1 Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; and 2 Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45219-0581
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ABSTRACT |
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Ischemic injury to the kidney, a
major cause of acute renal failure, leads to the detachment and loss of
numerous tubular epithelial cells. Integrin-laminin interactions may
promote regeneration of the damaged epithelium by influencing kidney
epithelial cell adhesion and differentiation. Laminins are major
structural components of basement membranes. Of the various laminin
isoforms, laminin-5 is of particular interest because of its proposed
role in the healing of skin wounds. In this study, we investigate the
expression of laminin-5 in rat kidney after unilateral
ischemia. Using a polyclonal antibody generated against
laminin-5, we find that immunostaining is confined to the basement
membranes of collecting ducts in the papilla and the major and minor
calyces in normal kidney. With injury and regeneration, however,
immunostaining becomes much more intense and widespread in basement
membranes along the nephron. Immunoblotting of ischemic kidney
extracts reveals significantly increased expression of a polypeptide of ~220 kDa, possibly corresponding to a precursor of one of the three
laminin-5 chains. Immunoblotting and immunostaining also demonstrate
significantly increased expression and altered localization of the
3-integrin subunit, a receptor for laminin-5. These
results indicate that there is induction of a laminin isoform, possibly laminin-5, and
3
1-integrin in the
ischemic kidney and may implicate this receptor-ligand
combination in the pathogenesis of acute renal failure and/or repair of
the injured kidney epithelium.
extracellular matrix; epithelial regeneration; acute renal failure
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INTRODUCTION |
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ISCHEMIC INJURY TO
THE KIDNEY, a major cause of acute renal failure
(53), leads to the detachment and loss of significant numbers of cells from the tubular epithelium (43-45).
Cells that remain adherent to the basement membrane dedifferentiate and
spread to fill gaps in the epithelium and then repolarize and
redifferentiate to reconstitute the normal architecture and function of
the tubular epithelium (6, 7, 9, 13, 35, 36, 47, 53, 55, 62). During this process of repair and regeneration, significant changes occur in the extracellular matrix and its integrin receptors (1, 10, 18, 58, 62, 63). In particular, the
1-integrin subunit, which is normally found on the basal
surface, newly appears on the lateral surfaces and, in some cases, the
apical plasma membrane of tubular epithelial cells (62).
In addition, large amounts of cellular and plasma fibronectin are
deposited along the basement membrane and in the tubular lumen
(63).
Of the extracellular matrix proteins likely to play significant roles
in regeneration of the epithelium after injury, laminins are of
particular interest. Laminins are heterotrimers of -,
-, and
-chains linked by disulfide bonds to form cruciform molecules (4, 8, 33). To date, five different
-chains, three
-chains, and three
-chains have been identified with different
combinations resulting in as many as 12 different laminin isoforms.
Laminins are major structural components of basement membranes that
mediate cell adhesion but also generate differentiation signals when
bound to their cell surface receptors.
The major group of laminin receptors are integrins (4).
The integrins are a superfamily of transmembrane glycoproteins that
consist of unrelated - and
-subunits (17, 54). Eight different
-families have been identified that can combine with 14 different
-subunits, resulting in >20 different
combinations. Association of
- and
-subunits into noncovalent
heterodimers is necessary for ligand binding and cell surface
expression. Depending on the
-heterodimer, ligand specificity for
laminin or other extracellular matrix molecules is conferred.
In the kidney, integrins with the 1-subunit are
expressed throughout the nephron (21, 22), in which they
interact with a variety of specific laminin isoforms (4, 32, 33,
51, 57) and other extracellular matrix proteins. The precise
roles of
1-integrin-laminin interactions in the adult
kidney are poorly understood but certainly involve cell differentiation
along with adhesion. In the embryonic kidney,
6
1-integrin, a laminin receptor, is
required for polarization of the condensed mesenchyme into the tubular
epithelium (50) and is also a major laminin receptor in
the mature kidney. Antibodies against the laminin-A-chain (now called
laminin-
1) also prevent the formation of a polarized
epithelium when added to organ cultures of developing embryonic kidney
(20).
Of the various laminin isoforms, laminin-5 is potentially significant
in the injured kidney because it has been previously implicated in
regeneration of skin wounds (25, 38-40, 42). Laminin-5 (also called kalinin, BM600/nicein, epiligrin, or ladsin) is
composed of the 3-,
3-, and
2-chains and is a component of the anchoring filaments
of the skin basement membrane (2, 4, 48, 56). In many cell
types,
3
1- and
6
4-integrins act as laminin-5 receptors
(2, 4, 28, 41). Laminin-5 is required for
epithelialization of skin after wounding (14, 25, 38, 40)
and is also important in the differentiation of enterocytes during
migration from the crypt to the villus tip (16, 26). In
the kidney, laminin-5 is necessary for branching of the ureteric bud
during development (60). Its expression in the adult
kidney has not been widely examined, although it has been observed in
the renal papilla (32, 33). Some reports also place it in
other parts of the kidney (32).
