Induction of a laminin isoform and alpha 3beta 1-integrin in renal ischemic injury and repair in vivo

Anna Zuk1 and Karl S. Matlin2

1 Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; and 2 Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45219-0581


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of ~220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha 3-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha 3beta 1-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.

extracellular matrix; epithelial regeneration; acute renal failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC INJURY TO THE KIDNEY, a major cause of acute renal failure (53), leads to the detachment and loss of significant numbers of cells from the tubular epithelium (43-45). Cells that remain adherent to the basement membrane dedifferentiate and spread to fill gaps in the epithelium and then repolarize and redifferentiate to reconstitute the normal architecture and function of the tubular epithelium (6, 7, 9, 13, 35, 36, 47, 53, 55, 62). During this process of repair and regeneration, significant changes occur in the extracellular matrix and its integrin receptors (1, 10, 18, 58, 62, 63). In particular, the beta 1-integrin subunit, which is normally found on the basal surface, newly appears on the lateral surfaces and, in some cases, the apical plasma membrane of tubular epithelial cells (62). In addition, large amounts of cellular and plasma fibronectin are deposited along the basement membrane and in the tubular lumen (63).

Of the extracellular matrix proteins likely to play significant roles in regeneration of the epithelium after injury, laminins are of particular interest. Laminins are heterotrimers of alpha -, beta -, and gamma -chains linked by disulfide bonds to form cruciform molecules (4, 8, 33). To date, five different alpha -chains, three beta -chains, and three gamma -chains have been identified with different combinations resulting in as many as 12 different laminin isoforms. Laminins are major structural components of basement membranes that mediate cell adhesion but also generate differentiation signals when bound to their cell surface receptors.

The major group of laminin receptors are integrins (4). The integrins are a superfamily of transmembrane glycoproteins that consist of unrelated alpha - and beta -subunits (17, 54). Eight different beta -families have been identified that can combine with 14 different alpha -subunits, resulting in >20 different alpha beta combinations. Association of alpha - and beta -subunits into noncovalent heterodimers is necessary for ligand binding and cell surface expression. Depending on the alpha beta -heterodimer, ligand specificity for laminin or other extracellular matrix molecules is conferred.

In the kidney, integrins with the beta 1-subunit are expressed throughout the nephron (21, 22), in which they interact with a variety of specific laminin isoforms (4, 32, 33, 51, 57) and other extracellular matrix proteins. The precise roles of beta 1-integrin-laminin interactions in the adult kidney are poorly understood but certainly involve cell differentiation along with adhesion. In the embryonic kidney, alpha 6beta 1-integrin, a laminin receptor, is required for polarization of the condensed mesenchyme into the tubular epithelium (50) and is also a major laminin receptor in the mature kidney. Antibodies against the laminin-A-chain (now called laminin-alpha 1) also prevent the formation of a polarized epithelium when added to organ cultures of developing embryonic kidney (20).

Of the various laminin isoforms, laminin-5 is potentially significant in the injured kidney because it has been previously implicated in regeneration of skin wounds (25, 38-40, 42). Laminin-5 (also called kalinin, BM600/nicein, epiligrin, or ladsin) is composed of the alpha 3-, beta 3-, and gamma 2-chains and is a component of the anchoring filaments of the skin basement membrane (2, 4, 48, 56). In many cell types, alpha 3beta 1- and alpha 6beta 4-integrins act as laminin-5 receptors (2, 4, 28, 41). Laminin-5 is required for epithelialization of skin after wounding (14, 25, 38, 40) and is also important in the differentiation of enterocytes during migration from the crypt to the villus tip (16, 26). In the kidney, laminin-5 is necessary for branching of the ureteric bud during development (60). Its expression in the adult kidney has not been widely examined, although it has been observed in the renal papilla (32, 33). Some reports also place it in other parts of the kidney (32).

Because of the importance of laminin in epithelial cell differentiation, we hypothesized that expression and distribution of laminin isoforms and their integrin receptors might be altered by ischemic injury to the kidney. Here, we report that the expression of a member of the laminin family, possibly the laminin-5 isoform, is dramatically increased in the basement membranes of the S3 segment and other regions of the kidney after ischemic injury in vivo. In addition, expression of the alpha 3-integrin subunit, which with beta 1 forms a receptor for laminin-5, is also significantly upregulated, in parallel with the laminin isoform. These results indicate that laminin-integrin interaction may play an important role in the regeneration of the renal tubular epithelium after injury, analogous to their functions in the epidermis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Surgery

A unilateral rat model of renal ischemia was used for these studies (59, 62, 63). In this model, blood flow to one kidney is surgically interrupted for 40 min without manipulation of the contralateral kidney, followed by release of the vascular clamp and reperfusion for various times up to several days. This procedure results in only mild systemic changes in blood urea nitrogen and serum creatinine (27, 59). In addition to the contralateral kidney, which served as an internal control, additional controls consisted of kidneys from animals that underwent sham surgery without ischemia.

Immunohistochemistry

Tissue harvesting. Left postischemic and right contralateral kidneys were fixed in situ for immunohistochemistry. Anesthetized animals were first perfused with warm Hank's balanced salt solution until the kidney was cleared of blood. The perfusate was then switched to 2% (wt/vol) paraformaldehyde-75 mM L-lysine-10 mM sodium periodate (PLP fixative) (31) at room temperature. Kidneys were removed, cleaned of adherent connective tissue, and sliced incompletely from the cortex to the medulla at ~2-mm intervals to facilitate penetration of fixative. Incompletely sliced, whole kidneys were transferred into fixative for 1 h at room temperature and refrigerated overnight. The following day, they were rinsed in PBS and stored in PBS containing sodium azide (0.02% wt/vol) at 4°C.

