1 Renal Unit, Massachusetts
General Hospital, Charlestown 02129; Departments of
2 Medicine and
4 Pathology, Acute renal
failure (ARF) as a consequence of ischemic injury is a common disease
affecting 5% of the hospitalized population. Despite the fact that
mortality from ARF is high, there has been little improvement in
survival rates over the last 40 years. The pathogenesis of ARF may be
related to substantial changes in cell-cell and cell-extracellular
matrix interactions mediated by
THE CELLULAR AND MOLECULAR responses of the kidney to
ischemic insult are complex and incompletely understood (for review, see Refs. 15, 19, 63, 72). In the early phases after injury, the
pathophysiological processes leading to cell damage and exfoliation predominate. Because the postischemic kidney has the ability to completely restore its structure and function, the second phase, which
overlaps with the first, involves repair of the damaged kidney. In many
instances, however, recovery is delayed or does not occur at all.
Indeed, the mortality rate from renal failure is high (~40%) and has
not changed significantly over 40 years (72). Therapeutic approaches
that minimize injury and hasten recovery can be developed only when the
cellular and molecular mechanisms of renal failure are more completely
understood.
Recent attention has focused on the role of the integrins in the
pathophysiology of acute renal failure (36, 54). The integrins are a
superfamily of heterodimeric transmembrane glycoproteins found on every
eukaryotic cell except erythrocytes; they are the major receptors for
the extracellular matrix (ECM) and, along with cadherins, are believed
to mediate cell-cell interactions (for review, see Ref. 31). Each
integrin consists of unrelated In the kidney, integrins possessing the
Genetic and biochemical studies implicate integrins containing the
Just as integrins containing In addition to contributing to injury, integrins may also be important
in repair. Because integrins mediate transmembrane signaling (for
review, see Refs. 10, 17, 22, 30, 31), influencing diverse cell
functions including cell behavior, differentiation, and gene
expression, integrin-mediated cell-cell and cell-ECM interactions may
also be critical to repair of the damaged epithelium. Tubular
epithelial cells that remain attached alter their cytoskeletal organization and polarization (7, 48-50); they also
dedifferentiate and proliferate (77). They may use integrins to spread
and migrate over the underlying basement membrane to reestablish
contacts with neighboring cells. Signals from these cell-matrix or
cell-cell interactions may then initiate redifferentiation.
In this study, we used an in vivo model of unilateral ischemia
in which rat kidneys are made ischemic by clamping the renal artery to
investigate the role of
Animals
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1-integrins. On the basis of in
vitro and in vivo studies, reorganization of
1-integrins from basal to
apical surfaces of injured tubular epithelia has been suggested to
facilitate epithelial detachment, contributing to tubular obstruction
and backleak of glomerular filtrate. In this study, we examine integrin
and extracellular matrix dynamics during epithelial injury and repair
using an in vivo rat model of unilateral ischemia. We find
that, soon after reperfusion,
1-integrins newly appear on
lateral borders in epithelial cells of the S3 segment but are not on
the apical surface. At later times, as further injury and regeneration
coordinately occur, epithelia adherent to the basement membrane
localize
1 predominantly to
basal surfaces even while the polarity of other marker proteins is
lost. At the same time, amorphous material consisting of depolarized exfoliated cells fills the luminal space. Notably,
1-integrins are not detected on
exfoliated cells. A novel finding is the presence of fibronectin, a
glycoprotein of plasma and the renal interstitium, in tubular spaces of
the distal nephron and to a lesser extent S3 segments. These results
indicate that
1-integrins
dramatically change their distribution during ischemic injury and
epithelial repair, possibly contributing to cell exfoliation initially
and to epithelial regeneration at later stages. Together with the appearance of large amounts of fibronectin in tubular lumens, these
alterations may play a significant role in the pathophysiology of ARF.
1-integrin; epithelial
polarity; fibronectin; acute renal failure
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
- and
-subunits whose association
into noncovalent heterodimers is necessary for ligand binding and cell
surface expression. The
-chains can associate with one or more
-subunits, forming receptors for laminins, collagens, fibronectin,
and vitronectin.
1-subunit are the most common.
During development of the metanephros,
1-integrins localize to all
cell surfaces (37-39). As the developing nephron elongates and
matures,
1-integrins polarize
to basal surfaces of tubular epithelia, where they interact with ECM
components of the basement membrane, including laminin and type IV
collagen. The surrounding interstitium is rich in fibronectin secreted
by fibroblasts on which
1-integrins are localized to
all cell surfaces.
1-subunit in kidney epithelial
differentiation and polarization. The
8-,
6-, and
3-integrin subunits complex
with
1. Knockout of the
8-subunit (51) impairs the
ability of kidney mesenchymal cells to epithelialize, whereas
antibodies recognizing
6 (68) prevent polarization by tubular epithelia. Knockout of
3 (40) or
8 (51) decreases branching of
the collecting duct system and, in the case of
3 (40), results in the
disruption of the glomerular basement membrane.