Because of the importance of laminin in epithelial cell
differentiation, we hypothesized that expression and distribution of
laminin isoforms and their integrin receptors might be altered by
ischemic injury to the kidney. Here, we report that the
expression of a member of the laminin family, possibly the laminin-5
isoform, is dramatically increased in the basement membranes of the S3 segment and other regions of the kidney after ischemic injury in vivo. In addition, expression of the 3-integrin
subunit, which with
1 forms a receptor for laminin-5, is
also significantly upregulated, in parallel with the laminin isoform.
These results indicate that laminin-integrin interaction may play an
important role in the regeneration of the renal tubular epithelium
after injury, analogous to their functions in the epidermis.
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MATERIALS AND METHODS |
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Animal Surgery
A unilateral rat model of renal ischemia was used for these studies (59, 62, 63). In this model, blood flow to one kidney is surgically interrupted for 40 min without manipulation of the contralateral kidney, followed by release of the vascular clamp and reperfusion for various times up to several days. This procedure results in only mild systemic changes in blood urea nitrogen and serum creatinine (27, 59). In addition to the contralateral kidney, which served as an internal control, additional controls consisted of kidneys from animals that underwent sham surgery without ischemia.Immunohistochemistry
Tissue harvesting. Left postischemic and right contralateral kidneys were fixed in situ for immunohistochemistry. Anesthetized animals were first perfused with warm Hank's balanced salt solution until the kidney was cleared of blood. The perfusate was then switched to 2% (wt/vol) paraformaldehyde-75 mM L-lysine-10 mM sodium periodate (PLP fixative) (31) at room temperature. Kidneys were removed, cleaned of adherent connective tissue, and sliced incompletely from the cortex to the medulla at ~2-mm intervals to facilitate penetration of fixative. Incompletely sliced, whole kidneys were transferred into fixative for 1 h at room temperature and refrigerated overnight. The following day, they were rinsed in PBS and stored in PBS containing sodium azide (0.02% wt/vol) at 4°C.
Cryosectioning.
Kidney pieces were prepared by extending the cut that terminated at the
medulla to the hilus to completely sever a segment of the kidney from
the rest of the tissue. They were then rinsed extensively in PBS and
infiltrated overnight at 4°C with 0.6 M sucrose/PBS. Samples were
then embedded in optimal cutting temperature compound (Miles
Laboratories, Naperville, IL), positioned horizontally on a sample
chuck for frozen sectioning, and flash frozen in liquid nitrogen.
Five-micrometer sections were cut on a Leica CM1850 cryostat (Leica,
Deerfield, IL), placed on SuperFrost Plus glass slides (Fisher
Scientific, Boston, MA), and stored at 20°C until use.
Immunofluorescence. The procedure of Zuk et al. (62) was used. Briefly, freshly prepared fixative was applied to each section for 10 min to minimize section loss during the procedure, followed by extensive rinsing in PBS. Autofluorescence of tissue sections was quenched with sodium borohydride in PBS (1 mg/ml) followed by additional rinsing. Samples were then permeabilized in 0.1% Triton X-100/PBS, rinsed, and incubated in 10% normal goat serum in PBS to block nonspecific binding sites. Diluted primary antibody was then applied to each sample, and the sections were incubated overnight at 4°C. Samples were further rinsed and incubated in secondary antibody at room temperature in the dark. After additional rinses, sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed by epifluorescence microscopy with either a Zeiss Axioplan, Axiovert, or Axioskop 2Plus Microscope (Carl Zeiss, Thornwood, NY). In some instances, sections were incubated in 1% SDS/PBS for 5 min before quenching to expose latent antigenic sites.
Images were recorded with TMAX 400 film (Eastman Kodak, Rochester, NY) pushed to ASA 800 or with a Zeiss AxioCam MR digital camera. To demonstrate increased expression of theImmunoblotting
Tissue harvesting.
Anesthetized animals were first perfused with sterile PBS at 0°C
containing 0.16× Complete protease inhibitors (Boehringer-Mannheim, Indianapolis, IN). Kidneys were then removed from the animal, placed on
ice, and dissected free of adherent connective tissue. Kidney pieces
were cut along the cortex-hilus axis, flash frozen in liquid nitrogen,
and stored at 80°C.