Cryosectioning. Kidney pieces were prepared by extending the cut that terminated at the medulla to the hilus to completely sever a segment of the kidney from the rest of the tissue. They were then rinsed extensively in PBS and infiltrated overnight at 4°C with 0.6 M sucrose/PBS. Samples were then embedded in optimal cutting temperature compound (Miles Laboratories, Naperville, IL), positioned horizontally on a sample chuck for frozen sectioning, and flash frozen in liquid nitrogen. Five-micrometer sections were cut on a Leica CM1850 cryostat (Leica, Deerfield, IL), placed on SuperFrost Plus glass slides (Fisher Scientific, Boston, MA), and stored at -20°C until use.

Immunofluorescence. The procedure of Zuk et al. (62) was used. Briefly, freshly prepared fixative was applied to each section for 10 min to minimize section loss during the procedure, followed by extensive rinsing in PBS. Autofluorescence of tissue sections was quenched with sodium borohydride in PBS (1 mg/ml) followed by additional rinsing. Samples were then permeabilized in 0.1% Triton X-100/PBS, rinsed, and incubated in 10% normal goat serum in PBS to block nonspecific binding sites. Diluted primary antibody was then applied to each sample, and the sections were incubated overnight at 4°C. Samples were further rinsed and incubated in secondary antibody at room temperature in the dark. After additional rinses, sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed by epifluorescence microscopy with either a Zeiss Axioplan, Axiovert, or Axioskop 2Plus Microscope (Carl Zeiss, Thornwood, NY). In some instances, sections were incubated in 1% SDS/PBS for 5 min before quenching to expose latent antigenic sites.

Images were recorded with TMAX 400 film (Eastman Kodak, Rochester, NY) pushed to ASA 800 or with a Zeiss AxioCam MR digital camera. To demonstrate increased expression of the alpha 3-integrin subunit, a standard exposure time was detemined from an automatic exposure of the 120-h postischemic time point, which exhibited the greatest fluorescence intensity. All other ischemic, contralateral, and mock kidney samples were then recorded at this setting. Controls for immunostaining included omitting the primary or secondary antibody or immunostaining tissue sections known to contain the antigen of interest. In all instances, kidney sections serving as controls were from the same animal and time point. Immunostaining was repeated at least three times for each time point and condition on a minimum of two animals.

Immunoblotting

Tissue harvesting. Anesthetized animals were first perfused with sterile PBS at 0°C containing 0.16× Complete protease inhibitors (Boehringer-Mannheim, Indianapolis, IN). Kidneys were then removed from the animal, placed on ice, and dissected free of adherent connective tissue. Kidney pieces were cut along the cortex-hilus axis, flash frozen in liquid nitrogen, and stored at -80°C.

Tissue preparation. The procedure of Zuk et al. (63) involving repeated homogenization, and extraction in solutions containing SDS, DTT, and protease inhibitors was used. For gel electrophoresis, aliquots of extracts containing equal amounts of protein were diluted with 1× sample buffer [200 mM Tris · Cl, pH 8.8, 5 mM EDTA, 1% (wt/vol) SDS, 20 mM DTT, 20% sucrose, 0.2% bromphenol blue] and alkylated with 50 mM iodoacetamide before loading.

Western blotting. Aliquots (25 µg) of kidney extract protein were resolved by 6% SDS-PAGE as previously described (63). Gels were then equilibrated in transfer buffer containing SDS, and proteins were transferred onto Immobilin-P membranes (Millipore, Bedford, MA) with a semidry transfer apparatus (Bio-Rad, Hercules, CA) at 90 mA for 42 min. Membranes were quickly stained in Coomassie blue to confirm equal protein loading and then destained. After a rinsing with wash buffer [10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween-20 (TBST)], membranes were blocked in 5% nonfat dry milk/TBST. Primary antibody was incubated with membranes sealed in plastic bags at 4°C overnight with rotation. Membranes were then washed extensively at room temperature and reacted with secondary antibody conjugated to horseradish peroxidase. They were washed again and peroxidase moieties reacted with Renaissance chemiluminescence reagents (NEN Life Science Products, Boston, MA). Membranes were exposed to X-OMAT Blue film (Eastman Kodak), which was then developed with a Kodak X-OMAT processor.

Antibodies

Rabbit polyclonal anti-human 8LN-5 antibody was kindly provided by Dr. Robert Burgeson (Cutaneous Biology Research Center, Massachusetts General Hospital, Boston, MA). This antibody was prepared against affinity-purified laminin-5 from human keratinocyte culture supernatants injected into rabbits. When used to probe Western blots of laminin-5 purified by affinity chromatography from collagenase-solubilized human amnion- or human keratinocyte-conditioned medium, this antibody recognizes the mature alpha 3-, beta 3-, and gamma 2-chains of laminin-5 that migrate at 165, 140, and 105 kDa, respectively (Fig. 1A) (29) In blots of human keratinocyte cell extracts, 8LN-5 also recognizes the alpha 3 precursor (lane 2, Fig. 1A). The antibody also immunostains the basement membrane underlying the epithelium in cryostat sections of human foreskin, in agreement with previous studies on localization of laminin-5 in skin (Fig. 1B) (2, 48). The antibody was used at a dilution of 1:1,500 to Western blot rat kidney extracts and at a concentration of 10 µg/ml for immunofluorescence of kidney sections.


