Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney

Anna Zuk1,2, Joseph V. Bonventre1,2,3, Dennis Brown1,4, and Karl S. Matlin1,2

1 Renal Unit, Massachusetts General Hospital, Charlestown 02129; Departments of 2 Medicine and 4 Pathology, Harvard Medical School, Boston 02115; and 3 Division of Health Sciences and Technology, Harvard Medical School- Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta 1-integrins. On the basis of in vitro and in vivo studies, reorganization of beta 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, beta 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 beta 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, beta 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 beta 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.

beta 1-integrin; epithelial polarity; fibronectin; acute renal failure

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha - and beta -subunits whose association into noncovalent heterodimers is necessary for ligand binding and cell surface expression. The beta -chains can associate with one or more alpha -subunits, forming receptors for laminins, collagens, fibronectin, and vitronectin.

In the kidney, integrins possessing the beta 1-subunit are the most common. During development of the metanephros, beta 1-integrins localize to all cell surfaces (37-39). As the developing nephron elongates and matures, beta 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 beta 1-integrins are localized to all cell surfaces.

Genetic and biochemical studies implicate integrins containing the beta 1-subunit in kidney epithelial differentiation and polarization. The alpha 8-, alpha 6-, and alpha 3-integrin subunits complex with beta 1. Knockout of the alpha 8-subunit (51) impairs the ability of kidney mesenchymal cells to epithelialize, whereas antibodies recognizing alpha 6 (68) prevent polarization by tubular epithelia. Knockout of alpha 3 (40) or alpha 8 (51) decreases branching of the collecting duct system and, in the case of alpha 3 (40), results in the disruption of the glomerular basement membrane.

Just as integrins containing beta 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 alpha 3-integrin subunit redistributed from basolateral to apical plasma membranes. On the basis of this observation, it was hypothesized (25, 54) that beta 1-integrins likewise redistributed after injury from basal to apical cell surfaces of epithelial cells in vivo. According to this model, the decrease of beta 1 on basal surfaces contributes to detachment of tubular epithelia (exfoliation) into the lumen, whereas the appearance of beta 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.

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 beta 1-integrins in ischemic injury and repair. We found that as early as 1-3 h after reperfusion, the beta 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 beta 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, beta 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 beta 1-integrins dynamically change during the injury phase, possibly contributing to epithelial detachment and tubular obstruction. During regeneration, beta 1-integrins may direct the redifferentiation process.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Male Sprague-Dawley rats (190-275 g) were purchased from Charles River Breeding Laboratories (Wilmington, MA) and housed in the Animal Facility at Massachusetts General Hospital under alternating 12 h cycles of light and dark. Water and food were given ad libitum. In preparation for surgery involving unilateral ischemia without removal of the contralateral kidney, 8-10 ml of sterile saline at 37°C were injected intraperitoneally. Animals were then anesthetized with pentobarbital sodium (6.5 mg/100 g body wt ip). An incision was made over the left flank, and the renal artery and vein were dissected and clamped with a microaneurysm clamp (Roboz Surgical Instrument, Washington, DC). The incision was temporarily closed until 40 min later, when the clamp was removed and the kidney was reperfused. The incision was then permanently sutured and, after the stated times of reperfusion, both kidneys (left postischemic and right contralateral) were fixed in situ for immunocytochemistry. Other animals underwent sham surgery without ischemia. Both sham-operated and right contralateral kidneys were used as controls.

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(-)] and stored in PBS(-) containing 0.02% sodium azide until they were prepared for cryosectioning.

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(-). To quench autofluorescence of the tissue, sections were incubated twice for 8 min each in a freshly prepared solution of 1 mg/ml sodium borohydride in PBS(-). After a rinse in PBS(-), tissue sections were treated for 4 min in 0.1% Triton X-100-PBS(-) and rinsed. Nonspecific background was quenched for 45-60 min in 10% normal goat serum (NGS) in PBS(-), followed by incubation overnight at 4°C in primary antibody (see Table 1) diluted in 10% NGS-PBS(-). Slides were flooded three times for 4 min each with PBS(-), and then secondary antibody in PBS(-) was added for 45 min at room temperature. Secondary antibodies were either goat anti-rabbit FITC adsorbed against mouse and human Ig (1:200; BioSource International, Camarillo, CA), goat anti-mouse FITC adsorbed against human Ig (1:200; BioSource International), or goat anti-mouse indocarbocyanine (CY3) adsorbed against rat, human, bovine, and horse serum proteins (1:800; Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were washed, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and stored in the dark at 4°C until use. Images were viewed on a Zeiss Axioplan microscope (Carl Zeiss, Thornwood, NY) and recorded on Kodak TMAX 400 film pushed to 800 (Eastman Kodak, Rochester, NY).

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 beta 1-integrin subunit, ECM proteins, and apical and basolateral membrane markers are listed in Table 1.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Antibodies used in the study

Initially, many different antibodies that recognize beta 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, alpha 5beta 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 beta 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 beta 1-integrins from MDCK cell lysates. The staining pattern and repertoire of beta 1-integrins expressed by MDCK are well established (55, 64, 80). The three different lots of polyclonal antibody recognizing alpha 5beta 1 used in this study immunoprecipitated from MDCK cell lysates a beta 1-integrin profile identical to that obtained with an antibody generated against a 37-amino acid sequence in the cytoplasmic domain of beta 1 (64). Because the alpha 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 beta 1-integrin subunit, antibody staining with these antibodies identifies only the beta 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 beta 1-Integrin Subunit

Quantification of the cellular localization of beta 1 was determined in tubules in the outer stripe of the outer medulla (Table 2). The time 0 point represented ischemia without reperfusion, the 1-h time point represented the beginning stages of injury and regeneration, and the 48-h time point represented the later stages. At 120 h postischemia, regeneration was apparent, albeit incomplete. Contralateral kidneys served as controls for quantification of beta 1 localization.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Cellular localization of beta 1-integrin subunit in outer stripe of outer medulla

Using representative images, the total number of tubules was counted, and the localization of beta 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 beta 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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Distribution of beta 1-Integrin

On the basis of previous in vitro and in vivo studies, beta 1-integrins have been hypothesized to play a role in tubular obstruction following injury induced by renal ischemia and reperfusion. To investigate this possibility in more detail, we examined the distribution of beta 1-integrin and ECM proteins in a rat model of unilateral renal ischemia.