1
appear to mediate normal kidney development, they may also have a role
in ischemic injury and repair. Using an in vitro model of kidney
epithelial cells exposed to oxidative stress, Gailit et al. (21)
demonstrated that the
3-integrin subunit
redistributed from basolateral to apical plasma membranes. On the basis
of this observation, it was hypothesized (25, 54) that
1-integrins likewise
redistributed after injury from basal to apical cell surfaces of
epithelial cells in vivo. According to this model, the decrease of
1 on basal surfaces contributes
to detachment of tubular epithelia (exfoliation) into the lumen,
whereas the appearance of
1 on apical plasma membranes of adherent epithelia and on surfaces of
exfoliated cells mediates cell-cell adhesion. This hypothesis provided
a potential mechanism to explain epithelial detachment (28, 44, 56,
60), tubular obstruction (3, 13, 45, 71), and backleak of
glomerular filtrate that have been documented to occur following renal
artery occlusion and reperfusion.
1-integrins in ischemic injury and repair. We found that as early as 1-3 h after reperfusion, the
1-integrin subunit is newly
found on lateral surfaces of tubular epithelia most prone to ischemic
injury. Damaged exfoliated cells obstructing tubular lumens do not
appear to express
1 even though
an apical membrane marker, leucine aminopeptidase, and a basolateral
membrane marker,
Na+-K+-ATPase,
are present on the plasma membranes of these cells. Surprisingly, tubular lumens are frequently filled with the ECM molecule fibronectin. During regeneration,
1-integrins persist on lateral
and basal surfaces of regenerating epithelia, with some cells
expressing them apically as well. The data indicate that cell-cell and
cell-ECM interactions mediated by
1-integrins dynamically change
during the injury phase, possibly contributing to epithelial detachment and tubular obstruction. During regeneration,
1-integrins may direct the
redifferentiation process.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Because one of the animal's kidneys is functionally intact, this model of unilateral ischemia results in only mild increases in plasma creatinine from 0.52 mg/dl before to 0.68 mg/dl after ischemia. Blood urea nitrogen also slightly increases from 17.5 mg/dl before to 20.5 mg/dl after ischemia (77). Glomerular filtration rate in the postischemic kidney, however, significantly decreases (4.2% of preischemic values) as measured by inulin clearance 1-3 h after reperfusion (43).
Preparation of Tissue
To fix kidneys, rats were first perfused via the left ventricle with Hanks' Balanced Salt Solution and then with 2% paraformaldehyde-75 mM L-lysine-10 mM sodium periodate (PLP; Ref. 46). After an initial fixation of 10 min, the kidneys were removed and stored in fixative overnight at 4°C. Kidneys were then rinsed in PBS without Ca2+ or Mg2+ [PBS(For cryosectioning, tissue pieces were equilibrated overnight at
4°C in 0.6 M sucrose-PBS(). Kidney pieces were then embedded in OCT medium (Miles Laboratories, Naperville, IL), frozen in liquid
nitrogen, and sectioned using a Reichert Frigocut cryostat (Leica,
Deerfield, IL). Five-micrometer sections were placed on SuperFrost Plus
glass slides (Fisher Scientific, Boston, MA) and were either used
immediately or stored at
20°C.
Immunohistochemistry
For immunohistochemistry, sections were fixed to the slides by incubation in PLP for 15 min, followed by three washes of 4 min each in PBS(For immunostaining with antibody to Na+-K+-ATPase, sections were incubated for 5 min in 1% SDS immediately after PLP fixation (8). Controls for immunohistochemistry consisted of staining some sections with rabbit preimmune serum in place of the polyclonal primary antibody. Alternatively, the primary and/or secondary antibodies were omitted. In all instances, sections used for controls were of the identical time point and condition (ischemic, contralateral, or sham-operated kidney) used for primary and secondary antibody staining. Immunostaining was repeated a minimum of three times for each time point and condition on three sets of animals.
Antibodies
Monoclonal and polyclonal antibodies used in the study that recognize the
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Initially, many different antibodies that recognize
1 were tried. Many of the
monoclonal and/or polyclonal antibodies either did not
cross-react with rat or gave questionable immunostaining results. The
rabbit polyclonal antibody generated against the integrin fibronectin
receptor,
5
1,
used in this study was purified from human placenta (Telios
Pharmaceuticals, San Diego, CA; GIBCO BRL, Grand Island, NY). This
antibody, which was used to detect the
1-integrin subunit in rat
kidneys, was simultaneously screened by immunostaining Madin-Darby
canine kidney (MDCK) cells, a distal tubule epithelial cell line, and
immunoblotting and/or immunoprecipitating
1-integrins from MDCK cell
lysates. The staining pattern and repertoire of
1-integrins expressed by MDCK
are well established (55, 64, 80). The three different lots of
polyclonal antibody recognizing
5
1
used in this study immunoprecipitated from MDCK cell lysates a
1-integrin profile identical to
that obtained with an antibody generated against a 37-amino acid
sequence in the cytoplasmic domain of
1 (64). Because the
5-subunit is not expressed by
tubular epithelia in adult kidney (11, 37-39, 67) and the
fibronectin receptor antibody recognizes an extracellular epitope of
the
1-integrin subunit,
antibody staining with these antibodies identifies only the
1-subunit in the kidney.