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Fig. 1.   Rabbit polyclonal anti-human 8LN-5 is specific for laminin-5. A: Western blotting of purified laminin-5 (lane 1), human keratinocyte cell lysates (lane 2), and human keratinocyte-conditioned medium (lane 3) indicates that this antibody recognizes the three chains of laminin-5, mature alpha 3 (alpha 3m), beta 3, and gamma 2, at 165, 140, and 105 kDa, respectively, and the alpha 3 precursor (alpha 3p) at 190 kDa. B: immunofluorescent staining of cryostat sections of human foreskin with antibody 8LN-5 demonstrates reactivity of the basement membrane underlying the epithelium (arrow). Bar = 40 µm.

Rabbit polyclonal anti-human beta 1-integrin antibody was generated against a 37-amino acid sequence of the cytoplasmic domain of human beta 1-integrin (Research Genetics, Huntsville, AL); for Western blots, a 1:1,000 dilution was used. Rabbit polyclonal anti-human beta 4-integrin antibody was kindly provided by Arthur Mercurio (Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA). This antibody, generated against a synthetic peptide corresponding to a 31-amino acid sequence of the cytoplasmic domain of human-beta 4, was used for immunoblots at a dilution of 1:1,000. Mouse monoclonal anti-mouse alpha 3-integrin antibody was purchased from Transduction Laboratories (Lexington, KY). This antibody was generated against a synthetic peptide corresponding to a 215-amino acid residue of the extracellular domain of the mouse alpha 3-integrin subunit; for immunoblots and immunostaining, it was used at 1:250 and 1:10, respectively. Rabbit polyclonal anti-alpha 3-integrin subunit antibody was generated against a synthetic peptide corresponding to the COOH-terminal cytoplasmic domain of human alpha 3-integrin (Chemicon International, Temecula, CA); for Western blots, a 1:500 dilution was used.

Secondary antibodies for immunostaining included goat anti-rabbit FITC adsorbed against mouse and human Ig (1:200; BioSource International, Camarillo, CA) or goat anti-mouse indocarbocyanine (CY3) adsorbed against rat, human, bovine, and horse serum proteins (1:800; Jackson ImmunoResearch Laboratories, Westgrove, PA).

Secondary antibodies for immunoblotting included affinity-purified peroxidase-conjugated donkey anti-rabbit (1:5,000) or donkey anti-mouse (1:1,000) IgG (Jackson ImmunoResearch Laboratories).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunostaining of Kidney Sections with 8LN-5 Antibody

When frozen sections of paraformaldehyde-fixed normal rat kidney were immunostained with antibody 8LN-5, which was raised against purified laminin-5, followed by a fluorescent secondary antibody, reactivity was observed in the basement membrane of the epithelium lining the major and minor calyces (Fig. 2A) and collecting ducts of the papilla (Fig. 2B). Basement membranes of collecting ducts in the cortex or outer and inner stripes of the outer medulla did not stain (data not shown). In addition, basement membranes of proximal and distal tubules in the cortex and medulla were not immunoreactive (data not shown).


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Fig. 2.   In cryostat sections of normal rat kidney, rabbit polyclonal anti-human 8LN-5 reacts with the basement membrane of the uroepithelium lining the calyx (A) and the collecting ducts of the papilla (B). Bar = 40 µm.

Ischemic injury is most prevalent in the S3 segment of proximal tubules in the outer stripe of the outer medulla, apparently because hemodynamic factors lead to impaired reperfusion of this region of the kidney (6, 13, 47). In sections taken from kidneys made ischemic by clamping of the renal artery, additional staining with 8LN-5 appeared in the outer stripe of the outer medulla. At 0 (Fig. 3A), 1 (Fig. 3B), and 3 h (data not shown) postischemia, immunostaining of this region with 8LN-5 revealed no reactivity. However, at 24 (data not shown) and 48 h postischemia, faint reactivity of basement membranes of S3 proximal tubule segments was seen (Fig. 3C). Stronger localization to basement membranes was present in other tubules at this time, identified as collecting ducts (CT, Fig. 3C) by their distinct lateral borders visible in parallel Nomarski images. At 120 h postischemia (Fig. 3D), strong reactivity with antibody 8LN-5 in the outer stripe was very evident in the basement membranes of regenerating proximal tubules (PT, Fig. 3D). In some instances, the intensity of staining varied within a given tubule, with some regions of the basement membrane showing stronger immunoreactivity than other regions of the same tubule (Fig. 3D). In general, staining of proximal tubules was variable, with a range of reactivities within a single field. Basement membrane localization was also observed in collecting tubules, with some diffuse cytoplasmic staining (CT, Fig. 3D). At all times after reperfusion, antibody 8LN-5 reactivity was not detected in the interstitial matrix. In contralateral (Fig. 3, E and F) and mock-treated (Fig. 3, G and H) kidney tissue, immunostaining with 8LN-5 was not present in the outer stripe of the outer medulla at any time (0-120 h) after reperfusion or mock surgery.


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Fig. 3.   Immunostaining of kidney cryosections of the outer stripe of the outer medulla with the 8LN5 antibody. At 0 (A) and 1 h (B) postischemia, no staining is evident. At 48 h postischemia (C), collecting tubules (CT) are clearly outlined and staining of basement membranes of proximal tubules (PT, outlined with arrowheads) are faintly visible. By 120 h postischemia (D and inset), both distended proximal tubules and collecting tubules are strongly outlined, although staining is variable from tubule to tubule and even within the same tubule. In contralateral controls from the same animal at both 48 (E) and 120 h (F) postischemia, no staining is visible. In kidney sections from mock surgical controls examined either 48 (G) or 120 h (H) after surgery, no immunolabeling with 8LN5 can be discerned. Bars = 40 µm.