In normal adult rat kidneys (see Fig. 1B) or contralateral controls (see Figs. 2E, 3D, 4C, 5C, and 5E) the beta 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 beta 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).


View larger version (107K):
[in this window]
[in a new window]
 
Fig. 1.   Immunofluorescent staining of cryostat sections of postischemic, nonreperfused (A) and normal (B) adult rat kidneys reacted with polyclonal antibody to the beta 1-integrin subunit. In the outer stripe of the outer medulla, beta 1 localizes only to basal plasma membranes (arrows, A and B). Dashed lines in A outline tubular lumen. * Tubular profiles sectioned tangentially. Bar, 40 µm.

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 beta 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-beta 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 beta 1-integrin subunit is detected on both basal and lateral surfaces (arrows, Fig. 2A) of some epithelia in S3 segments. Quantitation of beta 1 localization indicates that 63.6% of tubules localize beta 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 beta 1 on some epithelia coexists with only basal membrane signal on other cells (Fig. 2A). In other tubules in the outer stripe, beta 1 is expressed only basally (~36.4% of tubular profiles; Table 2).


View larger version (160K):
[in this window]
[in a new window]
 
Fig. 2.   Immunofluorescent staining of cryostat sections of postischemic (A, C, D) and contralateral nonischemic (E) kidneys taken 1 h after reperfusion and reacted with polyclonal antibody to the beta 1-integrin subunit. Corresponding Nomarski image of A is shown in B. In the outer stripe of the outer medulla (A, B), beta 1-integrin localizes to both basal and lateral surfaces of S3 proximal tubule segments (arrows). In other epithelia of the same tubule, beta 1 is also detected basally. Epithelia are cuboidal in shape and apical microvilli are absent (B). Cellular debris fill some tubular lumens (asterisks, B); beta 1 is not expressed by cellular debris (asterisks, A); it is also absent in regions where contact of luminal material with one another or with the adherent epithelium appears to be made (A, B). Bracketed area (A, B) highlights broken basement membrane. In the cortex (C) and inner stripe of the outer medulla (D), beta 1 localizes to basal plasma membranes, although infrequent lateral staining of proximal tubules in the cortex (arrow, C) is observed. In the outer stripe of control contralateral nonischemic kidney (E), beta 1 is found only on basal plasma membranes (arrowheads); microvillar brush border (E) stains nonspecifically. GL, glomerulus; TAL, thick ascending limb; CD, collecting duct. Bar, 40 µm.

Cellular debris of varying size, possibly representing apical membrane blebs or shed microvilli (13, 73), congest the lumen (asterisks, Fig. 2B). Approximately 55.5% of tubules in the outer stripe contain cellular debris at this time. Cellular debris, however, are negative for beta 1 (asterisks, Fig. 2A and Table 2); beta 1 is also absent from areas where contact of luminal material appears to be made with apical membranes of adherent cells (Fig. 2, A and B).

In the cortex and inner stripe, localization of the beta 1-subunit to basal cell surfaces does not change (Fig. 2, C and D, respectively). In a few proximal tubules in the cortex, however, faint distribution to lateral borders is sometimes observed (arrow, Fig. 2C).

At 3 h after reperfusion, immunostaining for beta 1 in the outer stripe, inner stripe, and cortex is comparable to that at 1 h after reperfusion (data not shown).

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, beta 1 appears to localize primarily to basal plasma membranes of adherent epithelia (Fig. 3A). Quantitation of beta 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 beta 1 is observed (Table 2).


View larger version (190K):
[in this window]
[in a new window]
 
Fig. 3.   Immunofluorescent staining of cryostat sections of postischemic (A, C) and contralateral nonischemic control (D) kidneys taken 48 h after reperfusion and reacted with polyclonal antibody to beta 1-integrin subunit. Corresponding Nomarski image of A is shown in B. Amorphous material (asterisks, B) that fills tubular lumens of S3 segments in outer stripe of outer medulla is negative for beta 1 (asterisks, A), whereas the adherent epithelium localizes beta 1 primarily to basal plasma membranes (A); beta 1 is also not detected between the adherent epithelium and luminal material (arrows, A, B). At bottom right of B, a disrupted tubule appears to have been infiltrated by inflammatory cells and/or fibroblasts immunoreactive for beta 1 (A). In outer stripe of contralateral control kidney (D), beta 1 is found only on basal cell surfaces. In the cortex (C), distal tubules (DT) localize beta 1 to basal and lateral cell surfaces, whereas proximal tubules (PT) express it primarily on basal plasma membranes. Bar, 40 µm.

Apical staining of beta 1 in combination with basal and/or lateral localization is rare. Of the total number of tubular profiles, only 5.3% contain on the average two cells (of a mean of 9.4 cells/tubule) in which beta 1 is distributed to both basal and apical plasma membranes (Table 2); 0.9% of tubular profiles randomize beta 1 to all cell surfaces, and, in these, approximately two cells (of 9.4 cells/tubule) localize beta 1 to apical, basal, and lateral plasma membranes (Table 2). In the outer stripe of contralateral control kidneys (Fig. 3D and Table 2), beta 1 is detected primarily on basal surfaces (91.2% of tubules).

Amorphous material (asterisks, Fig. 3B) possibly representing cellular debris and/or exfoliated cells fills tubular lumens of damaged S3 segments. Staining of kidneys at these time points with antibodies to leucine aminopeptidase (Fig. 7B) and Na+-K+-ATPase (Fig. 7A) confirms that the amorphous material consists of exfoliated cells (see Distribution of Apical and Basolateral Membrane Markers). Contrary to a previously published report (62), however, beta 1 does not appear between exfoliated epithelial cells (asterisks, Fig. 3A). It is also not detected between exfoliated cells and the adherent epithelium (arrow, Fig. 3A). Infrequently, however, weak diffuse staining is observed in the luminal space (Table 2); of the 82.3% of tubular profiles that contain exfoliated cells, 7.5% show weak reactivity for beta 1, possibly representing its degradation (Table 2).