Tissue preparation. The procedure of Zuk et al. (63) involving repeated homogenization, and extraction in solutions containing SDS, DTT, and protease inhibitors was used. For gel electrophoresis, aliquots of extracts containing equal amounts of protein were diluted with 1× sample buffer [200 mM Tris · Cl, pH 8.8, 5 mM EDTA, 1% (wt/vol) SDS, 20 mM DTT, 20% sucrose, 0.2% bromphenol blue] and alkylated with 50 mM iodoacetamide before loading.
Western blotting. Aliquots (25 µg) of kidney extract protein were resolved by 6% SDS-PAGE as previously described (63). Gels were then equilibrated in transfer buffer containing SDS, and proteins were transferred onto Immobilin-P membranes (Millipore, Bedford, MA) with a semidry transfer apparatus (Bio-Rad, Hercules, CA) at 90 mA for 42 min. Membranes were quickly stained in Coomassie blue to confirm equal protein loading and then destained. After a rinsing with wash buffer [10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween-20 (TBST)], membranes were blocked in 5% nonfat dry milk/TBST. Primary antibody was incubated with membranes sealed in plastic bags at 4°C overnight with rotation. Membranes were then washed extensively at room temperature and reacted with secondary antibody conjugated to horseradish peroxidase. They were washed again and peroxidase moieties reacted with Renaissance chemiluminescence reagents (NEN Life Science Products, Boston, MA). Membranes were exposed to X-OMAT Blue film (Eastman Kodak), which was then developed with a Kodak X-OMAT processor.
Antibodies
Rabbit polyclonal anti-human 8LN-5 antibody was kindly provided by Dr. Robert Burgeson (Cutaneous Biology Research Center, Massachusetts General Hospital, Boston, MA). This antibody was prepared against affinity-purified laminin-5 from human keratinocyte culture supernatants injected into rabbits. When used to probe Western blots of laminin-5 purified by affinity chromatography from collagenase-solubilized human amnion- or human keratinocyte-conditioned medium, this antibody recognizes the mature
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Rabbit polyclonal anti-human 1-integrin antibody was
generated against a 37-amino acid sequence of the cytoplasmic domain of
human
1-integrin (Research Genetics, Huntsville, AL);
for Western blots, a 1:1,000 dilution was used. Rabbit polyclonal anti-human
4-integrin antibody was kindly provided by
Arthur Mercurio (Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA). This antibody, generated against a synthetic peptide corresponding to a 31-amino acid sequence of the cytoplasmic domain of human-
4, was used for immunoblots at a
dilution of 1:1,000. Mouse monoclonal anti-mouse
3-integrin antibody was purchased from Transduction
Laboratories (Lexington, KY). This antibody was generated against a
synthetic peptide corresponding to a 215-amino acid residue of the
extracellular domain of the mouse
3-integrin subunit;
for immunoblots and immunostaining, it was used at 1:250 and 1:10,
respectively. Rabbit polyclonal anti-
3-integrin subunit
antibody was generated against a synthetic peptide corresponding to the
COOH-terminal cytoplasmic domain of human
3-integrin
(Chemicon International, Temecula, CA); for Western blots, a 1:500
dilution was used.
Secondary antibodies for immunostaining included goat anti-rabbit FITC adsorbed against mouse and human Ig (1:200; BioSource International, Camarillo, CA) or goat anti-mouse indocarbocyanine (CY3) adsorbed against rat, human, bovine, and horse serum proteins (1:800; Jackson ImmunoResearch Laboratories, Westgrove, PA).
Secondary antibodies for immunoblotting included affinity-purified peroxidase-conjugated donkey anti-rabbit (1:5,000) or donkey anti-mouse (1:1,000) IgG (Jackson ImmunoResearch Laboratories).
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RESULTS |
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Immunostaining of Kidney Sections with 8LN-5 Antibody
When frozen sections of paraformaldehyde-fixed normal rat kidney were immunostained with antibody 8LN-5, which was raised against purified laminin-5, followed by a fluorescent secondary antibody, reactivity was observed in the basement membrane of the epithelium lining the major and minor calyces (Fig. 2A) and collecting ducts of the papilla (Fig. 2B). Basement membranes of collecting ducts in the cortex or outer and inner stripes of the outer medulla did not stain (data not shown). In addition, basement membranes of proximal and distal tubules in the cortex and medulla were not immunoreactive (data not shown).
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Ischemic injury is most prevalent in the S3 segment of proximal
tubules in the outer stripe of the outer medulla, apparently because
hemodynamic factors lead to impaired reperfusion of this region of the
kidney (6, 13, 47). In sections taken from kidneys made
ischemic by clamping of the renal artery, additional staining
with 8LN-5 appeared in the outer stripe of the outer medulla. At 0 (Fig. 3A), 1 (Fig.