In the inner stripe of the outer medulla, 8LN-5 immunostaining revealed no reactivity in renal tubules at 0 (Fig. 4A), 1 (Fig. 4B), and 3 h (data not shown) postischemia. However, at 24 (data not shown) and 48 h (Fig. 4C) postischemia, the antibody highlighted basement membranes of collecting tubules (CT, Fig. 4C) and thick limbs (TL, Fig. 4C); similar staining persisted at 120 h after reperfusion (Fig. 4D). In contralateral (Fig. 4, E and F) and mock (Fig. 4, G and H) surgical controls at equivalent times, reactivity with antibody 8LN-5 was absent from renal tubules of the inner stripe of the outer medulla.


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Fig. 4.   Immunostaining of kidney cryosections of the inner stripe of the outer medulla with the 8LN5 antibody. At 0 (A) and 1 h (B) postischemia, no staining is evident. At 48 h postischemia (C), collecting tubules and thick limbs (TL) are clearly outlined. By 120 h postischemia (D), staining of both collecting tubules and thick limbs is even more intense. In contralateral controls from the same animal at both 48 (E) and 120 h (F) postischemia, no staining is visible. In kidney sections from mock surgical controls examined either 48 (G) or 120 h (H) after surgery, no immunolabeling with 8LN5 can be discerned. Bar = 40 µm.

In the papilla of ischemic kidneys as well as contralateral and mock surgical controls (data not shown), 8LN-5 immunostaining was distributed to the basement membranes lining the major and minor calcyes and collecting tubules, similar to that observed in the normal kidney (see Fig. 2, A and B).

In the cortex, antibody 8LN-5 did not immunostain any renal tubules in response to ischemia-reperfusion (0-120 h postischemia; data not shown). Reactivity was also not observed in the cortex of contralateral and mock kidneys at equivalent times (0-120 h postischemia; data not shown).

In summary, unilateral ischemic injury to the kidney induced 8LN-5 immunoreactivity in the basement membrane of the renal tubular epithelium in the outer and inner stripes of the outer medulla, including the S3 segment of the proximal tubule, in which no staining was observed under normal or control conditions. Immunostaining was not induced in the cortical nephron, and normal reactivity in the papilla was not appreciably increased with ischemic injury.

Immunoblotting of Kidney Extracts with the 8LN-5 Antibody

To provide biochemical evidence for the induction of antibody 8LN-5 reactivity, kidney extracts representing various times after reperfusion were immunoblotted with the same antibody used for immunostaining. In ischemic kidneys at all times after reperfusion (Fig. 5A), a polypeptide chain of ~220 kDa was detected that dramatically increased in level of expression from 24 to 120 h postischemia. This polypeptide may correspond to the precursor of the laminin-5 alpha 3-chain, which is reported to have a molecular mass of 190-200 kDa in humans (4, 29). Beginning at 24 h postischemia, a 140-kDa polypeptide chain was also seen, possibly corresponding to the beta 3-chain (Fig. 5A). No polypeptides migrating at 165 kDa, corresponding to the mature laminin-5 alpha 3-chain, or 105 kDa, corresponding to the mobility of the mature laminin-5 gamma 2-chain, were detected at any time. No reactivity was detected in samples of contralateral (Fig. 5B) or mock (Fig. 5C) kidneys at any time after reperfusion by immunoblotting.


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Fig. 5.   Immunoblotting of kidney extracts with the 8LN5 antibody. In the ischemic kidney (A), a high-molecular-mass 220-kDa polypeptide chain is readily detected after reperfusion and increases in expression at subsequent times thereafter. A 140-kDa band also appears at 24 h postischemia, possibly representing the beta 3-chain of laminin-5. No polypeptides are detected in contralateral (B) or mock (C) kidney. A: purified laminin-5 was used as positive control (+C). B and C: ischemic kidney lysate was used as positive control. Arrowheads, relative molecular mass.

In summary, immunoblotting with 8LN-5 demonstrated induction in ischemic kidneys of a high-molecular-weight polypeptide, possibly corresponding to the precursor of the laminin alpha 3-chain, and another polypeptide comigrating with laminin-beta 3, with peak intensity between 24 and 120 h postischemia.

Induction of Integrin Subunits

If laminin-5 is induced in the kidney by ischemic injury, then it is possible that the expression of cognate integrin-laminin receptors, including the laminin-5 receptors alpha 3beta 1 and alpha 6beta 4 (2, 4, 28, 41), might also be increased. To test this, kidney extracts were immunoblotted with specific antibodies against the beta 1-, beta 4-, and alpha 3-integrin subunits.

Immunoblotting with a rabbit polyclonal anti-human beta 1-integrin antibody raised against a synthetic peptide of the cytoplasmic domain indicated that there was no increased expression from 0 to 120 h postischemia in response to injury and repair (Fig. 6A). In addition, the amount of the beta 1-subunit in ischemic-reperfused kidneys did not appear appreciably different from contralateral (Fig. 6B) or mock (Fig. 6C) surgical controls at equivalent times. However, in samples from ischemic animals, a lower molecular weight band was observed from 24 to 120 h postischemia (arrow, Fig. 6A), which was absent in contralateral (Fig. 6B) or mock (Fig. 6C) kidneys at the same times after reperfusion. This may correspond to an immature, intracellular precursor of beta 1, which has been observed to accumulate in some cells (49, 61).