The cortex is less susceptible to experimental ischemic injury (13, 23, 61); here, beta 1continues to localize primarily to basal surfaces of proximal tubular cells and to outline the epithelia of the glomerulus and capillary tuft (Fig. 3C). In some distal tubules, however, basal and lateral localization is observed (Fig. 3C).

Cellular distribution of beta 1 in the inner stripe of the outer medulla also appears to be affected. At 24-48 h postischemia, discrete localization of beta 1 to basal plasma membranes of epithelia of thick ascending limbs (TAL; Fig. 4A) cannot be discerned compared with corresponding contralateral controls (Fig. 4C); beta 1 localization appears to be more diffuse. In some collecting tubules, beta 1 localizes to basal and lateral surfaces (CT, Fig. 4A). Amorphous material, representing cellular debris and/or exfoliated cells (see Fig. 7, C and E) dislodged from proximal nephron segments, fills the lumen of the distal nephron (Fig. 4B) and does not stain for beta 1 (Fig. 4A).


View larger version (77K):
[in this window]
[in a new window]
 
Fig. 4.   Immunofluorescent staining of postischemic (A) and nonischemic contralateral control (C) kidneys taken 24 h after reperfusion and incubated with polyclonal antibody to beta 1-integrin. Corresponding Nomarski image of A is shown in B. Appearance of beta 1 on basal surfaces of TAL of inner stripe of outer medulla is less distinct after ischemia (A) than in nonischemic contralateral control (C); beta 1 is also detected on basal and lateral surfaces of collecting tubules (CT). Bar, 40 µm.

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 beta 1-subunit varies. In some epithelia of regenerating S3 segments, strong localization of beta 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 beta 1 both basally and laterally (Table 2). In either the same tubule or other tubules in the same region, beta 1 is also found on apical plasma membranes (long solid arrows, Fig. 5, A and B). 22.2% of tubular profiles depolarize beta 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).


View larger version (156K):
[in this window]
[in a new window]
 
Fig. 5.   Immunofluorescent staining for beta 1-integrin in cryostat sections of postischemic (A, B, D) and nonischemic contralateral control (C, E) kidneys taken 120 h after reperfusion. In the outer stripe of the outer medulla of the postischemic kidney (A, B), some epithelial cells of S3 segments localize beta 1 to basal and lateral surfaces (short solid arrows), whereas others randomize it to apical, basal, and lateral plasma membranes (long solid arrows). Other epithelia show only a basal distribution (open arrows). Few exfoliated cells are found in tubular lumens. In these cells, beta 1 is either intracellular (A, B), absent (A), or randomized (A) on plasma membranes. Star in A highlights localization of beta 1-integrin between an exfoliated and an adherent tubular epithelial cell. In the outer stripe (C) of nonischemic contralateral kidneys, beta 1 is found exclusively on basal plasma membranes; the microvillar brush border in C stains nonspecifically. The inner stripe is not completely regenerated after reperfusion injury (D) compared with its corresponding contralateral control (E); beta 1 is basally localized (D), albeit weakly, on thick ascending limbs (TAL), and collecting tubules (CT) organize beta 1 basolaterally (D). Bar, 40 µm.

Distribution of beta 1 to basal plasma membranes (open arrows, Fig. 5, A and B) is also detected in S3 segments of the outer stripe of the outer medulla; 46.5% of tubules show this localization (Table 2). In 7.1% of tubular profiles, an average of 1.7 cells (of 23.9 cells/tubule) distribute beta 1 both basally and apically (Table 2). Thus it appears that significantly more tubules at 120 h postischemia contain cells with apical, basal, and/or lateral localization of beta 1 than at earlier times. In contrast to this spectrum of staining patterns in the outer stripe of the postischemic kidney, only basal plasma membrane staining (Fig. 5C) is observed in corresponding regions of contralateral control kidneys (94.6% of tubular profiles; Table 2).

By 120 h after reperfusion, little material is seen in tubular lumens. The luminal material that is present appears to be either individual cells or small groups of cells. Some luminal cells are negative for beta 1-integrin (Fig. 5A), whereas others are strongly immunoreactive. Of the 44.4% of tubules that contain exfoliated material, 27.2% are immunopositive for beta 1 (Table 2). In these cases, beta 1 is either randomly expressed on the plasma membrane (Fig. 5A) or is present intracellularly (Fig. 5, A and B). Infrequently detached cells in tubular spaces appear to adhere to apical surfaces of epithelia residing on basement membranes (star, Fig. 5A); beta 1 localizes at the junction where the luminal cell contacts the adherent epithelial cell.

The inner stripe still appears to be affected by 120 h postischemia; beta 1 is weakly expressed on basal surfaces of thick ascending limbs (TAL, Fig. 5D) and localizes to basolateral borders of collecting tubules (CT, Fig. 5D). In corresponding contralateral controls (Fig. 5E), beta 1 distinctly localizes only to basal plasma membranes. In the cortex, beta 1 is seen on basal plasma membranes of distal and proximal tubules (data not shown), similar to the pattern of staining in contralateral controls (data not shown).

In summary, the data indicate that in response to ischemia and reperfusion, the beta 1-integrin subunit reorganizes predominantly in S3 segment epithelia that remain attached to basement membranes. Within 1 h of reperfusion, as the injury phase begins, beta 1 newly appears on lateral surfaces of adherent epithelia; cellular debris congesting tubular lumens are negative for beta 1. By 24-48 h after reperfusion, when the injury phase is complete and regeneration predominates, beta 1 is expressed primarily on basal surfaces of remaining tubular epithelia and is not detected on exfoliated cells (see below). At 120 h after reperfusion, as regeneration proceeds, a wide spectrum of staining patterns is observed in the adherent epithelium and the few luminal cells that are present.

Distribution of Apical and Basolateral Membrane Markers

The observation that localization of beta 1-integrin changes in adherent epithelia after ischemiareperfusion and is absent on cellular debris and amorphous material that occlude luminal spaces raised the question of whether other plasma membrane proteins change their distribution. To determine this, we examined the distribution of the Na+-K+-ATPase, a basolateral membrane marker, and leucine aminopeptidase, an apical marker of the proximal tubule, after ischemia-reperfusion.