The rabbit anti-rat fibronectin polyclonal antibody (GIBCO BRL) was generated against rat plasma fibronectin. On Western blots it recognizes a band of ~220 kDa (data not shown), the molecular weight of fibronectin under reducing conditions. To verify the specificity of staining, competition experiments between the antibody and purified antigen were carried out. Purified rat fibronectin ranging in concentration up to 200 µg/ml was incubated for 1 h at 4°C with fibronectin antibody diluted 1:750, the concentration used to stain tissue sections. The antigen-antibody mixture was then added to the slides and incubated for 45-60 min at room temperature before being processed as described above. At a fibronectin concentration of 12.5 µg/ml, immunostaining was significantly diminished (data not shown); at 25 µg/ml, it was obliterated (see Fig. 10E).
Culture supernatants containing mouse monoclonal antibody against the ECM proteins, laminin (2E8) or type IV collagen (M3F7), were acquired from the Developmental Studies Hybridoma Bank; culture supernatants were used undiluted. Mouse monoclonal antibodies against the apical membrane marker, leucine aminopeptidase (1:800), and the basolateral membrane marker, Na+-K+-ATPase (1:100), were supplied by Drs. A. Quaroni (Cornell University, New York, NY) and D. Fambrough (Johns Hopkins University, Baltimore, MD), respectively.
Quantification of Cellular Localization of the
1-Integrin Subunit
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Using representative images, the total number of tubules was counted,
and the localization of 1 to
either basal or basal and lateral surfaces in the majority of cells was
scored. Values were expressed as a percentage of the total number of
tubules. Those tubules demonstrating intraluminal
1 staining were also counted,
and the value was expressed as a percentage of the number of tubules
with luminal debris and/or cells.
Tubules sectioned tangentially were omitted because of the difficulty of imaging the luminal space. In addition, only discrete tubules either with or without exfoliated debris and/or cells were scored. At 48 h postischemia, in particular, when damage to tubular architecture is extensive, it was sometimes difficult to identify distinct tubules either because of the damage or because of the frequent presence of what appeared to be infiltrating inflammatory cells and/or fibroblasts. These tubules were not included.
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RESULTS |
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Distribution of 1-Integrin
In normal adult rat kidneys (see Fig.
1B) or contralateral controls (see
Figs. 2E,
3D,
4C,
5C, and
5E) the
1-integrin subunit localizes
exclusively to basal cell surfaces of proximal and distal tubules in
the cortex, outer stripe, and inner stripe, in agreement with
observations in normal adult human kidneys (11, 37-39). After 40 min of renal ischemia induced by renal artery clamp in the
absence of reperfusion (0 h postischemia), distribution of the
1-subunit does not change.
Basal plasma membranes of tubular epithelia, including those in the
outer stripe of the outer medulla (Fig.
1A
and Table 2), are strongly outlined, as are epithelia of
the glomerulus and capillary tuft (data not shown).
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Distribution 1-3 h after reperfusion.
Ischemic injury is most apparent in the S3 segment of proximal tubules
in the outer stripe of the outer medulla (13, 23, 61), most likely due
to hemodynamic factors leading to impaired reperfusion (4a). In this
region, localization of the
1-subunit changes 1 h after
reperfusion (Fig.
2A
and Table 2) compared with corresponding regions of contralateral
control (Fig. 2E and Table 2) or
normal adult kidneys (Fig. 1B). In
S3 segments of control (arrowheads, Fig.
2E) or normal (Fig.
1B) kidneys,
anti-
1 antibody exclusively
stains basal plasma membranes; 97.1% of tubular profiles in the outer
stripe of contralateral control kidneys show this distribution (Table
2). In contrast, by 1 h after reperfusion, the
1-integrin subunit is detected
on both basal and lateral surfaces (arrows, Fig.
2A) of some epithelia in S3
segments. Quantitation of
1
localization indicates that 63.6% of tubules localize
1 to both basal and lateral
surfaces (Table 2). Epithelial cells are cuboidal in shape (Fig.
2B), apical microvilli are absent (Fig. 2B) and regions of
tubulorrhexis representing severed basement membrane (brackets, Fig.
2B) are observed. Within a given
tubule, basal and lateral plasma membrane staining of
1 on some epithelia coexists
with only basal membrane signal on other cells (Fig. 2A). In other tubules in the outer
stripe,
1 is expressed only basally (~36.4% of tubular profiles; Table 2).
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Distribution 24-48 h after reperfusion.