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Fig. 6.   Immunoblotting of kidney extracts with antibodies specific for beta 1-integrin. In extracts derived from ischemic (A), contralateral (B), or mock surgical control (C) kidneys, the level of beta 1-integrin expression does not change significantly after surgical intervention. In postischemic kidneys at 24-120 h (A), a lower molecular weight band appears (arrow), which is not present in contralateral (B) or mock (C) samples and possibly represents the biosynthetic precursor of beta 1. Postischemic kidney extracts served as positive controls in B and C. Arrowheads, molecular mass markers.

There are conflicting reports on whether beta 4-integrin is present in the kidney (24, 37, 60). Immunoblotting of kidney extracts of ischemic (Fig. 7A), contralateral (Fig. 7B), and mock (Fig. 7C) surgical controls indicated low levels of expression of the beta 4-subunit, with no significant change in ischemic vs. contralateral or mock surgical controls.


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Fig. 7.   Immunoblotting of kidney extracts with antibodies specific for beta 4-integrin. In extracts derived from ischemic (A), contralateral (B), or mock surgical control (C) kidneys, the level of beta 4-integrin expression does not change significantly after ischemic injury or mock surgery. Madin-Darby canine kidney cell lysate served as positive control. Arrowhead, molecular mass marker.

To determine whether the levels of the alpha 3-integrin subunit changed as a result of ischemic injury, extracts were immunoblotted with a rabbit polyclonal anti-alpha 3-integrin antibody generated against a synthetic peptide corresponding to the COOH-terminal cytoplasmic domain of human alpha 3-integrin. Levels of the alpha 3-integrin subunit significantly increased from 3 to 120 h postischemia (Fig. 8A) compared with contralateral (Fig. 8B) and mock (Fig. 8C) kidney extracts at the same time points. Comparable results were obtained with a monoclonal antibody recognizing mouse alpha 3-integrin (data not shown).


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Fig. 8.   Immunoblotting of kidney extracts with antibodies specific for alpha 3-integrin. Levels of the alpha 3-integrin subunit significantly increase from 3 to 120 h in postischemic kidneys (A), whereas in contralateral (B) and mock (C) kidneys, levels appear roughly the same from 0 to 120 h postischemia. Postischemic kidney extracts served as positive controls in B and C. Arrowheads, molecular mass markers.

The alpha 3-integrin subunit was next immunolocalized by using a monoclonal antibody that recognizes the NH2-terminal region of alpha 3 to determine whether its distribution in the ischemic kidney corresponded to observed immunostaining with the 8LN-5 antibody. At 0 (arrow, Fig. 9, A and B), 1 (arrow, Fig. 8, C and D), and 3 h (data not shown) postischemia, alpha 3-integrin immunoreactivity faintly localized to basal plasma membranes of renal tubules in the outer stripe of the outer medulla, similar to the immunostaining pattern seen in corresponding regions of contralateral (data not shown) and mock (data not shown) kidneys at equivalent times. Cellular debris that filled the lumenal space at 1 (*, Fig. 9, C and D) and 3 h (data not shown) postischemia, consisting in part of shed microvilli or apical membrane blebs (62), were unreactive, as expected. Reactivity was also absent from sites in which luminal material contacted apical surfaces of surviving, adherent cells (arrowhead, Fig. 9, C and D).


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Fig. 9.   Immunolocalization of the alpha 3-integrin subunit in the outer stripe of the outer medulla after ischemic injury. A, C, E, and G: immunofluorescent staining of cryostat sections of postischemic kidneys. B, D, F, and H: Nomarski images of identical fields. At 0 (A and B) and 1 h (C and D) postischemia, the alpha 3-integrin subunit faintly localizes to basal plasma membranes (arrows) but is not detected on cellular debris in the tubular space (*; C and D) or between cellular debris and adherent epithelia (arrowhead; C and D). A and B: residual red blood cells (rbc) trapped in the tissue immediately after the vascular clamp are strongly autofluorescent. E and F: at 24 h postischemia, alpha 3 localizes to basal plasma membranes but is not detected on detached cells in the luminal space (*) or on apical surfaces of adherent epithelia (arrowheads). G and H: at 120 h postischemia, when regeneration predominates, alpha 3 strongly localizes to basal and lateral plasma membranes and infrequently to apical cell surfaces (arrows). Luminal material (*) is sometimes immunoreactive. The alpha 3-integrin subunit is not detected in the interstitial space at any time after reperfusion. Exposure of all fluorescent images was identical to that of G. Bar = 40 µm.

At 24 (Fig. 9, E and F) and 48 h (data not shown) postischemia, alpha 3-integrin immunostaining localized to basal plasma membranes of all renal tubules in the outer stripe of the outer medulla; lateral localization was infrequently seen and apical staining of alpha 3 in combination with basal and/or lateral localization was rare. Luminal material representing cellular debris and/or depolarized exfoliated cells (62) did not react with the alpha 3-integrin antibody (*, Fig. 9, E and F). Reactivity was also not detected between detached cells and the adherent epithelium (arrowheads, Fig. 9, E and F). In the outer stripe of contralateral (data not shown) and mock (Fig. 10A) surgical controls, immunoreactivity was faintly seen on basal cell surfaces.


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Fig. 10.   Immunolocalization of alpha 3-integrin in cryosections of a mock surgical kidney (A) and a contralateral kidney (B). Kidneys were removed 24 (A) or 120 h (B) after surgical intervention. In both cases, faint staining is confined to the basal plasma membranes of the renal tubular epithelial cells (arrows). Exposure of both images was identical to that of Fig. 9G. Bar = 40 µm.