As 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).


View larger version (146K):
[in this window]
[in a new window]
 
Fig. 6.   Immunofluorescent staining of cryostat sections of postischemic kidney reacted with monoclonal antibody to either Na+-K+-ATPase (A, C, E) or leucine aminopeptidase (B, D, F). In the absence of reperfusion (A, B) in the outer stripe of the outer medulla, the postischemic kidney localizes Na+-K+-ATPase (A) and leucine aminopeptidase (B) to basolateral surfaces and to apical microvilli of S3 segments, respectively. One hour after reperfusion, Na+-K+-ATPase intensely stains distal tubules and to a lesser extent S3 proximal tubules (C), where it localizes predominantly to basolateral surfaces; some staining of apical surfaces and what appear to be cytoplasmic vesicles is also seen (C). In contrast, aminopeptidase highlights the apical membrane, apical cytoplasm, and luminal space (D). Dashed lines in D mark basal surfaces of tubular epithelia. At 3 h after reperfusion, Na+-K+-ATPase is depolarized to all cell surfaces (E) in the majority of S3 proximal tubules. Punctate staining for leucine aminopeptidase (F) in apical cytoplasms predominates. Bar, 40 µm.

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 beta 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 beta 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 beta 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.


View larger version (147K):
[in this window]
[in a new window]
 
Fig. 7.   Immunofluorescent staining of cryostat sections of postischemic kidney taken 48 h after reperfusion and reacted with monoclonal antibody to either Na+-K+-ATPase (A, C) or leucine aminopeptidase (B, E). Corresponding Nomarski images of C and E are shown in D and F, respectively. In S3 proximal tubules in the outer stripe of the outer medulla, Na+-K+-ATPase (A) and leucine aminopeptidase (B) strongly randomize to plasma membranes of exfoliated cells, whereas in adherent epithelia (arrows) weaker depolarized staining is observed. In the inner stripe, thick ascending limbs are immunoreactive for Na+-K+-ATPase (C, D), as is luminal material of dislodged cells, which also contain aminopeptidase (E, F). Bar, 40 µm.

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).


View larger version (118K):
[in this window]
[in a new window]
 
Fig. 8.   Immunofluorescent staining of cryostat sections of postischemic kidney taken 120 h after reperfusion and reacted with monoclonal antibody to either Na+-K+-ATPase (A, B) or leucine aminopeptidase (C, D). Regenerating tubules in the outer stripe show heterogeneous staining. Some epithelia randomize Na+-K+-ATPase (A) and aminopeptidase (C) to all cell surfaces. Others localize Na+-K+-ATPase to lateral and/or basal plasma membranes (A, B) and distribute aminopeptidase to apical domains (C, D). Na+-K+-ATPase and aminopeptidase are weakly expressed along some regenerating tubules (B, D, respectively). Bar, 40 µm.

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 beta 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 of beta 1-integrins, a major group of ECM receptors, was altered by ischemia and reperfusion, we also examined the localization of specific ECM proteins. Defective cell-ECM interactions have been hypothesized to contribute to renal failure by one or more mechanisms (26, 54, 66).

Kidney 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.


View larger version (164K):
[in this window]
[in a new window]
 
Fig. 9.   Immunofluorescent staining of cryostat sections of postischemic kidney taken 24 h after reperfusion and reacted with monoclonal antibody to laminin. In the outer stripe, laminin outlines tubular basement membranes, highlighting regions of tubulorrhexis (arrows, A). Only rarely is laminin detected in tubular lumens (asterisk, B). Bar, 40 µm.

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).


View larger version (134K):
[in this window]
[in a new window]
 
Fig. 10.   Immunofluorescent staining of cryostat sections of postischemic kidney taken 24 h after reperfusion (A, C, E) and reacted with polyclonal antibody to fibronectin. Corresponding Nomarski images of A and C are shown in B and D, respectively. Contralateral control kidney is shown in F; normal rat kidney is shown in G. In the outer stripe of the outer medulla, hazy fibronectin staining is seen in tubules containing exfoliated cells (single asterisks, A, B). Tubules may represent either the S3 segment or more proximal regions of the distal nephron. Some tubular lumens in which detached cells are not observed (double asterisk, B) contain fibronectin (double asterisk, A). In comparable regions in normal kidney (G), fibronectin outlines tubules and the renal interstitium. In the inner stripe of the outer medulla, strong fibronectin immunoreactivity is detected in luminal compartments of the distal nephron (C, D) but is not detected in contralateral control kidney (F). Staining of luminal fibronectin is obliterated (E) by preincubating anti-rat fibronectin antibody with purified rat fibronectin (25 µg/ml). Bar, 40 µm.


View larger version (111K):
[in this window]
[in a new window]
 
Fig. 11.   Immunofluorescent staining of cryostat sections of postischemic kidney taken 120 h after reperfusion and reacted with polyclonal antibody to fibronectin. Fibronectin is strongly expressed in the interstitium of the outer (A) and inner (B) stripes, suggesting an incipient fibrosis. However, it is absent from tubular spaces of proximal S3 (A) and distal nephron segments (B). Bar, 40 µm.

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta 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 beta 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 beta 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 beta 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 beta 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.

beta 1-Integrin in Renal Ischemic Injury and Repair

Previous experiments examined the distribution of the alpha 3-integrin subunit, which forms a heterodimeric complex with beta 1, in an in vitro model of oxidative stress. Nonlethal exposure of BS-C-1 cells, a renal epithelial cell line, to hydrogen peroxide resulted in reorganization of the alpha 3-integrin subunit from basal to apical surfaces (21). Focal cell contacts were disrupted, and talin, a cytoskeletal protein that binds integrin cytoplasmic domains, disappeared from basal plasma membranes. No independent evidence, however, was provided that BS-C-1 cells were apically-to-basally polarized before stress. In addition, distribution of the beta 1-subunit was not examined. Because association of alpha - and beta -subunits into heterodimers is necessary for cell surface expression and ligand binding, it is unlikely that alpha 3 alone, without beta 1, was present on apical plasma membranes.