Although regeneration of the tubular epithelium commences immediately
after injury (73), at 24 h postischemia injury predominates, based on disruption of normal tubular architecture. By 48 h after reperfusion, the injury phase appears to be complete and regeneration of the damaged kidney is the primary response. During this time in
damaged S3 tubules, 1 appears
to localize primarily to basal plasma membranes of adherent epithelia
(Fig.
3A).
Quantitation of
1 localization
indicates that 85.8% of tubular profiles in the outer stripe express
this integrin subunit only on basal cell surfaces (Table 2). The
epithelial lining is sometimes difficult to identify because the
adhering cells are flat (arrow, Fig.
3A; Ref. 32), filling in gaps of
denuded basement membrane. The adherent epithelium thus is not detected
in every section. In the remaining tubules (14.2%), however, basal and
lateral staining of
1 is
observed (Table 2).
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Distribution 120 h after reperfusion.
By 120 h after reperfusion, regeneration of the damaged kidney
continues and in some areas appears to be complete. Flattened cells
indicative of regeneration are observed, as are cuboidal cells
characteristic of normal kidney epithelia. Among these cells, localization of the 1-subunit
varies. In some epithelia of regenerating S3 segments, strong
localization of
1 is observed
on basal and lateral surfaces (short solid arrows, Fig.
5,
A and
B). Quantitation of its cellular
localization indicates that 53.5% of tubules in the outer stripe of
the outer medulla express
1
both basally and laterally (Table 2). In either the same tubule or
other tubules in the same region,
1 is also found on apical
plasma membranes (long solid arrows, Fig. 5,
A and
B). 22.2% of tubular profiles depolarize
1 to all cell
surfaces; basal, lateral, and apical localization is observed in ~5.8
cells/tubule (containing on average 23.9 cells; Table 2).
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Distribution of Apical and Basolateral Membrane Markers
The observation that localization ofAs expected, in normal adult or ischemic nonreperfused (Fig. 6A) rat kidneys, Na+-K+-ATPase localizes to basolateral surfaces of proximal and distal tubular epithelia, with distal tubules being more strongly reactive (33). In contrast, leucine aminopeptidase is found only on apical brush borders of proximal tubule cells (Fig. 6B; Ref. 12).
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One hour after reperfusion,
Na+-K+-ATPase
(Fig. 6C) persists on basolateral
surfaces; however, in some S3 segments it localizes to apical plasma
membranes (Fig. 6C), in agreement
with Molitoris and colleagues (48-50). The apical marker
aminopeptidase is observed in a punctate pattern (Fig.
6D), possibly in the apical
cytoplasm. Interestingly, cellular debris negative for
1-integrin (asterisks, Fig.
2A) and
Na+-K+-ATPase
(data not shown) richly express aminopeptidase (Fig.
6D), consistent with previous
studies demonstrating that cellular debris in tubular lumens at this
time are composed of shed microvilli and/or apical membrane
blebs (13, 73). As damage to the kidney increases after 3 h of
reperfusion,
Na+-K+-ATPase
frequently localizes to all cell surfaces (Fig.
6E). In contrast, aminopeptidase has
a strong punctate distribution in apical cytoplasms (Fig.
6F), reminiscent of endocytic
vesicles; it also continues to be observed on cellular debris in the
lumen (Fig. 6F).
In striking contrast to the lack of
1 immunostaining in exfoliated
cells at 24-48 h after reperfusion (Fig.
3A), S3 segment epithelial cells
dislodged into tubular lumens randomly express both
Na+-K+-ATPase
(Fig.
7A) and
aminopeptidase (Fig. 7B) on the
plasma membrane. This observation indicates that the amorphous material
congesting the lumen at this time consists of exfoliated cells and not
only shed microvilli or other cellular debris seen earlier (1-3 h
after reperfusion; Figs. 6, D and
F). Nevertheless, as mentioned
previously, these cells do not express
1-integrin (Fig.
3A). Although epithelial cells
adhering to basement membrane are not visible in every section at
24-48 h postischemia due to their flattened cell shape
(see Fig. 3B),
Na+-K+-ATPase
and aminopeptidase are faintly detected on all cell surfaces of
flattened epithelia adhering to basement membrane (arrows, Fig. 7,
A and
B). Dislodged cells, immunoreactive
for
Na+-K+-ATPase
(Fig. 7, C and
D) and aminopeptidase (Fig. 7,
E and
F), are also detected in tubular
lumens of distal nephron segments in the inner stripe where
Na+-K+-ATPase
localizes to basolateral surfaces (Fig.
7C). Because of the aminopeptidase
staining (Fig. 7E), these cells most
likely are proximal in origin, having been propelled through the
nephron to the distal segments.
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At 120 h after reperfusion, localization of Na+-K+-ATPase and aminopeptidase varies among regenerating tubules. In some instances, Na+-K+-ATPase localizes to lateral and/or basal surfaces, with some cells expressing it apically (Fig. 8A). Aminopeptidase is detected primarily on apical surfaces (Fig. 8, C and D) but in some instances localizes to apical, lateral, and basal plasma membranes (Fig. 8C). Interestingly, Na+-K+-ATPase and aminopeptidase are weakly expressed by some tubules (Fig. 8, B-D). Intensity of staining also varies within a given segment (Fig. 8, A, C, and D).