At 120 h postischemia, strong alpha 3-integrin immunoreactivity was observed on basal and/or lateral surfaces of tubular epithelial cells in the outer stripe (Fig. 9, G and H). In some instances, staining also appeared apically (arrow, Fig. 9, G and H). Staining intensity sometimes varied among cells within the same tubule. Luminal material, which at this time, represents individual cells or small groups of cells (62), was sometimes immunoreactive (*, Fig. 9, G and H). In contralateral (Fig. 10B) and mock (data not shown) kidneys at this time point, alpha 3 staining was detected only basally, and the intensity of staining was much less than that of the corresponding ischemic samples.

In the inner stripe of the outer medulla at 0 (Fig. 11A), 1 (Fig. 11B), and 3 h (data not shown) postischemia, alpha 3-integrin immunostaining was observed on basal plasma membranes of epithelia lining the distal nephron segment. In collecting ducts (CT, Fig. 11, A and B), faint lateral localization was also observed. At 24 to 48 h postischemia, basal localization remained, but more tubules also showed lateral distribution (Fig. 11C) than at earlier times. A similar pattern persisted at 120 h after ischemic injury, but staining was much more intense (Fig. 11D). In contralateral (Fig. 11F) and mock (Fig. 11E) surgical controls, only relatively faint basal immunostaining of the alpha 3-integrin subunit was detected at all time points.


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Fig. 11.   Immunolocalization of the alpha 3-integrin subunit in the inner stripe of the outer medulla in cryostat sections of postischemic (A-D), contralateral (F), and mock (E) kidneys. At 0 (A) and 1 h (B) postischemia, the alpha 3-integrin subunit localizes to the basal plasma membrane of thick limbs and basolateral surfaces of collecting tubules. At 24 (C) and 120 h (D) postischemia, the alpha 3-integrin subunit is found more frequently on basal and/or lateral plasma membranes of renal tubules. In contralateral (E) and mock (F) kidneys at 24- and 120-h reperfusion, respectively, faint alpha 3 staining is detected only basally. A-C and F: red blood cells are autofluorescent. All images were exposed identically to D. Bar = 40 µm.

In the cortex of ischemic, contralateral, and mock surgical control kidneys, alpha 3 immunostaining was detected in the glomerular capillary tuft, Bowman's capsule, and epithelia lining the distal tubule (Fig. 12), in agreement with published reports (21, 23, 24). However, it was absent from proximal tubular epithelia (Fig. 12), as reported by others (21, 23, 24, 46). There was no staining of the alpha 3-integrin in the interstitium of the kidney cortex (data not shown) and medulla of ischemic, contralateral, or mock kidneys at any time (for outer and inner stripes of ischemic, contralateral, and mock kidneys, see Figs. 9-11).


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Fig. 12.   Immunolocalization of alpha 3-integrin in the renal cortex in a cryosection from a 120-h postischemic kidney. Staining is confined to Bowman's capsule and the capillary tuft of the glomerulus, distal tubule, and collecting duct. Identification of the latter is evident from localization to both basal and straight lateral borders of the cells (CT). Proximal tubules adjacent to the glomerulus react very weakly with the antibody. This overall pattern of staining is identical to that seen in images from contralateral and mock surgical control kidneys and from kidneys taken at earlier times postischemia. A: fluorescent image. B: Nomarski image of the same field. Bar = 40 µm.

In summary, alpha 3-integrin immunostaining significantly increased in the ischemic kidney, particularly from 24 to 120 h postischemia, with peak reactivity at 120 h postischemia. Most of this increase was evident in the outer stripe of the outer medulla and involved regenerating proximal tubules. However, ischemic injury also induced increased immunoreactivity in the inner stripe of the outer medulla in both distal tubules and collecting ducts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this article, we report that unilateral ischemic injury to the rat kidney, caused by clamping of the renal vasculature for 40 min, leads to the induction of a laminin variant, most likely laminin-5, and a laminin receptor, alpha 3beta 1-integrin, in epithelial cells and the underlying matrix of the outer and inner stripe of the outer medulla. Induction begins ~24 h postischemia, with the highest expression noted at 120 h postischemia, the last time point examined. Because induction is so late after the ischemic episode and affects areas of the kidney most damaged, including the S3 segment, it is likely that the new production of this laminin and integrin are related to regeneration of the injured epithelium.

For these studies, we used rabbit polyclonal antibody 8LN-5 generated against affinity-purified laminin-5 to examine the expression of this laminin isoform in both normal and postichemic rat kidneys. By immunoblotting, this antibody recognizes polypeptide chains of 165, 140, and 105 kDa in purified laminin-5- and human keratinocyte-conditioned medium corresponding to the mature, proteolytically processed forms of the alpha 3-, beta 3-, and gamma 2-chains (48). In addition, antibody 8LN-5 recognized the alpha 3 precursor in extracts of human keratinocytes from the immunoblotted and immunostained basement membrane of skin (29). Thus the data indicate that antibody 8LN-5 is specific for the laminin-5 isoform of the laminin family. We cannot, however, rule out the possibility that 8LN-5 reacts with epitopes shared between laminin-5 chains and subunits of other laminin isoforms.

In immunoblots of ischemic kidney extracts, the 8LN-5 antibody recognizes a polypeptide with mobility slightly slower than 200 kDa and one at 140 kDa. In keratinocytes and a variety of other cell types, the laminin-5 alpha 3-subunit is synthesized and secreted as a precursor of ~200 kDa in a complex with the beta 3-chain and a 155-kDa precursor of the gamma 2-subunit (15, 29, 30). At some undetermined point after secretion, the alpha 3 and gamma 2 precursors are processed to their mature size by extracellular proteases, possibly plasmin, metalloendoproteases, or a combination of enzymes (14, 15, 30, 38, 40). On the basis of this biosynthetic pathway and the established specificity of the 8LN5 antibody, we believe that the polypeptides induced in the ischemic kidney are the alpha 3 precursor and the beta 3-chain of the laminin-5 molecule (Burgeson R and Koch M, Cutaneous Biology Research Center, Massachusetts General Hospital, personal communication). Unfortunately, multiple attempts to confirm the identity of the high-molecular- weight polypeptide and the laminin alpha 3-subunit were unsuccessful because appropriate cross-reacting antibodies could not be obtained. Future work utilizing a PCR-based approach and possibly new antibodies will be necessary to confirm our interpretation.