On the basis of the redistribution of alpha 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, beta 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 beta 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 beta 1-integrins in renal ischemic injury. In our experiments, beta 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 beta 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 beta 1 in Renal Ischemic Injury and Repair

On the basis of our observations, we propose a model for the involvement of beta 1-integrins in the injury and repair processes resulting from renal ischemia and reperfusion (Fig. 12). We suggest that, after ischemia and reperfusion, basally localized integrins are deactivated, resulting in their disengagement from ligands in the basement membrane. Under these conditions, some integrins would be free to diffuse in the plane of the membrane and appear on the lateral surface. In the distal tubule, where the degree of deactivation might not be as severe, cells would remain attached and, within a period of time, basal localization of beta 1 would be recovered. In the S3 segment, however, integrin deactivation would result in the detachment of some cells. In these cases, the deactivated integrins, now devoid of ligand and possibly damaged by the deactivation process, would be rapidly endocytosed and degraded. As the tubular epithelium regenerated, cells would flatten, spread, and migrate over denuded areas of the basement membrane while dedifferentiating and depolarizing. Under these circumstances, beta 1-integrins would play a more dynamic role in facilitating the attachment to and detachment from the basement membrane that would accompany cell extension and movement. At the same time, new integrin alpha - and beta -subunits might be expressed to mediate attachment to interstitial matrix proteins, such as fibronectin, or to components of basement membrane expressed as part of the repair process. By transmitting a variety of signals intracellularly, these integrins might also be important both in dedifferentiation in the initial stages of regeneration and in redifferentiation and repolarization at the end. As a new epithelium reformed, integrins would become reactivated and, ultimately, return to their exclusively basal location.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 12.   Model of beta 1-integrin in renal ischemic injury and repair. After ischemia and reperfusion, beta 1-integrins that are basally localized are deactivated, resulting in their disengagement from ligands in the basement membrane. Deactivated integrins are then free to diffuse to lateral surfaces. In distal tubules, where the degree of deactivation is not as severe, cells remain attached and basal localization of beta 1 is eventually recovered. In S3 segments, however, integrin deactivation results in exfoliation of some cells. Deactivated integrins, now devoid of ligand, are endocytosed and degraded. As the tubular epithelium regenerates, cells use beta 1 to flatten, spread, and migrate over denuded areas of basement membrane while dedifferentiating and depolarizing. New integrin alpha - and beta -subunits may also be expressed to mediate attachment either to fibronectin, an interstitial matrix protein, or to other components of basement membrane newly expressed as part of the repair process. By transmitting a variety of intracellular signals, these integrins may be important in redifferentiation and repolarization. As a new epithelium reforms, beta 1-integrins are reactivated, ultimately returning to an exclusively basal location.

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 beta 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 beta 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 beta 1-molecule, it is unlikely that the loss of immunoreactivity was due solely to degradation of the cytoplasmic part of beta 1. Finally, our findings of a lack of polarity of both apical and basolateral markers as well as, in some cases, beta 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 beta 1 family may contribute to intraluminal obstruction and oliguria. Kidney epithelial cells may express integrins containing the beta 3, beta 5, or beta 6 families in addition to beta 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 integrin alpha IIbbeta 3 is expressed on the cell surface but incapable of binding its ligand fibrinogen until the platelet is activated by thrombin (for review, see Ref. 31). Conversely, in keratinocytes, the fibronectin receptor alpha 5beta 1 is expressed on the cell surface, where it binds to fibronectin (2). During terminal differentiation in vitro, alpha 5beta 1 remains on the cell surface but is deactivated, releasing cells from their adhesion to the fibronectin matrix (2).

Activation of integrins is regulated by the cytoplasmic tails of both alpha - and beta -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 alpha -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 alpha - and beta -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 beta 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 alpha - and beta -subunits (14). Indeed, in activated platelets, calpain has been shown to cleave the cytoplasmic tail of the beta 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 beta 1-integrins has often been considered evidence of their role in cell-cell adhesion (41). In keratinocytes, beta 1-, alpha 2-, and alpha 3-integrin subunits were initially localized to points of cell-cell contact (34), and an antibody against the alpha 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 (alpha 2-alpha 3; Ref. 70) or homophilic (alpha 2-alpha 2; Ref. 69) integrin-integrin interactions. However, recent work examining adhesion in a strictly cellular environment indicates that beta 1-integrins do not mediate cell-cell adhesion (75). Even though various transfected integrin subunits belonging to the beta 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

beta 1-Integrins expressed by the adherent epithelium may direct regeneration and repair of the damaged kidney epithelium. Initially, integrins may facilitate spreading and migration of cells into areas of basement membrane denuded by exfoliation. While some of these may interact with laminins and collagens that make up the basement membrane, new integrins may be expressed that bind to interstitial matrix proteins such as fibronectin exposed by the injury process. These may include not only alpha 5beta 1, which is seen in other tissues after wounding (29, 52) but also alpha vbeta 6, a fibronectin receptor expressed by both damaged skin and lung epithelia (5, 74). Our observations, which have been made with an antibody directed primarily against the beta 1-subunit, would not exclude either of these possibilities.

As regeneration proceeds, beta 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, beta 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 beta 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 beta  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 beta 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 beta 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.

    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.

    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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abrahamson, D. R., and V. Leardkamolkarn. Development of kidney tubular basement membranes. Kidney Int. 39: 382-393, 1991[Medline].

2.   Adams, J. C., and F. M. Watt. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5beta 1 integrin loss from the cell surface. Cell 63: 425-435, 1990[Medline].

3.   Arendshorst, W. J., W. F. Finn, C. W. Gottschalk, and H. K. Lucas. Micropuncture study of acute renal failure following temporary renal ischemia in the rat. Kidney Int. Suppl. 10: S100-S105, 1976.

4.   Argraves, W. S., S. Suzuki, H. Arai, K. Thompson, M. D. Pierschbacher, and E. Ruoslahti. Amino acid sequence of the human fibronectin receptor. J. Cell Biol. 105: 1183-1190, 1987[Abstract].