3B), and 3 h (data not shown) postischemia,
immunostaining of this region with 8LN-5 revealed no reactivity.
However, at 24 (data not shown) and 48 h postischemia, faint reactivity of basement membranes of S3 proximal tubule segments was seen (Fig. 3C). Stronger localization to basement
membranes was present in other tubules at this time, identified as
collecting ducts (CT, Fig. 3C) by their distinct lateral
borders visible in parallel Nomarski images. At 120 h
postischemia (Fig. 3D), strong reactivity with
antibody 8LN-5 in the outer stripe was very evident in the basement
membranes of regenerating proximal tubules (PT, Fig. 3D). In
some instances, the intensity of staining varied within a given tubule,
with some regions of the basement membrane showing stronger
immunoreactivity than other regions of the same tubule (Fig.
3D). In general, staining of proximal tubules was variable,
with a range of reactivities within a single field. Basement membrane
localization was also observed in collecting tubules, with some diffuse
cytoplasmic staining (CT, Fig. 3D). At all times after
reperfusion, antibody 8LN-5 reactivity was not detected in the
interstitial matrix. In contralateral (Fig. 3, E and
F) and mock-treated (Fig. 3, G and H)
kidney tissue, immunostaining with 8LN-5 was not present in the outer
stripe of the outer medulla at any time (0-120 h) after
reperfusion or mock surgery.
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In the inner stripe of the outer medulla, 8LN-5 immunostaining revealed
no reactivity in renal tubules at 0 (Fig.
4A), 1 (Fig. 4B),
and 3 h (data not shown) postischemia. However, at 24 (data not shown) and 48 h (Fig. 4C)
postischemia, the antibody highlighted basement membranes of
collecting tubules (CT, Fig. 4C) and thick limbs (TL, Fig.
4C); similar staining persisted at 120 h after reperfusion (Fig. 4D). In contralateral (Fig. 4,
E and F) and mock (Fig. 4, G and
H) surgical controls at equivalent times, reactivity with
antibody 8LN-5 was absent from renal tubules of the inner stripe of the
outer medulla.
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In the papilla of ischemic kidneys as well as contralateral and mock surgical controls (data not shown), 8LN-5 immunostaining was distributed to the basement membranes lining the major and minor calcyes and collecting tubules, similar to that observed in the normal kidney (see Fig. 2, A and B).
In the cortex, antibody 8LN-5 did not immunostain any renal tubules in response to ischemia-reperfusion (0-120 h postischemia; data not shown). Reactivity was also not observed in the cortex of contralateral and mock kidneys at equivalent times (0-120 h postischemia; data not shown).
In summary, unilateral ischemic injury to the kidney induced 8LN-5 immunoreactivity in the basement membrane of the renal tubular epithelium in the outer and inner stripes of the outer medulla, including the S3 segment of the proximal tubule, in which no staining was observed under normal or control conditions. Immunostaining was not induced in the cortical nephron, and normal reactivity in the papilla was not appreciably increased with ischemic injury.
Immunoblotting of Kidney Extracts with the 8LN-5 Antibody
To provide biochemical evidence for the induction of antibody 8LN-5 reactivity, kidney extracts representing various times after reperfusion were immunoblotted with the same antibody used for immunostaining. In ischemic kidneys at all times after reperfusion (Fig. 5A), a polypeptide chain of ~220 kDa was detected that dramatically increased in level of expression from 24 to 120 h postischemia. This polypeptide may correspond to the precursor of the laminin-5
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In summary, immunoblotting with 8LN-5 demonstrated induction in
ischemic kidneys of a high-molecular-weight polypeptide,
possibly corresponding to the precursor of the laminin
3-chain, and another polypeptide comigrating with
laminin-
3, with peak intensity between 24 and 120 h postischemia.
Induction of Integrin Subunits
If laminin-5 is induced in the kidney by ischemic injury, then it is possible that the expression of cognate integrin-laminin receptors, including the laminin-5 receptorsImmunoblotting with a rabbit polyclonal anti-human
1-integrin antibody raised against a synthetic peptide
of the cytoplasmic domain indicated that there was no increased
expression from 0 to 120 h postischemia in response to
injury and repair (Fig. 6A). In addition, the amount of the
1-subunit in
ischemic-reperfused kidneys did not appear appreciably
different from contralateral (Fig. 6B) or mock (Fig.
6C) surgical controls at equivalent times. However, in
samples from ischemic animals, a lower molecular weight band
was observed from 24 to 120 h postischemia (arrow, Fig.