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The data indicate that after ischemic-reperfusion injury, the adherent
epithelium continues to express apical and basolateral membrane
proteins; however, distribution is not polarized. The loss of
epithelial polarity is observed by 1 h after reperfusion and persists
to some extent 120 h later, when a spectrum of localization patterns
and expression levels are observed, ranging from polarized to
nonpolarized and faint to strong staining. In contrast to the absence
of 1 staining, amorphous
material in tubular lumens at 24-48 h after reperfusion expresses
apical and basolateral membrane markers, indicating that it consists of
exfoliated cells. These proteins are uniformly found on all cell
surfaces.
ECM Expression
Because the distribution and expression ofKidney sections from animals killed various times after reperfusion or animals made ischemic but not reperfused (0 h postischemia) were probed with monoclonal antibodies recognizing the major constituents of basement membrane: laminin and type IV collagen. In all cases, laminin and type IV collagen localized to tubular basement membranes (data not shown); however, as early as 1 h after reperfusion, immunostaining highlighted breaks in basement membranes (data not shown; also see Fig. 2B) not observed in corresponding contralateral controls (data not shown). Tubules with disrupted basement membranes were confined to the outer stripe. Tubulorrhexis appeared to be most prevalent at 24 h after reperfusion (Fig. 9A), declining thereafter (data not shown). Interestingly, in a few tubules, laminin localized to the luminal compartment (Fig. 9B). In contralateral controls (data not shown), tubulorrhexis was not observed and laminin was not found in the luminal space. Profiles of severed basement membranes were completely absent at 120 h postischemia (data not shown), indicating that by this time the basement membrane had regenerated.
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The existence of tubulorrhexis raised the possibility that interstitial fluid could enter the tubular lumen via plasma from congested capillaries. In addition, tubulorrhexis might promote interactions between epithelia at the wound edge and underlying interstitial matrix molecules.
To test this, the distribution of fibronectin, an important constituent of both plasma and the renal interstitium, was examined. In normal (see Fig. 10G) and contralateral control kidneys (data not shown) or kidneys that were made ischemic but not reperfused (0 h postischemia; data not shown), fibronectin outlines tubules, presumably localizing to the renal interstitium (1). This localization persists after ischemiareperfusion. However, in response to reperfusion injury, fibronectin also is detected in lumens of S3 segments and the distal nephron. In S3 segments, staining with anti-fibronectin antibody initially highlights regions of tubulorrhexis between 1 and 3 h after reperfusion (data not shown). By 24 h postischemia, hazy fibronectin staining of S3 segments and/or more proximal regions of the distal nephron is observed (single asterisks, Fig. 10A). Nomarski imaging shows that the hazy pattern of staining coincides with exfoliated cells and cellular material occluding the lumen (single asterisks, Fig. 10B). In a few instances, fibrils immunoreactive for fibronectin are detected between exfoliated cells (data not shown). By 120 h, fibronectin staining of luminal contents is no longer observed (Fig. 11A).
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The majority of staining for fibronectin is confined to the lumen of the distal nephron after ischemic injury and reperfusion. At 3 h postischemia, fibronectin is detected in tubular lumens of distal nephrons running throughout the cortex (data not shown), outer stripe (data not shown), and inner stripe (data not shown). At 24 h postischemia, luminal fibronectin dramatically increases in distal nephrons of the inner stripe (Fig. 10C) but is also detected at significant levels in these segments in the cortex (data not shown) and outer stripe (double asterisk, Fig. 10A). The number of distal tubules with luminal fibronectin begins to decrease 48 h postischemia, but profiles are still found throughout the kidney (data not shown). By 120 h postischemia, distal luminal fibronectin is no longer observed (Fig. 11B). Localization of fibronectin in the lumen of the distal nephron is not detected in normal kidneys (data not shown), kidneys that were not reperfused (data not shown), or corresponding contralateral controls (Fig. 10F). In addition, fibronectin staining throughout the kidney is not detected when immunoreactive sites on the antibody are competed out with purified antigen (Fig. 10E) or sections are reacted with preimmune sera (data not shown; see MATERIALS AND METHODS).
Thus, after ischemic insult, the distribution of laminin and collagen IV to the basement membrane is largely unaltered, although the basement membrane itself may be severed. In contrast, fibronectin is detected in S3 segments of the proximal tubule and in substantial amounts in distal nephron segments.
