTS |
We thank Eileen Roach for expert technical help, Dr. Peter
Yurchenco for the mouse laminin
1-chain cDNA, and Drs. Lawrence Holzman and Roger Wiggins for antibodies against nephrin and GLEPP-1, respectively.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-34972 and DK-52483.
Address for reprint requests and other correspondence: D. R. Abrahamson, Dept. of Anatomy and Cell Biology, Univ. of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7400 (Email: dabrahamson{at}kumc.edu).
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. Section 1734 solely to indicate this fact.
Received 21 September 2000; accepted in final form 5 December 2000.
 |
REFERENCES |
1.
Abrahamson, DR.
Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys.
J Cell Biol
100:
1988-2000,
1985[Abstract].
2.
Abrahamson, DR.
Glomerulogenesis in the developing kidney.
Semin Nephrol
11:
375-389,
1991[ISI][Medline].
3.
Abrahamson, DR,
Irwin MH,
St. John PL,
Perry EW,
Accavitti MA,
Heck LE,
and
Couchman JR.
Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains.
J Cell Biol
109:
3477-3491,
1989[Abstract].
4.
Abrahamson, DR,
and
St. John PL.
Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.
J Histochem Cytochem
40:
1943-1953,
1992[Abstract/Free Full Text].
5.
Abrahamson, DR,
St. John PL,
Pillion DJ,
and
Tucker DC.
Glomerular development in intraocular and intrarenal grafts of fetal kidneys.
Lab Invest
64:
629-639,
1991[ISI][Medline].
6.
Avner, ED,
Villee DB,
Schneeberger EE,
and
Grupe WE.
An organ culture model for the study of metanephric development.
J Urol
129:
660-664,
1983[ISI][Medline].
7.
Bernstein, J,
Cheng F,
and
Roszka J.
Glomerular differentiation in metanephric culture.
Lab Invest
45:
183-190,
1981[ISI][Medline].
8.
Desjardins, M,
and
Bendayan M.
Ontogenesis of glomerular basement membrane: structure and functional properties.
J Cell Biol
113:
689-700,
1991[Abstract].
9.
Durbeej, M,
Fecker L,
Hjalt T,
Zhang H,
Salmivirta K,
Klein G,
Timpl R,
Sorokin L,
Ebendal T,
Ekblom P,
and
Ekblom M.
Expression of laminin
1,
5 and
2 chains during embryogenesis of the kidney and vasculature.
Matrix Biol
15:
397-413,
1996[ISI][Medline].
10.
Durkin, ME,
Gautam M,
Loechel F,
Sanes JR,
Merlie JP,
Albrechtsen R,
and
Wewer UM.
Structural organization of the human and mouse beta2 chain genes, and alternative splicing at the 5' end of the human transcript.
J Biol Chem
271:
13407-13416,
1996[Abstract/Free Full Text].
11.
Hansen, K,
and
Abrass CK.
Role of laminin isoforms in glomerular structure.
Pathobiology
67:
84-91,
1999[ISI][Medline].
12.
Holzman, LB,
St. John PL,
Kovari LA,
Verma R,
Holthofer H,
and
Abrahamson DR.
Nephrin localizes to the slit pore of the glomerular epithelial cell.
Kidney Int
56:
1481-1491,
1999[ISI][Medline].
13.
Kanwar, YS,
Carone FA,
Kuma A,
Wada J,
Ota K,
and
Wallner EI.
Role of extracellular matrix, growth factors, and proto-oncogenes in metanephric development.
Kidney Int
52:
589-606,
1997[ISI][Medline].
14.
Martin, PT,
Ettinger AJ,
and
Sanes JR.
A synaptic localization domain in the synaptic cleft protein laminin
2 (s-laminin).
Science
269:
413-416,
1995[ISI][Medline].
15.
Miner, JH.
Developmental biology of glomerular basement membrane components.
Curr Opin Nephrol Hypertens
7:
13-19,
1998[ISI][Medline].