6A), which was absent in contralateral (Fig. 6B)
or mock (Fig. 6C) kidneys at the same times after
reperfusion. This may correspond to an immature, intracellular
precursor of
1, which has been observed to accumulate in
some cells (49, 61).
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There are conflicting reports on whether 4-integrin is
present in the kidney (24, 37, 60). Immunoblotting of
kidney extracts of ischemic (Fig.
7A), contralateral (Fig.
7B), and mock (Fig. 7C) surgical controls
indicated low levels of expression of the
4-subunit,
with no significant change in ischemic vs. contralateral or
mock surgical controls.
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To determine whether the levels of the 3-integrin
subunit changed as a result of ischemic injury, extracts were
immunoblotted with a rabbit polyclonal anti-
3-integrin
antibody generated against a synthetic peptide corresponding to the
COOH-terminal cytoplasmic domain of human
3-integrin. Levels of the
3-integrin subunit significantly increased from 3 to
120 h postischemia (Fig.
8A) compared with
contralateral (Fig. 8B) and mock (Fig. 8C) kidney extracts at the same time points. Comparable results were obtained with
a monoclonal antibody recognizing mouse
3-integrin (data not shown).
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The 3-integrin subunit was next immunolocalized by using
a monoclonal antibody that recognizes the NH2-terminal
region of
3 to determine whether its distribution in the
ischemic kidney corresponded to observed immunostaining with
the 8LN-5 antibody. At 0 (arrow, Fig. 9,
A and B), 1 (arrow, Fig. 8, C and
D), and 3 h (data not shown) postischemia,
3-integrin immunoreactivity faintly localized to basal
plasma membranes of renal tubules in the outer stripe of the outer
medulla, similar to the immunostaining pattern seen in corresponding
regions of contralateral (data not shown) and mock (data not shown)
kidneys at equivalent times. Cellular debris that filled the lumenal
space at 1 (*, Fig. 9, C and D) and 3 h
(data not shown) postischemia, consisting in part of shed
microvilli or apical membrane blebs (62), were unreactive,
as expected. Reactivity was also absent from sites in which luminal
material contacted apical surfaces of surviving, adherent cells
(arrowhead, Fig. 9, C and D).
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At 24 (Fig. 9, E and F) and 48 h (data not
shown) postischemia, 3-integrin immunostaining
localized to basal plasma membranes of all renal tubules in the outer
stripe of the outer medulla; lateral localization was infrequently seen
and apical staining of
3 in combination with basal
and/or lateral localization was rare. Luminal material representing
cellular debris and/or depolarized exfoliated cells (62)
did not react with the
3-integrin antibody (*, Fig. 9,
E and F). Reactivity was also not detected
between detached cells and the adherent epithelium (arrowheads, Fig. 9, E and F). In the outer stripe of contralateral
(data not shown) and mock (Fig.
10A) surgical controls,
immunoreactivity was faintly seen on basal cell surfaces.
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At 120 h postischemia, strong 3-integrin
immunoreactivity was observed on basal and/or lateral surfaces of
tubular epithelial cells in the outer stripe (Fig. 9, G and
H). In some instances, staining also appeared apically
(arrow, Fig. 9, G and H). Staining intensity
sometimes varied among cells within the same tubule. Luminal material,
which at this time, represents individual cells or small groups of
cells (62), was sometimes immunoreactive (*, Fig. 9,
G and H). In contralateral (Fig. 10B)
and mock (data not shown) kidneys at this time point,
3
staining was detected only basally, and the intensity of staining was
much less than that of the corresponding ischemic samples.
In the inner stripe of the outer medulla at 0 (Fig.
11A), 1 (Fig.
11B), and 3 h (data not shown) postischemia,
3-integrin immunostaining was observed on basal plasma
membranes of epithelia lining the distal nephron segment. In collecting
ducts (CT, Fig. 11, A and B), faint lateral
localization was also observed. At 24 to 48 h
postischemia, basal localization remained, but more tubules also showed lateral distribution (Fig. 11C) than at earlier
times. A similar pattern persisted at 120 h after ischemic
injury, but staining was much more intense (Fig. 11D). In
contralateral (Fig. 11F) and mock (Fig. 11E)
surgical controls, only relatively faint basal immunostaining of the
3-integrin subunit was detected at all time points.