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DISCUSSION |
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Injury of rat kidneys by ischemia followed by reperfusion
initiates changes in cell-cell and cell-ECM interactions and
alterations in epithelial cell polarity (for review, see Refs. 19, 48). As we have shown here, one of the molecules most significantly affected
is the 1-integrin subunit,
which is an element of receptors for collagens, laminins, and
fibronectin in the kidney. Between 1 and 3 h after reperfusion,
localization of
1-integrin in
adherent epithelia of proximal tubule S3 segments changes from
exclusively basal to basal and lateral. Polarity begins to be lost, as
evidenced by detection of the basolateral membrane marker
Na+-K+-ATPase
on the apical surface and internalization of the apical membrane marker
leucine aminopeptidase. Cellular material, representing apical but not
basolateral membrane debris, occupies tubular lumens. By
24-48 h after reperfusion, damage to the kidney
increases. The basal lamina of the S3 segments is denuded by
exfoliation of cells and is sometimes broken. Despite the fact that the
Na+-K+-ATPase
and aminopeptidase are weakly but uniformly distributed on both apical
and basolateral cell surfaces, adherent epithelia of S3 segments
continue to express
1 primarily
on basal and infrequently on lateral and apical surfaces. At the same
time, depolarized exfoliated cells expressing both leucine
aminopeptidase and the Na+-K+-ATPase
fill the luminal space. These cells, however, do not express the
1-subunit. Strikingly, large
amounts of fibronectin are also observed in lumens of the distal
nephron. At 120 h after reperfusion, repair from ischemic injury is
evident. The
1-subunit is again basally located in some regenerating epithelia; in others it is both
basal and lateral or is also detected on the apical surface. Apical and
basolateral membrane markers are polarized in some cells; in others
they are weakly but uniformly expressed on all cell surfaces. Basement
membranes of S3 segments are intact, and luminal fibronectin in distal
nephron segments is no longer observed.
1-Integrin in Renal
Ischemic Injury and Repair
On the basis of the redistribution of
3, the authors proposed a role
for defective cell-cell and cell-ECM interactions in the
pathophysiology of acute renal failure (26). Loss of integrin receptor
on basal cell surfaces was hypothesized to cause exfoliation of
epithelial cells into tubular lumens. Redistribution of integrins from
basal to apical plasma membranes on remaining epithelia was also
suggested to facilitate attachment of dislodged cells and fragments of
basement membrane released during injury. Under these circumstances,
1-integrins expressed by
exfoliated cells were thought to promote cell-cell adhesion either
directly or via bridging ECM molecules, culminating in tubular
obstruction and, ultimately, renal failure.
In subsequent experiments using an in vivo rat model, Goligorsky and
colleagues (62) observed 1 on
the apical surface of adherent cells and on the plasma membrane of
exfoliated cells occupying the lumen. In contrast, our results reported
here do not support a functional role for apical expression of
1-integrins in renal ischemic
injury. In our experiments,
1-integrins were not seen on
the apical surfaces of adherent epithelia at times soon after
ischemia and only rarely on apical surfaces at later times when
regeneration of the epithelium was underway (24-48 h
postischemia). In addition, the lack of
1 on exfoliated cells suggests
that integrins neither mediate aggregation of exfoliated cells nor
anchor them to the adherent epithelium.
Our findings are also inconsistent with a significant role for the basement membrane ECM molecules collagen and laminin in bridging cell interactions that could contribute to tubular occlusion. Although tubulorrhexis was observed, fragments or soluble components of the basement membrane, including laminin, were rarely seen in the tubular lumen. The absence of laminin in the luminal space further indicates that, within the sensitivity of our analysis, it is not secreted apically by the regenerating epithelium or randomly by the depolarized exfoliated cells. In contrast, fibronectin was abundantly expressed in luminal compartments. Fibronectin assembles into fibrils when bound by integrins on plasma membranes (76, 78, 79). Because fibronectin fibrils were not observed to outline exfoliated cells, we believe that it is unlikely to serve as a bridging molecule between adherent and exfoliated cells and to contribute in this way to tubular occlusion.
Model of 1 in Renal
Ischemic Injury and Repair
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This model is consistent with our observations in the postischemic
kidney. In both the S3 segment and the distal tubule in the cortex, we
observed integrins on the lateral cell surfaces of attached cells early
in the injury phase; in distal tubules in the inner stripe, we observed
a decrease in 1 levels on basal surfaces, consistent with its proposed degradation. In the distal tubule, where no exfoliation occurs, integrin localization returned to
basal plasma membranes, suggesting that reactivation, whether by
signaling mechanisms or resynthesis, resulted in reengagement of
integrins with ligands of the basement membrane. In the S3 segment,
considerable exfoliation was seen. No
1-integrins were detected in
the exfoliated cells, however, consistent with the proposal that
degradation occurred. Because the antibodies used in our studies
reacted with the extracellular region of the
1-molecule, it is unlikely that
the loss of immunoreactivity was due solely to degradation of the
cytoplasmic part of
1. Finally,
our findings of a lack of polarity of both apical and basolateral
markers as well as, in some cases,
1 at later times after
reperfusion are more indicative of a morphogenetic response to
regeneration than an immediate reaction to injury.