4a.  Bonventre, J. V., M. Brezis, N. Siegel, S. Rosen, M. Venkatachalam, and D. Portilla. The relative importance of proximal vs. distal tubular injury in acute renal failure. In: Controversies related to acute renal failure, edited by W. Lieberthal and S. K. Nigam. Am. J. Physiol. 275 (Renal Physiol. 44). In press.

5.   Breuss, J. M., J. Gallo, H. M. DeLisser, I. V. Klimanskaya, H. G. Folkesson, J. F. Pittet, S. L. Nishimura, K. Aldape, D. V. Landers, W. Carpenter, N. Gillett, D. Sheppard, M. A. Matthay, S. M. Albelda, R. H. Kramer, and R. Pytela. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J. Cell Sci. 108: 2241-2251, 1995[Abstract/Free Full Text].

6.   Bronk, S. F., and G. J. Gores. pH-dependent nonlysosomal proteolysis contributes to lethal anoxic injury of rat hepatocytes. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G744-G751, 1993[Abstract/Free Full Text].

7.   Brown, D., R. Lee, and J. V. Bonventre. Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion. Am. J. Physiol. 273 (Renal Physiol. 42): F1003-F1012, 1997[Abstract/Free Full Text].

8.   Brown, D., J. Lydon, M. McLaughlin, A. Stuart-Tilley, R. Tyszkowski, and S. Alper. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem. Cell Biol. 105: 261-267, 1996[Medline].

9.   Carter, W. G., E. A. Wayner, T. S. Bouchard, and P. Kaur. The role of integrins alpha 2beta 1 and alpha 3beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J. Cell Biol. 110: 1387-1404, 1990[Abstract].

10.   Clark, E. A., and J. S. Brugge. Integrins and signal transduction pathways: the road taken. Science 268: 233-239, 1995[Medline].

11.   Cosio, F. G., D. D. Sedmak, and N. J. Nahman. Cellular receptors for matrix proteins in normal human kidney and human mesangial cells. Kidney Int. 38: 886-895, 1990[Medline].

12.   Desnuelle, P. Intestinal and renal aminopeptidase: a model of a transmembrane protein. Eur. J. Biochem. 101: 1-11, 1979[Medline].

13.   Donohoe, J. F., M. A. Venkatachalam, D. B. Bernard, and N. G. Levinsky. Tubular leakage and obstruction after renal ischemia: structural-functional correlations. Kidney Int. 13: 208-222, 1978[Medline].

14.   Du, X., T. C. Saido, S. Tsubuki, F. E. Indig, M. J. Williams, and M. H. Ginsberg. Calpain cleavage of the cytoplasmic domain of the beta3 subunit. J. Biol. Chem. 270: 26146-26151, 1995[Abstract/Free Full Text].

15.   Edelstein, C. L., H. Ling, and R. W. Schrier. The nature of renal cell injury. Kidney Int. 51: 1341-1351, 1997[Medline].

16.   Edelstein, C. L., E. D. Wieder, M. M. Yaqoob, P. E. Gengaro, T. J. Burke, and R. W. Schrier. The role of cysteine proteases in hypoxia-induced renal proximal tubular injury. Proc. Natl. Acad. Sci. USA 92: 7662-7666, 1995[Abstract].

16a.   Engvall, E., G. E. Davis, K. Dickerson, E. Ruoslahti, S. Varon, and M. Manthorpe. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J. Cell Biol. 103: 2457-2465, 1986[Abstract].

17.   Faull, R. J., and M. H. Ginsberg. Inside-out signaling through integrins. J. Am. Soc. Nephrol. 7: 1091-1097, 1996[Abstract].

18.   Ffrench-Constant, C., L. Van De Water, H. F. Dvorak, and R. O. Hynes. Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J. Cell Biol. 109: 903-914, 1989[Abstract].

19.   Fish, E. M., and B. A. Molitoris. Alterations in epithelial cell polarity and the pathogenesis of disease states. N. Engl. J. Med. 330: 1580-1588, 1994[Free Full Text].

19a.   Foellmer, H. G., J. A. Madri, and H. Furthmayr. Methods in laboratory investigation. Monoclonal antibodies to type IV collagen: probes for the study of structure and function of basement membranes. Lab. Invest. 48: 639-649, 1983[Medline].

20.   Fujikawa, L. S., C. S. Foster, T. J. Harrist, J. M. Lanigan, and R. B. Colvin. Fibronectin in healing rabbit corneal wounds. Lab. Invest. 45: 120-129, 1981[Medline].

21.   Gailit, J., D. Colflesh, I. Rabiner, J. Simone, and M. S. Goligorsky. Redistribution and dysfunction of integrins in cultured renal epithelial cells exposed to oxidative stress. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F149-F157, 1993[Abstract/Free Full Text].

22.   Ginsberg, M. H. Integrins: dynamic regulation of ligand binding. Biochem. Soc. Trans. 23: 439-447, 1995[Medline].

23.   Glaumann, G., H. Glaumann, and B. F. Trump. Studies of cell recovery from injury. III. Ultrastructural studies of the recovery of the pars recta of the proximal tubule (P3) segment of the rat kidney from temporary ischemia. Virchows Arch. 25: 281-308, 1977.

24.   Göke, M., A. Zuk, and D. K. Podolsky. Regulation and function of extracellular matrix in intestinal epithelial restitution in vitro. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G729-G740, 1996[Abstract/Free Full Text].

25.   Goligorsky, M. S., and G. F. DiBona. Pathogenetic role of Arg-Gly-Asp recognizing integrins in acute renal failure. Proc. Natl. Acad. Sci. USA 90: 5700-5704, 1993[Abstract].

26.   Goligorsky, M. S., W. Lieberthal, L. Racusen, and E. E. Simon. Integrin receptors in renal tubular epithelium: new insights into pathophysiology of acute renal failure. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F1-F8, 1993[Abstract/Free Full Text].

27.   Goligorsky, M. S., E. Noiri, H. Kessler, and V. Romanov. Therapeutic potential of RGD peptides in acute renal injury. Kidney Int. 51: 1487-1492, 1997[Medline].

28.   Graber, M., B. Lane, R. Lamia, and E. Pastoriza-Munoz. Bubble cells: renal tubular cells in the urinary sediment with characteristics of viability. J. Am. Soc. Nephrol. 1: 999-1004, 1991[Abstract].