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In the cortex of ischemic, contralateral, and mock surgical
control kidneys, 3 immunostaining was detected in the
glomerular capillary tuft, Bowman's capsule, and epithelia lining the
distal tubule (Fig. 12), in agreement
with published reports (21, 23, 24). However, it was
absent from proximal tubular epithelia (Fig. 12), as reported by others
(21, 23, 24, 46). There was no staining of the
3-integrin in the interstitium of the kidney cortex
(data not shown) and medulla of ischemic, contralateral, or
mock kidneys at any time (for outer and inner stripes of
ischemic, contralateral, and mock kidneys, see Figs.
9-11).
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In summary, 3-integrin immunostaining significantly
increased in the ischemic kidney, particularly from 24 to
120 h postischemia, with peak reactivity at 120 h
postischemia. Most of this increase was evident in the outer
stripe of the outer medulla and involved regenerating proximal
tubules. However, ischemic injury also induced increased immunoreactivity in the inner stripe of the outer medulla in
both distal tubules and collecting ducts.
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DISCUSSION |
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In this article, we report that unilateral ischemic injury
to the rat kidney, caused by clamping of the renal vasculature for 40 min, leads to the induction of a laminin variant, most likely
laminin-5, and a laminin receptor,
3
1-integrin, in epithelial cells and the
underlying matrix of the outer and inner stripe of the outer medulla.
Induction begins ~24 h postischemia, with the highest
expression noted at 120 h postischemia, the last time point examined. Because induction is so late after the ischemic episode and affects areas of the kidney most damaged, including the S3
segment, it is likely that the new production of this laminin and
integrin are related to regeneration of the injured epithelium.
For these studies, we used rabbit polyclonal antibody 8LN-5 generated
against affinity-purified laminin-5 to examine the expression of this
laminin isoform in both normal and postichemic rat kidneys. By
immunoblotting, this antibody recognizes polypeptide chains of 165, 140, and 105 kDa in purified laminin-5- and human
keratinocyte-conditioned medium corresponding to the mature,
proteolytically processed forms of the 3-,
3-, and
2-chains (48). In
addition, antibody 8LN-5 recognized the
3 precursor in
extracts of human keratinocytes from the immunoblotted and
immunostained basement membrane of skin (29). Thus
the data indicate that antibody 8LN-5 is specific for the laminin-5
isoform of the laminin family. We cannot, however, rule out the
possibility that 8LN-5 reacts with epitopes shared between laminin-5
chains and subunits of other laminin isoforms.
In immunoblots of ischemic kidney extracts, the 8LN-5 antibody
recognizes a polypeptide with mobility slightly slower than 200 kDa and
one at 140 kDa. In keratinocytes and a variety of other cell types, the
laminin-5 3-subunit is synthesized and secreted as a
precursor of ~200 kDa in a complex with the
3-chain and a 155-kDa precursor of the
2-subunit (15, 29,
30). At some undetermined point after secretion, the
3 and
2 precursors are processed to their
mature size by extracellular proteases, possibly plasmin,
metalloendoproteases, or a combination of enzymes (14, 15, 30,
38, 40). On the basis of this biosynthetic pathway and the
established specificity of the 8LN5 antibody, we believe that the
polypeptides induced in the ischemic kidney are the
3 precursor and the
3-chain of the
laminin-5 molecule (Burgeson R and Koch M, Cutaneous Biology Research
Center, Massachusetts General Hospital, personal communication).
Unfortunately, multiple attempts to confirm the identity of the
high-molecular- weight polypeptide and the laminin
3-subunit were unsuccessful because appropriate
cross-reacting antibodies could not be obtained. Future work utilizing
a PCR-based approach and possibly new antibodies will be necessary to
confirm our interpretation.
Another confounding factor is the apparently poor recovery in kidney
extracts of the 3-subunit, as well as the failure to detect the
2-chain. There are several possible
explanations for this discrepancy. When laminin-5 was originally
isolated from amniotic membranes, very stringent extraction procedures
involving extensive collagenase digestion were carried out
(48). In our case, kidneys were homogenized and extracted
with SDS- and DTT-containing buffers, but without collagenase
digestion. Thus it is possible that the three laminin-5 chains, which
would have been dissociated by reduction and denaturation, nevertheless
were differentially extracted. It is also possible that some chains
were proteolyzed during or after harvesting and processing of the
kidneys, despite our efforts to inhibit proteolytic enzymes. Finally,
it is conceivable that another laminin isoform consisting of the
3-chain found in laminin-5 along with
- and
-chains other than those recognized by the 8LN5 antibody, is induced
in the kidney by ischemic injury (32). This could
be a completely new isoform or one or both of the other two known
laminin isoforms that express
3 (3, 32).