Integrins other than those of the
1 family may contribute to
intraluminal obstruction and oliguria. Kidney epithelial cells may
express integrins containing the
3,
5, or
6 families in addition to
1, either constitutively (37,
39, 58) or in response to injury (5, 74). Any or all of these could
redistribute early on to the apical surfaces of attached cells and
remain on the plasma membranes of exfoliated cells. Because fibronectin can bind to each of these integrin families, it could act as a bridging
molecule. Although we do not favor this possibility in S3 segments
because assembly of fibronectin into fibrils (via interactions with
integrins) was not observed, our limited observations and analysis at
this time do not permit us to exclude the possibility that fibronectin,
present in apparently high concentrations in lumens of distal tubules,
could be interacting with newly expressed integrins on apical surfaces
of adherent epithelia. Also, because the interactions between these
integrins and fibronectin are mediated by Arg-Gly-Asp (RGD) sequences
in the fibronectin molecule, this scenario might help to explain the
reported effectiveness of RGD peptides in ameliorating renal failure in
vivo (25, 27, 53, 62). Clearly additional studies are required to
address these points.
Integrin Activation and Deactivation
Integrin activation and deactivation are well-known but poorly understood processes. In platelets, the integrinActivation of integrins is regulated by the cytoplasmic tails of both
- and
-subunits. Deletion of specific sequential motifs inactivates integrins, making them unable to bind ligand (17, 22, 35,
57). In contrast, elimination of the entire tail of the
-subunit can
render integrins constitutively active (17, 22, 35, 57). On the basis
of these types of experiments, a hingelike model for regulation of
integrin affinity has been proposed that postulates that the
interaction between integrin
- and
-subunit cytoplasmic tails
renders integrins unable to bind ligand until this interaction is
relaxed by association with an "integrin-activator complex" (IAC)
(22, 57).
Although integrin activation may be mediated by signal transduction
pathways (so-called "inside-out signaling"), integrin deactivation may occur either reversibly through signal transduction and dissociation of postulated IACs or irreversibly by integrin degradation. In the kidney, one candidate signal transduction pathway
is the one that leads to stress-activated protein kinase (SAPK; also
called Jun kinase-1). SAPK is a mitogen-activated protein kinase that
is closely related to the extracellular signal-regulated kinases (ERK)
ERK1 and ERK2 but is activated by distinct mechanisms. After unilateral
renal ischemia in the rat, SAPK activity increases within 5 min
after reperfusion and remains elevated 90 min later (59). This
increased activity of SAPK coincides with the change in
1-integrin localization
observed within 1 h of reperfusion in our study.
Irreversible integrin deactivation may occur through the action of the
protease calpain. Calpain is known to be a mediator of ischemic injury
in renal proximal tubules (16), brain, liver, and heart (6, 42, 65).
After renal ischemia and reperfusion, increasing amounts of
cytoplasmic Ca2+ may activate
calpain, permitting it to cleave cytoplasmic tail residues from
integrin - and
-subunits (14). Indeed, in activated platelets,
calpain has been shown to cleave the cytoplasmic tail of the
3-subunit, possibly affecting
integrin signaling and cytoskeletal association (14). Alternatively,
calpain may digest cytoskeletal proteins or integrin-associated
proteins directly, untethering integrins from their cytoplasmic
linkages. Under these conditions, integrins might release ligand and be
degraded by endocytosis into lysosomes.
It is likely that the integrins that we observe on the lateral borders
of tubular epithelial cells soon after reperfusion represent
deactivated integrins rather than integrins engaged in cell-cell
interactions with neighboring cells. The lateral appearance of
1-integrins has often been
considered evidence of their role in cell-cell adhesion (41). In
keratinocytes,
1-,
2-, and
3-integrin subunits were
initially localized to points of cell-cell contact (34), and an
antibody against the
3-subunit
inhibited Ca2+-induced aggregation
(9). Studies using immobilized integrins, affinity chromatography, and
blocking monoclonal antibodies have also supported the idea that
cell-cell adhesion is possibly due to heterophilic
(
2-
3;
Ref. 70) or homophilic
(
2-
2;
Ref. 69) integrin-integrin interactions. However, recent work examining adhesion in a strictly cellular environment indicates that
1-integrins do not mediate
cell-cell adhesion (75). Even though various transfected integrin
subunits belonging to the
1
family were expressed on cell surfaces and bound matrix ligand, no
direct evidence for homophilic or heterophilic integrin-integrin
interactions was obtained from cell-cell adhesion assays.