16.
Miner, JH,
Cunningham J,
and
Sanes JR.
Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin
5 chain.
J Cell Biol
143:
1713-1723,
1998[Abstract/Free Full Text].
17.
Miner, JH,
and
Li C.
Defective glomerulogenesis in the absence of laminin
5 demonstrates a developmental role for the kidney glomerular basement membrane.
Dev Biol
217:
278-289,
2000[ISI][Medline].
18.
Miner, JH,
Patton BL,
Lentz SI,
Gilbert DJ,
Snider WD,
Jenkins NA,
Copeland NG,
and
Sanes JR.
The laminin
chains: expression, developmental transitions, and chromosomal locations of
1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel
3 isoform.
J Cell Biol
137:
685-701,
1997[Abstract/Free Full Text].
19.
Miner, JH,
and
Sanes JR.
Collagen IV
3,
4,
5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches.
J Cell Biol
127:
879-991,
1994[Abstract].
20.
Moscovici, C,
Moscovici MG,
Jimenez H,
Lai MM,
Hayman MJ,
and
Vogt PK.
Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail.
Cell
11:
95-103,
1977[ISI][Medline].
21.
Noakes, PG,
Miner JH,
Gautam M,
Cunningham JM,
Sanes JR,
and
Merlie JP.
The renal glomerulus of mice lacking s-laminin/laminin
2: nephrosis despite molecular compensation by laminin
1.
Nat Genet
10:
400-406,
1995[ISI][Medline].
22.
Nagata, M,
and
Watanabe T.
Podocytes in metanephric organ culture express characteristic in vivo phenotypes.
Histochem Cell Biol
108:
17-25,
1997[ISI][Medline].
23.
Robert, B,
St. John PL,
and
Abrahamson DR.
Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice.
Am J Physiol Renal Physiol
275:
F164-F172,
1998[Abstract/Free Full Text].
24.
Robert, B,
St. John PL,
Hyink DP,
and
Abrahamson DR.
Evidence that embryonic kidney cells expressing Flk-1 are intrinsic, vasculogenic angioblasts.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F744-F753,
1996[Abstract/Free Full Text].
25.
Sanes, JR,
Engvall E,
Butkowski R,
and
Hunter DD.
Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere.
J Cell Biol
111:
1685-1699,
1990[Abstract].
26.
Sasaki, M,
Kato S,
Kohno K,
Martin GR,
and
Yamada Y.
Sequence of the cDNA encoding the laminin
1 chain reveals a multidomain protein containing cysteine-rich repeats.
Proc Natl Acad Sci USA
84:
935-939,
1987[Abstract].
27.
Saxén, L.
Organogenesis of the Kidney. Cambridge, UK: Cambridge Univ. Press, 1987.
28.
Sorokin, LM,
Pausch F,
Durbeej M,
and
Ekblom P.
Differential expression of five laminin
(1-5) chains in developing and adult mouse kidney.
Dev Dyn
210:
446-462,
1997[ISI][Medline].
29.
Steadman, R,
Irwin MH,
St. John PL,
Blackburn WD,
Heck LW,
and
Abrahamson DR.
Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties.
J Leukoc Biol
53:
354-365,
1993[Abstract].
30.
Vanden Heuvel, GB,
Leardkamolkarn V,
St. John PL,
and
Abrahamson DR.
Carboxy terminal sequence and synthesis of rat kidney laminin
1 chain.
Kidney Int
49:
752-760,
1996[ISI][Medline].
31.
Wang, R,
Moorer-Hickman D,
St. John PL,
and
Abrahamson DR.
Binding of injected laminin to developing kidney glomerular mesangial matrices and basement membranes in vivo.
J Histochem Cytochem
46:
291-300,
1998[Abstract/Free Full Text].
32.
Wang, R,
St. John PL,
Kretzler M,
Wiggins RC,
and
Abrahamson DR.
Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.
Kidney Int
57:
1847-1859,
2000[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 280(4):F695-F705
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