Both laminin-6 (3
1
1) and
-7 (
3
2
1) contain the
3-chain (3, 32). However, both are reported
to covalently associate with laminin-5 by means of disulfide bonding,
and their expression in the absence of laminin-5 has not been seen
(3). The
1-chain of laminin is a component
of most of the 12 known forms of laminin and is expressed throughout
the basement membranes of kidney tubules both before and after
ischemic injury (32, 57, 58, 62). Because of this
and because we have not examined the ischemic kidney for
expression of the laminin-
2-chain, we cannot rule out
the possibility that laminin-6 as well as laminin-7 are induced in the
ischemic kidney together with, or even instead of, laminin-5.
Expression of laminin-5 has been reported in the collecting duct system
of embryonic mouse and adult human kidney (32, 34, 52).
Our immunohistochemical analysis of the renal cortex and medulla of
normal rat kidney clearly demonstrates that laminin-5 is expressed only
in the basement membranes of papillary collecting ducts and not those
of the cortex and outer medulla. This limited distribution is probably
the reason we are unable to detect laminin-5 polypeptides by
immunoblotting in the nonischemic kidney, given that our
extracts are derived from whole kidneys. In the skin, which is a
stratified epithelium, laminin-5 plays a structural role, anchoring the
epidermis to the dermis via hemidesomosomes (48). Given
that the epithelium of the papillary collecting duct stratifies in its
distal portions, it is perhaps reasonable to conclude that laminin-5 is
playing a somewhat similar role under nonpathological conditions.
Furthermore, in contrast to published studies on embryonic mouse and
adult human kidneys (34, 52), we do not detect laminin-5
in basement membranes of proximal and distal tubules of normal or
control (contralateral and mock) rat kidneys. These reports used
polyclonal and monoclonal antibodies generated against the
2-chain of laminin-5, which antibody 8LN-5 also
identifies (see Fig. 1A).
After ischemic injury, laminin-5 induction occurs within 24 h of reperfusion, with increasing expression evident up to 120 h postischemia, the last time point examined in this study. Expression occurs in the S3 segment of the proximal tubule, in which most cellular injury is evident, and in the thick limb of the inner stripe of the outer medulla and continues in the collecting ducts. Expression in the collecting ducts becomes more widespread as a result of injury, extending from the papilla to tubules of the inner and outer stripe. The latter is notable because no overt morphological damage is observed in this part of the nephron, on the basis of Nomarski images and immunostaining with antibodies against integrin receptors, extracellular matrix molecules, and apical and basolateral membrane markers (62, 63). In nephrogenesis, laminin-5 promotes branching of the ureteric bud (60), the progenitor of the collecting duct system. Thus it is possible that laminin-5 provides some form of differentiation signal to the cells of the collecting duct, which is initiated by the ischemic insult, perhaps to repair latent defects or strengthen cell-substratum contacts of the epithelium. It is also of interest that no induction of laminin-5 was seen in cortical proximal tubules, which depolarize in response to ischemia but quickly regenerate polarity on reperfusion.
In the S3 segment, it is likely that laminin-5, along with the laminin
receptor integrin 3
1 (see below), play
important roles in the repair and regeneration of tubular epithelium.
In vitro studies have established that the laminin-5 precursor, which contains the 200-kDa form of the
3-subunit as well as a
precursor of the
2-chain (15, 29, 30),
stimulates migration of keratinocytes to close wounds (5, 14, 15,
38-40). In this process, laminin-5 interacts with
3
1-integrin, generating signals that
regulate interaction of cells with collagen via
2
1-integrin (40).
2
1-Integrin may function as a receptor
for laminin-5 in this process as well (5). Processing of
laminin-5 chains may then inhibit migration, possibly due to a switch
of laminin-5 from
3
1 to
6
4 and its incorporation in contacts
mediated by hemidesmosomes (14, 15, 38-40, 42).
Laminin-5 processing is also involved in migration of breast epithelial
cells (11, 12). Similarly, it has been postulated that
laminin-5 modulates
3
1- and
2
1-integrins to facilitate enterocytic
differentiation of the colon carcinoma cell line HT29
(16). Therefore, it seems reasonable to propose that in
the postischemic kidney, the induction of laminin-5 and its
integrin receptor
3
1 are related to
redifferentiation and repolarization of the cells to restore normal
function. Under these circumstances, we would not expect to see a
conversion of the laminin
3-subunit to its mature form
because no hemidesmosomes are present in the kidney; in fact, no such
conversion is observed.
Integrin receptors for laminin-5 include
3
1,
6
4, and
6
1 (2, 4, 28, 41). We
hypothesized that increased expression of laminin-5 in response to
ischemic injury might also induce increased or more widespread
expression of its cognate integrin receptors. In fact, ischemia
dramatically upregulated the expression of the
3-integrin subunit, although no changes in the levels of
the
1-subunit were observed. In the normal kidney,
3-integrin is expressed in epithelial cells of distal
tubules and collecting ducts but not in proximal tubules (21,
46). Although we generally agree with these observations, we do
detect
3-integrin in epithelial cells of proximal
tubules in the outer stripe of the outer medulla, albeit at low levels.