29.   Grinnell, F. Wound repair, keratinocyte activation and integrin modulation. J. Cell Sci. 101: 1-5, 1992[Medline].

30.   Hemler, M. E., J. B. Weitzman, R. Pasqualini, S. Kawaguchi, P. D. Kassner, and F. B. Berdichevsky. Structure, biochemical properties, and biological functions of integrin cytoplasmic domains. In: The Integrins: The Biological Problems, edited by Y. Takada. Boca Raton, FL: CRC, 1995.

31.   Hynes, R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25, 1992[Medline].

32.   Kartha, S., and F. G. Toback. Migration of kidney epithelial cells during recovery from acute renal failure. In: Acute Renal Failure: New Concepts and Therapeutic Strategies, edited by M. S. Goligorsky, and J. H. Stein. New York: Churchill Livingstone, 1995, p. 287-319.

33.   Kashgarian, M., D. Biemesderfer, M. Caplan, and B. I. Forbush. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int. 28: 899-913, 1985[Medline].

34.   Kaufmann, R., D. Frosch, C. Westphal, L. Weber, and C. E. Klein. Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures. J. Cell Biol. 109: 1807-1815, 1989[Abstract].

35.   Kawaguchi, S., and M. E. Hemler. Role of the alpha subunit cytoplasmic domain in regulation of adhesive activity mediated by the integrin VLA-2. J. Biol. Chem. 268: 16279-16285, 1993[Abstract/Free Full Text].

36.   Kelly, K. J., W. W. Williams, R. B. Colvin, and J. V. Bonventre. Antibody to intercellular adhesion molecule 1 protects against ischemic injury. Proc. Natl. Acad. Sci. USA 91: 812-816, 1994[Abstract].

37.   Korhonen, M. Normal distribution of adhesion molecules in the kidney. In: Acute Renal Failure: New Concepts and Therapeutic Strategies, edited by M. S. Goligorsky, and J. H. Stein. New York: Churchill Livingstone, 1995, p. 231-244.

38.   Korhonen, M., J. Ylanne, L. Laitinen, and I. Virtanen. The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J. Cell Biol. 111: 1245-1254, 1990[Abstract].

39.   Korhonen, M., J. Ylanne, L. Laitinen, and I. Virtanen. Distribution of beta 1 and beta 3 integrins in human fetal and adult kidneys. Lab. Invest. 62: 616-625, 1990[Medline].

40.   Kreidberg, J. A., M. J. Donovan, S. L. Goldstein, H. Rennke, K. Shepherd, R. C. Jones, and R. Jaenisch. Alpha3beta1 integrin has a crucial role in kidney and lung organogenesis. Development 122: 3537-3547, 1996[Abstract/Free Full Text].

41.   Larjava, H., J. Peltonen, S. K. Akiyama, S. S. Yamada, H. R. Gralnick, J. Uitto, and K. M. Yamada. Novel function for beta 1 integrins in keratinocyte cell-cell interactions. J. Cell Biol. 110: 803-815, 1990[Abstract].

42.   Lizuka, K., H. Kawaguchi, and H. Yasuda. Calpain is activated during hypoxic myocardial cell injury. Biochem. Med. Metab. Biol. 46: 427-431, 1991[Medline].

43.   Malis, C. D., J. Y. Cheung, A. Leaf, and J. V. Bonventre. Effects of verapamil in models of ischemic acute renal failure in the rat. Am. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 14): F735-F742, 1983[Abstract/Free Full Text].

44.   Mandal, A. K., A. H. Sklar, and J. B. Hudson. Transmission electron microscopy of urinary sediment in human acute renal failure. Kidney Int. 28: 58-63, 1985[Medline].

45.   Mason, J., C. Olbricht, T. Takabatake, and K. Thurau. The early phase of experimental acute renal failure. I. Intratubular pressure and obstruction. Pflügers Arch. 370: 155-163, 1977[Medline].

46.   McLean, I. W., and P. K. Nakane. Periodate-lysine-paraformaldehyde fixative. A new fixative for electron microscopy. J. Histochem. Cytochem. 22: 1077-1083, 1974[Medline].

47.   Mendrick, D. L., D. C. Chung, and H. G. Rennke. Heymann antigen gp330 demonstrates affinity for fibronectin, laminin, and type I collagen and mediates rat proximal tubule epithelial cell adherence to such matrices in vitro. Exp. Cell Res. 188: 23-35, 1990[Medline].

48.   Molitoris, B. A. Ischemia-induced loss of epithelial polarity: potential role of the actin cytoskeleton. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F769-F778, 1991[Abstract/Free Full Text].

49.   Molitoris, B. A., R. Dahl, and A. Geerdes. Cytoskeleton disruption and apical redistribution of proximal tubule Na+-K+-ATPase during ischemia. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F488-F495, 1992[Abstract/Free Full Text].

50.   Molitoris, B. A., C. A. Hoilien, R. Dahl, D. J. Ahnen, P. D. Wilson, and J. Kim. Characterization of ischemia-induced loss of epithelial polarity. J. Membr. Biol. 106: 233-242, 1988[Medline].

51.   Müller, U., D. Wang, S. Denda, J. J. Meneses, R. A. Pedersen, and L. F. Reichardt. Integrin alpha 8beta 1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell 88: 603-614, 1997[Medline].

52.   Murakami, J., T. Nishido, and T. Otori. Coordinated appearance of beta 1 integrins and fibronectin during corneal wound healing. J. Lab. Clin. Med. 120: 86-93, 1991.

53.   Noiri, E., J. Gailit, D. Sheth, H. Magazine, M. Gurrath, G. Muller, H. Kessler, and M. S. Goligorsky. Cyclic RGD peptides ameliorate ischemic acute renal failure in rats. Kidney Int. 46: 1050-1058, 1994[Medline].

54.   Noiri, E., V. Romanov, T. Forest, J. Gailit, G. F. DiBona, F. Miller, P. Som, Z. H. Oster, and M. S. Goligorsky. Pathophysiology of renal tubular obstruction: therapeutic role of synthetic RGD peptides in acute renal failure. Kidney Int. 48: 1375-1385, 1995[Medline].