Another confounding factor is the apparently poor recovery in kidney extracts of the beta 3-subunit, as well as the failure to detect the gamma 2-chain. There are several possible explanations for this discrepancy. When laminin-5 was originally isolated from amniotic membranes, very stringent extraction procedures involving extensive collagenase digestion were carried out (48). In our case, kidneys were homogenized and extracted with SDS- and DTT-containing buffers, but without collagenase digestion. Thus it is possible that the three laminin-5 chains, which would have been dissociated by reduction and denaturation, nevertheless were differentially extracted. It is also possible that some chains were proteolyzed during or after harvesting and processing of the kidneys, despite our efforts to inhibit proteolytic enzymes. Finally, it is conceivable that another laminin isoform consisting of the alpha 3-chain found in laminin-5 along with beta - and gamma -chains other than those recognized by the 8LN5 antibody, is induced in the kidney by ischemic injury (32). This could be a completely new isoform or one or both of the other two known laminin isoforms that express alpha 3 (3, 32).

Both laminin-6 (alpha 3beta 1gamma 1) and -7 (alpha 3beta 2gamma 1) contain the alpha 3-chain (3, 32). However, both are reported to covalently associate with laminin-5 by means of disulfide bonding, and their expression in the absence of laminin-5 has not been seen (3). The gamma 1-chain of laminin is a component of most of the 12 known forms of laminin and is expressed throughout the basement membranes of kidney tubules both before and after ischemic injury (32, 57, 58, 62). Because of this and because we have not examined the ischemic kidney for expression of the laminin-beta 2-chain, we cannot rule out the possibility that laminin-6 as well as laminin-7 are induced in the ischemic kidney together with, or even instead of, laminin-5.

Expression of laminin-5 has been reported in the collecting duct system of embryonic mouse and adult human kidney (32, 34, 52). Our immunohistochemical analysis of the renal cortex and medulla of normal rat kidney clearly demonstrates that laminin-5 is expressed only in the basement membranes of papillary collecting ducts and not those of the cortex and outer medulla. This limited distribution is probably the reason we are unable to detect laminin-5 polypeptides by immunoblotting in the nonischemic kidney, given that our extracts are derived from whole kidneys. In the skin, which is a stratified epithelium, laminin-5 plays a structural role, anchoring the epidermis to the dermis via hemidesomosomes (48). Given that the epithelium of the papillary collecting duct stratifies in its distal portions, it is perhaps reasonable to conclude that laminin-5 is playing a somewhat similar role under nonpathological conditions. Furthermore, in contrast to published studies on embryonic mouse and adult human kidneys (34, 52), we do not detect laminin-5 in basement membranes of proximal and distal tubules of normal or control (contralateral and mock) rat kidneys. These reports used polyclonal and monoclonal antibodies generated against the gamma 2-chain of laminin-5, which antibody 8LN-5 also identifies (see Fig. 1A).

After ischemic injury, laminin-5 induction occurs within 24 h of reperfusion, with increasing expression evident up to 120 h postischemia, the last time point examined in this study. Expression occurs in the S3 segment of the proximal tubule, in which most cellular injury is evident, and in the thick limb of the inner stripe of the outer medulla and continues in the collecting ducts. Expression in the collecting ducts becomes more widespread as a result of injury, extending from the papilla to tubules of the inner and outer stripe. The latter is notable because no overt morphological damage is observed in this part of the nephron, on the basis of Nomarski images and immunostaining with antibodies against integrin receptors, extracellular matrix molecules, and apical and basolateral membrane markers (62, 63). In nephrogenesis, laminin-5 promotes branching of the ureteric bud (60), the progenitor of the collecting duct system. Thus it is possible that laminin-5 provides some form of differentiation signal to the cells of the collecting duct, which is initiated by the ischemic insult, perhaps to repair latent defects or strengthen cell-substratum contacts of the epithelium. It is also of interest that no induction of laminin-5 was seen in cortical proximal tubules, which depolarize in response to ischemia but quickly regenerate polarity on reperfusion.

In the S3 segment, it is likely that laminin-5, along with the laminin receptor integrin alpha 3beta 1 (see below), play important roles in the repair and regeneration of tubular epithelium. In vitro studies have established that the laminin-5 precursor, which contains the 200-kDa form of the alpha 3-subunit as well as a precursor of the gamma 2-chain (15, 29, 30), stimulates migration of keratinocytes to close wounds (5, 14, 15, 38-40). In this process, laminin-5 interacts with alpha 3beta 1-integrin, generating signals that regulate interaction of cells with collagen via alpha 2beta 1-integrin (40). alpha 2beta 1-Integrin may function as a receptor for laminin-5 in this process as well (5). Processing of laminin-5 chains may then inhibit migration, possibly due to a switch of laminin-5 from alpha 3beta 1 to alpha 6beta 4 and its incorporation in contacts mediated by hemidesmosomes (14, 15, 38-40, 42). Laminin-5 processing is also involved in migration of breast epithelial cells (11, 12). Similarly, it has been postulated that laminin-5 modulates alpha 3beta 1- and alpha 2beta 1-integrins to facilitate enterocytic differentiation of the colon carcinoma cell line HT29 (16). Therefore, it seems reasonable to propose that in the postischemic kidney, the induction of laminin-5 and its integrin receptor alpha 3beta 1 are related to redifferentiation and repolarization of the cells to restore normal function. Under these circumstances, we would not expect to see a conversion of the laminin alpha 3-subunit to its mature form because no hemidesmosomes are present in the kidney; in fact, no such conversion is observed.