Regeneration of the Kidney Epithelium
As regeneration proceeds,
1-integrins appear to
depolarize along with other apical and basolateral membrane markers. At
120 h after reperfusion, when the injured kidney is still regenerating,
1 distributes to basal
and/or lateral and in some instances apical cell surfaces. The
epithelium appears to have dedifferentiated, recapitulating certain
aspects of normal renal development (77). In embryonic and fetal
kidneys, tubular cells also randomly distribute
1-integrins, which, as
development proceeds, become confined to basal plasma membranes
(37-39). Indeed, the spectrum of staining patterns and expression
levels for aminopeptidase and
Na+-K+-ATPase,
ranging from depolarized to polarized and faint to strong, further
suggests that the regenerating kidney resembles a less differentiated
state. On the basis of our earlier in vitro results (80), it is clear
that "ectopic" integrin expression can have significant effects
on epithelial morphogenesis. Additional studies in vivo are now
required to determine whether the depolarization of integrins observed
during regeneration of the tubular epithelium is merely a sign of
dedifferentiation or whether it plays an important role in the process
of epithelial restitution.
Fibronectin and Tubular Obstruction
A striking finding of this study is that fibronectin was abundantly expressed in tubular lumens. Fibronectin was observed in distal tubules as early as 3 h postischemia, reached a maximum at 24 h, and declined thereafter. It is unclear from our study whether the source of fibronectin in luminal compartments is the plasma, the tubular epithelial cells, or a combination of both. Permeability changes in endothelial capillaries after ischemic hypoxia in combination with tubular damage may promote leakage of plasma constituents, including fibronectin, into luminal spaces, congregating in more distal nephron segments. Indeed, the hazy fibronectin staining of damaged tubules in the outer medulla suggests that fibronectin may be percolating through the interstitium from damaged blood vessels into injured tubules. A potential source of luminal fibronectin may also be the damaged tubular epithelium. Synthesis of fibronectin by injured epithelia has been demonstrated in restitution of wounded skin, cornea, and intestinal epithelium (18, 20, 24).As described previously, we do not believe it likely that fibronectin
facilitates tubular obstruction by acting as a bridging molecule
between adherent and exfoliated epithelial cells. Indeed, the
observation that fibronectin is not assembled into a fibrillar matrix
and thus is not retained by epithelia in S3 segments may explain why
the majority of it is seen in the lumens of the distal nephron. Here,
high concentrations of fibronectin may directly block tubules. As its
name implies, fibronectin is a large, fibrous protein capable of
assembling into multimolecular complexes. Normally, the structures
formed are fibrils and require association with cells for their
assembly (76, 78, 79). However, under the abnormal conditions of renal
injury, the formation of aberrant structures cannot be ruled out. This
may be facilitated by interaction with other proteins in the tubular
lumen. The Heyman antigen gp330, for example, is found at the base of
the brush border of proximal tubular epithelial cells, where it can act
as a receptor mediating attachment to fibronectin and other matrix
molecules (47). After injury, when the luminal domains of proximal
tubule cells are shed, gp330 may interact with fibronectin to create
insoluble and obstructive complexes. Alternatively, as mentioned
earlier, fibronectin in the lumen of the distal nephron may interact
with other families newly expressed on apical surfaces of adherent epithelia. A scenario such as this may explain the therapeutic potential of RGD peptides in ameliorating renal failure in vivo (25,
27, 53, 62).
In summary, we have demonstrated that in the injury phase of renal
ischemia and reperfusion, the
1-integrin subunit reorganizes from exclusively basal to basal and lateral surfaces as polarization begins to be lost. These changes in location may reflect integrin deactivation and disengagement from ligands in the basement membrane. This, in turn, may facilitate exfoliation of tubular epithelial cells
into the lumen. Although it remains possible that some integrins mediate attachment of the exfoliated cells to the luminal walls, our
results indicate that
1-integrins are not involved.
Notably, the accumulation of fibronectin in distal tubules at this time might itself be a contributing factor in tubular obstruction and renal
failure. Later, during recovery from ischemic injury, viable cells
dedifferentiate and depolarize integrins to all cell surfaces, recapitulating some events seen in normal renal development.
Reestablishment of integrin polarity may contribute to restoration of a
normal tubular epithelium.
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ACKNOWLEDGEMENTS |
---|
We thank Suzanne Miller for excellent technical assistance, Robert Tyszkowski for perfusion fixation of animals, and Drs. James Casanova and Gregory Zinkl for helpful discussions.
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FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants RO1-DK-46768 (to K.S. Matlin), PO1-DK-38452 (to K. S. Matlin, J. V. Bonventre), RO1-DK-42956 (to D. Brown), and R37-DK-39773 (to J. V. Bonventre). Mouse monoclonal antibodies 2E8 (anti-laminin) and M3F7 (anti-type IV collagen), developed by Drs. E. Engvall and H. Furthmayr, respectively, were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Dept. of Biological Sciences, Iowa City, IA 52242) under National Institute of Child Health and Human Development Contract NO1-HD-7-3263.
Present address of K. S. Matlin: Dept. of Surgery, Beth Israel Deaconess Medical Center East Campus, 330 Brookline Ave., Boston, MA 02215.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and present address of A. Zuk: Dept. of Surgery, Beth Israel Deaconess Medical Center East Campus, 330 Brookline Ave., RW, 873, Boston, MA 02215.
Received 23 February 1998; accepted in final form 20 May 1998.
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