55.   Ojakian, G. K., and R. Schwimmer. Regulation of epithelial cell surface polarity by beta 1 integrins. J. Cell Sci. 107: 561-576, 1994[Abstract/Free Full Text].

56.   Olsen, T. S., H. S. Olsen, and H. E. Hansen. Tubular ultrastructure in acute renal failure in man: epithelial necrosis and regeneration. Virchows Arch. 406: 75-89, 1985.

57.   O'Toole, T. E., Y. Katagiri, J. Faull, K. Peter, R. Tamura, V. Quaranta, J. C. Loftus, S. J. Shattil, and M. H. Ginsberg. Integrin cytoplasmic domains mediate inside-out signal transduction. J. Cell Biol. 124: 1047-1059, 1994[Abstract].

58.   Pasqualini, R., J. Bodorova, S. Ye, and M. E. Hemler. A study of the structure, function and distribution of beta 5 integrins using novel anti-beta 5 monoclonal antibodies. J. Cell Sci. 105: 101-111, 1993[Abstract/Free Full Text].

59.   Pombo, C. M., J. V. Bonventre, J. Avruch, J. R. Woodgett, J. M. Kyriakis, and T. Force. The stress-activated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion. J. Biol. Chem. 269: 26546-26551, 1994[Abstract/Free Full Text].

60.   Racusen, L. C., B. A. Fivush, Y.-L. Li, I. Slatnik, and K. Solez. Dissociation of tubular cell detachment and tubular cell death in clinical and experimental "acute tubular necrosis". Lab. Invest. 64: 546-556, 1991[Medline].

61.   Reimer, K. A., C. E. Ganote, and R. B. Jennings. Alterations in renal cortex following ischemic injury. III. Ultrastructure of proximal tubule after ischemia or autolysis. Lab. Invest. 26: 347-363, 1972[Medline].

62.   Romanov, V., E. Noiri, G. Czerwinski, D. Finsinger, H. Kessler, and M. S. Goligorsky. Two novel probes reveal tubular and vascular Arg-Gly-Asp (RGD) binding sites in the ischemic rat kidney. Kidney Int. 52: 93-102, 1997[Medline].

63.   Safirstein, R. L., and J. V. Bonventre. Molecular response to ischemic and nephrotoxic acute renal failure. In: Molecular Nephrology: Kidney Function in Health and Disease, edited by D. Schlondorff, and J. V. Bonventre. New York: Marcel-Dekker, 1995, p. 839-854.

64.   Schoenenberger, C., A. Zuk, G. M. Zinkl, D. Kendall, and K. S. Matlin. Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity. J. Cell Sci. 107: 527-541, 1994[Abstract/Free Full Text].

65.   Seubert, P., K. Lee, and G. Lynch. Ischemia triggers NMDA receptor linked cytoskeletal proteolysis in hippocampus. Brain Res. 492: 366-370, 1989[Medline].

66.   Simon, E. E. Potential role of integrins in acute renal failure. Nephrol. Dial. Transplant. 9: 26-33, 1994[Medline].

67.   Simon, E. E., and J. A. McDonald. Extracellular matrix receptors in the kidney cortex. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F783-F792, 1990[Abstract/Free Full Text].

68.   Sorokin, L., A. Sonnenberg, M. Aumailley, R. Timpl, and P. Ekblom. Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules. J. Cell Biol. 111: 1265-1274, 1990[Abstract].

69.   Sriramarao, P., P. Steffner, and K. R. Gehlsen. Biochemical evidence for a homophilic interaction of the alpha 3beta 1 integrin. J. Biol. Chem. 268: 22036-22041, 1993[Abstract/Free Full Text].

70.   Symington, B. E., Y. Takada, and W. G. Carter. Interaction of integrins alpha 3beta 1 and alpha 2beta 1: potential role in keratinocyte intercellular adhesion. J. Cell Biol. 120: 523-535, 1993[Abstract].

71.   Tanner, G. A., and S. Sophasan. Kidney pressures after temporary artery occlusion in the rat. Am. J. Physiol. 230: 1173-1181, 1976[Medline].

72.   Thadhani, R., M. Pascual, and J. V. Bonventre. Acute renal failure. N. Engl. J. Med. 334: 1448-1460, 1996[Free Full Text].

73.   Venkatachalam, M. A., D. B. Bernard, J. F. Donohoe, and N. G. Levinsky. Ischemic damage and repair in the rat proximal tubule: differences among the S1, S2, and S3 segments. Kidney Int. 14: 31-49, 1978[Medline].

74.   Wang, A., Y. Yokosaki, R. Ferrando, J. Balmes, and D. Sheppard. Differential regulation of airway epithelial integrins by growth factors. Am. J. Respir. Cell Mol. Biol. 15: 664-672, 1996[Abstract].

75.   Weitzman, J. B., A. Chen, and M. E. Hemler. Investigation of the role of beta 1 integrins in cell-cell adhesion. J. Cell Sci. 108: 3635-3644, 1995[Abstract/Free Full Text].

76.   Wennerberg, K., L. Lohikangas, D. Gullberg, M. Pfaff, S. Johansson, and R. Fassler. beta 1 Integrin-dependent and -independent polymerization of fibronectin. J. Cell Biol. 132: 227-238, 1996[Abstract].

77.   Witzgall, R., D. Brown, C. Schwarz, and J. V. Bonventre. Localization of proliferating cell nuclear antigen, vimentin, c-fos and clusterin in the postischemic kidney. J. Clin. Invest. 93: 2175-2188, 1994[Medline].

78.   Wu, C., J. S. Bauer, R. L. Juliano, and J. A. McDonald. The alpha 5beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly. J. Biol. Chem. 268: 21883-21888, 1993[Abstract/Free Full Text].

79.   Wu, C., V. M. Keivens, T. E. O'Toole, J. A. McDonald, and M. H. Ginsberg. Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix. Cell 83: 715-724, 1995[Medline].

80.   Zuk, A., and K. S. Matlin. Apical beta 1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I collagen overlay. J. Cell Sci. 109: 1875-1889, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(3):C711-C731
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society