With ischemic injury, 3-integrin expression in
proximal tubules of the outer stripe significantly increases.
Expression also continues to be evident and may be increased in the
glomerulus, distal tubules, and collecting ducts.
3
1-Integrin, in addition to being a
receptor for laminin-5, is also a receptor for types of collagen and
other laminin isotypes, including laminin-10 (4, 19).
Although we consider it likely that one of its roles in the
postischemic proximal tubule is as a receptor for laminin-5, which is coordinately induced, we cannot prima facie rule out the
involvement of
3
1-integrin as a receptor
for other extracellular matrix ligands. Indeed,
3
1 is clearly playing other, distinctive roles in other regions of the nephron under normal circumstances and,
most likely, after injury (23, 24). This conclusion is reinforced by the observation that the breadth and intensity of the
increase in
3
1 expression in the
ischemic kidney appears to be greater than that of the induced
laminin isoform.
As described previously, 6
4 is also a
laminin-5 receptor that forms part of the hemidesmosomal complex in
skin. On this basis, it would seem unlikely that
4
expression would be increased in the postischemic kidney. On
the other hand, the
4-integrin subunit is the major
receptor for laminin-5 during embryonic development of the collecting
ducts (60) and therefore might play a role in the
regeneration process. Nevertheless, we did not detect any change in
4-integrin levels, which are low in the normal kidney, suggesting that the receptor for the upregulated laminin variant was
not
6
4-integrin.
We also attempted to examine the relative expression of the
6-integrin subunit but were unable to find suitable
antibodies that cross-reacted with the rat protein. Given that
6
1 is a major laminin receptor expressed
throughout the tubules of the adult kidney (24), it is
possible that it may also play a major role in recovery from
ischemic injury. In the future, when better reagents are
available, its function in epithelial regeneration will have to be reevaluated.
The distribution of the 3-integrin subunit in the
injured and regenerating kidney agrees with our published data on
1-integrin, the only
-subunit with which
3 is known to dimerize. We did not detect the
3-integrin subunit on exfoliated cells in the luminal
space after ischemic injury, supporting our laboratory's observation that members of the
1-integrin family are
absent on detached cells (62). In addition, in the injury
phase, the
3-integrin subunit was not detected on apical
surfaces of the surviving epithelium, also supporting our laboratory's
earlier finding that the
1-integrin family is absent
from this location (62). These observations support our
laboratory's previous conclusion that members of the
1-integrin family, including
3
1, do not mediate cell-cell adhesion
between attached and exfoliated cells and therefore do not contribute
in this manner to tubular obstruction.
In conclusion, our immunocytochemical analysis demonstrates that a
laminin variant and 3
1-integrin are
induced in the tubular basement membrane and epithelium of the kidney
by ischemic injury during the phase at which epithelial cell
redifferentiation and repolarization are known to occur. On the basis
of immunoblotting and the known processing pathway of laminin-5, we
believe that the laminin isoform induced is laminin-5, although we
cannot rule out other possibilities at the present time. Overall, these
results suggest that fundamental elements of epithelial regeneration
may depend on novel laminin-integrin interactions in epithelial cells from tissues as diverse as those of the kidney, intestine, and skin.
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ACKNOWLEDGEMENTS |
---|
We thank the Morphology Core of the Harvard Digestive Disease Center (DK-34854) for use of the Leica cryostat, Dr. Elizabeth D. Hay, Department of Cell Biology, Harvard Medical School, for use of the Zeiss Axiophot microscope, and Dr. Manuel Koch, Cutaneous Biology Research Center, Massachusetts General Hospital, for helpful discussions. We are also grateful to Holly Waechter for technical assistance and Dr. Steven Boyce of the Shriners Burns Institute, University of Cincinnati College of Medicine, for providing cultured human keratinocytes.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-46768 (K. S. Matlin) and a Scholars in Medicine Grant and William F. Milton Fund Award (A. Zuk).
Address for reprint requests and other correspondence: K. S. Matlin, The Vontz Ctr., ML0581, Univ. of Cincinnati College of Medicine, 3125 Eden Ave., Cincinnati, OH 45219-0581 (E-mail: karl.matlin{at}uc.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.
July 16, 2002;10.1152/ajprenal.00176.2002
Received 3 May 2002; accepted in final form 9 July 2002.
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