Integrin receptors for laminin-5 include alpha 3beta 1, alpha 6beta 4, and alpha 6beta 1 (2, 4, 28, 41). We hypothesized that increased expression of laminin-5 in response to ischemic injury might also induce increased or more widespread expression of its cognate integrin receptors. In fact, ischemia dramatically upregulated the expression of the alpha 3-integrin subunit, although no changes in the levels of the beta 1-subunit were observed. In the normal kidney, alpha 3-integrin is expressed in epithelial cells of distal tubules and collecting ducts but not in proximal tubules (21, 46). Although we generally agree with these observations, we do detect alpha 3-integrin in epithelial cells of proximal tubules in the outer stripe of the outer medulla, albeit at low levels.

With ischemic injury, alpha 3-integrin expression in proximal tubules of the outer stripe significantly increases. Expression also continues to be evident and may be increased in the glomerulus, distal tubules, and collecting ducts. alpha 3beta 1-Integrin, in addition to being a receptor for laminin-5, is also a receptor for types of collagen and other laminin isotypes, including laminin-10 (4, 19). Although we consider it likely that one of its roles in the postischemic proximal tubule is as a receptor for laminin-5, which is coordinately induced, we cannot prima facie rule out the involvement of alpha 3beta 1-integrin as a receptor for other extracellular matrix ligands. Indeed, alpha 3beta 1 is clearly playing other, distinctive roles in other regions of the nephron under normal circumstances and, most likely, after injury (23, 24). This conclusion is reinforced by the observation that the breadth and intensity of the increase in alpha 3beta 1 expression in the ischemic kidney appears to be greater than that of the induced laminin isoform.

As described previously, alpha 6beta 4 is also a laminin-5 receptor that forms part of the hemidesmosomal complex in skin. On this basis, it would seem unlikely that beta 4 expression would be increased in the postischemic kidney. On the other hand, the beta 4-integrin subunit is the major receptor for laminin-5 during embryonic development of the collecting ducts (60) and therefore might play a role in the regeneration process. Nevertheless, we did not detect any change in beta 4-integrin levels, which are low in the normal kidney, suggesting that the receptor for the upregulated laminin variant was not alpha 6beta 4-integrin.

We also attempted to examine the relative expression of the alpha 6-integrin subunit but were unable to find suitable antibodies that cross-reacted with the rat protein. Given that alpha 6beta 1 is a major laminin receptor expressed throughout the tubules of the adult kidney (24), it is possible that it may also play a major role in recovery from ischemic injury. In the future, when better reagents are available, its function in epithelial regeneration will have to be reevaluated.

The distribution of the alpha 3-integrin subunit in the injured and regenerating kidney agrees with our published data on beta 1-integrin, the only beta -subunit with which alpha 3 is known to dimerize. We did not detect the alpha 3-integrin subunit on exfoliated cells in the luminal space after ischemic injury, supporting our laboratory's observation that members of the beta 1-integrin family are absent on detached cells (62). In addition, in the injury phase, the alpha 3-integrin subunit was not detected on apical surfaces of the surviving epithelium, also supporting our laboratory's earlier finding that the beta 1-integrin family is absent from this location (62). These observations support our laboratory's previous conclusion that members of the beta 1-integrin family, including alpha 3beta 1, do not mediate cell-cell adhesion between attached and exfoliated cells and therefore do not contribute in this manner to tubular obstruction.

In conclusion, our immunocytochemical analysis demonstrates that a laminin variant and alpha 3beta 1-integrin are induced in the tubular basement membrane and epithelium of the kidney by ischemic injury during the phase at which epithelial cell redifferentiation and repolarization are known to occur. On the basis of immunoblotting and the known processing pathway of laminin-5, we believe that the laminin isoform induced is laminin-5, although we cannot rule out other possibilities at the present time. Overall, these results suggest that fundamental elements of epithelial regeneration may depend on novel laminin-integrin interactions in epithelial cells from tissues as diverse as those of the kidney, intestine, and skin.


    ACKNOWLEDGEMENTS

We thank the Morphology Core of the Harvard Digestive Disease Center (DK-34854) for use of the Leica cryostat, Dr. Elizabeth D. Hay, Department of Cell Biology, Harvard Medical School, for use of the Zeiss Axiophot microscope, and Dr. Manuel Koch, Cutaneous Biology Research Center, Massachusetts General Hospital, for helpful discussions. We are also grateful to Holly Waechter for technical assistance and Dr. Steven Boyce of the Shriners Burns Institute, University of Cincinnati College of Medicine, for providing cultured human keratinocytes.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-46768 (K. S. Matlin) and a Scholars in Medicine Grant and William F. Milton Fund Award (A. Zuk).

Address for reprint requests and other correspondence: K. S. Matlin, The Vontz Ctr., ML0581, Univ. of Cincinnati College of Medicine, 3125 Eden Ave., Cincinnati, OH 45219-0581 (E-mail: karl.matlin{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 16, 2002;10.1152/ajprenal.00176.2002

Received 3 May 2002; accepted in final form 9 July 2002.


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